National Renewable Energy Laboratory 

NREL/TP-580-24190 

A Look Back at the 

U.S. Department of Energy’s 

Aquatic Species Program: 

Biodiesel from Algae 

Close-Out Report 
NREL/TP-580-24190

A Look Back at the U.S. Department of Energy’s Aquatic Species

Program—Biodiesel from Algae

July 1998

By

John Sheehan

Terri Dunahay

John Benemann

Paul Roessler

Prepared for:

U.S. Department of Energy’s

Office of Fuels Development

Prepared by: the

National Renewable Energy Laboratory

1617 Cole Boulevard

Golden, Colorado 80401-3393

A national laboratory of the U.S. Department of Energy

Operated by Midwest Research Institute

Under Contract No. DE-AC36-83CH10093
Executive Summary

From 1978 to 1996, the U.S. Department of Energy’s Office of Fuels Development funded a program to

develop renewable transportation fuels from algae. The main focus of the program, know as the Aquatic

Species Program (or ASP) was the production of biodiesel from high lipid-content algae grown in ponds,

utilizing waste CO2 from coal fired power plants. Over the almost two decades of this program,

tremendous advances were made in the science of manipulating the metabolism of algae and the

engineering of microalgae algae production systems. Technical highlights of the program are summarized

below:

Applied Biology

A unique collection of oil-producing microalgae.

The ASP studied a fairly specific aspect of algae—their ability to produce natural

oils. Researchers not only concerned themselves with finding algae that produced a

lot of oil, but also with algae that grow under severe conditions—extremes of

temperature, pH and salinity. At the outset of the program, no collections existed that

either emphasized or characterized algae in terms of these constraints. Early on,

researchers set out to build such a collection. Algae were collected from sites in the

west, the northwest and the southeastern regions of the continental U.S., as well as

Hawaii. At its peak, the collection contained over 3,000 strains of organisms. After

screening, isolation and characterization efforts, the collection was eventually

winnowed down to around 300 species, mostly green algae and diatoms. The

collection, now housed at the University of Hawaii, is still available to researchers.

This collection is an untapped resource, both in terms of the unique organisms

available and the mostly untapped genetic resource they represent. It is our sincere

hope that future researchers will make use of the collection not only as a source of

new products for energy production, but for many as yet undiscovered new products

and genes for industry and medicine.

Shedding light on the physiology and biochemistry of algae.

Prior to this program, little work had been done to improve oil production in algal

organisms. Much of the program’s research focused attention on the elusive “lipid

trigger.” (Lipids are another generic name for TAGs, the primary storage form of

natural oils.) This “trigger” refers to the observation that, under environmental stress,

many microalgae appeared to flip a switch to turn on production of TAGs. Nutrient

deficiency was the major factor studied. Our work with nitrogen-deficiency in algae

and silicon deficiency in diatoms did not turn up any overwhelming evidence in

support of this trigger theory. The common thread among the studies showing

increased oil production under stress seems to be the observed cessation of cell

division. While the rate of production of all cell components is lower under nutrient

starvation, oil production seems to remain higher, leading to an accumulation of oil in

the cells. The increased oil content of the algae does not to lead to increased overall

productivity of oil. In fact, overall rates of oil production are lower during periods of

nutrient deficiency. Higher levels of oil in the cells are more than offset by lower

rates of cell growth.
National Renewable Energy Laboratory

A Look Back at the Aquatic Species Program—Executive Summary ii

Breakthroughs in molecular biology and genetic engineering.

Plant biotechnology is a field that is only now coming into its own. Within the field of plant

biotechnology, algae research is one of the least trodden territories. The slower rate of advance in this field

makes each step forward in our research all the more remarkable. Our work on the molecular biology and

genetics of algae is thus marked with significant scientific discoveries. The program was the first to isolate

the enzyme Acetyl CoA Carboxylase (ACCase) from a diatom. This enzyme was found to catalyze a key

metabolic step in the synthesis of oils in algae. The gene that encodes for the production of ACCase was

eventually isolated and cloned. This was the first report of the cloning of the full sequence of the ACCase

gene in any photosynthetic organism. With this gene in hand, researchers went on to develop the first

successful transformation system for diatoms—the tools and genetic components for expressing a foreign

gene. The ACCase gene and the transformation system for diatoms have both been patented. In the

closing days of the program, researchers initiated the first experiments in metabolic engineering as a means

of increasing oil production. Researchers demonstrated an ability to make algae over-express the ACCase

gene, a major milestone for the research, with the hope that increasing the level of ACCase activity in the

cells would lead to higher oil production. These early experiments did not, however, demonstrate increased

oil production in the cells.

Algae Production Systems

Demonstration of Open Pond Systems for Mass Production of Microalgae.

Over the course of the program, efforts were made to establish the feasibility of large-scale algae

production in open ponds. In studies conducted in California, Hawaii and New Mexico, the ASP proved

the concept of long term, reliable production of algae. California and Hawaii served as early test bed sites.

Based on results from six years of tests run in parallel in California and Hawaii, 1,000 m2 pond systems

were built and tested in Roswell, New Mexico. The Roswell, New Mexico tests proved that outdoor ponds

could be run with extremely high efficiency of CO2 utilization. Careful control of pH and other physical

conditions for introducing CO2 into the ponds allowed greater than 90% utilization of injected CO2. The

Roswell test site successfully completed a full year of operation with reasonable control of the algal species

grown. Single day productivities reported over the course of one year were as high as 50 grams of algae

per square meter per day, a long-term target for the program. Attempts to achieve consistently high

productivities were hampered by low temperature conditions encountered at the site. The desert conditions

of New Mexico provided ample sunlight, but temperatures regularly reached low levels (especially at

night). If such locations are to be used in the future, some form of temperature control with enclosure of

the ponds may well be required.

The high cost of algae production remains an obstacle.

The cost analyses for large-scale microalgae production evolved from rather

superficial analyses in the 1970s to the much more detailed and sophisticated studies

conducted during the 1980s. A major conclusion from these analyses is that there is

little prospect for any alternatives to the open pond designs, given the low cost

requirements associated with fuel production. The factors that most influence cost

are biological, and not engineering-related. These analyses point to the need for

highly productive organisms capable of near-theoretical levels of conversion of

sunlight to biomass. Even with aggressive assumptions about biological

productivity, we project costs for biodiesel which are two times higher than current

petroleum diesel fuel costs.
National Renewable Energy Laboratory

A Look Back at the Aquatic Species Program—Executive Summary iii

Resource Availability

Land, water and CO2 resources can support substantial biodiesel production and CO2

savings.

The ASP regularly revisited the question of available resources for producing biodiesel from microalgae.

This is not a trivial effort. Such resource assessments require a combined evaluation of appropriate climate,

land and resource availability. These analyses indicate that significant potential land, water and CO2

resources exist to support this technology. Algal biodiesel could easily supply several “quads” of

biodiesel—substantially more than existing oilseed crops could provide. Microalgae systems use far less

water than traditional oilseed crops. Land is hardly a limitation. Two hundred thousand hectares (less than

0.1% of climatically suitable land areas in the U.S.) could produce one quad of fuel. Thus, though the

technology faces many R&D hurdles before it can be practicable, it is clear that resource limitations are not

an argument against the technology.
A Look Back at the U.S.

Department of Energy’s

Aquatic Species Program:

Biodiesel from Algae

Part I:

Program Summary
Background

Origins of the Program

This year marks the 20th anniversary of the National Renewable Energy Laboratory

(NREL). In 1978, the Carter Administration established what was then called the

Solar Energy Research Institute (SERI) in Golden, CO. This was a first-of-its kind

federal laboratory dedicated to the development of solar energy. The formation of

this lab came in response to the energy crises of the early and mid 1970s. At the

same time, the Carter Administration consolidated all federal energy activities under

the auspices of the newly established U.S. Department of Energy (DOE).

Among its various programs established to develop all forms of solar energy, DOE

initiated research on the use of plant life as a source of transportation fuels. Today,

this program—known as the Biofuels Program—is funded and managed by the

Office of Fuels Development (OFD) within the Office of Transportation

Technologies under the Assistant Secretary for Energy Efficiency and Renewable

Energy at DOE. The program has, over the years, focused on a broad range of

alternative fuels, including ethanol and methanol (alcohol fuel substitutes for

gasoline), biogas (methane derived from plant materials) and biodiesel (a natural oilderived diesel fuel substitute). The Aquatic Species Program (ASP) was just one

component of research within the Biofuels Program aimed at developing alternative

sources of natural oil for biodiesel production.

Close-out of the Program

The Aquatic Species Program (ASP) was a relatively small research effort intended

to look at the use of aquatic plants as sources of energy. While its history dates back

to 1978, much of the research from 1978 to 1982 was focused on using algae to

produce hydrogen. The program switched emphasis to other transportation fuels, in

particular biodiesel, beginning in the early 1980s. This report provides a summary of

the research activities carried out from 1980 to 1996, with an emphasis on algae for

biodiesel production.

In 1995, DOE made the difficult decision to eliminate funding for algae research

within the Biofuels Program. Under pressure to reduce budgets, the Department

chose a strategy of more narrowly focusing its limited resources in one or two key

areas, the largest of these being the development of bioethanol. The purpose of this

report is to bring closure to the Biofuels Program’s algae research. This report is a

summary and compilation of all the work done over the last 16 years of the program.

It includes work carried out by NREL researchers at our labs in Golden, as well as

subcontracted research and development activities conducted by private companies

and universities around the country. More importantly, this report should be seen not

as an ending, but as a beginning. When the time is right, we fully expect to see

renewed interest in algae as a source of fuels and other chemicals. The highlights

presented here should serve as a foundation for these future efforts.

A Look Back at the Aquatic Species Program—Program Summary
What is the technology?

Biological Concepts

Photosynthetic organisms include plants, algae and some photosynthetic bacteria.

Photosynthesis is the key to making solar energy available in useable forms for all

organic life in our environment. These organisms use energy from the sun to

combine water with carbon dioxide (CO2) to create biomass. While other elements of

the Biofuels Program have focused on terrestrial plants as sources of fuels, ASP was

concerned with photosynthetic organisms that grew in aquatic environments. These

include macroalgae, microalgae and emergents. Macroalgae, more commonly known

as “seaweed,” are fast growing marine and freshwater plants that can grow to

considerable size (up to 60m in length). Emergents are plants that grow partially

submerged in bogs and marshes. Microalgae are, as the name suggests, microscopic

photosynthetic organisms. Like macroalgae, these organisms are found in both

marine and freshwater environments. In the early days of the program, research was

done on all three types of aquatic species. As emphasis switched to production of

natural oils for biodiesel, microalgae became the exclusive focus of the research.

This is because microalgae generally produce more of the right kinds of natural oils

needed for biodiesel (see the discussion of fuel concepts presented later in this

overview).

In many ways, the study of microalgae is a relatively limited field of study. Algae

are not nearly as well understood as other organisms that have found a role in today’s

biotechnology industry. This is part of what makes our program so valuable. Much

of the work done over the past two decades represents genuine additions to the

scientific literature. The limited size of the scientific community involved in this

work also makes it more difficult, and sometimes slower, compared to the progress

seen with more conventional organisms. The study of microalgae represents an area

of high risk and high gains.

These photosynthetic organisms are far from monolithic. Biologists have categorized

microalgae in a variety of classes, mainly distinguished by their pigmentation, life

cycle and basic cellular structure. The four most important (at least in terms of

abundance) are:

• The diatoms (Bacillariophyceae). These algae dominate the

phytoplankton of the oceans, but are also found in fresh and

brackish water. Approximately 100,000 species are known to

exist. Diatoms contain polymerized silica (Si) in their cell walls.

All cells store carbon in a variety of forms. Diatoms store

carbon in the form of natural oils or as a polymer of

carbohydrates known as chyrsolaminarin.

• The green algae (Chlorophyceae). These are also quite

abundant, especially in freshwater. (Anyone who owns a

swimming pool is more than familiar with this class of algae).

They can occur as single cells or as colonies. Green algae are the

evolutionary progenitors of modern plants. The main storage

compound for green algae is starch, though oils can be produced

under certain conditions.

2 A Look Back at the Aquatic Species Program—Program Summary
• The blue-green algae (Cyanophyceae). Much closer to bacteria

in structure and organization, these algae play an important role

in fixing nitrogen from the atmosphere. There are approximately

2,000 known species found in a variety of habitats.

• The golden algae (Chrysophyceae). This group of algae is

similar to the diatoms. They have more complex pigment

systems, and can appear yellow, brown or orange in color.

Approximately 1,000 species are known to exist, primarily in

freshwater systems. They are similar to diatoms in pigmentation

and biochemical composition. The golden algae produce natural

oils and carbohydrates as storage compounds.

The bulk of the organisms collected and studied in this program fall in the first two

classes—the diatoms and the green algae.

Microalgae are the most primitive form of plants. While the mechanism of

photosynthesis in microalgae is similar to that of higher plants, they are generally

more efficient converters of solar energy because of their simple cellular structure.

In addition, because the cells grow in aqueous suspension, they have more efficient

access to water, CO2, and other nutrients. For these reasons, microalgae are capable

of producing 30 times the amount oil per unit area of land, compared to terrestrial

oilseed crops.

Put quite simply, microalgae are remarkable and efficient biological factories capable of

taking a waste (zero-energy) form of carbon (CO2) and converting it into a high density

liquid form of energy (natural oil). This ability has been the foundation of the research

program funded by the Office Fuels Development.

Algae Production Concepts

Like many good ideas (and certainly many of the concepts that are now the basis for

renewable energy technology), the concept of using microalgae as a source of fuel is

older than most people realize. The idea of producing methane gas from algae was

proposed in the early 1950s1. These early researchers visualized a process in which

wastewater could be used as a medium and source of nutrients for algae production.

The concept found a new life with the energy crisis of the 1970s. DOE and its

predecessors funded work on this combined process for wastewater treatment and

energy production during the 1970s. This approach had the benefit of serving

multiple needs—both environmental and energy-related. It was seen as a way of

introducing this alternative energy source in a near-term timeframe.

In the 1980s, DOE’s program gradually shifted its focus to technologies that could

have large-scale impacts on national consumption of fossil energy. Much of DOE’s

publications from this period reflect a philosophy of energy research that might,

somewhat pejoratively, be called “the quads mentality.” A quad is a short-hand name

for the unit of energy often used by DOE to describe the amounts of energy that a

given technology might be able to displace. Quad is short for “quadrillion Btus”—a

unit of energy representing 1015 (1,000,000,000,000,000) Btus of energy. This

perspective led DOE to focus on the concept of immense algae farms.

A Look Back at the Aquatic Species Program—Program Summary 3 
Such algae farms would be based on the use of open, shallow ponds in which some

source of waste CO2 could be efficiently bubbled into the ponds and captured by the

algae (see the figure below).

Water

Nutrients

Algae

Waste CO2

Motorized 

paddle 

wheel

The ponds are “raceway” designs, in which the algae, water and nutrients circulate

around a racetrack. Paddlewheels provide the flow. The algae are thus kept

suspended in water. Algae are circulated back up to the surface on a regular

frequency. The ponds are kept shallow because of the need to keep the algae

exposed to sunlight and the limited depth to which sunlight can penetrate the pond

water. The ponds are operated continuously; that is, water and nutrients are

constantly fed to the pond, while algae-containing water is removed at the other end.

Some kind of harvesting system is required to recover the algae, which contains

substantial amounts of natural oil.

The concept of an “algae farm” is illustrated on the next page. The size of these

ponds is measured in terms of surface area (as opposed to volume), since surface area

is so critical to capturing sunlight. Their productivity is measured in terms of

biomass produced per day per unit of available surface area. Even at levels of

productivity that would stretch the limits of an aggressive research and development

program, such systems will require acres of land. At such large sizes, it is more

appropriate to think of these operations on the scale of a farm.

There are quite a number of sources of waste CO2. Every operation that involves

combustion of fuel for energy is a potential source. The program targeted coal and

other fossil fuel-fired power plants as the main sources of CO2. Typical coal-fired

power plants emit flue gas from their stacks containing up to 13% CO2. This high

concentration of CO2 enhances transfer and uptake of CO2 in the ponds. The concept

of coupling a coal-fired power plant with an algae farm provides an elegant approach

to recycle of the CO2 from coal combustion into a useable liquid fuel.

4 A Look Back at the Aquatic Species Program—Program Summary
CO2 

Recovery 

System

Algae/Oil 

Recovery

System

Fuel 

Production

Other system designs are possible. The Japanese, French and German governments

have invested significant R&D dollars on novel closed bioreactor designs for algae

production. The main advantage of such closed systems is that they are not as

subject to contamination with whatever organism happens to be carried in the wind.

The Japanese have, for example, developed optical fiber-based reactor systems that

could dramatically reduce the amount of surface area required for algae production.

While breakthroughs in these types of systems may well occur, their costs are, for

now, prohibitive—especially for production of fuels. DOE’s program focused

primarily on open pond raceway systems because of their relative low cost.

The Aquatic Species Program envisioned vast arrays of algae ponds covering acres of

land analogous to traditional farming. Such large farms would be located adjacent to

power plants. The bubbling of flue gas from a power plant into these ponds provides a

system for recycling of waste CO2 from the burning of fossil fuels.

Fuel Production Concepts

The previous sections have alluded to a number of potential fuel products from algae.

The ASP considered three main options for fuel production:

• Production of methane gas via biological or thermal gasification.

• Production of ethanol via fermentation

A Look Back at the Aquatic Species Program—Program Summary 5
• Production of biodiesel

A fourth option is the direct combustion of the algal biomass for production of steam

or electricity. Because the Office of Fuels Development has a mandate to work on

transportation fuels, the ASP did not focus much attention on direct combustion. The

concept of algal biomass as a fuel extender in coal-fired power plants was evaluated

under a separate program funded by DOE’s Office of Fossil Fuels. The Japanese

have been the most aggressive in pursuing this application. They have sponsored

demonstrations of algae production and use at a Japanese power plant.

Algal biomass contains three main components:

• Carbohydrates

• Protein

• Natural Oils

The economics of fuel production from algae (or from any biomass, for that matter)

demands that we utilize all the biomass as efficiently as possible. To achieve this, the

three fuel production options listed previously can be used in a number of

combinations. The most simplistic approach is to produce methane gas, since the

both the biological and thermal processes involved are not very sensitive to what

form the biomass is in. Gasification is a somewhat brute force technology in the

sense that it involves the breakdown of any form of organic carbon into methane.

Ethanol production, by contrast, is most effective for conversion of the carbohydrate

fraction. Biodiesel production applies exclusively to the natural oil fraction. Some

combination of all three components can also be utilized as an animal feed. Process

design models developed under the program considered a combination of animal feed

production, biological gasification and biodiesel production.

The main product of interest in the ASP was biodiesel. In its most general sense,

biodiesel is any biomass-derived diesel fuel substitute. Today, biodiesel has come to

mean a very specific chemical modification of natural oils. Oilseed crops such as

rapeseed (in Europe) and soybean oil (in the U.S.) have been extensively evaluated as

sources of biodiesel. Biodiesel made from rapeseed oil is now a substantial

commercial enterprise in Europe. Commercialization of biodiesel in the U.S. is still

in its nascent stage.

The bulk of the natural oil made by oilseed crops is in the form of triacylglycerols

(TAGs). TAGs consist of three long chains of fatty acids attached to a glycerol

backbone. The algae species studied in this program can produce up to 60% of their

body weight in the form of TAGs. Thus, algae represent an alternative source of

biodiesel, one that does not compete with the existing oilseed market.

As a matter of historical interest, Rudolph Diesel first used peanut oil (which is

mostly in the form of TAGs) at the turn of the century to demonstrate his patented

diesel engine2. The rapid introduction of cheap petroleum quickly made petroleum

the preferred source of diesel fuel, so much so that today’s diesel engines do not

operate well when operated on unmodified TAGs. Natural oils, it turns out, are too

viscous to be used in modern diesel engines.

6 A Look Back at the Aquatic Species Program—Program Summary
In the 1980s, a chemical modification of natural oils was introduced that helped to

bring the viscosity of the oils within the range of current petroleum diesel3. By

reacting these TAGs with simple alcohols (a chemical reaction known as

“transesterification” already commonplace in the oleochemicals industry), we can

create a chemical compound known as an alkyl ester4, but which is known more

generically as biodiesel (see the figure below). Its properties are very close to those

of petroleum diesel fuel.

HC

HC

HC COOH

COOH

COOH

3(CH2OH)

O CH2

O CH2

O CH2

+

1 molecule of glycerol

1 TAG 3 molecules of biodiesel

3 molecules of alcohol HCOH

HCOH

HCOH

+

Commercial experience with biodiesel has been very promising5. Biodiesel performs

as well as petroleum diesel, while reducing emissions of particulate matter, CO,

hydrocarbons and SOx. Emissions of NOx are, however, higher for biodiesel in many

engines. Biodiesel virtually eliminates the notorious black soot emissions associated

with diesel engines. Total particulate matter emissions are also much lower6,7,8.

Other environmental benefits of biodiesel include the fact that it is highly

biodegradable9 and that it appears to reduce emissions of air toxics and carcinogens

(relative to petroleum diesel)10. A proper discussion of biodiesel would require much

more space than can be accommodated here. Suffice it to say that, given many of its

environmental benefits and the emerging success of the fuel in Europe, biodiesel is a

very promising fuel product.

High oil-producing algae can be used to produce biodiesel, a chemically modified

natural oil that is emerging as an exciting new option for diesel engines. At the same

time, algae technology provides a means for recycling waste carbon from fossil fuel

combustion. Algal biodiesel is one of the only avenues available for high-volume re-use

of CO2 generated in power plants. It is a technology that marries the potential need for

carbon disposal in the electric utility industry with the need for clean-burning

alternatives to petroleum in the transportation sector.

Why microalgae technology?

There are a number of benefits that serve as driving forces for developing and

deploying algae technology. Some of these benefits have already been mentioned.

Four key areas are outlined here. The first two address national and international

issues that continue to grow in importance—energy security and climate change. The

A Look Back at the Aquatic Species Program—Program Summary 7
remaining areas address aspects of algae technology that differentiate it from other

technology options being pursued by DOE.

Energy Security

Energy security is the number one driving force behind DOE’s Biofuels Program.

The U.S. transportation sector is at the heart of this security issue. Cheap oil prices

during the 1980s and 1990s have driven foreign oil imports to all time highs. In

1996, imports reached an important milestone—imported oil consumption exceeded

domestic oil consumption. DOE’s Energy Information Administration paints a dismal

picture of our growing dependence on foreign oil. Consider these basic points11:

• Petroleum demand is increasing, especially due to new demand

from Asian markets.

• New demand for oil will come primarily from the Persian Gulf.

• As long as prices for petroleum remain low, we can expect our

imports to exceed 60% of our total consumption ten years from

now.

• U.S. domestic supplies will likewise remain low as long as prices

for petroleum remain low.

Not everyone shares this view of the future, or sees it as a reason for concern. The

American Petroleum Institute12 does not see foreign imports as a matter of national

security. Others have argued that the prediction of increasing Mideast oil

dependence worldwide is wrong. But the concern about our foreign oil addiction is

widely held by a broad range of political and commercial perspectives13.

While there may be uncertainty and even contention over when and if there is a

national security issue, there is one more piece to the puzzle that influences our

perspective on this issue. This is the fact that, quite simply, 98% of the transportation

sector in the U.S. relies on petroleum (mostly in the form of gasoline and diesel fuel).

The implication of this indisputable observation is that even minor hiccups in the

supply of oil could have crippling effects on our nation. This lends special

significance to the Biofuels Program as a means of diversifying the fuel base in our

transportation sector.

Our almost complete reliance on petroleum in transportation comes from the demand for

gasoline in passenger vehicles and the demand for diesel fuel in commerce. Bioethanol

made from terrestrial energy crops offers a future alternative to gasoline, biodiesel made

from algal oils could do the same for diesel fuel.

Climate Change

CO2 is recognized as the most important (at least in quantity) of the atmospheric

pollutants that contribute to the “greenhouse effect,” a term coined by the French

mathematician Fourier in the mid-1800s to describe the trapping of heat in the

Earth’s atmosphere by gases capable of absorbing radiation. By the end of the last

century, scientists were already speculating on the potential impacts of anthropogenic

8 A Look Back at the Aquatic Species Program—Program Summary
CO2. The watershed event that brought the question of global warming to the

forefront in the scientific community was the publication of Revelle’s data in 1957,

which quantified the geologically unprecedented build-up of atmospheric CO2 that

began with the advent of the industrial revolution. Revelle14 characterized the

potential risk of global climate change this way:

“Human beings are carrying out a large scale geophysical experiment of

a kind that could not have happened in the past nor be produced in the

future. Within a few centuries, we are returning to the atmosphere and

the oceans the concentrated organic carbon stored in sedimentary rocks

over hundreds of millions of years.”

Despite 40 years of research since Revelle first identified the potential risk of global

warming, the debate over the real impacts of the increased CO2 levels still rages. We

may never be able to scientifically predict the climatic effects of increasing carbon

dioxide levels due to the complexity of atmospheric and meteorological modeling.

Indeed, Revelle’s concise statement of the risks at play in global climate change

remains the best framing of the issue available for policy makers today. The question

we face as a nation is how much risk we are willing to take on an issue like this. That

debate has never properly taken place with the American public.

As Revelle’s statement implies, the burning of fossil fuels is the major source of the

current build up of atmospheric CO2. Thus, identifying alternatives to fossil fuels must be

a key strategy in reducing greenhouse gas emissions. While no one single fuel can

substitute for fossil fuels in an all of the energy sectors, we believe that biodiesel made

from algal oils is a fuel which can make a major contribution to the reduction of CO2

generated by power plants and commercial diesel engines.

The Synergy of Coal and Microalgae

Many of our fossil fuel reserves, but especially coal, are going to play significant

roles for years to come. On a worldwide basis, coal is, by far, the largest fossil energy

resource available. About one-fourth of the world’s coal reserves reside in the

United States. To put this in perspective, consider the fact that, at current rates of

consumption, coal reserves could last for over 200 years.

Regardless of how much faith you put in future fossil energy projections, it is clear

that coal will continue to play an important role in our energy future—especially

given the relatively large amounts of coal that we control within our own borders.

DOE’s Energy Information Administration estimates that electricity will become an

increasingly large contributor to future U.S. energy demand. How will this new

demand be met? Initially, low cost natural gas will grow in use. Inevitably, the

demand for electricity will have to be met by coal. Coal will remain the mainstay of

U.S. baseline electricity generation, accounting for half of electricity generation by

the year 2010.

The long term demand for coal brings with it a demand for technologies that can

mitigate the environmental problems associated with coal. While control

technologies will be used to reduce air pollutants associated with acid rain, no

technologies exist today which address the problem of greenhouse gas emissions.

Coal is the most carbon-intensive of the fossil fuels. In other words, for every Btu of

energy liberated by combustion, coal emits more CO2 than either petroleum or

A Look Back at the Aquatic Species Program—Program Summary 9
natural gas. As pressure to reduce carbon emissions grows, this will become an

increasingly acute problem for the U.S.

One measure of how serious this problem could be is the absurdity of some of the

proposals being developed for handling carbon emissions from power plants. The

preferred option offered by researchers at MIT is ocean disposal, despite the expense

and uncertainty of piping CO2 from power plants and injecting the CO2 in the

ocean15.

Commonsense suggests that recycling of carbon would be more efficacious than deep

ocean disposal. No one clearly understands the long-term effects of injecting large

amounts of CO2 into our oceans. Beyond these environmental concerns, such largescale disposal schemes represent an economic sinkhole. Huge amounts of capital and

operating dollars would be spent simply to dispose of carbon. While such Draconian

measures may ultimately be needed, it makes more sense to first re-use stationary

sources of carbon as much as possible. Algae technology is unique in its ability to

produce a useful, high-volume product from waste CO2.

Consumption of coal, an abundant domestic fuel source for electricity generation, will

continue to grow over the coming decades, both in the U.S. and abroad. Algae

technology can extend the useful energy we get from coal combustion and reduce carbon

emissions by recycling waste CO2 from power plants into clean-burning biodiesel. When

compared to the extreme measures proposed for disposing of power plant carbon

emissions, algal recycling of carbon simply makes sense.

Terrestrial versus Aquatic Biomass

Algae grow in aquatic environments. In that sense, algae technology will not

compete for the land already being eyed by proponents of other biomass-based fuel

technologies. Biomass power and bioethanol both compete for the same land and for

similar feedstocks—trees and grasses specifically grown for energy production.

More importantly, many of the algal species studied in this program can grow in

brackish water—that is, water that contains high levels of salt. This means that algae

technology will not put additional demand on freshwater supplies needed for

domestic, industrial and agricultural use.

The unique ability of algae to grow in saline water means that we can target areas of

the country in which saline groundwater supplies prevent any other useful application

of water or land resources. If we were to draw a map showing areas best suited for

energy crop production (based on climate and resource needs), we would see that

algae technology needs complement the needs of both agriculture and other biomassbased energy technologies.

In a world of ever more limited natural resources, algae technology offers the

opportunity to utilize land and water resources that are, today, unsuited for any other

use. Land use needs for microalgae complement, rather than compete, with other

biomass-based fuel technologies.

10 A Look Back at the Aquatic Species Program—Program Summary
Technical Highlights of the Program

Applied Biology

A unique collection of oil-producing microalgae.

The ASP studied a fairly specific aspect of algae—their ability to produce natural

oils. Researchers not only concerned themselves with finding algae that produced a

lot of oil, but also with algae that grow under severe conditions—extremes of

temperature, pH and salinity. At the outset of the program, no collections existed that

either emphasized or characterized algae in terms of these constraints. Early on,

researchers set out to build such a collection. Algae were collected from sites in the

west, the northwest and the southeastern regions of the continental U.S., as well as

Hawaii. At its peak, the collection contained over 3,000 strains of organisms. After

screening, isolation and characterization efforts, the collection was eventually

winnowed down to around 300 species, mostly green algae and diatoms. The

collection, now housed at the University of Hawaii, is still available to researchers.

This collection is an untapped resource, both in terms of the unique organisms

available and the mostly untapped genetic resource they represent. It is our sincere

hope that future researchers will make use of the collection not only as a source of

new products for energy production, but for many as yet undiscovered new products

and genes for industry and medicine.

Shedding light on the physiology and biochemistry of algae.

Prior to this program, little work had been done to improve oil production in algal

organisms. Much of the program’s research focused attention on the elusive “lipid

trigger.” (Lipids are another generic name for TAGs, the primary storage form of

natural oils.) This “trigger” refers to the observation that, under environmental stress,

many microalgae appeared to flip a switch to turn on production of TAGs. Nutrient

deficiency was the major factor studied. Our work with nitrogen-deficiency in algae

and silicon deficiency in diatoms did not turn up any overwhelming evidence in

support of this trigger theory. The common thread among the studies showing

increased oil production under stress seems to be the observed cessation of cell

division. While the rate of production of all cell components is lower under nutrient

starvation, oil production seems to remain higher, leading to an accumulation of oil in

the cells. The increased oil content of the algae does not to lead to increased overall

productivity of oil. In fact, overall rates of oil production are lower during periods of

nutrient deficiency. Higher levels of oil in the cells are more than offset by lower

rates of cell growth.

Breakthroughs in molecular biology and genetic engineering.

Plant biotechnology is a field that is only now coming into its own. Within the field

of plant biotechnology, algae research is one of the least trodden territories. The

slower rate of advance in this field makes each step forward in our research all the

more remarkable. Our work on the molecular biology and genetics of algae is thus

marked with significant scientific discoveries. The program was the first to isolate

A Look Back at the Aquatic Species Program—Program Summary 11
the enzyme Acetyl CoA Carboxylase (ACCase) from a diatom. This enzyme was

found to catalyze a key metabolic step in the synthesis of oils in algae. The gene that

encodes for the production of ACCase was eventually isolated and cloned. This was

the first report of the cloning of the full sequence of the ACCase gene in any

photosynthetic organism. With this gene in hand, researchers went on to develop

the first successful transformation system for diatoms—the tools and genetic

components for expressing a foreign gene. The ACCase gene and the transformation

system for diatoms have both been patented. In the closing days of the program,

researchers initiated the first experiments in metabolic engineering as a means of

increasing oil production. Researchers demonstrated an ability to make algae overexpress the ACCase gene, a major milestone for the research, with the hope that

increasing the level of ACCase activity in the cells would lead to higher oil

production. These early experiments did not, however, demonstrate increased oil

production in the cells.

Algae Production Systems

Demonstration of Open Pond Systems for Mass Production of Microalgae.

Over the course of the program, efforts were made to establish the feasibility of

large-scale algae production in open ponds. In studies conducted in California,

Hawaii and New Mexico, the ASP proved the concept of long term, reliable

production of algae. California and Hawaii served as early test bed sites. Based on

results from six years of tests run in parallel in California and Hawaii, 1,000 m2 pond

systems were built and tested in Roswell, New Mexico. The Roswell, New Mexico

tests proved that outdoor ponds could be run with extremely high efficiency of CO2

utilization. Careful control of pH and other physical conditions for introducing CO2

into the ponds allowed greater than 90% utilization of injected CO2. The Roswell

test site successfully completed a full year of operation with reasonable control of the

algal species grown. Single day productivities reported over the course of one year

were as high as 50 grams of algae per square meter per day, a long-term target for the

program. Attempts to achieve consistently high productivities were hampered by low

temperature conditions encountered at the site. The desert conditions of New Mexico

provided ample sunlight, but temperatures regularly reached low levels (especially at

night). If such locations are to be used in the future, some form of temperature

control with enclosure of the ponds may well be required.

A disconnect between the lab and the field.

An important lesson from the outdoor testing of algae production systems is the

inability to maintain laboratory organisms in the field. Algal species that looked very

promising when tested in the laboratory were not robust under conditions

encountered in the field. In fact, the best approach for successful cultivation of a

consistent species of algae was to allow a contaminant native to the area to take over

the ponds.

The high cost of algae production remains an obstacle.

12 A Look Back at the Aquatic Species Program—Program Summary
The cost analyses for large-scale microalgae production evolved from rather

superficial analyses in the 1970s to the much more detailed and sophisticated studies

conducted during the 1980s. A major conclusion from these analyses is that there is

little prospect for any alternatives to the open pond designs, given the low cost

requirements associated with fuel production. The factors that most influence cost

are biological, and not engineering-related. These analyses point to the need for

highly productive organisms capable of near-theoretical levels of conversion of

sunlight to biomass. Even with aggressive assumptions about biological

productivity, we project costs for biodiesel which are two times higher than current

petroleum diesel fuel costs.

Resource Availability

Land, water and CO2 resources can support substantial biodiesel production and CO2

savings.

The ASP regularly revisited the question of available resources for producing

biodiesel from microalgae. This is not a trivial effort. Such resource assessments

require a combined evaluation of appropriate climate, land and resource availability.

These analyses indicate that significant potential land, water and CO2 resources exist

to support this technology. Algal biodiesel could easily supply several “quads” of

biodiesel—substantially more than existing oilseed crops could provide. Microalgae

systems use far less water than traditional oilseed crops. Land is hardly a limitation.

Two hundred thousand hectares (less than 0.1% of climatically suitable land areas in

the U.S.) could produce one quad of fuel. Thus, though the technology faces many

R&D hurdles before it can be practicable, it is clear that resource limitations are not

an argument against the technology.

A Brief Chronology of the Research Activities

Part II of this report details the specific research accomplishments of the program on

a year-to-year basis. In order to provide a consistent context and framework for

understanding this detail, we have attempted to outline the major activities of the

program as they unfolded over the course of the past two decades. The timeline on

the following page shows the major activities broken down into two main

categories—laboratory studies and outdoor testing/systems analysis. For the sake of

clarity and brevity, many of the research projects and findings from the program are

not presented here. Instead, only those findings that form a thread throughout the

work are highlighted. There were many other studies and findings of value in the

program. The reader is encouraged to review Part II of this report for a more

comprehensive discussion of the research.

Laboratory Studies

The research pathway in the lab can be broken down into three types of activities:

• Collection, screening and characterization of algae.

• Biochemical and physiological studies of lipid production

• Molecular biology and genetic engineering studies

A Look Back at the Aquatic Species Program—Program Summary 13 
There is a logic to the sequence of these activities. Researchers first identified a need

to collect and identify algae that met minimal requirements for this technology.

Collection and screening occurred over a seven-year period from 1980 to 1987.

Once a substantial amount of information was available on the types of oil-producing

algae and their capabilities, the program began to switch its emphasis to

understanding the biochemistry and physiology of oil production in algae. A natural

next step was to use this information to identify approaches to genetically manipulate

the metabolism of algae to enhance oil production.

Algae collection efforts initially focused on shallow, inland saline habitats,

particularly in western Colorado, New Mexico and Utah. The reasoning behind

collecting strains from these habitats was that the strains would be adapted to at least

some of the environmental conditions expected in mass culture facilities located in

the southwestern U.S. (a region identified early on as a target for deployment of the

technology). Organisms isolated from shallow habitats were also expected to be

more tolerant to wide swings in temperature and salinity. In the meantime,

subcontractors were collecting organisms from the southeastern region of the U.S.

(Florida, Mississippi, and Alabama). By 1984, researchers in the program had

developed improved tools and techniques for collecting and screening organisms.

These included a modified rotary screening apparatus and statistically designed saline

media formulations that mimicked typical brackish water conditions in the southwest.

In 1985, a rapid screening test was in place for identifying high oil-producing algae.

In the last years of the collection effort, the focus switched to finding algae that were

tolerant to low temperature. This expanded the reach of the collection activities into

the northwest. By 1987, the algae collection contained over 3,000 species.

As the collection efforts began to wind down, it became apparent that no one single

species was going to be found that met all of the needs of the technology. As a

result, about midway through the collection efforts, the program began studies on the

biochemistry and physiology of oil production in algae in hopes of learning how to

improve the performance of existing organisms. A number of ASP subcontractors

struggled to identify the so-called “lipid trigger.” These studies confirmed

observations that deficiencies in nitrogen could lead to an increase in the level of oil

present in many species of algae. Observations of cellular structure also supported

the notion of a trigger that caused rapid build up of oil droplets in the cells during

periods of nitrogen depletion.

14 A Look Back at the Aquatic Species Program—Program Summary
Pre-1980 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996

Lab Studies

Outdoor Culture Studies and Systems Analysis

Collection, 

Screening, 

Characterization

CO, AL,MI

Biochemistry, Physiology 

of Lipid Production 

Artificial saline 

media; TempSalinity Gradient 

Screening

CO, UT

Carribean, NM,CO, 

UT, FL, HI, NE, AL

UT, WA, CO, CA, 

NV

Nile Red lipid 

screening

AZ,CA, 

NV,NM,

TX,UT

Genetic Engineering 

of Algae 

N-deficiency 

lipid trigger Si-deficiency 

lipid trigger

By 1987, over 

3,000 strains of 

algae had been 

collected. 

Future efforts 

Isolation and 

characterization 

of ACCase 

enzyme

Link between 

Si-deficiency 

and ACCase

Transient 

expression of 

foreign gene in 

algae using 

protoplasts

1st successful 

genetic 

transformation of 

Algae Production in Wastewater diatom

Treatment

<100 sq.m. Pond Studies (CA, HI)

1000 sq.m. Pond Study (NM)

Systems Analysis and Resource Assessment
In the end, however, the studies conducted both by NREL researchers and program

subcontractors concluded that no simple trigger for lipid production exists. Instead,

we found that environmental stresses like nitrogen depletion lead to inhibition of cell

division, without immediately slowing down oil production. It appeared that no

simple means existed for increasing oil production, without a penalty in overall

productivity due to a slowing down of cell growth. The use of nutrient depletion as a

means of inducing oil production may still have merit. Some experiments conducted

at NREL suggested that the kinetics of cell growth and lipid accumulation are very

subtle. With a better understanding of these kinetics, it may be possible to provide a

net increase in total oil productivity by carefully controlling the timing of nutrient

depletion and cell harvesting.

In 1986, researchers at NREL reported on the use of Si depletion as a way to increase

oil levels in diatoms. They found that when Si was used up, cell division slowed

down since Si is a component of the diatoms’ cell walls. In the diatom C. cryptica,

the rate of oil production remained constant once Si depletion occurred, while growth

rate of the cells dropped. Further studies identified two factors that seemed to be at

play in this species:

1. Si-depleted cells direct newly assimilated carbon more toward

lipid production and less toward carbohydrate production.

2. Si-depleted cells slowly convert non-lipid cell components to

lipids.

Diatoms store carbon in lipid form or in carbohydrate form. The results of these

experiments suggested that it might be possible to alter which route the cells used for

storage (see schematic below):

CO2

C

Photosynthesis

Lipid Synthesis Carbohydrate

Synthesis

16 A Look Back at the Aquatic Species Program—Program Summary
Through the process of photosynthesis, algae cells assimilate carbon. There are

numerous metabolic pathways through which the carbon can go, resulting in

synthesis of whatever compounds are needed by the cell. These pathways consist of

sequences of enzymes, each of which catalyzes a specific reaction. Two possible

pathways for carbon are shown on the previous page. They represent the two storage

forms that carbon can take.

Researchers at NREL began to look for key enzymes in the lipid synthesis pathway.

These would be enzymes whose level of activity in the cell influences the rate at

which oils are formed. Think of these enzymes as valves or spigots controlling the

flow of carbon down the pathway. Higher enzyme activity leads to higher rates of oil

production. When algae cells increase the activity of active enzymes, they are

opening up the spigot to allow greater flow of carbon to oil production. Finding such

critical enzymes was key to understanding the mechanisms for controlling oil

production.

By 1988, researchers had shown that increases in the levels of the enzyme Acetyl

CoA Carboxylase (ACCase) correlated well with lipid accumulation during Si

depletion. They also showed that the increased levels correlated with increased

expression of the gene encoding for this enzyme. These findings led to a focus on

isolating the enzyme and cloning the gene responsible for its expression. By the end

of the program, not only had researchers successfully cloned the ACCase gene, but

they had also succeeded in developing the tools for expressing foreign genes in

diatoms.

In the 1990s, genetic engineering had become the main focus of the program. While

we have highlighted the successes of over-expressing ACCase in diatoms, other

approaches were also developed for foreign gene expression—in green algae as well

as in diatoms. Another interesting sideline in the research involved studies aimed at

identifying key enzymes involved in the synthesis of storage carbohydrates. Instead

of over-expressing these enzymes, researchers hoped to inactivate them. Returning

to our “spigot” analogy, this approach was like shutting off the flow of carbon to

carbohydrates, in the hopes that it would force carbon to flow down the lipid

synthesis pathway (again, see the schematic on the previous page). This work led to

the discovery of a unique multifunctional enzyme in the carbohydrate synthesis

pathway. This enzyme and its gene were both patented by NREL in 1996.

Outdoor Testing and Systems Analysis

The first work done in earnest by DOE on demonstration of algae technology for

energy production predates the Aquatic Species Program. In 1976, the Energy

Research and Development Administration (before it was folded into DOE) funded a

project at the University of California Berkeley’s Richmond Field Station to evaluate

a combined wastewater treatment/fuel production system based on microalgae. Over

the course of several years, the Richmond Field Station demonstrated techniques for

algae harvesting and for control of species growing in open ponds.

By the time the Aquatic Species Program took on microalgae research, emphasis had

already moved from wastewater treatment based systems to dedicated algae farm

operations. From 1980 to 1987, the program funded two parallel efforts to develop

large scale mass culture systems for microalgae. One effort was at the University of

California, and it was based on a so-called “High Rate Pond” (HRP) design. The

other effort was carried out at the University of Hawaii, where a patented “Algae

A Look Back at the Aquatic Species Program—Program Summary 17
Raceway Production System” (ARPS). Both designs utilized open raceway designs.

The HRP design was based on a shallow, mixed raceway concept developed at

Berkeley in 1963 and successfully applied in wastewater treatment operations in

California. The ARPS was really a variation on the same concept. Both efforts

carried out their test work in ponds of 100 square meters or less. They studied a

variety of fundamental operational issues, such as the effects of fluid flow patterns,

light intensity, dissolved oxygen levels, pH and algae harvesting methods.

At the conclusion of the smaller scale tests conducted in California and Hawaii, the

program engaged in a competitive bidding process to select a system design for scale

up of algae mass culture. The HRP design evaluated at UC Berkeley was selected for

scale-up. The “Outdoor Test Facility” (OTF) was designed and built at the site of an

abandoned water treatment plant in Roswell, New Mexico. From 1988 to 1990,

1,000 square meter ponds were successfully operated at Roswell. This project

demonstrated how to achieve very efficient (>90%) utilization of CO2 in large ponds.

The best results were obtained using native species of algae that naturally took over

in the ponds (as opposed to using laboratory cultures). The OTF also demonstrated

production of high levels of oil in algae using both nitrogen and silica depletion

strategies. While daily productivities did reach program target levels of 50 grams per

square per day, overall productivity was much lower (around 10 grams per square

meter per day) due to the number of cold temperature days encountered at this site.

Nevertheless, the project established the proof-of-concept for large scale open pond

operations. The facility was shut down in 1990, and has not been operated since.

A variety of other outdoor projects were funded over the course of the program,

including a three-year project on algal biodiesel production conducted in Israel. In

addition, research at the Georgia Institute of Technology was carried out in the late

1980s. This work consisted of a combination of experimental and computer

modeling work. This project resulted in the development of the APM (Algal Pond

Model), a computer modeling tool for predicting performance of outdoor pond

systems.

Two types of systems analysis were conducted frequently over the course of the

program—resource assessments and engineering design/cost analyses. The former

addresses the following important question: how much impact can algae technology

have on petroleum use within the limits of available resources? Engineering designs

provide some input to this question as well, since such designs tell us something

about the resource demands of the technology. These designs also tell us how much

the technology will cost.

As early as 1982, the program began to study the question of resource availability for

algae technology. Initial studies scoped out criteria and methodology that should be

used in the assessment. In 1985, a study done for Argonne National Lab produced

maps of the southwestern U.S. which showed suitable zones for algae production

based on climate, land and water availability. In 1990, estimates of available CO2

supplies were completed for the first time. These estimates suggested that that there

was enough waste CO2 available in the states where climate conditions were suitable

to support 2 to 7 quads of fuel production annually. The cost of the CO2 was

estimated to range anywhere from $9 to $90 per ton of CO2. This study did not

consider any regionally specific data, but drew its conclusions from overall data on

CO2 availability across a broad region. Also in 1990, a study was funded to assess

land and water availability for algae technology in New Mexico. This study took a

more regionally specific look at the resource question, but did so by sacrificing any

18 A Look Back at the Aquatic Species Program—Program Summary
consideration of available CO2 supplies. This last study sums up the difficulties

faced in these types of studies. The results obtained on resource availability are

either able to provide a complete, but general, perspective on resources or they are

more detailed in approach, but incomplete in the analysis of all resources.

Engineering design and cost studies have been done throughout the course of the

ASP, with ever increasing realism in the design assumptions and cost estimates. The

last set of cost estimates for the program was developed in 1995. These estimates

showed that algal biodiesel cost would range from $1.40 to $4.40 per gallon based on

current and long-term projections for the performance of the technology. Even with

assumptions of $50 per ton of CO2 as a carbon credit, the cost of biodiesel never

competes with the projected cost of petroleum diesel.

Program Funding History

Like all of the renewable fuels programs, the ASP has always been on a fiscal roller

coaster

0

500

1000

1500

2000

2500

3000

$1000s per year

1978198019821984198619881990199219941996

Funding History for the Aquatic Species Program

In its heyday, this program leaped to levels of $2 to $2.75 million in annual funding.

In most cases, these peaks came in sudden bursts in which the funding level of the

program would double from one year to the next. After the boom years of 1984 and

1985, funding fell rapidly to its low of $250,000 in 1991. The last three years of the

program saw a steady level of $500,000 (not counting FY 1996, which were mostly

used to cover the cost of employee terminations). Ironically, these last three years

were among the most productive in the history of the program (given the

breakthroughs that occurred in genetic engineering). Though funding levels were

A Look Back at the Aquatic Species Program—Program Summary 19
relatively low, they were at least steady—providing a desperately needed stability for

the program. The years of higher spending are, for the most part, dominated by

costly demonstration work (the tests carried out in California, Hawaii and

culminating in New Mexico), engineering analysis and culture collection activities.

High Return for a Small Investment of DOE Funds

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

$1000s per year

1978198019821984198619881990199219941996

Aquatic Species

Program

Total Biofuels Program

The total cost of the Aquatic Species Program is $25.05 million over a twenty-year

period. Compared to the total spending under the Biofuels Program ($458 million

over the same period), this has not been a high cost research program. At its peak,

ASP accounted for 14% of the annual Biofuels budget; while, on average, it

represented only 5.5% of the total budget. Given that relatively small investment,

DOE has seen a tremendous return on its research dollars.

Future Directions

Put less emphasis on outdoor field demonstrations and more on basic biology

Much work remains to be done on a fundamental level to maximize the overall

productivity of algae mass culture systems. The bulk of this work is probably best

done in the laboratory. The results of this program’s demonstration activities have

proven the concept of outdoor open pond production of algae. While it is important

to continue a certain amount of field work, small scale studies and research on the

20 A Look Back at the Aquatic Species Program—Program Summary
basic biological issues are clearly more cost effective than large scale demonstration

studies.

Take Advantage of Plant Biotechnology

We have only scratched the surface in the area of genetic engineering for algae. With

the advances occurring in this field today, any future effort on modifying algae to

increase natural oil production and overall productivity are likely to proceed rapidly.

The genetic engineering tools established in the program serve as a strong foundation

for further genetic enhancements of algae.

Start with what works in the field

Select strains that work well at the specific site where the technology is to be used.

These native strains are the most likely to be successful. Then, focus on optimizing

the production of these native strains and use them as starting points for genetic

engineering work.

Maximize photosynthetic efficiency.

Not enough is understood about what the theoretical limits of solar energy conversion

are. Recent advances in our understanding of photosynthetic mechanisms at a

molecular level, in conjunction with the advances being made in genetic engineering

tools for plant systems, offer exciting opportunities for constructing algae which do

not suffer the limitations of light saturation photoinhibition.

Set realistic expectations for the technology

Projections for future costs of petroleum are a moving target. DOE expects

petroleum costs to remain relatively flat over the next 20 years. Expecting algal

biodiesel to compete with such cheap petroleum prices is unrealistic. Without some

mechanism for monetizing its environmental benefits (such as carbon taxes), algal

biodiesel is not going to get off the ground.

Look for near term, intermediate technology deployment opportunities such as

wastewater treatment.

Excessive focus on long term energy displacement goals will slow down

development of the technology. A more balanced approach is needed in which more

near term opportunities can be used to launch the technology in the commercial

arena. Several such opportunities exist. Wastewater treatment is a prime example.

The economics of algae technology are much more favorable when it is used as a

waste treatment process and as a source of fuel. This harks back to the early days of

DOE’s research.

A Look Back at the Aquatic Species Program—Program Summary 21
Footnotes

 

1

 Meier, R.L. (1955) “Biological Cycles in the Transformation of Solar Energy into Useful Fuels.” In

Solar Energy Research (Daniels, F.; Duffie, J.A.; eds), Madison University Wisconsin Press, pp. 179-

183.

2

 Peterson, C. L. (1986) “Vegetable Oil as a Diesel Fuel: Status and Research Priorities,” Transactions

of the ASAE, pp 1413-1422. American Society of Agricultural Engineers, St. Joseph, MO.

3

 Bruwer, J.; van D. Boshoff, B.; du Plessis, L.; Fuls, J.; Hawkins, C,; van der Walt, A.; Engelbrecht, A.,

(1980)“Sunflower Seed Oil As an Extender for Diesel Fuel in Agricultural Tractors,” presented at the

1980 Symposium of the South African Institute of Agricultural Engineers.

4

 Markley, K. (1961) “Chapter 9: Esters and Esterfication,” in Fatty Acids: Their Chemistry, Properties,

Production and Uses Part 2, 2nd Edition (Markley, K.; ed.). Interscience Publications, New York.

5

 European engine manufacturers have had very positive experience using rapeseed oil-derived

biodiesel. In the U.S., engine manufacturers have expressed tentative support for blends of soy-derived

biodiesel of up to 20%. See Alternative Fuels Committee of the Engine Manufacturers Association

(1995) Biodiesel Fuels and Their Use in Diesel Engine Applications Engine Manufacturers’

Association, Chicago, IL.

6

 Graboski, M.; McCormick, R. (1994) Final Report: Emissions from Biodiesel Blends and Neat

Biodiesel from a 1991 Model Series 60 Engine Operating at High Altitude. Colorado Institute for High

Altitude Fuels and Engine Research. Subcontractor’s report to National Renewable Energy Laboratory,

Golden,CO.

7

 FEV Engine Technology, Inc. (1994) Emissions and Performance Characteristics of the Navistar

T444E DI Diesel Engine Fueled with Blends of Biodiesel and Low Sulfur Diesel Fuel: Phase I final

Report. Contractor’s report to the National Biodiesel Board, Jefferson City, MO.

8

 Fosseen Manufacturing and Development, Ltd. (1994) Emissions and Performance Characteristics of

the Navistar T444E DI Diesel Engine Fueled with Blends of Biodiesel and Low Sulfur Diesel Fuel:

Phase I final Report. Contractor’s report to National Biodiesel Board, Jefferson City, MO.

9

 Peterson, C.; Reece, D. (1994) “Toxicology, Biodegradability and Environmental Benefits of

Biodiesel,” in Biodiesel '94 (Nelson, R.; Swanson. D.; Farrell, J.;eds). Western Regional Biomass

Energy Program, Golden, CO.

10 Sharpe, Chris, Southwest Research Institute (1998). Presentation on speciated emissions presented at

the Biodiesel Environmental Workshop.

11 Annual Energy Outlook 1996 with Projections to 2015. U.S. Department of Energy, Energy

Information Administration, DOE/EIA-0383(96), Washington, D.C. 1996.

12 Reinventing Energy: Making the Right Choices. The American Petroleum Institute, Washington, DC.

1996.

13 See Romm, J. The Atlantic Monthly, April 1996, pp 57-74.

14 Revelle, R.; Suess, H. Tellus, 9/1, pp 18-21, 1957.

15 Herzog, H., et al (1993) The Capture, Utilization and Disposal of Carbon Dioxide from Fossil

FuelPower Plants. Report to the U.S. Department of Energy DOE/ER-30194.

22 A Look Back at the Aquatic Species Program—Program Summary
A Look Back at the U.S.

Department of Energy’s

Aquatic Species Program:

Biodiesel from Algae

Part II:

Technical Review
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 

Table of Contents 

I. INTRODUCTION............................................................................................................................1

II. LABORATORY STUDIES............................................................................................................. 5

II.A. COLLECTION, SCREENING, AND CHARACTERIZATION OF MICROALGAE 5

II.A.1. Collection, Screening, and Characterization of Microalgae by SERI In-House Researchers.......5

II.A.1.a. Introduction ...................................................................................................................................5

II.A.1.b. Collection and Screening Activities - 1983...................................................................................8

II.A.1.c. Collection and Screening Activities - 1984...................................................................................8

II.A.1.d. Collection and Screening Activities - 1985.................................................................................17

II.A.1.e. Collection and Screening Activities - 1986 and 1987 .................................................................19

II.A.1.f. Development of a Rapid Screening Procedure for Growth and Lipid Content of Microalgae ....21

II.A.1.g. Statistical Analysis of Multivariate Effects on Microalgal Growth and Lipid Content ..............27

II.A.1.h. Detailed Analyses of Microalgal Lipids......................................................................................28

II.A.2. Collection, Screening, and Characterization of Microalgae: Research by SERI 

Subcontractors.............................................................................................................................32

II.A.2.a. Introduction .................................................................................................................................32

II.A.2.b. Yields, Photosynthetic Efficiencies, and Proximate Chemical Composition of Dense 

Cultures of Marine Microalgae....................................................................................................33

II.A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments ................................36

II.A.2.d. Screening and Characterizing Oleaginous Microalgal Species from the Southeastern United 

States ...........................................................................................................................................40

II.A.2.e. Collection of High Energy Strains of Saline Microalgae from Southwestern States ..................43

II.A.2.f. Collection of High Energy Yielding Strains of Saline Microalgae from the Hawaiian Islands ..45

II.A.2.g. Characterization of Hydrocarbon Producing Strains of Microalgae ...........................................46

II.A.2.h. Collection of High Energy Yielding Strains of Saline Microalgae from South Florida..............48

II.A.2.i. Collection and Selection of High Energy Thermophilic Strains of Microalgae ..........................50

II.A.3. The SERI Microalgae Culture Collection....................................................................................50

II.A.3.a. History of SERI Microalgae Culture Collection .........................................................................51

II.A.3.b. Current status of the SERI/NREL Microalgae Culture Collection..............................................55

II.A.4. Collection and Screening of Microalgae—Conclusions and Recommendations.........................64

II.B. MICROALGAL STRAIN IMPROVEMENT 67

II.B.1. Physiology, Biochemistry, and Molecular Biology of Lipid Production: Work by SERI 

Subcontractors.............................................................................................................................67

II.B.1.a. Introduction .................................................................................................................................67

II.B.1.b. Chrysophycean Lipids: Effects of Induction Strategy in the Quantity and Types of Lipids .......68

II.B.1.c. Genetic Variation in High Energy Yielding Microalgae .............................................................70

II.B.1.d. Ultrastructure Evaluation of Lipid Producing Microalgae ..........................................................75

II.B.1.e. Improvement of Microalgal Lipid Production by Flow Cytometry.............................................78

II.B.1.f. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ...........................................81

II.B.1.g. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae ...........................................83

II.B.1.h. Transformation and Somatic Cell Genetics for the Improvement of Energy Production in 

Microalgae...................................................................................................................................87

II.B.2. Physiology, Biochemistry, and Molecular Biology of Lipid Production: NREL In-House 

Researchers .................................................................................................................................95

II.B.2.a. Introduction .................................................................................................................................95
National Renewable Energy Laboratory 

ii A Look Back at the Aquatic Species Program—Technical Review 

II.B.2.b. Lipid Accumulation Induced by Nitrogen Limitation .................................................................96

II.B.2.c. Studies on Photosynthetic Efficiency in Oleaginous Algae ........................................................97

II.B.2.d. Lipid Accumulation in Silicon-Deficient Diatoms......................................................................98

II.B.2.e. Isolation and Characterization of Acetyl-CoA Carboxylase from C. cryptica ..........................102

II.B.2.f. Cloning of the Acetyl-CoA Carboxylase Gene from C. cryptica ..............................................105

II.B.2.g. Biochemistry of Lipid Synthesis in Nannochloropsis ...............................................................108

II.B.2.h. Biochemistry and Molecular Biology of Chrysolaminarin Synthesis .......................................108

II.B.3 Manipulation of Lipid Production in Microalgae via Genetic Engineering..............................113

II.B.3.a. Introduction ...............................................................................................................................113

II.B.3.b. Mutagenesis and Selection ........................................................................................................114

II.B.3.c. Development of a Genetic Transformation System for Microalgae..........................................116

II.B.3.d. Attempts to Manipulate Microalgal Lipid Composition via Genetic Engineering....................137

II.B.3.e. The Effect of Different Promoters on Expression of Luciferase in Cyclotella..........................139

II.B.4. Microalgal Strain Improvement – Conclusions and Recommendations .............................................142

III. OUTDOOR STUDIES AND SYSTEMS ANALYSIS...............................................................145

III.A. PROJECTS FUNDED BY ERDA/DOE 1976-1979 145

III.A.1. Introduction...............................................................................................................................145

III.A.2. Species Control in Large-Scale Algal Biomass Production ......................................................147

III.A.3 An Integrated System for the Conversion of Solar Energy with Sewage-Grown Microalgae ...152

III.A.4. Large-Scale Freshwater Microalgal Biomass Production for Fuel and Fertilizer ...................156

III.A.5. Other Microalgae Projects During the ERDA/DOE Period .....................................................161

III.B. THE ASP MICROALGAL MASS CULTURE 162

III.B.1. Introduction .......................................................................................................................................162

III.B.2. The ARPS Project in Hawaii, 1980-1987..................................................................................165

III.B.2.a. Hawaii ARPS Project Initiation, 1980-1981 .............................................................................165

III.B.2 b. Second Year of the Hawaii ARPS Project, 1981-1982 .............................................................166

III.B.2.c. Third Year of the Hawaii ARPS Project, 1982-1983 ................................................................169

III.B.2.d. Fourth Year of the Hawaii ARPS Project, 1983-1984...............................................................170

III.B.2.e. Fifth Year of the ARPS Project, 1984-1985..............................................................................172

III.B.2.f. Sixth Year of the Hawaii ARPS Project, 1985-1986.................................................................172

III.B.2.g. Seventh Year of the Hawaii ARPS Project, 1986-1987 ............................................................173

III.B.2.h. Hawaii ARPS Project, Conclusions...........................................................................................174

III.B.3. High Rate Pond (HRP) Operations in California, 1981-1986 ..................................................176

III.B.3.a. HRP Design and Construction Phase, 1981 ..............................................................................176

III.B.3.b. HRP Operations in California, Oct-Nov. 1982..........................................................................179

III.B.3.c. Continuing California HRP Pond Operations, 1983-1984 ........................................................179

III.B.3.d. Completion of the California HRP Project, 1985-1986.............................................................185

III.B.4. The Israeli Microalgae Biodiesel Production Project...............................................................190

III.B.5. Design and Operation of a Microalgae Outdoor Test Facility (OTF) in New Mexico .............193

III.B.5.a. Facility Design and Construction. .............................................................................................193

III.B.5.b. First Year OTF Experiments .....................................................................................................195

III.B.3.c. Full OTF System Operations.....................................................................................................195

III.B.5.d. Conclusions...............................................................................................................................198

III.B.6. The Effects of Environmental Fluctuation on Laboratory Cultures ..........................................199

III.B.6.a. Species Control and Productivity ..............................................................................................199

III.B.6.b. The Algal Pond Growth Model. ................................................................................................202
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review iii 

III.B.6.c. Microalgae Competition under Fluctuating Conditions in the Laboratory................................205

III.B.6.d. Lipid Productivity of Microalgae ..............................................................................................205

III.B.6.e. Competition Studies with Continuous and Semicontinuous Cultures .......................................208

III.C. RESOURCE ANALYSES 211

III.C.1. Introduction...............................................................................................................................211

III.C.2. The Battelle Columbus 1982 Resource Assessment Report.......................................................212

III.C.3. The 1982 Argonne Study of CO2 Availability ............................................................................212

III.C.4. The 1985 SERI Resource Evaluation Report.............................................................................213

III.C.5. The 1990 SERI Study on CO2 Sources.......................................................................................215

III.C.6. The 1990 SERI Study of Water Resources in New Mexico ........................................................217

III.C.7. Conclusions ...............................................................................................................................219

III.D. ENGINEERING SYSTEMS AND COST ANALYSES 219

III.D.1. Introduction...............................................................................................................................219

III.D.2. The Algal Pond Subsystem of the “Photosynthesis Energy Factory” .......................................220

III.D.3. Cost Analysis of Microalgae Biomass Systems..........................................................................221

III.D.4. Cost Analysis of Aquatic Biomass Systems................................................................................224

III.D.5. Microalgae as a Source of Liquid Fuels....................................................................................225

III.D.6. Fuels from Microalgae Technology Status, Potential and Research Requirements..................229

III.D.7. Design and Analysis of Microalgae Open Pond Systems ..........................................................233

III.D.8. Systems and Economic Analysis of Microalgae Ponds for Conversion of CO2 to Biomass ......237

III.D.9. NREL Studies of Flue Gas CO2 Utilization by Microalgae.......................................................241

III.D.10. Conclusions. ..............................................................................................................................245

IV. CONCLUSIONS AND RECOMMENDATIONS.....................................................................248

IV.A. MICROALGAL STRAIN IMPROVEMENT 248

IV.A.1. Conclusions ...............................................................................................................................248

IV.A.2. R & D Recommendations ..........................................................................................................250

IV.A.2.a. General Considerations .............................................................................................................250

IV.A.2.b. Maximum Efficiency of Photosynthesis....................................................................................250

IV.A.2.c. Overcoming Light Saturation, Photooxidation, and Other Limitations.....................................252

IV.A.2.d. Microalgal Strains for Mass Culture: Source and Genetic Improvements ................................253

IV.B. MICROALGAL MASS CULTURE 255

IV.B.1. Conclusions ...............................................................................................................................255

IV.B.1.a. Cost and Productivity Goals......................................................................................................255

IV.B.1.b. Higher Value Byproducts and Coproducts ................................................................................256

IV.B.1.c. The Japanese R&D Program for Microalgae CO2 Utilization...................................................257

IV.B.1.d. Resource Projections and Microalgae Biodiesel R&D..............................................................259

IV.B.1.e. Summary of Major Conclusions from the ASP Microalgal Mass Culture Work ......................260

IV.B.2. R & D Recomendations .............................................................................................................260

IV.B.2 a. Biodiesel Production and Algal Mass Culture for Wastewater Treatment ................................260

IV.B.3. Conclusions ...............................................................................................................................261

V. BIBLIOGRAPHY ........................................................................................................................263

V.A. SERI/NREL/DOE REPORTS AND PUBLICATIONS 263

V.B. ADDITIONAL REFERENCES 293

V.C. PATENTS 294
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 

 I. Introduction

Photosynthetic organisms, including plants, algae, and some photosynthetic bacteria, efficiently 

utilize the energy from the sun to convert water and CO2 from the air into biomass. The Aquatic 

Species Program (ASP) at SERI1

 was initiated as a long term, basic research effort to produce 

renewable fuels and chemicals from biomass. It emphasized the use of photosynthetic organisms 

from aquatic environments, expecially species that grow in environments unsuitable for crop 

production. Early in the program, macroalgae, microalgae, and emergents were investigated for 

their ability to make lipids (as a feedstock for liquid fuel or chemical production) or 

carbohydrates (for fermentation into ethanol or anaerobic digestion for methane production). 

Macroalgae (seaweeds) are fast-growing marine or freshwater plants that can reach considerable 

size; for example, the giant brown kelp can grow a meter in 1 day and as long as 60 m. 

Emergents are plants such as cattails or rushes that grow partially submerged in bogs or marshes. 

Macroalgae and emergents were found to produce small amounts of lipid, which function mainly 

as structural components of the cell membranes, and produce carbohydrate for use as their 

primary energy storage compound. In contrast, many microalgae, (microscopic, photosynthetic 

organisms that live in saline or freshwater environments), produce lipids as the primary storage 

molecule. By the early 1980s, the decision was made to focus ASP research efforts on the use of 

microalgal lipids for the production of fuels and other energy products. The studies on the 

growth and chemical composition of macroalgae and emergents will not be discussed in this 

report. However, interested readers are referred to reports by subcontractors J.D. Ryther, Harbor 

Branch Foundation, Florida (seaweeds), and D. Pratt, from the University of Minnesota, St. Paul 

(emergents) listed in the Bibliography. 

Microalgae, like higher plants, produce storage lipids in the form of triacyglycerols (TAGs). 

Although TAGs could be used to produce of a wide variety of chemicals, work at SERI focused 

on the production of fatty acid methyl esters (FAMEs), which can be used as a substitute for 

fossil-derived diesel fuel. This fuel, known as biodiesel, can be synthesized from TAGs via a 

simple transesterification reaction in the presence of acid or base and methanol. Biodiesel can be 

used in unmodified diesel engines, and has advantages over conventional diesel fuel in that it is 

renewable, biodegradable, and produces less SOX and particulate emissions when burned. The 

technology is available to produce biodiesel from TAGs, and there are growing biodiesel 

industries both in the United States and Europe that use soybean or rapeseed oil as the biodiesel 

feedstock. However, the potential market for biodiesel far surpasses the availability of plant oils 

not designated for other markets. Thus, there was significant interest in the development of 

microalgal lipids for biodiesel production. 

Microalgae exhibit properties that make them well suited for use in a commercial-scale biodiesel 

production facility. Many species exhibit rapid growth and high productivity, and many microalgal 

species can be induced to accumulate substantial quantities of lipids, often greater than 60% of 

 1

 The Solar Energy Research Institute (SERI) became the National Renewable Energy Laboratory (NREL) 

in 1990. In this report, the laboratory may be referred to as either SERI or NREL, depending on the time 

period during which the work being described was performed. 
National Renewable Energy Laboratory 

2 A Look Back at the Aquatic Species Program—Technical Review 

their biomass. Microalgae can also grow in saline waters that are not suitable for agricultural 

irrigation or consumption by humans or animals. The growth requirements are very simple, 

primarily carbon dioxide (CO2) and water, although the growth rates can be accelerated by 

sufficient aeration and the addition of nutrients. A brief overview of the characteristics of the 

major microalgal classes can be found in Section II.A.2. 

A major undertaking by ASP researchers in the early stages of the program was to identify 

candidate microalgal species that exhibited characteristics desirable for a commercial production 

strain. Resource analyses carried out by SERI (discussed in Section III.C.) indicated that the 

desert regions of the southwestern United States were attractive areas in which to locate 

microalgal-based biofuel production facilities. This, in part, dictated the required strain 

characteristics. These characteristics included the ability of the strains to grow rapidly and have 

high lipid productivity when growing under high light intensity, high temperature, and in saline 

waters indigenous to the area in which the commercial production facility is located. In addition, 

because it is not possible to control the weather in the area of the ponds, the best strains should 

have good productivity under fluctuating light intensity, temperature, and salinity. 

A multi-faceted effort was carried out to: 

• isolate microalgae from a variety of saline habitats (including oceans, lakes, ponds, 

and various ephemeral water bodies), 

• screen those isolates for the ability to grow under a variety of conditions, 

• analyze the biochemical components of the strains (especially with respect to lipids), 

and 

• determine the effects of environmental variables on the growth and lipid composition 

of selected strains. 

This effort involved in-house researchers and subcontractors from academia, industry, and other 

government laboratories. Section II.A.1. documents the efforts of SERI in-house researchers in 

the area of microalgal strain isolation and screening. It also describes the methodologies 

developed and employed during the isolation, screening, and characterization phases of the work. 

Section II.A.2. describes parallel efforts conducted by SERI subcontractors. An account of the 

history and current status of the NREL Microalgae Culture Collection is presented in Section 

II.A.3. 

Although the collection and screening efforts produced a number of viable candidate strains, no 

one algal strain was identified that exhibited the optimal properties of rapid growth and high 

constitutive lipid production. Many microalgae can be induced to accumulate lipids under 

conditions of nutrient deprivation. If this process could be understood, it might be possible to 

manipulate either the culture conditions, or to manipulate the organisms themselves, to increase 

lipid accumulation in a particular strain. Therefore, studies were initiated both at SERI and by 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 3 

ASP subcontractors to study the biochemistry and physiology of lipid production in oleaginous 

(oil-producing) microalgae. Work performed by several ASP subcontractors was designed to 

understand the mechanism of lipid accumulation. In particular, these researchers tried to 

determine whether there is a specific “lipid trigger” that is induced by factors such as nitrogen 

(N) starvation. Subcontractors also studied ultrastructural changes induced in microalgae during 

lipid accumulation. They also initiated efforts to produce improved algae strains by looking for 

genetic variability between algal isolates, attempting to use flow cytometry to screen for 

naturally-occurring high lipid individuals, and exploring algal viruses as potential genetic 

vectors. The work performed by ASP subcontractors is described in Section II.B.1. 

Although some of the efforts of the in-house SERI researchers were also directed toward 

understanding the lipid trigger induced by N starvation, they showed that silica (Si) depletion in 

diatoms also induced lipid accumulation. Unlike N, Si is not a major component of cellular 

molecules, therefore it was thought that the Si effect on lipid production might be less complex 

than the N effect, and thus easier to understand. This initiated a major research effort at SERI to 

understand the biochemistry and molecular biology of lipid accumulation in Si-depleted diatoms. 

 This work led to the isolation and characterization of several enzymes involved in lipid and 

carbohydrate synthesis pathways, as well as the cloning of the genes that code for these enzymes. 

 One goal was to genetically manipulate these genes in order to optimize lipid accumulation in 

the algae. Therefore, reseach was performed simultaneously to develop a genetic transformation 

system for oleaginous microalgal strains. The successful development of a method to genetically 

engineer diatoms was used in attempts to manipulate microalgal lipid levels by overexpressing or 

down-regulating key genes in the lipid or carbohydrate synthetic pathways. Unfortunately, 

program funding was discontinued before these experiments could be carried out beyond the 

prelimilary stages. 

Cost-effective production of biodiesel requires not only the development of microalgal strains 

with optimal properties of growth and lipid production, but also an optimized pond design and a 

clear understanding of the available resources (land, water, power, etc.) required. Section III 

reviews the R&D on outdoor microalgae mass culture for production of biodiesel, as well as 

supporting engineering, economic and resource analyses, carried out and supported by ASP 

during the 1980s and early 1990s. It also covers work supported by DOE and its predecessor 

agency, the Energy Research and Development Administration (ERDA), during the 1970s and 

some recent work on utilization of CO2 from power plant flue gases. 

From 1976 to 1979, researchers at the University of California-Berkeley used shallow, paddle 

wheel mixed, raceway-type (high-rate) ponds to demonstrate a process for the simultaneous 

treatment of wastewater and production of energy (specifically methane). Starting in 1980, the 

ASP supported outdoor microalgal cultivation projects in Hawaii and California, using fresh and 

seawater supplies, respectively, in conjunction with agricultural fertilizers and CO2. The two 

projects differed in the types of algae cultivated and the design of the mass culture system, with 

the project in California continuing to develop the high-rate pond design, and the Hawaii project 

studying an (initially) enclosed and intensively mixed system. From 1987 to 1990, an “Outdoor 

Test Facility” was designed, constructed and operated in Roswell, New Mexico, including two 
National Renewable Energy Laboratory 

4 A Look Back at the Aquatic Species Program—Technical Review 

1,000 m2 high-rate ponds. This last project represented the culmination of ASP R&D in 

large-scale algal mass culture R&D. These studies are described in Section III.A. Some 

supporting laboratory studies and development of an “Algal Pond Model” (APM) are also 

reviewed at the end of that section. The conclusion from these extensive outdoor mass culture 

studies was that the use of microalgae for the low-cost production of biodiesel is technically 

feasible, but still requires considerable long-term R&D to achieve the high productivities 

required. 

Section III.B. reviews the resource assessments, for water, land, CO2, etc., carried out by the 

ASP, primarily for the southwestern United States. These studies demonstrated the potential 

availability of large brackish and saline water resources suitable for microalgae mass cultures, 

large land and CO2 resources. They suggest that the potential production of microalgae-derived 

biodiesel may represent more than 10% of U.S. transportation fuels, although full resource 

exploitation would be significantly constrained in practice. Several engineering and economic 

cost analyses were also supported by DOE and the ASP, and these are reviewed in Section III.C., 

including recent work by the ASP and DOE on power plant flue gas utilization for greenhouse 

gas (CO2) mitigation. 

The overall conclusion of these studies was that in principle and practice large-scale microalgae 

production is not limited by design, engineering, or net energy considerations and could be 

economically competitive with other renewable energy sources. However, long-term R&D 

would be required to actually achieve the very high productivities and other assumptions made in 

such cost analyses. Section III.D. provides recommendations for future research that could make 

this technology commercially feasible. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 5 

 II. Laboratory Studies 

II.A. Collection, Screening, and Characterization of Microalgae 

II.A.1. Collection, Screening, and Characterization of Microalgae by SERI 

In-House Researchers

II.A.1.a. Introduction 

This chapter describes the research performed at SERI in the area of microalgal collection and 

screening. In addition to performing actual research in this area, SERI personnel were 

responsible for coordinating the efforts of the many subcontractors performing similar activities, 

and for standardizing certain procedures and analyses. These efforts ultimately resulted in the 

development of the SERI Microalgal Culture Collection, which is more fully described in 

Chapter II.A.3. 

Brief review of algal taxonomic groups and characteristics: 

For the purposes of this report, microalgae are defined as microscopic organisms that can grow 

via photosynthesis. Many microalgae grow quite rapidly, and are considerably more productive 

than land plants and macroalgae (seaweed). Microalgae reproduction occurs primarily by 

vegetative (asexual) cell division, although sexual reproduction can occur in many species under 

appropriate growth conditions. 

There are several main groups of microalgae, which differ primarily in pigment composition, 

biochemical constituents, ultrastructure, and life cycle. Five groups were of primary importance 

to the ASP: diatoms (Class Bacillariophyceae), green algae (Class Chlorophyceae), goldenbrown algae (Class Chrysophyceae), prymnesiophytes (Class Prymnesiophyceae), and the 

eustigmatophytes (Class Eustigmatophyceae). The blue-green algae, or cyanobacteria (Class 

Cyanophyceae), were also represented in some of the collections. A brief description of these 

algal groups follows. 

• Diatoms. Diatoms are among the most common and widely distributed groups 

of algae in existence; about 100,000 species are known. This group tends to 

dominate the phytoplankton of the oceans, but is commonly found in fresh- and 

brackish-water habitats as well. The cells are golden-brown because of the 

presence of high levels of fucoxanthin, a photosynthetic accessory pigment. 

Several other xanthophylls are present at lower levels, as well as β-carotene, 

chlorophyll a and chlorophyll c. The main storage compounds of diatoms are 

lipids (TAGs) and a β-1,3-linked carbohydrate known as chrysolaminarin. A 

distinguishing feature of diatoms is the presence of a cell wall that contains 

substantial quantities of polymerized Si. This has implications for media costs 

in a commercial production facility, because silicate is a relatively expensive 

chemical. On the other hand, Si deficiency is known to promote storage lipid 
National Renewable Energy Laboratory 

6 A Look Back at the Aquatic Species Program—Technical Review 

accumulation in diatoms, and thus could provide a controllable means to induce 

lipid synthesis in a two-stage production process. Another characteristic of 

diatoms that distinguishes them from most other algal groups is that they are 

diploid (having two copies of each chromosome) during vegetative growth; 

most algae are haploid (with one copy of each chromosome) except for brief 

periods when the cells are reproducing sexually. The main ramification of this 

from a strain development perspective is that it makes producing improved 

strains via classical mutagenesis and selection/screening substantially more 

difficult. As a consequence, diatom strain development programs must rely 

heavily on genetic engineering approaches. 

• Green Algae. Green algae, often referred to as chlorophytes, are also abundant; 

approximately 8,000 species are estimated to be in existence. This group has 

chlorophyll a and chlorophyll b. These algae use starch as their primary storage 

component. However, N-deficiency promotes the accumulation of lipids in 

certain species. Green algae are the evolutionary progenitors of higher plants, 

and, as such, have received more attention than other groups of algae. A 

member of this group, Chlamydomonas reinhardtii (and closely related species) 

has been studied very extensively, in part because of its ability to control sexual 

reproduction, thus allowing detailed genetic analysis. Indeed, Chlamydomonas

was the first alga to be genetically transformed. However, it does not 

accumulate lipids, and thus was not considered for use in the ASP. Another 

common genus that has been studied fairly extensively is Chlorella. 

• Golden-Brown Algae. This group of algae, commonly referred to as 

chrysophytes, is similar to diatoms with respect to pigments and biochemical 

composition. Approximately 1,000 species are known, which are found 

primarily in freshwater habitats. Lipids and chrysolaminarin are considered to 

be the major carbon storage form in this group. Some chysophytes have lightly 

silicified cell walls. 

• Prymnesiophytes. This group of algae, also known as the haptophytes, consists 

of approximately 500 species. They are primarily marine organisms, and can 

account for a substantial proportion of the primary productivity of tropical 

oceans. As with the diatoms and chrysophytes, fucoxanthin imparts a brown 

color to the cells, and lipids and chrysolaminarin are the major storage products. 

This group includes the coccolithophorids, which are distinguished by 

calcareous scales surrounding the cell wall. 

• Eustigmatophytes. This group represents an important component of the 

“picoplankton”, which are very small cells (2-4 μm in diameter). The genus 

Nannochloropsis is one of the few marine species in this class, and is common 

in the world’s oceans. Chlorophyll a is the only chlorophyll present in the cells, 

although several xanthophylls serve as accessory photosynthetic pigments. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 7 

• Cyanobacteria. This group is prokaryotic, and therefore very different from all 

other groups of microalgae. They contain no nucleus, no chloroplasts, and have 

a different gene structure. There are approximately 2,000 species of 

cyanobacteria, which occur in many habitats. Although this group is 

distinguished by having members that can assimilate atmospheric N (thus 

eliminating the need to provide fixed N to the cells), no member of this class 

produces significant quantities of storage lipid; therefore, this group was not 

deemed useful to the ASP. 

Collection and Screening of Microalgae: Programmatic Rationale 

The in-house collection effort was focused on collecting strains from inland saline habitats, 

particularly in Colorado, New Mexico, and Utah. The reasoning behind collecting strains from 

these habitats was that the strains would be adapted to at least some of the environmental 

conditions in mass culture facilities in the southwestern United States (i.e., high light intensity 

and high temperatures). They would also be well suited for growth in the saline waters available 

for use in such facilities. In addition, many of the aquatic habitats in this region are shallow, and 

therefore subject to large variations in temperature and salinity; thus, the strains collected in this 

region might be expected to better withstand the fluctuations that would occur in a commercial 

production pond. Cyanobacteria, chrysophytes, and diatoms often dominate inland saline 

habitats. The latter were of particular interest to the program because of their propensity to 

accumulate lipids. There had never been a large-scale effort to collect strains with this 

combination of characteristics; therefore, they were not available from culture collections. 

The stated objectives2

 of the SERI culture collection and screening effort were to: 

• Assemble and maintain a set of viable mono-specific algal cultures stored under 

conditions best suited to the maintenance of their original physiological and 

biochemical characteristics. 

• Develop storage techniques that will help maintain the genetic variability and 

physiological adaptability of the species. 

• Collect single species cultures of microalgae from the arid regions of Colorado, 

Utah, and New Mexico for product and performance screening. 

• Develop media which are suitable for their growth. 

• Evaluate each species for its temperature and salinity tolerances, and quantify 

growth rates and proximate chemical composition for each species over the 

range of tolerated conditions. 

 2 Taken from the Proceedings of the April 1984 Aquatic Species Program Principal Investigators’ Meeting. 
National Renewable Energy Laboratory 

8 A Look Back at the Aquatic Species Program—Technical Review 

Each objective was met during the course of research within the ASP. The following pages 

describe in detail the major findings of the work conducted by SERI researchers. 

II.A.1.b. Collection and Screening Activities - 1983 

The first collecting trips made by SERI researchers took place in the fall of 1983. Five saline hot 

springs in western Colorado were selected for sampling because of their abundant diatom 

populations, and because a variety of water types was represented. Water samples were used to 

inoculate natural collection site water that had been enriched with N (ammonium and nitrate) and 

phosphate (P) and then filter sterilized. Water samples were also taken for subsequent chemical 

analyses. The temperature and conductivity of the site water were determined at the time of 

collection. Conductivity ranged from 1.9 mmhos•cm-2 at South Canyon Spring to 85.0 

mmhos•cm-2 (nearly three times the conductivity of seawater) at Piceance Spring. Water 

temperature at the time of collection ranged from 11o to 46oC. 

In the laboratory, researchers tried to isolate the dominant diatoms from the enriched water 

samples. Cyanobacteria and other contaminants were removed primarily with agar plating. 

Approximately 125 unialgal diatom strains were isolated. The predominant genera found were 

Achnanthes, Amphora, Caloneis, Camphylodiscus, Cymbella, Entomoneis, Gyrosigma, Melosira, 

Navicula, Nitzschia, Pleurosigma, and Surirella. 

A standardized lipid analysis protocol was not yet in place to screen these strains. However, 

many algal strains were known to accumulate lipids under conditions of nutrient stress. 

Microscopic analysis of cells grown under N-deficient conditions revealed lipid droplets in 

several of the strains, particularly in Amphora and Cymbella. 

In addition to yielding several promising algal strains, this initial collection trip was useful for 

identifying areas for improving the collection and screening protocols. Some of these 

improvements were implemented for the 1984-collecting season, and are described in the next 

section. 

Publications: 

Barclay, W.R. (1984) “Microalgal technology and research at SERI: Species collection and 

characterization.” Aquatic Species Program Review: Proceedings of the April 1984 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341; 

pp. 152-159. 

II.A.1.c. Collection and Screening Activities - 1984 

The screening and characterization protocols used by SERI researchers were refined for the 1984 

collecting season. Included in these refinements was the development of a modified “rotary 

screening apparatus”, a standard type of motorized culture mixing wheel for 16x150-mm culture 

tubes. The rotating wheel was constructed of Plexiglas to allow better light exposure (see Figure 

II.A.1). The wheel was typically illuminated with a high-intensity tungsten stage lamp, and 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 9 

could be placed inside a box behind a CuSO4-water heat filter for temperature control. The 

Plexiglas wheel allowed all the cultures to receive equal illumination. Another technological 

advance used a temperature-salinity gradient table to characterize the thermal and salinity 

preferences and tolerances of the isolates. 

Development of artificial saline media. 

One of the most significant contributions made by SERI researchers during 1984 was the 

development of media that mimicked the saline water in shallow aquifers in the southwestern 

United States. This was an important undertaking because it allowed algal strains to be screened 

for growth in the types of water that would likely be available in an outdoor mass culture facility. 

To identify the major water types available in the southwestern United States, state and federal 

reports that described the chemical characteristics of water from 85 saline wells in New Mexico 

were studied. The data were statistically analyzed to identify the relationships between the 

various ionic constituents. (Data from wells deeper than 83 m was not used in this analysis, 

because the cost of pumping water from those depths was prohibitive.) R-mode factor analysis 

indicated that two factors were largely responsible for the differences between the waters 

examined (Barclay et al. 1988). The first factor, monovalent ion concentration, was responsible 

for 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of these 

factors against each other clearly delineated two primary water types, referred to as “Type I” and 

“Type II”. Type I waters were characterized by a low monovalent-to-divalent ion ratio (average 

value = 0.4), whereas Type II waters had a higher level of monovalent ions (monovalent-todivalent ion ratio of 9.4). The major ions present in Type I water were Na+

, Cl-, Mg2+, and Ca2+. 

The major ions of Type II water were Na+

, Cl-, SO4

2-, and HCO3-

. Type II water is consequently 

termed a “sodium bicarbonate class” of water. Approximately three-fourths of the saline well 

waters were of the Type II variety, and one-fourth could be characterized as Type I. 

The survey indicated that both types of water exhibited a range of conductivities; the researchers 

believed that the higher-conductivity waters resulted from evaporation of the lower conductivity 

waters. In addition, they recognized that the conductivity of the water in an outdoor production 

pond would increase with time because of the high rates of evaporation in the southwestern 

United States (as high as 1 cm•day-1). Therefore, artificial media that covered a wide range of 

conductivities had to be developed. To this end, an experiment was conducted in which media 

that contained the salts typically present in low-conductivity Type I and Type II waters were 

allowed to evaporate with stirring at 35oC. Samples were removed at various times and filtered. 

The ions still dissolved in the waters were quantified using an inductively coupled plasma 

spectrometer and a high-performance liquid chromatograph. In this manner, media formulations 

were derived at SERI that covered a range of conductivities (from 10 to 70 mmho•cm-1) for both 

media types. The media most commonly used were designated SERI Type I/10, Type I/25, Type 

I/55, Type I/70, Type II/10, Type II/25, Type II/55, and Type II/70, in which the number 

following the slash indicates the specific conductivity of the medium. The compositions of these 

media are given in Figure II.A.2. 
National Renewable Energy Laboratory 

10 A Look Back at the Aquatic Species Program—Technical Review 

In order to assess whether these media formulations accurately reflected the types of water in 

desert region surface waters, samples of the water at numerous algal collection sites in the 

southwestern United States were chemically analyzed. The relative compositions of the anionic 

and cationic constituents were then plotted on separate trilinear plots, which allowed a graphical 

representation of the various water samples relative to SERI Type I and Type II media (Figure 

II.A.3). This analysis indicated that Type I water has higher proportions of Mg2+ and Ca2+ than 

most surface waters examined, whereas Type II water was fairly representative of the sampled 

waters with respect to these cations. On the other hand, natural surface waters often had an anion 

composition similar to both SERI Type I and Type II media. The researchers concluded that 

these artificial media would serve well as standardized media for testing newly acquired strains, 

thereby allowing all ASP researchers (both in-house personnel and subcontractors) to screen 

strains for growth potential in waters similar to those that would be available for commercial 

production. 

Collection activities. 

Collecting trips made by SERI researchers in 1984 focused on shallow saline habitats, including 

ephemeral ponds, playas, and springs in the arid regions of Colorado and Utah. After collection, 

the water and sediment samples were kept under cool, dark conditions for 1 to 3 days until they 

could be further treated in the laboratory. The pH, temperature, conductivity, redox potential, 

and alkalinity of the collection site waters were determined, and water samples were taken for 

subsequent ion analysis. In the laboratory, the samples were enriched with 300 μM urea, 30 μM 

PO4, 36 μM Na2SiO3, 3 μM NaFeEDTA, trace metals (5 mL/L PII stock, see Figure II.A.2), and 

vitamins. The enrichment tubes were then placed in the rotary screening apparatus (maintained 

at 25oC or 30oC) and illuminated at ~400 μE•m-2

•s

-1. Over a 5-day period, the illumination 

provided by the stage lamp was gradually increased to 1,000 μE•m-2

•s

-1. The predominant strains 

present in the tubes were isolated as unialgal cultures by agar plating or by serial dilution in 

liquid media. 

The isolated strains were then tested for their ability to grow in incubators at 25oC at 150-200 

μE•m-2

•s

-1 in the standard media types described above. and in artificial seawater (termed “Rila 

Salts ASW,” using Rila Marine Mix, an artificial sea salt mixture produced by Rila Products, 

Teaneck, NJ. The strains that grew well in at least one of these media were further characterized 

with respect to growth on a temperature-salinity gradient table at a light intensity of 200 

μE•m-2

•s

-1. Thirty combinations of temperature (10o to 35oC) and salinity (10 to 70 mmho•cm-1) 

were included in this analysis, representing the ranges that might be expected in actual outdoor 

production systems. Once again, the cultures were enriched with nutrients to maximize growth 

rates. The cultures used to inoculate the test cultures were preconditioned by growth in the 

media at 17° and 27oC. The optical density at 750 nm (OD750) of the cultures was measured 

twice daily for 5 days, and the growth rates were calculated from the increase in culture density 

during the exponential phase of growth. A refinement of this method was to measure the growth 

rates in semicontinuous cultures, wherein the cultures were periodically diluted by half with fresh 

medium; this method provided more reproducible results than the batch mode experiments. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 11 

Figure II.A.3 gives an example of the type of growth data generated by the use of temperaturesalinity gradient tables. The contour lines in the plot are interpolations indicating where a 

particular combination of temperature and salinity would result in a given growth rate. Many 

such plots were generated for various strains, and are shown in the culture collection catalogs and 

ASP annual reports. 

Approximately 300 strains were collected from the 1984 trips to Utah and Colorado. Of these, 

only 15 grew well at temperatures ≥30oC and conductivities greater than 5 mmho•cm-1. Nine 

were diatoms, including the genera Amphora, Cymbella, Amphipleura, Chaetoceros, Nitzschia, 

Hantzschia, and Diploneis. Several chlorophytes (Chlorella, Scenedesmus, Ankistrodesmus, and 

Chlorococcum) were also identified as promising strains, along with one chrysophyte 

(Boekelovia). 

Two strains isolated as a result of the 1984 collecting effort (Ankistrodesmus sp. and Boekelovia 

sp.) were characterized in greater detail using the temperature-salinity matrix described earlier. 

Boekelovia exhibited a wide range of temperature and salinity tolerance, and grew faster than one 

doubling•day-1 from 10 to 70 mmho•cm-1 conductivity and from 10o to 32oC, exhibiting maximal 

growth of 3.5 doublings•day-1 in Type II/25 medium. Reasonable growth rates were also 

achieved in SERI Type I and ASW-Rila salts media (as many as 1.73 and 2.6 doublings•day-1, 

respectively). Ankistrodesmus was also able to grow well in a wide range of salinities and 

temperatures, with maximal growth rates occurring in Type II/25 medium (3.0 doublings•day-1). 

Boekelovia and Ankistrodesmus were also examined with regard to their lipid accumulation 

potential. Two-liter cultures were grown in media that contained high (600 μM) and low (300 

μM) urea concentrations at a light intensity of 200 μE•m-2

•s

-1. Half of each culture was harvested 

2 days after the low-N culture entered stationary phase to determine the lipid content of Nsufficient cells and cells that were just entering N-deficient growth. After 10 days of N-limited 

growth, the remainder of the low-N culture was harvested. Lipids were extracted via a 

modification of the method of Bligh and Dyer (1959) and lipid mass was determined 

gravimetrically. The lipid content of Boekelovia was 27% of the organic mass in N-sufficient 

cells, increasing to 42% and 59% after 2 days and 10 days of N-deficiency, respectively. There 

was less effect of N starvation on the lipid content of Ankistrodesmus; the lipid content only 

increased from 23% in N-sufficient cells to 29% in cells that were N-deficient for 10 days. 

In conclusion, research at SERI in 1984 led to the development of artificial media that mimicked 

the saline groundwater typically found in the desert regions of the southwestern United States. 

This allowed the strains isolated during collecting trips at various ionic concentrations to be 

systematically screened and provided standardized media that could be used in different 

laboratories performing ASP-sponsored research. Numerous strains were characterized with 

respect to growth at several temperatures and salinities using these new media. 
National Renewable Energy Laboratory 

12 A Look Back at the Aquatic Species Program—Technical Review 

Publications: 

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae 

Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP232-3079, 147 pp. 

Barclay, B.; Nagle, N.; Terry, K. (1987) “Screening microalgae for biomass production potential: 

Protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 23-40. 

Barclay, B.; Nagle, N.; Terry, K.; Roessler, P. (1985) “Collecting and screening microalgae from 

shallow, inland saline habitats.” Aquatic Species Program Review: Proceedings of the March 

1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-2700; pp. 52-68. 

Barclay, W.R.; Nagle, N.J.; Terry, K.L.; Ellingson, S.B.; Sommerfeld, M.R. (1988) 

“Characterization of saline groundwater resource quality for aquatic biomass production: A 

statistically-based approach.” Wat. Res. 22:373-379. 

Sommerfeld, M.R.; Ellingson, S.B. (1987) “Collection of high energy yielding strains of saline 

microalgae from southwestern states.” FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-3071; pp. 53-66. 

Additional References: 

Bligh, E.G.; Dyer, D.J. (1959) “A rapid method for total lipid extraction and purification.” Can. 

J. Biochem. Physiol. 37:911-917. 

Siver, P. (1983) “A new thermal gradient device for culturing algae.” British J. Phycol. 18:159-

163. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 13 

Figure II.A.1. Rotary screening apparatus used for microalgal screening. 
National Renewable Energy Laboratory 

14 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.A.2. Formulations for SERI Type I and Type II artificial inland saline waters. 

Recipes for the preparation of Type I and Type II media at five different salinities, expressed as 

conductivity of the final solution. Formulas for these media were developed by statistical 

analysis of saline groundwater data for the state of New Mexico. For each salt, necessary 

additions in mg/L are listed. (Source: Barclay et al. 1986). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 15 

Figure II.A.3. Trilinear plots showing the ionic constitutents of various water samples 

relative to SERI Type I and SERI Type II artificial saline media. (Source: Sommerfeld and 

Ellingson 1987.) 
National Renewable Energy Laboratory 

16 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.A.4. Growth contour plots. Examples of growth contour plots generated from 

data obtained by the use of a temperature-gradient table. The contour lines represent 

interpolated values indicating where a particular combination of temperature and salinity 

would result in a given growth rate. The data shown, given as doublings•day) represent the 

exponential growth of Monoraphidium sp. (S/MONOR-2) in semicontinuous culture. Each 

point represents the mean of at least five separate daily growth rate determinations. (Source: 

Barclay et al. 1987). 

A: Type I inland saline water 

B: Type II inland saline water 

C: Seawater 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 17 

II.A.1.d. Collection and Screening Activities - 1985 

In 1985, the strain enrichment procedure utilizing the rotary screening apparatus described 

previously was modified to include incubation of samples in SERI Type I and Type II media (25 

and 55 mmho•cm-1 conductivity) and in artificial seawater, in addition to the original site water. 

The cultures that exhibited substantial algal growth were further treated to isolate the 

predominant strains as unialgal (clonal) isolates. These strains were then tested for growth using 

the temperature-salinity matrix described earlier. 

Collection activities. 

Collection efforts by SERI researchers in 1985 again focused on shallow inland saline habitats. 

This time collecting trips were also made to New Mexico and Nebraska, in addition to Colorado 

and Utah. Eighty-six sites were sampled during the year, 53 of which were sampled in the 

spring. From these 53 sites, 17 promising strains were isolated. An analysis was conducted 

comparing the results of the new protocol with those that would have resulted from the protocol 

used in prior years. This analysis indicated that the revised protocol was in fact superior to the 

older protocol. For example, only six of the 17 strains selected via the new protocol would also 

have been selected using the old protocol. Only three of the 17 strains grew best in the artificial 

medium type that most closely resembled the collection site water; in fact, only six strains were 

even considered to grow well in the collection site water relative to growth in at least one of the 

artificial medium. This analysis clearly indicated the value of performing the initial screening 

and enrichment in a variety of relevant media. The results suggest that the shallow saline 

environments sampled probably contain a large number of species whose metabolism is arrested 

at any given time. In other words, the water quality of such sites varies greatly, depending on 

precipitation and evaporation, so probably only a few of the many species present are actively 

growing at any given time. This also may explain the wide range of salinities and temperatures 

tolerated by many of these strains. 

Growth rates. 

Six promising strains were analyzed in SERI Type I, Type II, and ASW (Rila) using the 

temperature-salinity gradient described previously. These included the diatoms Chaetoceros

muelleri (CHAET14), Navicula (NAVIC1), Cyclotella (CYCLO2), Amphora (AMPHO1 and 

AMPHO2), and the chlorophyte Monoraphidium minutum (MONOR2). (NAVIC1 and 

CYCLO2 were actually collected from the Florida keys; the remaining strains were collected in 

Colorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in at 

least two media types. Furthermore, all strains exhibited temperature optima of 30oC or higher. 

Maximal growth rates of these strains, along with the optimal temperature, conductivity, and 

media type determined in these experiments are shown in Table II.A.1. (Higher growth rates 

were determined for some of these strains in subsequent experiments; see results presented in 

Barclay et al. [1987]). Temperature-salinity growth contours are provided for these strains in the 

1986 ASP Annual Report (Barclay et al. 1986). 
National Renewable Energy Laboratory 

18 A Look Back at the Aquatic Species Program—Technical Review 

Table II.A.1. Growth characteristics of various microalgal strains collected in 1985. 

Strain Maximum 

Growth Rate 

(doublings•day-1) 

Optimal 

Temperature 

(oC) 

Optimal 

Conductivity 

(mmho•cm

-1) 

Optimal Medium Type 

(dependent on temperature 

and conductivity used) 

AMPHO1 1.7 30 10-25 Type I, ASW 

AMPHO2 2.48 30-35 40-70 Type I, Type II 

CHAET14 2.87 35 25-70 Type II, ASW 

CYCLO2 1.63 30-35 10-70 Type I, ASW 

MONOR2 2.84 25-30 25 Type I, II, ASW 

NAVIC1 2.77 30 10-40 Type I, Type II 

Experiments were also conducted in an attempt to identify the chemical components of SERI 

Type I and Type II media most important for controlling the growth of the various algal strains. 

Bicarbonate and divalent cation concentrations were found to be important determinants in 

controlling the growth of Boekelovia sp. (BOEKE1) and Monoraphidium (MONOR2). The 

growth rate of MONOR2 increased by more than five-fold as the bicarbonate concentration of 

Type II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 by 

approximately 60% over this range. These results make sense, since media enriched in 

bicarbonate would have more dissolved carbon available for photosynthesis. An unexpected 

finding was that there was a decrease of nearly 50% in the growth rate of BOEKE1 as the 

divalent cation concentration increased from 5 mM to 95 mM (in Type I/10 medium containing 

altered amounts of calcium and magnesium). The effects of magnesium and calcium 

concentration on the growth of MONOR2 were less pronounced. These results indicate that 

matching the chosen strain for a particular production site to the type of water available for mass 

cultivation will be important. 

Lipid content. 

The lipid contents of several strains were determined for cultures in exponential growth phase 

and for cultures that were N-limited for 7 days or Si-limited for 2 days. In general, nutrient 

deficiency led to an increase in the lipid content of the cells, but this was not always the case. 

The highest lipid content occurred with NAVIC1, which increased from 22% in exponential 

phase cells to 49% in Si-deficient cells and to 58% in N-deficient cells. For the green alga 

MONOR2, the lipid content increased from 22% in exponentially growing cells to 52% for cells 

that had been N-starved for 7 days. CHAET14 also exhibited a large increase in lipid content in 

response to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A more 

modest increase occurred for nutrient-deficient AMPHO1 cells, whereas the lipid content of 

CYCLO2 was similar in exponential phase and nutrient-deficient cells, and actually decreased in 

AMPHO2 as a result of nutrient deficiency. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 19 

These results suggested that high lipid content was indeed achievable in many strains by 

manipulating the nutrient levels in the growth media. However, these experiments did not 

provide information on actual lipid productivity in the cultures, which is the more important 

factor for developing a commercially viable biodiesel production process. This lack of lipid 

productivity data also occurred with most of the ASP subcontractors involved in strain screening 

and characterization, but was understandable because the process for maximizing lipid yields 

from microalgae grown in mass culture never was optimized. Therefore, there was no basis for 

designing experiments to estimate lipid productivity potential. 

Publications: 

Barclay, B.; Nagle, N.; Terry, K. (1986) “Screening microalgae for biomass production potential: 

protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 22-40. 

Barclay, W.R.; Terry, K.L.; Nagle, N.J.; Weissman, J.C.; Goebel, R.P. (1987) “Potential of new 

strains of marine and inland saline-adapted microalgae for aquaculture.” J. World. Aquaculture 

Soc. 18:218-228. 

II.A.1.e. Collection and Screening Activities - 1986 and 1987 

SERI in-house algal strain collection and screening efforts during 1986-1987 were focused in 

three separate areas. First, detailed characterization of previously collected strains continued. 

Second, because the desert southwest sites targeted for biodiesel production facilities can be quite 

cool during the winter, a new effort to collect strains from cold-water sites was initiated. Finally, 

a strategy was developed and implemented to reduce the number of strains that had accumulated 

as a result of in-house and subcontracted research efforts, which allowed researchers to focus on 

the most promising strains. 

Strain characterization. 

Eight additional strains collected previously from warm-water sites that grew well during the 

initial screening procedures were characterized with respect to temperature and salinity 

tolerances, growth rates, and lipid content under various conditions. These strains were 

Chaetoceros muelleri (strains CHAET6, CHAET9, CHAET10, CHAET15, and CHAET39), 

Cyclotella cryptica (CYCLO4), Pleurochrysis carterae (PLEUR1), and Thalassiosira weissflogii

(THALA2). Each strain was grown in a variety of temperature-salinity combinations by the use 

of a temperature-salinity gradient table. The maximal growth rate achieved under these 

conditions occurred with CHAET9, which exhibited a growth rate of 4.0 doublings•day-1. The 

remaining strains all had maximum growth rates that exceeded 1.4 doublings•day-1, and several 

grew at rates exceeding 2.5 doublings•day-1 (i.e., CHAET6, CHAET10, and CHAET39). All had 

an optimal temperature of 30°C or higher, except for PLEUR1 and THALA2, which had optimal 

temperatures of 25°C and 28°C, respectively. Most of the strains grew well in a wide range of 

salinities (e.g., five of the eight strains exhibited a growth rate greater than one doubling•day-1 at 
National Renewable Energy Laboratory 

20 A Look Back at the Aquatic Species Program—Technical Review 

conductivities between 10 and 70 mmho•cm-1). With respect to the effect of water type on 

growth, CHAET39, CYCLO4, and PLEUR1 grew best on SERI Type I medium. On the other 

hand, CHAET6, CHAET9, and CHAET10 grew best in SERI Type II medium, but also exhibited 

good growth on Type I medium and artificial seawater. CHAET15 and THALA2 achieved 

maximal growth rates on artificial seawater, and, along with PLEUR1, grew very poorly on Type 

II medium. These results again highlight the need to have a variety of algal strains available for 

the specific water resources that would be available for mass culture in various locations. 

The lipid contents of these 10 strains were also determined for exponentially growing cells, as 

well as for cells that were grown under nutrient-limited conditions. Nitrogen deficiency led to an 

increase in the lipid contents of CHAET6, CHAET9, CHAET10, CHAET15, CHAET39, and 

PLEUR1. The mean lipid content of these strains increased from 11.2% (of the total organic 

mass) in nutrient-sufficient cells to 22.7% after 4 days of N deficiency. Silicon deficiency led to 

an increase in the lipid content of all strains (although in some cases the increase was small and 

probably not statistically significant). The mean lipid content of the eight strains increased from 

12.2% in nutrient-sufficient cells to 23.4% in Si-deficient cells. A few strains were poor lipid 

producers, such as CHAET6, CYCLO4, and PLEUR1, which did not produce more than 20% 

lipid under any growth conditions. 

Cold water strain collection efforts. 

Most microalgal collection efforts carried out under the auspices of the ASP before 1987 focused 

on sites that were expected to naturally experience high temperatures; indeed, one subcontractor, 

Keith Cooksey (Montana State University) specifically searched for thermophilic strains isolated 

from hot springs. This was because the temperatures of production ponds in the southwestern 

United States during the prime growing season were expected to reach high levels; thus the 

production strains would have to thrive under such conditions. However, temperatures in this 

region are quite cool for several months of the year and can drop to below freezing at night. 

Consequently, an effort was initiated by SERI researchers to collect, screen, and characterize 

strains from cold-water habitats. 

Four collecting trips were made between October 1986 and March 1987 to various inland saline 

water sites in Utah and eastern Washington, and to the coastal lagoon waters in southern 

California. Water samples were enriched with N, P, trace metals, and vitamins; artificial media 

were not used in the initial selection protocol for these experiments. The rotary screening 

apparatus was maintained at 15°C for the duration of the screening process by including a copper 

cooling coil inside the screening chamber. The cultures were incubated for 5-10 days, which is 

longer than for warm-water strains because of the slower growth at the cooler temperature. This 

procedure created a problem, however, in that many more strains survived the selection process 

than when 30°C was used as the selection temperature. As a consequence, separating strains 

from each other and identifying which were the best for further characterization were more 

difficult. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 21 

An interesting finding from the cold water strain collection project was that many species that 

predominate after the enrichment procedure were the same as the warm water species selected in 

previous collection efforts. The genera and species that were commonly found in both the cold 

water and warm water screening projects were C. muelleri, Amphora coffeiformis, Cyclotella, 

Navicula, and Nitzschia. However, some ochromonids and green coccoid algae were also 

isolated from the cold water collection effort; these types of alga were less commonly isolated 

during the warm-water selection procedures. Additional work would be needed to characterize 

these strains with respect to lipid production potential. Future work should look at the fatty acid 

profiles of oil found in the cold-water strains. Such cold-water organisms often contain high 

levels of polyunsaturated fatty acids, which would perform poorly as a feedstock for biodiesel 

because of their low oxidative stability and tendency to polymerize during combustion 

(Harrington et al. 1986). 

Publications: 

Johansen, J.R.; Doucette, G.J.; Barclay, W.R.; Bull, J.D. (1988) “The morphology and ecology of 

Pleurochrysis carterae var dentata nov. (Prymnesiophyceae), a new coccolithophorid from an 

inland saline pond in New Mexico, USA.” Phycologica 27:78-88. 

Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, and 

characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987 

Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3206, pp. 27-42. 

Johansen, J.R.; Theriot, E. (1987) “The relationship between valve diameter and number of 

central fultoportulae in Thalassiosira weissflogii (Bacillariophyceae).” J. Phycol. 23:663-665. 

Tadros, M.G.; Johansen, J.R. (1988) “Physiological characterization of six lipid-producing 

diatoms from the southeastern United States.” J. Phycol. 24:445-452. 

Additional References: 

Harrington, K.J. (1986) Biomass 9:1-17. 

II.A.1.f. Development of a Rapid Screening Procedure for Growth and Lipid Content 

of Microalgae 

By 1987, SERI researchers and subcontractors had collected approximately 3,000 algal strains. 

Most of these strains had not been well characterized, especially with respect to lipid production 

capabilities. As a consequence, work commenced on the development of a simple screening 

procedure to estimate the lipid contents of cells to determine which strains had the best potential 

as biofuel production organisms. Ideally, the procedure should be simple and reproducible so 

that it could be used as a standard method in numerous laboratories. The researchers hoped that 

such a screening tool would allow the size of the strain collection to be reduced to a manageable 

number (~200) representing the most promising strains. 
National Renewable Energy Laboratory 

22 A Look Back at the Aquatic Species Program—Technical Review 

Development of a rapid screen for lipid content. 

In an attempt to develop a reproducible, easy-to-use screening procedure to identify algal strains 

with high lipid contents, Dr. Keith Cooksey (an ASP subcontractor at Montana State University) 

suggested that investigators explore the possibility of using the lipophilic dye Nile Red (9-

diethylamino-5H-benzo{a}phenoxazine-5-one) to stain cells. Nile Red was first isolated from 

Nile Blue by Greenspan et al. (1985), who showed that Nile Red will fluoresce in a nonpolar 

environment and could serve as a probe to detect nonpolar lipids in cells. Nile Red permeates all 

structures within a cell, but the characteristic yellow fluorescence (approximately 575 nm) only 

occurs when the dye is in a nonpolar environment, primarily neutral storage lipid droplets. Earlier 

work within the ASP by Dr. Steve Lien had shown the utility of Nile Blue in microscopically 

assessing the lipid content of algal cells (Lien 1981). The active ingredient in these Nile Blue 

preparations may in fact have been Nile Red. Parallel efforts to develop a Nile Red staining 

procedure were carried out by SERI researchers and ASP subcontractors, notably Drs. Cooksey 

and Sommerfeld. 

Cooksey et al. (1987) used the diatom Amphora coffeiformis to optimize the Nile Red staining 

procedure. The dye was dissolved in acetone and used at a concentration of 1 mg/mL of cell 

suspension. In this species, the fluorescence of the dye in live stained cells was stable for only 2-

7 minutes; fluorescence measurements had to be completed rapidly to ensure consistent results. 

The kinetics of fluorescence in stained cells varied in different species, presumably due to 

differences in the permeability of cell walls to the stain, and differences in how the lipid is stored 

in the cells, i.e., as large or small droplets. Fixing the stained cells with formaldehyde or ethanol 

preserved the Nile Red fluorescence for 2 hours, but cells that were chemically fixed before Nile 

Red staining did not exhibit the characteristic yellow fluorescence. When Nile Red fluorescence 

was measured in algal cultures over time, the fluorescence increased as the culture became N 

deficient. The fluorescence level was linearly correlated with an increase in the total lipid 

content, determined gravimetrically, in a growing culture of algal cells. Fractionation of the lipids 

by silicic acid column chromatography demonstrated that the increase in lipid was due primarily 

to an increase in neutral lipids rather than in the polar lipids or glycolipids, which are found 

primarily in cell membranes. 

Additional development of the Nile Red screening procedure occurred at SERI and at Milt 

Sommerfeld’s laboratory at Arizona State University. The resultant protocol involved taking a 

fixed volume of a diluted algal culture (typically 4 mL), adding 0.04 mL of a Nile Red solution 

(0.1 mg/mL in acetone), and determining the fluorescence after 5 min using a fluorometer 

equipped with the appropriate excitation and emission filters. 

Although the use of Nile Red allowed various microalgae to be rapidly screened for neutral lipid 

accumulation, interspecies comparisons may be subject to misinterpretation because of the 

species-specific staining differences described earlier. Nonetheless, before Nile Red was used, 

quantitating lipids from cells was very time consuming. It required the extraction of lipid from a 

large number of cells using organic solvents, evaporation of the solvent, and determination of the 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 23 

amount of lipid by weighing the dried extract. Consequently, the use of Nile Red as a rapid 

screening procedure can still have substantial value. 

Screening for growth in high conductivity media. 

The estimation of lipid content using a simple procedure such as the Nile Red assay is clearly an 

important component of a rapid screening procedure for identifying promising strains, but an 

equally important component is a means to identify strains that grow rapidly under the expected 

culture conditions. Reports detailing the amount and types of saline groundwater available in the 

southwestern United States, along with data concerning the high rates of evaporation in this 

region, indicated that tolerance of algal strains to high conductivity (higher than 50 mmho•cm-1) 

could be important. Therefore, an additional component of the secondary screening procedure 

developed to reduce the number of strains being maintained by ASP researchers. Algae were 

tested for the ability to grow at high conductivity (55 mmho•cm-1, both Type I and Type II 

media), high temperature (30oC), and high light intensity (average of 200 μE•m-2

•s

-1, 12 h 

light:12 h dark cycle) in cultures that were continually agitated via aeration. To prevent osmotic 

shock, strains were adapted to higher conductivities via a stepwise transfer into media with 

increasing conductivity at 2-day intervals. Tubes were used that could be placed directly in a 

spectrophotometer (i.e., 25 mm diameter, 50 mL volume), allowing the culture density to be 

measured without removing a sample. The tubes also held enough medium to allow samples to 

be taken for Nile Red lipid analysis (both for N-sufficient and N-deficient cells), and for ash-free 

dry mass determinations. The tubes were placed in a rack and illuminated by fluorescent lamps 

from below for the screening procedure. Optical density measurements were taken twice daily 

for 4 days during exponential growth to determine growth rates. Samples were removed for Nile 

Red fluorometric analysis during exponential growth and after 2 days (Arizona State University) 

or 4 days (SERI) of N deficiency. 

This newly developed rapid screening protocol was subsequently used both in Milt Sommerfeld’s 

laboratory and at SERI to screen many microalgal isolates. Keith Cooksey’s laboratory also 

examined numerous strains using this procedure. Sommerfeld’s laboratory examined 

approximately 800 strains that had been collected over the previous 2 years of the subcontract. 

Only 102 of these strains survived transfer into media having a conductivity of 55 mmho•cm-1. 

Of these strains, 40 grew in both Type I/55 and Type II/55 media, 42 grew only in Type I/55 

medium, and 19 grew only in Type II/55 medium. The 10 fastest-growing strains, along with 

their preferred media, are shown in Table II.A.2. 
National Renewable Energy Laboratory 

24 A Look Back at the Aquatic Species Program—Technical Review 

Table II.A.2 Fastest growing strains from Arizona State University collection. 

Strain Genus Class Growth Rate 

(doublings•day-1) 

Medium 

OSCIL2 Oscillatoria Cyanophyceae 4.23 I/55 

OSCIL3 Oscillatoria Cyanophyceae 3.50 I/55 

CHLOC4 Chlorococcum/ 

Eremosphaera

Chlorophyceae 3.47 I/55 

SYNEC5 Synechococcus Cyanophyceae 3.25 II/55 

ASU0735 Oscillatoria Cyanophyceae 3.06 I/55 

AMPHO46 Amphora Bacillariophyceae 2.81 I/55 

NANNO13 Nannochloris Chlorophyceae 2.78 I/55 

POLYC1 Synechococcus Cyanophyceae 2.73 I/55 

CHLOR23 Chlorella Chlorophyceae 2.66 I/55 

SYNEC3 Synechococcus Cyanophyceae 2.51 II/55 

The lipid production potential of these strains was evaluated by the use of the fluorometric Nile 

Red assay. In Type I/55 medium, 49 strains had a higher apparent lipid content after 2 days of 

N-deficient growth, whereas 13 strains had the same or lower apparent lipid levels in response to 

N deficiency. In Type II/55 medium, 42 strains had higher lipid levels, and 7 strains had lower 

or unchanged lipid levels as a consequence of N deficiency. Of note was that the mean lipid 

level in cells grown in Type II/55 medium was nearly twice that of Type I/55-grown cells. 

The strains exhibiting the highest Nile Red fluorescence levels are shown in Table II.A.3. All of 

these strains are diatoms, confirming the propensity of this group to accumulate lipids. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 25 

Table II.A.3. Strains from the Arizona State University collection having the highest 

Nile Red fluorescence. 

Strain Genus Class Triolein 

equivalents 

(mg•

L-1) 

Exponential 

growth 

Triolein 

equivalents 

(mg•

L-1) 

N-deficient 

growth 

NITZS54 Nitzschia Bacillariophyceae 8 1003 

NITZS53 Nitzschia Bacillariophyceae 17 934 

NITZS55 Nitzschia Bacillariophyceae 37 908 

ASU3004 Amphora Bacillariophyceae 9 593 

NAVIC36 Nitzschia Bacillariophyceae 61 579 

AMPHO45 Amphora Bacillariophyceae 39 308 

FRAGI2 Fragilaria Bacillariophyceae 6 304 

AMPHO27 Amphora Bacillariophyceae 38 235 

NITZS52 Nitzschia Bacillariophyceae 24 234 

These researchers also ranked strains according to the estimated lipid productivity of rapidly 

growing cells, based on the calculated growth rates and estimated lipid contents of exponential 

phase cells. The top strains resulting from this analysis are shown in Table II.A.4. However, the 

optimal strategy for maximizing lipid yield in actual mass culture facilities may require an 

“induction” step (i.e., manipulation of the culture environment, possibly involving nutrient 

deficiency). The ranking of strains would obviously be very different in that case. 
National Renewable Energy Laboratory 

26 A Look Back at the Aquatic Species Program—Technical Review 

Table II.A.4. Strains in the Arizona State University collection with the highest 

apparent lipid productivity during exponential growth, based on Nile Red staining. 

Strain Genus Class Triolein equivalents

(mg•L-1•day-1) 

AMPHO27 Amphora Bacillariophyceae 345 

CHLOC4 Eremosphaera/ 

Chlorococcum

Chlorophyceae 117 

SYNEC5 Synechococcus Cyanophyceae 86 

AMPHO46 Amphora Bacillariophyceae 71 

SYNEC4 Synechococcus Cyanophyceae 64 

AMPHO45 Amphora Bacillariophyceae 63 

NITZS55 Nitzschia Bacillariophyceae 48 

OOCYS9 Oocystis Chlorophyceae 46 

NITZS52 Nitzschia Bacillariophyceae 45 

SERI researchers also started to evaluate various strains by the rapid screening procedure. Initial 

work focused on 25 partially characterized strains. These strains were analyzed for growth and 

Nile Red fluorescence in exponentially growing cultures and in cultures grown under N-deficient 

conditions for 4 days. The results of the SERI and Sommerfeld laboratories cannot be compared 

directly, because Nile Red units are expressed differently and the time duration of N deficiency 

was not the same. The best strains of the 25 tested (based on the highest Nile Red fluorescence 

normalized to ash-free dry weight (AFDW) and rapid exponential growth) were determined to be 

CHAET9 (muelleri), NAVIC2 (Navicula saprophila), and NITZS12 (Nitzschia pusilla). 

Twenty-eight strains of Chaetoceros were also examined using this screening protocol. The best 

strains indentified were CHAET21, CHAET22, CHAET23, and CHAET25 (all muelleri). All 

but the latter strain were isolated from various regions of the Great Salt Lake in Utah. 

The departure of Dr. Bill Barclay and Dr. Jeff Johansen from the ASP, along with a greater 

emphasis on genetic improvement of strains, marked the end of the in-house collection and 

screening work. As a consequence, many of the 3,000 strains collected by ASP researchers 

during the course of this research effort were never analyzed via this rapid screening protocol. 

Nonetheless, enough strains had been analyzed at SERI and at the laboratories of various 

subcontractors to obtain a substantial number of promising strains. The next step was to 

determine their ability to grow in actual outdoor mass culture ponds. This work is described in 

Section III of this report. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 27 

Publications: 

Berglund, D.; Cooksey, B.; Cooksey, K.E.; Priscu, L.R. (1987) “Collection and screening of 

microalgae for lipid production: possible use of a flow cytometer for lipid analysis.” FY 1986 

Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3071, pp. 41-52. 

Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R. (1987) “Fluorometric determination of 

the neutral lipid content of microalgal cells using Nile Red”, J. Microbiol. Methods 6:333-345. 

Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, and 

characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987 

Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3206, pp. 27-42. 

Lien, S. (1981) In Proc. Brighton Int. Conf. On Energy from Biomass (Palz, W.; Chartier, P.; and 

Hall, D.O., eds.), Applied Science Publishers, London, pp. 697-702. 

Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987) “Screening microalgae isolated from the 

southwest for growth potential and lipid yield.” FY 1987 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 43-57. 

Additional References: 

Greenspan, P.; Mayer, E.P.; Fowler, S.D. (1985) “Nile Red: A selective fluorescent stain for 

intracellular lipid droplets.” J. Cell Biol 100:965-973. 

Greenspan, P.; Fowler, S.D. (1985) J. Lipid Res. 26:787 

II.A.1.g. Statistical Analysis of Multivariate Effects on Microalgal Growth and Lipid 

Content 

As discussed earlier, environmental variables (particularly nutrient status) can have great effects 

on growth and the quantity and quality of lipids in microalgae. To determine the effects of 

several environmental variables alone and in combination on the growth and lipid contents of 

microalgae, a multivariate, fractional factorial design experiment was carried out with two 

promising diatoms, Navicula saprophila (NAVIC1) and C. muelleri (CHAET9). For these 

experiments, cells were grown in modified SERI Type II/25 medium in which the alkalinity was 

adjusted by adding sodium carbonate and sodium bicarbonate and the conductivity was adjusted 

by adding sodium chloride. Cultures were grown on the temperature gradient table described 

previously at 200 μE•m-2

•s

-1. The following variables were tested in the multivariate analysis: 

conductivity (20 to 80 mmho•cm-1), temperature (17° to 32oC), N (urea) concentration (0 to 144 

mg•L-1), sodium silicate concentration (0 to 500 mg·L-1), and alkalinity (8.8 to 88 meq•L-1). In 

these experiments, growth was measured by changes in AFDW and lipid was measured by the 

use of the Nile Red fluorometric assay. 
National Renewable Energy Laboratory 

28 A Look Back at the Aquatic Species Program—Technical Review 

The results indicated that the N content and conductivity of the medium were the most important 

variables affecting lipid content (Nile Red fluorescence) of both NAVIC1 and CHAET9. As N 

levels and conductivity increased, the amount of neutral lipid per mg of AFDW decreased. The 

interaction of N and conductivity was an important determinant of lipid content as well. Silicon 

level and alkalinity were more important factors in determining the lipid content for CHAET9 

than for NAVIC1. N concentration was by far the most important factor in determining final cell 

mass for NAVIC1, and was a major factor for cell mass yield in CHAET9 (along with the 

interaction of conductivity and alkalinity, which had a large negative impact on growth). 

Alkalinity was a major factor for growth of both NAVIC1 and CHAET9. However, these 

experiments did not determine actual growth rates, but only the final cell yields; thus, how actual 

cell division rates compared with each other is not known. 

These experiments indicate the importance of examining the interactions of environmental 

variables in determining the effects on growth and lipid production. However, the models 

generated by these kinds of experiments are specific for the strains being studied, and the results 

cannot necessarily be used to predict the effects of these variables on other strains. Furthermore, 

for such models to be truly predictive of growth and lipid production in an actual mass culture, 

much more sophiticated (and realistic) experimental setups would be required. 

Publications: 

Chelf, P. (1990) “Environmental control of lipid and biomass production in two diatom species.” 

J. Appl. Phycol. 2:121-129. 

II.A.1.h. Detailed Analyses of Microalgal Lipids 

In addition to the in-house research being conducted in the area of strain collection and 

screening, there was an effort by Dr. Thomas Tornabene and others to characterize various 

strains via detailed lipid compositional analyses. Dr. Tornabene’s laboratory at SERI (and later 

at the Georgia Institute of Technology) served as the focal point for the analysis of lipids in algal 

samples supplied by various researchers in the ASP. This section will describe the results of 

these analyses, and will provide details about the analytical methods used, as these methods were 

the most comprehensive used in the program. An early report by Tornabene et al. (1980) 

described the lipids that were present in the halophilic alga Dunaliella that had been isolated 

from the Great Salt Lake in Utah. The cells were grown to late logarithmic phase, harvested, and 

extracted with chloroform/methanol via the method of Bligh and Dyer (1959). Additional 

extraction by acetate buffer, followed by refluxing with an alkaline methanol/water mixture was 

then performed, followed by partitioning of lipids into petroleum ether. The extracted lipids 

were fractionated on the basis of polarity using silicic acid columns via differential elution with 

hexane, benzene, chloroform, acetone, and methanol. In this procedure, the lipids are eluted as 

follows: 

1. hexane: acyclic hydrocarbons 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 29 

2. benzene: cyclic hydrocarbons, polyunsaturated acyclic hydrocarbons, sterols, 

and xanthophylls 

3. chloroform: mono-, di- and triacylglycerols, free fatty acids, and phaeophytin a

4. acetone: glycolipids, carotenoids, and chlorophyll a and b; and 

5. methanol: phospholipids and chlorophyll c. 

The various lipid classes were further analyzed via Si gel thin layer chromatography (both oneand two-dimensional), wherein lipids were detected via the use of iodine vapors (and 

autoradiography in the case of 14C-labeled lipids). In addition, lipids containing amino groups 

were detected via the ninhydrin reagent, and phospholipids were detected by the use of 

molybdate/H2SO4. Fatty acids were analyzed via gas chromatography using either flame 

ionization or mass spectroscopic detection after being converted to their methyl ester derivatives 

in the presence of methanolic HCl. The head groups of the polar lipids were identified via gas 

chromatography after being converted to alditol acetates. These and related methods were 

described by Tornabene et al. (1982). 

These analyses indicated that lipids comprised 45%-55% of the total organic mass of Dunaliella

cells. Based on the distribution of 14C after labeling the cells with 14C-bicarbonate, neutral lipids 

accounted for 58.5% of the lipid mass, whereas phospholipids and galactolipids were 22.9% and 

10.9% of the lipid mass, respectively. Isoprenoid hydrocarbons (including β-carotene) and 

aliphatic hydrocarbons (in which the major components were tentatively identified as straightchain and methyl-branched C17 and C19 hydrocarbons with various degrees of unsaturation) 

represented 7.0% and 5.2% of the lipids, respectively. The major fatty acids present were 

palmitic (20.6%), linolenic (12.5%), linoleic (10.7%) and palmitoleic (7.8%), but no attempt was 

made to ascertain whether any of these fatty acids predominated a particular lipid class. The high 

hydrocarbon content of this alga is rather atypical of most of the strains characterized in the ASP. 

These types of hydrocarbons would probably require catalytic conversion into a usable fuel 

source, which would perhaps limit their utility as a production organism. 

A detailed analysis of the lipids present in the green alga Neochloris oleoabundans was also 

carried out by Tornabene (who was later to hold a position at the Georgia Institute of 

Technology), along with G. Holzer (Colorado School of Mines), S. Lien and N. Burris (SERI) 

(Tornabene et al. 1983). The strain used in this study was obtained from the University of Texas 

Algal Culture Collection, and reportedly contained substantial quantities of lipid when grown 

under N-deficient conditions. (However, this is a freshwater strain). Exponentially growing cells 

were transferred into a low-N medium, and after 5 to 7 days of growth in stirred cultures that 

were bubbled with 1% CO2 in air, the cells were harvested and the lipids were extracted. 

Analytical methods were similar to those described earlier, and included the use of pyrrolidineacetic acid/mass spectrometry to determine the position of double bonds in the fatty acids. These 

analyses indicated that 35%-54% of the cellular dry weight was in the form of lipids in Ndeficient cells. Neutral lipids accounted for more than 80% of the total lipids, and were 
National Renewable Energy Laboratory 

30 A Look Back at the Aquatic Species Program—Technical Review 

predominantly in the form of TAGs. Small amounts of straight-chain hydrocarbons and sterols 

were also found (one sterol was identified as a ∆7

 sterol, but low quantities of material made 

identification of the sterols difficult). A number of polar lipids were also quantified, but all polar 

lipids combined accounted for less than 10% of the lipid mass. The fatty acids that comprised 

the TAGs were present in the following proportions: 36% oleic (18:1 ∆9), 15% palmitic (16:0), 

11% stearic (18:0), 8.4% iso-17:0 (an unusual fatty acid for microalgae), and 7.4% linoleic (18:2 

∆9,12). Other saturated and monounsaturated fatty acids were present in TAGs, but represented 

less than 5% each of the total fatty acids present. The high proportion of saturated and 

monounsaturated fatty acids in this alga is considered optimal from a fuel quality standpoint, in 

that fuel polymerization during combustion would be substantially less than what would occur 

with polyunsaturated fatty acid-derived fuel (Harrington, 1986). 

Additional research carried out in Tornabene’s laboratory (Ben-Amotz et al. 1985) examined the 

lipid composition of 7 algal species. Some were from existing culture collections and others 

were isolated by ASP researchers. The lipid contents of these strains were determined under 

conditions of N sufficiency, after 10 days of N deficiency, and under different salinity levels. 

Botryocooccus braunii has received considerable interest as a fuel production organism in other 

laboratories because of its high lipid content. This study confirmed the high lipid levels (55% of 

the organic mass for N-deficient cells). Most of this lipid was in the form of hydrocarbons, 

including C29 to C34 aliphatic hydrocarbons and a variety of branched and unsaturated 

isoprenoids. Glycerolipids were less abundant than the hydrocarbons, and were composed 

primarily of 16:0 and various C18 fatty acids. These data, coupled with the fact that this species 

grows very slowly (one doubling per 72 hours), indicated that Botryococcus would not function 

well as a feedstock for lipid-based fuel production. 

The other species examined in this study were the chlorophytes Ankistrodesmus, Dunaliella, and 

Nannochloris, the diatom Nitzschia, and the chrysophyte Isochrysis. N deficiency led to an 

increase in the lipid content of Ankistrodesmus (from 24.5% to 40.3%), Isochrysis (from 7.1% to 

26.0%), and Nannochloris (from 20.8% to 35.5%), but resulted in a decrease in the lipid content 

of Dunaliella (from 25.3% to 9.2%). Elevating the NaCl concentration of the medium had little 

effect on the lipid content of Botryococcus cells, but caused a slight decrease in the lipid content 

of Dunaliella salina (from 25.3% to 18.5% with an increase in [NaCl] from 0.5 to 2 M). 

Conversely, the lipid content of Isochrysis increased from 7.1% to 15.3% as the NaCl increased 

from 0.5 to 1 M. These results once again highlight the impact of culture conditions on the 

quantities of lipids present. However, as stated before, the most important characteristic of a 

lipid production strain is the overall lipid productivity for a given amount of time, which was not 

examined in this study. 

The polar lipid composition of the strains examined in this study were typical of photosynthetic 

microalgae, and included phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, 

phosphatidylglycerol, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 31 

Table II.A.5 indicates the major fatty acids (those at levels exceeding 5% of the total) present in 

these strains, both under N-sufficient and N-deficient growth conditions. 

In conclusion, the work carried out by Tornabene’s laboratory provided a detailed 

characterization of the lipids present in a variety of microalgae. No general conclusions could be 

made from the work except that the lipid composition of various microalgal strains can differ 

quite substantially. Because the nature of the lipids can have a large impact on the quality of the 

fuel product, characterizing the potential production strains is important to ensure that deleterious 

lipids (e.g., highly polyunsaturated fatty acids in the case of biodiesel fuel) are not present at high 

levels. 

Table II.A.5. Major fatty acids of various microalgae. (Fatty acids in bold are present 

at levels of 15% or higher) 

Strain Nitrogen-sufficient cells Nitrogen-deficient cells 

Ankistrodesmus 16:0, 16:4, 18:1, 18:3 16:0, 18:1, 18:3 

Botryococcus braunii 16:0, 18:1, 18:2, 18:3 16:0, 18:1, 18:3, 20:5 

Dunaliella bardawil not determined 12:0, 14:0/14:1, 16:0, 18:1, 18:2, 

18:3 

Dunaliella salina 14:0/14:1, 16:0, 16:3, 16:4, 

18:2, 18:3

16:0, 16:3, 18:1, 18:2, 18:3

Isochrysis sp. 14:0/14:1, 16:0, 16:1, 18:1, 

18:3, 18:4, 22:6 

14:0/14:1, 18:1, 18:2, 18:3, 18:4, 

22:6 

Nannochloris sp. 14:0/14:1, 16:0, 16:1, 16:2, 

16:3, 20:5 

not determined 

Nitzschia sp. 14:0/14:1, 16:0, 16:1, 16:2, 

16:3, 20:6 

not determined 

Publications: 

Ben-Amotz, A.; Tornabene, T.G. (1983) “Chemical profile of algae with emphasis on lipids of 

microalgae.” Aquatic Species Program Review: Proceedings of the March 1983 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, 

pp. 123-134. 

Ben-Amotz, A.; Tornabene, T.G.; Thomas, W.H. (1985) “Chemical profiles of selected species 

of microalgae with emphasis on lipids.” J. Phycol. 21:72-81. 

Tornabene, T.G.; Holzer, G.; Peterson, S.L. (1980) “Lipid profile of the halophilic alga, 

Dunaliella salina.” Biochem. Biophys. Res. Comm. 96:1349-1356. 
National Renewable Energy Laboratory 

32 A Look Back at the Aquatic Species Program—Technical Review 

Tornabene, T.G.; Ben-Amotz, A.; Hubbard, J.S. (1982) “Isolation, analysis and identification of 

lipids.” SERI ASP Report prepared under contract XK-2-02149-01. 

Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogen 

starved green alga Neochloris oleoabundans.” Enzyme Microb. Technol. 5:435-440. 

Tornabene, T.G. (1984) “Chemical profile of microalgae with emphasis on lipids.” Aquatic 

Species Program Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 64-78. 

Tornabene, T.G.; Benemann, J.R. (1985) “Chemical profiles on microalgae with emphasis on 

lipids.” Aquatic Species Program Review: Proceedings of the March 1985 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, 

pp. 83-99. 

Additional References: 

Harrington, K.J. (1986) Biomass 9:1-17. 

II.A.2. Collection, Screening, and Characterization of Microalgae: Research 

by SERI Subcontractors

II.A.2.a. Introduction 

Included in this section are summaries of the research conducted by various subcontractors 

within the ASP who contributed to the collection, screening, and characterization of microalgal 

strains for potential use in biofuel production facilities. Initially, a variety of strain isolation and 

screening procedures were carried out by the various research groups, as there was no established 

protocol. This lack of uniformity in the screening protocols made comparing the results from one 

laboratory with those of another difficult, and meant that the criteria for selecting the best strains 

differed between the laboratories. At the same time, however, this arrangement provided the 

opportunity for new ideas regarding collecting and screening to be pursued, thereby allowing 

individual creativity in a manner that might be beneficial to the entire program. 

Several subcontractors participated in the strain collection and screening effort. Dr. Bill Thomas 

and colleagues (Scripps Institution of Oceanography) collected a large number of strains from the 

desert regions of eastern California and western Nevada. Additional microalgal strains from 

desert waters in Arizona, New Mexico, California, Nevada, Utah, and Texas were obtained 

through the efforts of Dr. Milt Sommerfeld’s laboratory at Arizona State University. Dr. 

Mahasin Tadros (Alabama A&M University) collected strains habitats in the southeastern United 

States (Alabama, Mississippi, and Florida). Additional strains from the Florida Keys and 

Everglades were collected by John Rhyther (Harbor Branch Foundation); Richard York (Hawaii 

Institute of Marine Biology) isolated a number of strains from the Hawaiian islands and 

surrounding waters. Certain environmental niches were focused on as well; Dr. Keith Cooksey 

(Montana State University) isolated several strains from thermal springs in Yellowstone National 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 33 

Park; Dr. Ralph Lewin (Scripps Institution of Oceanography) focused on picopleustonic algae 

(“floating” algae at the air-sea interface). The results of these efforts are described below. 

II.A.2.b. Yields, Photosynthetic Efficiencies, and Proximate Chemical Composition 

of Dense Cultures of Marine Microalgae 

Subcontractor: University of California, San Diego 

Principal Investigator: William H. Thomas 

Period of Performance: 1980 - 1983 

Subcontract Number: XK-0-9111-1 

Work carried out under this subcontract represented one of the first attempts by an ASP 

subcontractor to characterize the productivity and lipid yields of various microalgae. Six algal 

strains (B. braunii, Dunaliella primolecta, Isochrysis sp., Monallanthus salina, Phaeodactylum 

tricornutum, and Tetraselmis sueica) were obtained from existing culture collections and 

analyzed with respect to lipid, protein, and carbohydrate content under various growth 

conditions. For these experiments, all cultures except for B. braunii were grown in natural 

seawater that was enriched with N, P, and trace metals. B. braunii was grown in an artificial 

seawater medium. Initial experiments to determine productivities of these species were 

performed using batch cultures in 9-L serum bottles. Of the strains tested, the highest growth 

rates were observed with P. tricornutum (Thomas strain) and M. salina. 

Additional experiments were performed in plexiglas vessels that were 5 cm thick, 39 cm deep, 

and 24 cm wide (surface area ~940 cm2

). The cultures were illuminated from the side with a 

2,000-watt tungsten-halide lamp, which was placed behind a water/CuSO4 thermal filter. In 

these experiments, the cultures were typically maintained for 40 to 90 days. In the early stages of 

an experiment, the cultures were maintained in a batch mode, and then converted to a continuous 

or semi-continuous dilution mode. Various culture parameters (including light intensity, dilution 

rate, and N status) were manipulated during the course of these experiments to determine their 

effects on the productivities and proximate chemical composition of the strains. The results of 

these experiments with each species tested are discussed below. These experiments are difficult 

to compare because the experiments were all carried out slightly differently (i.e., different light 

intensities, different culturing methods [batch, semi-continuous, and continuous], different means 

of obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted in 

differences in light quality and culture temperature). Nonetheless, the general conclusions of this 

study are of interest. 

P. tricornutum (Thomas strain): 

This strain has been used for several past studies, and was concomitantly being tested in outdoor 

mass culture by another subcontractor (the University of Hawaii; principal investigator Dr. 

Edward Laws; discussed in Section III). Therefore, this strain was subjected to more extensive 

testing than the other strains in this subcontract. In one experiment reported for this strain, the 
National Renewable Energy Laboratory 

34 A Look Back at the Aquatic Species Program—Technical Review 

effects of light intensity on productivity were determined in batch cultures (i.e., in the Plexiglas 

culture apparatus described earlier without culture replacement and dilution). The maximum 

productivity observed for this strain (21 to 22 g dry weight•m-2•d-1)

3

 was observed at a total daily 

illumination of 63-95 kcal (representing approximately 40%-60% of full sunlight in southern 

California during the summer). This value was slightly higher than the productivity observed 

with a total daily illumination of 70% full sunlight (17.1 g dry weight•m-2•d-1). Productivities 

under N-limiting, continuous growth mode conditions were between 7 and 11 g dry 

weight•m-2•d-1. Likewise, productivities under N-sufficient, continuous growth mode conditions 

were reduced relative to batch cultures. 

In addition to measuring overall productivities, the levels of protein, carbohydrate, lipid, and ash 

were determined for cells grown under the various conditions described earlier. Illumination of 

the cultures from 40% to 70% of full sunlight did not have a large impact on the cellular 

composition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reduction 

of the protein content from 55% (in N-sufficient cells) to 25% of the cellular dry weight. 

Carbohydrate content increased from 10.5% to 15.1%, and the mean lipid content increased from 

19.8% to 22.2%, although these differences in carbohydrate and lipid contents did not appear to 

be statistically significant. At one stage of the experiment, however, a time course of N 

deficiency led to a consistent rise in lipid content from 19.9% to 30.8% over the course of 7 days. 

The actual rate of lipid production did not increase, however, because the overall productivity of 

the cultures was reduced under N-deficient growth. 

D. primolecta: 

The maximum productivity observed for this species (12.0 g dry weight•m-2•d-1 ) occurred during 

continuous culture at 60% full sunlight under N-sufficient conditions. Doubling the light 

intensity lowered the productivity to 6.1 g dry weight•m-2•d-1. The chemical composition of Nsufficient cells (as an average percentage of total cell dry weight) was 64.2% protein, 12.6% 

carbohydrate, and 23.1% lipid. After 7 days of growth under N-deficient conditions, the 

composition was 26.8% protein, 59.7% carbohydrate, and 13.7% lipid. Therefore, this alga 

accumulates carbohydrates rather than lipids in response to nutrient deficiency, limiting its 

usefulness as a lipid production strain. 

M. salina: 

This alga reportedly contained high levels of lipids when grown under N-deficient conditions. 

The highest productivity (13.9 g dry weight•m-2•d-1) was observed under N-sufficient conditions 

at a light intensity of 50% full sunlight, although detailed experiments with regards to the effects 

of light intensity on productivity were not conducted. There was little difference in the lipid 

 3

Reporting of productivities in g dry weight·m-2 ·d-1 derives from the goal of mass culturing the algae in 

shallow open ponds. The objective would be to maximize biomass produced per area of pond. However, it 

is often difficult to compare results between experiments when the data are reported in this manner, as 

factors such as culture depth and vessel design would significantly affect productivity of the cultures.
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 35 

content of cells grown under N-sufficient and N-deficient conditions (20.7% and 22.1%, 

respectively). 

T. sueica: 

The highest productivity observed for this strain was 19.1 g dry weight•m-2•d-1, which occurred 

in N-sufficient batch cultures grown under a light intensity of 60% full sunlight. N deficiency 

resulted in a large increase in carbohydrate content (from a mean value of 10.7% to a mean value 

of 47.1%). On the other hand, protein content was reduced substantially (from 67.6% to 28.3%), 

and the lipid content decreased from 23.1% to 14.6% in response to N deficiency. 

Isochrysis sp.(Tahitian strain T-ISO):

This strain is commonly used as a feed organism in aquaculture production systems. A 

productivity of 11.5 g dry weight•m-2•d-1 was typical for batch cultures of this species, which was 

approximately 33% higher than the value recorded during semi-continuous growth (dilution of 

0.15 L/d). Productivity was lowered during N-deficient growth to 5.5-7.6 g dry weight•m-2•d-1. 

This strain accumulated carbohydrate in response to N deficiency (from a mean value of 23.1% 

to 56.9%). Lipid content also increased slightly (from 28.5% to 33.4%), whereas protein content 

was reduced from 44.9% to 27.3%. The higher lipid content of N-deficient cells did not translate 

to higher lipid productivities, however, because of the lower overall productivity of the stressed 

cultures. 

B. braunii: 

Some very limited experiments were conducted with this species, which is known to accumulate 

hydrocarbons. A culture grown under a light intensity of 60% full sunlight had a productivity of 

only 3.4 g dry weight•m-2•d-1. The lipid content of these cells was 29% of the cellular dry 

weight; the N status of the cells was not reported, but it is assumed that the cells were grown 

under N-sufficient conditions. 

Overall Conclusions 

Of the species examined, P. tricornutum and T. sueica had the highest overall productivities. 

These species also had the highest lipid productivities, which were 4.34 and 4.47 g lipid•m-2•d-1, 

respectively. For both species, the maximal productivities were obtained in batch cultures, as 

opposed to semi-continuous or continuous cultures. Although the lipid contents of cells were 

often higher in response to N deficiency, the lipid productivities of all species tested were 

invariably lower under N deficiency because of an overall reduction in the culture growth rates. 

For the species tested under continuous or semi-continuous growth conditions, lipid 

productivities were reduced from 14% to 45% of the values measured for N-sufficient cultures. 
National Renewable Energy Laboratory 

36 A Look Back at the Aquatic Species Program—Technical Review 

The results also pointed to the importance of identifying strains that are not photoinhibited at 

light intensities that would occur in outdoor ponds. Finally, this work highlighted the fact that 

some microalgae accumulate carbohydrates during nutrient-deficient growth; such strains are 

clearly not acceptable for use as a feedstock for lipid-based fuel production. 

Publications: 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A. (1981) “Effects of light intensity and 

nitrogen deficiency on yield, proximate composition, and photosynthetic efficiency of 

Phaeodactylum.” Proceedings of the Subcontractors’ Review Meeting—Aquatic Species 

Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 33-58. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984a) “Yields, 

photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. I. 

Introduction and Phaeodactylum tricornutum experiments.” Biomass 5:181-209. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984b) “Yields, 

photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. II. 

Dunaliella primolecta and Tetraselmis suecica experiments.” Biomass 5:211-225. 

(Also see references listed in the following section.) 

II.A.2.c. Selection of High-Yielding Microalgae from Desert Saline Environments 

Subcontractor: University of California, San Diego 

Principal Investigator: William H. Thomas 

Period of Performance: 1983 - 1986 

Subcontract Number: XK-2-02170-0-01 

The work carried out under this subcontract represented one of the first efforts to collect 

microalgae from inland saline habitats and to screen those strains for rapid growth rates and lipid 

content. Collecting trips were made to eastern California and western Nevada, and initial 

culturing efforts were conducted at the Sierra Nevada Aquatic Research Laboratory near 

Mammoth Lakes, California, and the Desert Studies Center (Zzyzx Springs), which is near 

Baker, California. Various saline waters and soils were sampled during these collecting trips. 

The collection sites included Pyramid Lake, Black Lake, Owens Lake, Walker Lake, Saline 

Valley, Zzyzx Springs, Armagosa River, Sperry River, Harper Lake, and Salt Creek. The water 

samples were enriched with N, P, Si, and trace metals, then incubated under natural conditions. 

The algae that grew up were isolated by the use of micropipettes. Soil samples were placed in 

“Zzyzx medium” before algal isolation (see Thomas et al. [1986] for media compositions). 

Diatoms, green algae, and cyanobacteria were the dominant types of algae isolated using these 

procedures. Of the 100 strains isolated, 42 were grown under standardized conditions in various 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 37 

artificial media that were designed to mimic the water from which the strains were originally 

obtained. The pH of these various media formulations was typically high because of the 

presence of high levels of carbonate and bicarbonate, and the total dissolved solids ranged from 

approximately 1.5 g•L-1 to over 260 g•L-1. The growth of the cultures was visually scored, and 

nine of the fastest growing strains were further analyzed with respect to growth under scaled-up 

conditions. 

For larger scale cultures, 6 L of medium that was enriched with N (nitrate, urea, or ammonium), 

phosphate, trace metals, and vitamins were placed in 9-L serum bottles, and the cultures were 

illuminated with fluorescent bulbs at a light intensity of 18% full sunlight. To enhance growth, 

the cultures were bubbled with 1% CO2 in air, and more nutrients were added as the cell density 

of the cultures increased. The strains tested in this manner included Nitzschia, Ankistrodesmus, 

Nannochloris, Oocystis (two strains), Chlorella (three strains), and Selenastrum. The estimated 

productivities ranged from 8.8 g dry weight•m-2•d-1 for for Nitzschia S-16 (NITZS14

) grown in 

the presence of urea to 45.8 g dry weight•m-2•d-1 for Oocystis pusilla 32-1, which was also grown 

with urea as the N source. (These productivity values were considered overestimates in that there 

was incidental side lighting of the flasks under the incubation conditions.) Also the productivity 

values did not always correlate with final biomass yield values, indicating that growth saturation 

was reached at different culture densities for the various strains. The maximum biomass yield 

was obtained with O. pusilla 32-1 (2.29 g dry weight•L-1). The results of these experiments 

indicated that certain strains had a clear preference for either urea or nitrate as the N source. 

Because urea is significantly less expensive than nitrate, these results have economic 

implications with respect to algal mass culture. However, the results regarding a preference for a 

particular N source were not always reproducible. 

Additional experiments were carried out to assess the combined effect of temperature and salinity 

on the growth of several of the isolates. A thermal gradient table was used for these experiments 

in which the incubation temperature at six stations on the table varied between 11°C and 35°C. 

Salinities of the media were varied in five increments along the other axis (as much as twice that 

of the natural waters), leading to a total of 30 different combinations of temperature and salinity. 

Growth was determined via optical density measurements, and contour lines were drawn upon a 

matrix chart of the various temperature/salinity combinations. This approach was later used by 

other subcontractors and SERI in-house researchers to determine the optimal growth 

characteristics of numerous promising algal strains. Results from this analysis were reported for 

eight different strains. In general, the strains grew better at higher salinities, indicating their 

halophilic nature, and had temperature optima for growth between 20°C and 30°C. Of the strains 

tested, the Mono Lake isolate NITZS1 had the highest temperature optimum (between 30 and 

36°C). 

The effect of light intensity on the growth of Ankistrodesmus falcatus 91-1 (ANKIS1, from 

Pyramid Lake) and O. pusilla 32-1 (from Walker Lake) was determined in 0.83-L cultures that 

were placed at varying distances from a tungsten lamp source. Neutral density filters were 

 4

NREL Microalgae Culture Collection strain designations are provided when relevant.
National Renewable Energy Laboratory 

38 A Look Back at the Aquatic Species Program—Technical Review 

placed between the light source and the cultures. This arrangement provided between 30% and 

70% of full sunlight. For ANKIS1, maximum productivity (21.9 g dry weight•m-2•d-1) was 

attained when the cells were subjected to 50% full sunlight. At 30% full sunlight, the 

productivity fell off to 14.7 g dry weight•m-2•d-1 (because of light limitation), and at 70% full 

sunlight, the productivity was reduced to 19.0 g dry weight•m-2•d-1 (most likely because of 

photoinhibition). For O. pusilla, a maximum productivity of 25.8 g dry weight•m-2•d-1 was also 

attained at 50% full sunlight, whereas productivity at 30% and 70% full sunlight was 18.7 and 

23.2 g dry weight•m-2•d-1, respectively. These productivity values are believed to be more 

accurate than those reported in the preceding section because light was able to enter the culture 

vessels only from one side. The relative high densities of these cultures (>1 g dry weight•L-1) 

permitted the cells to tolerate higher light intensities than would be possible in less dense 

cultures, because of the self-shading of the cells. 

Experiments were also conducted to determine the effects of varying the culture vessel width 

(i.e., culture depth) on overall productivities. ANKIS1 was grown in containers that were 5, 10, 

and 15 cm wide. The containers were illuminated with a tungsten lamp at 50% full sunlight, and 

were bubbled vigorously with 1% CO2 in air. The results of these experiments indicated that 

growth rate, when expressed as g dry weight•L-1•d-1, was highest in the 5-cm thick culture and 

lowest in the 15-cm thick culture. However, when productivities were expressed as g dry 

weight•m-2•d-1, which takes into account the actual surface area that is illuminated, the thicker 

cultures were more productive. For example, the volumetric productivities over 10 days were 

0.72, 0.35 and 0.31 g dry weight•L-1•d-1) for the 5-, 10-, and 15-cm thick cultures, respectively, 

whereas the corresponding areal productivities for these cultures were 41.1, 40.2, and 52.7 g dry 

weight•m-2•d-1. Because the economic constraints regarding an actual algal biodiesel production 

facility dictate the use of open pond systems, the areal productivity values are the more important 

consideration, although less water (and consequently less water handling) is required when 

dealing with more dense cultures. The productivities reported for these experiments may be 

overestimates of what these strains could achieve in outdoor mass culture because of the 

optimized mixing and aeration regime. 

In the final year of this subcontract, additional collecting trips were taken to gather more 

microalgal strains. These strains, along with some that had been collected during earlier trips, 

were screened more rigorously than before. In this revised selection process, the strains were 

subjected to higher light intensities and higher temperatures, and the abilities of the strains to 

grow in “SERI standard media5

“ were investigated. This selection procedure resulted in the 

isolation of 41 additional strains. Initial screening of strains involved incubating the isolates at 

25°C and 30°C under 40% full sunlight (provided by a 2000-W tungsten-halide lamp) in the 

SERI standard medium that most closely resembled the water from which they were originally 

isolated. Twelve of the strains that grew best under these conditions were then tested under the 

same temperature and light conditions in an early version of standard SERI media (Type I and 

Type II at low, medium, and high salinities; see media compositions in Thomas et al. [1985]). 

 5

The development of the SERI standard media is discussed in Chapter II.A.1. The compositions of these 

media are given in Table II.A.1.
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 39 

The results indicated that most of the strains had a definite preference for a particular medium 

type and level of salinity. The results also indicated some inconsistencies in the growth rates of 

cells grown in the two experiments. For example, Chlorella BL-6 (CHLOR2) grew very well in 

the preliminary experiments in Type II/low salinity medium (2.48 doublings•d-1), but grew much 

more poorly when grown in all five SERI media (including Type II/low salinity) in the second set 

of experiments. Conversely, Chlamydomonas HL-9 grew much more quickly in the second set 

of experiments than in the first set. The reasons for these discrepancies are unclear, as the culture 

conditions were essentially the same, and underscore the need to perform replicate experiments. 

Several marine microalgae were also tested for the ability to grow in SERI standard media. 

Phaeodactylum tricornutum, Chaetoceros gracilis, and Platymonas all grew well (>1.25 

doublings•d-1) in at least one SERI medium. Isochrysis T-ISO was unable to grow in any SERI 

medium, however. 

The combined effects of temperature and salinity on the growth rates of eight of these newly 

collected strains were determined by the use of a temperature-salinity gradient table. In general, 

the strains grew best in the range of salinity that was similar to that of the water from which they 

were originally isolated. The optimal temperature for growth was generally in the 25°C to 35°C 

range, although one Chlorella strain from Salt Creek grew well at 40°C. Based on the results of 

these experiments, two strains were selected for analysis of growth characteristics in larger scale 

(12 L) cultures at 50%-70% full sunlight. CHLOR2 achieved a productivity of 55.5 g dry 

weight•m-2•d-1 under these conditions, and Nannochloris MO-2A had a productivity of 31.9 g dry 

weight•m-2•d-1. 

This subcontract represented one of the first efforts in the ASP to collect and screen microalgal 

strains to identify suitable biofuel production strains. As a consequence, many of the screening 

and characterization protocols were still being developed; therefore, there is a substantial lack of 

uniformity in the testing of the various strains isolated. Nonetheless, a number of promising 

strains were isolated during the course of this research, and several methods were developed that 

helped establish standard screening protocols used by other ASP researchers. 

Publications: 

Thomas, W.H.; Gaines, S.R. (1982) “Algae from the arid southwestern United States: an 

annotated bibliography.” Report for Subcontract XK-2-0270-01. Solar Energy Research 

Institute, Golden, Colorado, October 1982. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983b) 

“Selection of high-yielding microalgae from desert saline environments.” Aquatic Species 

Program Review: Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 97-122. 

Thomas, W.H. (1983b) “Microalgae from desert saline waters as potential biomass producers.” 

Progress in Solar Energy 6:143-145. 
National Renewable Energy Laboratory 

40 A Look Back at the Aquatic Species Program—Technical Review 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1984c) “Cultural requirements, yields, 

and light utilization efficiencies of some desert saline microalgae.” Aquatic Species Program 

Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-2341, pp. 7-63. 

Thomas, W.H.; Tornabene, T.G.; Weissman, J. (1984d) “Screening for lipid yielding microalgae: 

Activities for 1983.” Final Subcontract Report. Solar Energy Research Institute, Golden, 

Colorado, SERI/STR-231-2207. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1985) “Selection of desert saline 

microalgae for high yields at elevated temperatures and light intensities and in SERI Standard 

artificial media.” Aquatic Species Program Review: Proceedings of the March 1985 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado SERI/CP-231-2700, 

pp. 5-27. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1986) “Cultural requirements, yields 

and light utilization efficiencies of some desert saline microalgae.” Nova Hedwigia 83:60-69. 

II.A.2.d. Screening and Characterizing Oleaginous Microalgal Species from the 

Southeastern United States 

Subcontractor: Alabama A&M University 

Principal Investigator: Mahasin Tadros 

Period of Performance: 1983 - 

Subcontract Number: XK-3-03-50-1 

The goal of this subcontract was to isolate and characterize strains of microalgae from the 

southeastern United States that have attributes desirable for a biodiesel production strain. During 

the first year of this work, field trips were made to several sites in Alabama to collect microalgal 

strains from a variety of habitats. Freshwater and brackish water strains were collected from 

rivers, lakes, estuaries, and ponds, and marine strains were collected from the waters surrounding 

Dauphin Island in the Gulf of Mexico. Collected samples were inoculated into various artificial 

media, including Bold’s Basal Medium, Chu no. 10, and “f/2” (Barclay et al. 1986). Artificial 

sea salts were used in place of seawater for the saltwater media. For initial strain selection, the 

cultures were incubated at 29-30°C with shaking at a light intensity of 100 to 125 μE•m-2

•s

-1 

provided by cool white fluorescent bulbs with a 14 h:10 h light:dark cycle. The fastest growing 

strains were isolated via micropipetting or by spreading samples on agar plates. In these 

preliminary experiments, the marine strains exhibiting the fastest growth were Cyclotella DI-35 

(CYCLO1), Hantzschia DI-160 (NITZS2), and Chlorococcum DI-34. The freshwater strains 

exhibiting the fastest growth rates were Chlorella MB-31, Scenedesmus TR-84, Ankistrodesmus

TR-87, and Nitzschia TR-114. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 41 

CYCLO1, Nitzschia TR-114, and Scenedesmus TR-84 were selected for more detailed growth 

analyses under various combinations of temperature, salinity, and light intensity. A temperature 

gradient table was employed for these experiments that was similar in design to the tables used 

by Dr. William Thomas (discussed earlier) and SERI researchers for screening purposes. Growth 

of standing cultures was determined by measuring final cell densities after 12 days of incubation. 

CYCLO1 achieved maximum cell density at a temperature of 30°C, a light intensity of 100 

μE•m-2

•s

-1, and a salinity of 15 ppt (parts per thousand). Growth was nearly as good at a light 

intensity of 200 μE•m-2

•s

-1 and a temperature of 35°C, and substantial growth occurred at a 

salinity of 32 ppt. Growth did not occur at 15° to 20°C. Nitzschia TR-114 achieved maximal 

cell density at 30°C, 15 ppt salinity, and 100 μE•m-2

•s

-1; growth was severely inhibited at 0 and 

45 ppt salinity. Growth was similar at 100 and 200 μE•m-2

•s

-1 for this strain, although the higher 

light intensity seemed to increase the thermal tolerance of the cells. The freshwater strain 

Scenedesmus TR-84 grew best at 25°C, and grew increasingly slower as the salt concentration of 

the medium was increased. 

The lipid contents of several strains isolated during the initial collecting trips were determined. 

For 14-day-old cultures that were reportedly N-limited (although no evidence is provided to 

support this), the lipid contents (as a percentage of the organic mass) were as follows: CYCLO1, 

42.1%; Nitzschia TR-114, 28.1%; Chlorella MB-31, 28.6%-32.4%; Scenedesmus TR-84, 44.7%; 

Ankistrodesmus TR-87, 28.1%; and Hantzschia DI-160 (NITZS2), 66%. 

Additional strains were collected the next year from intertidal waters near Biloxi, Mississippi and 

St. Joseph Bay, Florida. Preliminary screening experiments indicated that five strains (all of 

which were diatoms) had the best growth rates and lipid accumulation potential: Navicula 

acceptata (two strains, NAVIC6 and NAVIC8), N. saprophila (NAVIC7), Nitzschia dissipata

(NITZS13), and Amphiprora hyalina (ENTOM3). These strains and CYCLO1 were grown semicontinuously in media with six different salinities at 25°, 30°, and 35°C. Cells were grown at 

light intensities of 80 μE•m-2

•s

-1 and 160 μE•m-2

•s

-1 (approximately 4% and 8% of full sunlight, 

respectively). The media were produced by adding various quantities of artificial sea salts to 

“f/2” medium; the resulting conductivities were <1, 10, 20, 35, 45, and 60 mmho•cm-1. (Note: 

seawater is typically 35-45 mmho•cm-1.) All strains exhibited more rapid growth under 160 

μE•m-2

•s

-1 illumination than at 80 μE•m-2

•s

-1; and even higher growth rates might well have been 

obtained at light intensities greater than 160 μE•m-2

•s

-1. 

ENTOM3 grew best (2.0 to 2.3 doublings•d-1) at 30°C in media with conductivities of 20-60 

mmho•cm-1. Growth was better with urea or nitrate as the N source rather than with ammonium. 

The lipid content of nutrient-sufficient cells was 22.1% of the organic mass, and increased to 

37.1% and 30.2% under Si-deficient and N-deficient conditions, respectively. 

CYCLO1 achieved the highest growth rates (2.8 to 3.0 doublings•d-1) at 35°C between 10 and 35 

mmho·cm-1. Cells grew best with nitrate as a N source, followed by ammonium and then urea. 

The highest lipid content was observed in N-deficient cells (42.1%), but was also elevated in Sideficient cells (38.6%) relative to nutrient-sufficient cells (13.2%). 
National Renewable Energy Laboratory 

42 A Look Back at the Aquatic Species Program—Technical Review 

NAVIC8 grew most rapidly (3.8 doublings•d-1) at 35°C and 45 mmho•cm-1. Nitrate and 

ammonium were more suitable N sources than urea. Lipid contents of 21.8%, 48.5%, and 32.4% 

were observed for cells grown under nutrient-sufficient, Si-deficient, and N-deficient conditions, 

respectively. 

During the final year of this subcontract, additional promising strains were isolated. Included in 

this group was Navicula BB-324 (NAVIC9), which had a growth rate exceeding 2.5 

doublings•d-1 at 30°C in artificial seawater and SERI Types I/10, I/25, I/40, II/10, II/25, II/40, 

and II/55 media. Navicula SB-304 (NAVIC8) also exhibited excellent growth (1.5-3.0 

doublings•d-1) in each medium. These two strains had Si starvation-induced lipid contents of 

42.5% and 47.2%, respectively. Other notable strains were Nitzschia SB-307 (NITZS13), which 

had a maximal growth rate of 2.5 doublings•d-1 and a lipid content of 45%-47% under nutrientstressed conditions. Amphiprora BB-333 (ENTOM3), Chaetoceros BB-330 (CHAET66), and 

Cylindrotheca AB-204 also grew rapidly (2.3-6.0 doublings•d-1), with stress-induced lipid 

contents ranging from 16.5%-37.1%. 

In conclusion, many promising strains were isolated as a result of this subcontract. The nutrient 

status of the cells again was played an important role in lipid accumulation. Furthermore, the 

nature of the N source included in the medium had a substantial impact on growth of the cultures. 

 Several of these strains were further tested in outdoor mass culture, as described in Section III. 

Publications: 

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae 

Culture Collection 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP232-3079, 147 pp. 

Tadros, M.G. (1985) “Screening and characterizing oleaginous microalgal species from the 

southeastern United States.” Aquatic Species Program Review: Proceedings of the March 1985 

Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP231-2700, pp. 28-42. 

Tadros, M.G. (1987a) “Screening and characterizing oleaginous microalgal species from the 

southeastern United States.” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 67-89. 

Tadros, M.G. (1987b) “Conclusion of the warm-water algae collection and screening efforts 

conducted in the southeastern United States.” FY 1987 Aquatic Species Program Annual Report,

Solar Energy Research Institute, SERI/SP-231-3206, pp. 58-74. 

Tadros, M.G.; Johansen, J.R. (1988) “Physiological characterization of six lipid-producing 

diatoms from the southeastern United States.” J. Phycol. 24:445-452. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 43 

II.A.2.e. Collection of High Energy Strains of Saline Microalgae from Southwestern 

States 

Subcontractor: Arizona State University 

Principal Investigator: Milton Sommerfeld 

Period of Performance: 1985 - 1987 

Subcontract Number: N/A 

The objectives of this subcontract were to collect microalgal strains from a variety of locations in 

the desert regions of the Southwestern United States and to screen them for their ability to grow 

under conditions in a commercial microalgal biodiesel facility. Studies were also conducted to 

optimize a fluorometric procedure for estimating cellular lipid content, and to use this method to 

screen some of the strains. 

Collecting trips took place between April 1985 and June 1986. Water samples containing 

microalgae were collected from 125 sites in Arizona, California, Nevada, New Mexico, Texas, 

and Utah. Some samples were taken from saline surface waters in the regions of Arizona and 

New Mexico that were deemed suitable for microalgal mass culture, based in part on the 

availability of large quantities of saline groundwater (Lansford et al. 1987). Researchers 

believed that strains from these areas would be well adapted to the indigenous waters available 

for mass culture. These areas included the Palo Verde Irrigation District in Arizona, and the 

Pecos River Basin, the Crow Flats area, and the Tularosa Basin in New Mexico. The 

temperature, pH, specific conductance, and water depth were recorded at each collection site. 

Most of the waters sampled had a specific conductance exceeding 2 mmho•cm-1. Temperatures 

ranged from 18°C to 45°C (mean = 26.9°C), pH ranged from 6.1 to 10.2 (mean = 8.0), and 

specific conductance ranged from 0.45 mmho•cm-1 to 474 mmho•cm-1 (mean = 22.7 mmho•cm-1). 

Planktonic algae were the primary type of alga collected, although neustonic and benthic forms 

were also collected when algal growth in such habitats was clearly visible. From these samples, 

more than 1,700 strains of microalgae were obtained. From these strains, approximately 700 

unialgal cultures were established. Initial strain isolations were performed by streaking out 

samples onto 1.5% agar plates containing seawater, sterilized collection site water, or SERI Type 

I or Type II medium having a conductivity similar to that of the collection site water. In some 

cases, an enrichment step was performed before streaking out the cells, wherein the samples were 

placed in tubes on a rotary agitation wheel in liquid media at a light intensity of 1500 μE•m-2

•s

-1

(~75% of full sunlight) using a 12h:12h light:dark cycle. 

Of the 700 unialgal cultures, 120 were identified taxonomically; 24 genera were represented in 

this group, including 60 chlorophytes, 40 diatoms, and 20 cyanophytes. The most common 

genera were Dunaliella, Chlorococcum, Chlorosarcina, Amphora, Nitzschia, Navicula, 

Oscillatoria, and Chroococcus. Initial screening typically involved visual assessment of growth 

at 25°C at 200 μE•m-2

•s

-1 in tubes containing SERI Type I/40 and Type II/40 media. Strains that 

grew the most rapidly were subjected to further characterization, including analysis of the effects 
National Renewable Energy Laboratory 

44 A Look Back at the Aquatic Species Program—Technical Review 

of temperature, salinity, light intensity, and N source on growth rates. Cultures were grown in 

several different media (SERI Types I and II media at various conductivities, and artificial 

seawater) at 30°C and 500 μE•m-2

•s

-1 using a 12h:12h light:dark cycle on a rotary screening 

apparatus. Thirty-one diatoms were tested under these conditions, and 11 exhibited growth rates 

exceeding one doubling•d-1. The highest growth rate observed for a diatom under these 

conditions was 1.96 doublings•d-1 for Amphora ASU0308. Of the 50 chlorophyte species tested, 

17 strains exhibited growth rates exceeding one doubling•d-1; the highest growth rate (2.58 

doublings•d-1) was observed for a strain of Dunaliella (ASU0038). Of the strains that were tested 

for growth in all seven standard media, 80% of the cultures grew in the low salinity SERI Type I 

and Type II media and more than 50% of the strains grew in seawater. The highest growth rates 

were typically observed in SERI Type I/10 and Type II/10 media and seawater, although the 

mean growth rates of all strains combined at the highest salinities (70 mmho•cm-1) were 60% to 

80% of the mean growth rates obtained at the lower salinities. Most of the strains were isolated 

from waters with specific conductances below 40 mmho•cm-1, which may explain the lower 

growth rates in the media having higher salinities. A few strains, however, grew quite well in the 

higher salinity media. Amphora ASU0032 (AMPHO27), Synechococcus ASU0071 (CHLOC5), 

also referred to by the subcontractor as Chroococcus, and Navicula ASU0267 were the only 

strains that had a growth rate that exceeded one doubling•d-1 in Type II/70 medium. Certain 

strains had high growth rates in both Type I/70 and Type II/70 media; included in this group 

were the strains mentioned earlier along with Synechococcus ASU0075 (CHROC2) and 

Dunaliella ASU0038. Some strains were clearly euryhaline (i.e., able to tolerate a wide range of 

salinities), and could grow in all media tested (e.g., CHLOC5 had a growth rate that exceeded 

one doubling•d-1 in each of the seven media tested). Other strains were stenohaline, and grew 

much better at one particular salinity. Certain strains showed no real preference for SERI Type I 

versus SERI Type II medium, despite the very different ionic composition of these media types. 

Other strains exhibited a clear preference for one media type over the other (e.g., Dunaliella

ASU0038 grew much better in SERI Type I medium or seawater than in SERI Type II medium). 

Twenty-eight of the strains tested had growth rates exceeding one doubling•d-1 in at least one 

media type, three strains had growth rates that exceeded two doublings•d-1, and one strain (either

Eremosphaera or Chlorococcum ASU0132 [CHLOC6] or ASU0048 [CHLOC4]) had a growth 

rate higher than three doublings•d-1. 

The proximate chemical compositions of 11 isolates were also determined in this study. Total 

lipid was determined by the Bligh-Dyer procedure (Bligh and Dyer 1959). Protein was 

determined by the heated biuret-Folin assay, and total carbohydrate was estimated by the phenolsulfuric acid method (see Sommerfeld et al. [1987b] for details). Of the newly isolated strains 

tested, Franceia ASU0146 (FRANC1) had the highest lipid content under normal growth 

conditions (26.5% of the AFDW). The strains were not evaluated under nutrient-deficient 

conditions, which often increases the lipid content of microalgae. Because analyzing the lipid 

content of all the strains that had been isolated by this type of procedure would be very difficult, 

the strains were examined by the use of lipophilic dyes. The dyes Nile Blue A, Sudan Black B, 

Oil Red O, and Nile Red were used in conjunction with fluorescence microscopy to check for oil 

droplets within the cells. Inconsistent results were obtained when using the first three stains, but 

Nile Red appeared to give more reliable results. The stained cells were visually scored for the 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 45 

presence of fluorescing lipid droplets. Additional work was carried out to develop a Nile Red 

staining procedure that could (in theory) provide a quantitative measure of lipid content by the 

use of a fluorometer. This latter work was discussed in Section II.A.1. along with the results of 

similar efforts carried out by SERI researchers and other subcontractors. 

This subcontract was somewhat unusual in that many of the strains were collected from the 

actual areas in which the commercial microalgal biodiesel facilities could be. As a consequence, 

many of the strains that were isolated had characteristics that could make them good candidates 

for production strains. Additional results from this subcontract are presented in Section II.A.1. 

Publications: 

Sommerfeld, M.R.; Ellingson, S.B. (1987a) “Collection of high energy yielding strains of saline 

microalgae from southwestern states.” FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 53-66. 

Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987b) “Screening microalgae isolated from the 

southwest for growth potential and lipid yield.” FY 1987 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 43-57. 

Additional References: 

Lansford, R.R.; Hernandez, J.W.; Enis, P.J.(1987) “Evaluation of available saline water resources 

in New Mexico for the production of micoralgae.” FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 227-248. 

II.A.2.f. Collection of High Energy Yielding Strains of Saline Microalgae from the 

Hawaiian Islands 

Subcontractor: Hawaii Institute of Marine Biology 

Principal Investigator: Richard H. York, Jr. 

Period of Performance: 1985 

Subcontract Number: N/A 

Microalgae were collected from a variety of sites in the Hawaiian islands, including ocean sites 

and inland saline habitats. The conductivity, dissolved oxygen content, pH, and temperature of 

each site was recorded. Individual cells were isolated via micropipetting and placed into glass 

tubes or fluorohalocarbon plastic bags containing either the original sample water, offshore 

seawater, SERI Type I medium, or SERI Type II medium. The plastic bags, which transmit the 

full visible solar spectrum, were placed in full sunlight without temperature control. This 

treatment was therefore believed to provide a good selection for strains that would be able to 

thrive under outdoor mass culture conditions. The glass tubes were incubated at 25°-26°C at 40 

μE•m-2

•s

-1 under a 16h:8h light:dark regime; these conditions were less stressful than the outdoor 
National Renewable Energy Laboratory 

46 A Look Back at the Aquatic Species Program—Technical Review 

conditions, and therefore led to the recovery of less hardy strains. A large-scale outdoor 

enrichment culture was also prepared by pumping 1700 L of enriched seawater into a 5.5 m 

diameter open tank, then strains arising in this culture were isolated. 

As a result of these procedures, 100 of the most rapidly growing strains were selected and 

maintained for further analysis. This group included members of the Chlorophyceae, 

Cyanophyceae, Bacillariophyceae, and Pyrrophyceae. Two strains, Chaetoceros SH 9-1 

(CHAET38) and Cyclotella 14-89 (THALA6), were grown in outdoor cultures consisting of cells 

in 1-L fluorohalocarbon plastic bags. The highest growth rates measured for these strains were 

2.12 doublings•d-1 for CHAET38 and 1.43 doublings•d-1 for THALA6. These growth rates were 

reported to correspond to 31 and 33 g dry weight•m-2•d-1, respectively, although how these values 

were derived is not clear. 

Publications: 

York, Jr., R.H. (1987) “Collection of high energy yielding strains of saline microalgae from the 

Hawaiian Islands.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, pp. 90-104. 

II.A.2.g. Characterization of Hydrocarbon Producing Strains of Microalgae 

Subcontractor: Scripps Institution of Oceanography 

Principal Investigator: Ralph A. Lewin 

Period of Performance: 1985 - 1986 

Subcontract Number: N/A 

This subcontract focused on the collection and characterization of picopleustonic algae, which 

are defined as algae (including the prokaryotic cyanophytes) that are very small (1-5 μm) and 

that live on the surface of the water. In February 1985, water samples were taken from various 

sites in the Caribbean Sea, including sites near the U.S. Virgin Islands (St. John, St. Thomas, and 

St. Croix), Tortola, Puerto Rico, Curaçao, Panama, and the Florida Keys. 130 samples (250 mL 

each) were collected and filtered through a 3-8-μm filter to remove larger cells. Smaller cells 

were collected on a 0.45 μm nitrocellulose filter, which was rolled up and placed in the original 

sampling water that had passed through the filter. These samples were placed under natural 

lighting at 20°C to 25°C until transported to the laboratory. The filters were then transferred into 

a tube of sterile enriched seawater (containing additional N and other nutrients) and incubated at 

25°C under continuous illumination from a fluorescent lamp at 30 μE•m-2

•s

-1. In an attempt to 

stimulate lipid accumulation via nutrient deficiency, a portion of each culture was transferred 

after 4 weeks of growth to a fresh tube of unenriched seawater and then allowed to grow under 

the same conditions. After 4 more weeks, a film of cells was often observed floating on the 

surface of the cultures. Small samples of these cells were transferred to fresh enriched seawater. 

After incubation for an additional 2 weeks, the cells in these cultures were microscopically 

examined, and cultures that were dominated by diatoms, cyanophytes, and flagellates were 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 47 

discarded, leaving approximately 60 cultures of small (1-5-μm) green cells. Unialgal cultures 

were established from these cells by isolating colonies on agar plates. Of these purified cultures, 

there were 14 isolates of Stichococcus, 21 isolates of Nannochloris, four strains of Chlorella, and 

several representatives of other genera. Stichococcus, Nannochloris, and Chlorella are all 

chlorophytes. Because cyanophytes typically do not accumulate lipids, they were eliminated 

from further study in this subcontract. The researchers anticipated that isolating strains in this 

manner would enrich for lipid-accumulating microalgae. 

The isolated strains were tested for the ability to grow in freshwater; all the Stichococcus and 

Chlorella strains grew well in freshwater, suggesting perhaps a brackish water origin for these 

strains. Only six of the 21 Nannochloris strains could grow on non-marine media. 

To quantitatively determine lipid content in the isolated strains, 1-L cultures were grown for 3 

weeks in enriched seawater under continuous illumination at 30 μE•m-2

•s

-1. The cultures were 

bubbled with 0.5% CO2 in air. Cells were harvested, frozen and lyophilized, and then extracted 

three times with a chloroform/methanol mixture (2:1 v/v). After the solvents evaporated, the 

lipid mass was determined gravimetrically and normalized to the cellular AFDW. The 13 

Stichococcus strains had lipid contents ranging from 9% to 59% of the AFDW, with an average 

of 33%. The lipid contents of the 21 Nannochloris strains ranged from 6% to 63%, with an 

average of 31%. Data were not presented for the lipid contents of the four Chlorella strains, 

although three strains of the eustigmatophyte Nannochloropsis that were isolated from Qingdao, 

China were examined; for this genus, the lipid content ranged from 31% to 68%, with an average 

of 46%. These reported lipid contents may be slight overestimates, in that there was apparently 

no attempt to remove somewhat polar materials that may have also been extracted via the use of 

an aqueous washing step. 

Some preliminary experiments were also conducted during the course of this subcontract 

regarding the growth of the eustigmatophyte Nannochloropsis (strain Nanno-Q, one of the 

Qingdao strains). This strain is euryhaline, and is able to grow in seawater as well as brackish 

water with one-tenth the salinity of seawater. The cells grew to a higher final yield with nitrate 

as the N source than with ammonium, and the lipid content rose substantially when the N source 

was initially supplied at levels below 200 μM (as determined by the percentage of cells that were 

floating due to elevated lipid levels). A number of Nannochloropsis strains that had been 

obtained primarily from the Culture Collection of Marine Phytoplankton at Bigelow Laboratory 

(West Boothbay Harbor, Maine) were analyzed with respect to maximum cell yields after 4 

weeks of growth at different temperatures. Most of the strains had temperature optima at or 

below 25°C, although one strain that had been collected near Long Island, New York had a 

temperature optimum of 33°C. 

This subcontract examined a group of microalgae that had not received much attention in the 

ASP until that point. The small size of picopleustonic algae could hinder harvesting efficiency in 

a mass culture facility, which would have a negative impact on the economics of biodiesel 

production. However, if the cells could be made to consistently float due to high lipid levels, this 
National Renewable Energy Laboratory 

48 A Look Back at the Aquatic Species Program—Technical Review 

property might facilitate harvesting. Outdoor testing of the most promising strains would help to 

evaluate this group of microalgae. 

Publications: 

Lewin, R.A. (1985) “Production of hydrocarbons by micro-algae; isolation and characterization 

of new and potentially useful algal strains.” Aquatic Species Program Review: Proceedings of the 

March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2700, pp. 43-51. 

Lewin, R.A.; Burrascano, C; Cheng, L. (1987) “Some picopleuston algae from the Caribbean 

region.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3071, pp. 105-121. 

II.A.2.h. Collection of High Energy Yielding Strains of Saline Microalgae from South 

Florida 

Subcontractor: Harbor Branch Foundation 

Principal Investigator: John H. Ryther 

Period of Performance: 1985 - 1986 

Subcontract Number: N/A 

The goal of the work performed under this subcontract was to collect and screen microalgal 

species from southern Florida. It emphasized collecting chromophytic algae (e.g., diatoms, 

chrysophytes, and prymnesiophytes), because this group of algae was known to often accumulate 

lipids. Collection trips were made in June and September 1985, and in February 1986 to the 

Florida Keys and the Everglades. Samples were taken from 123 sites, including various 

mangrove swamps, salt flats, canals, ditches, and shallow ponds. The basic physicochemical 

characteristics of the collection site waters were determined. The mean temperature was 29°-

30°C both for sites in the Florida Keys and the Everglades. The mean conductivity of the water 

from the Keys (35.6 mmho•cm-1) was somewhat higher than that of the Everglades (25.7 

mmho•cm-1), whereas the pH values were similar (~8). To select for the fastest growing 

microalgal strains in the water samples, the original samples were enriched with nitrate, trace 

metals, and vitamins, and incubated under continuous light (880 μE•m-2

•s

-1, or 45% of full 

sunlight) at 30°C. The strains that became dominant in the cultures were isolated into unialgal 

cultures via micropipetting, serial dilution, and spreading onto agar plates. As a consequence of 

these experiments, 61 unialgal cultures were produced. 

Preliminary evaluation of the growth of these strains in various media was performed in test 

tubes containing enriched seawater, SERI Type I/25, Type I/40, Type II/25, and Type II/40 

media. The test tubes were incubated at 30°C under constant illumination at 300 μE•m-2

•s

-1. 

Growth rates were determined by measuring the OD750 every day for 5 days, and the final culture 

density was measured after 10 days. One hundred ten strains (including some strains already in 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 49 

the Harbor Branch algal collection) were screened in this manner. In general, the strains that 

were newly isolated under the selection scheme outlined above grew more rapidly than the 

culture collection strains. Members of the Prymnesiophyceae, particularly coccolithophorids and 

ochromonads, tended to grow well in most media types, but the dinoflagellates isolated via these 

procedures did not grow well in the SERI standard media. Most species grew better in Type II 

medium than in Type I medium, although there were certainly exceptions to this. The highest 

growth rate (3.26 doublings•d-1) was observed with a strain of Hymenomonas HB152 

(HYMEN3) in Type II/25 medium. Seven strains had growth rates that exceeded 2 doublings•d-1

in at least one media type; included in this group were Dunaliella HB37 (DUNAL2), 

Nannochloris HB44 (NANNO2), a yellow green unicell HB54 (UNKNO4), Chlorella HB82 

(CHLOR7) and HB87, Pyramimonas HB133 (PYRAM2), and HYMEN3. Nine strains had 

growth rates of at least one doubling•d-1 in all five media (including Chlorella HB84 (CHLOR8) 

and HB97 (CHLOR9), Nannochloris HB85 (NANO3), and all the strains mentioned earlier in 

this paragraph except for HYMEN3). 

Several of the most promising strains were examined in more detail; they were grown in a matrix 

of five different salinities (8-60 mmho•cm-1) at five different temperatures (15°-35°C) by the use 

of a temperature-salinity gradient table, as described in previous sections. An artificial seawater 

medium (ASP-2) diluted with varying amounts of distilled water was used for these experiments. 

The cultures were exposed to constant illumination at 180 μE•m-2

•s

-1. Each of the four strains 

tested (Tetraselmis HB47 [TETRA4], PYRAM2, UNKNO4, and an olive green unicell HB154 

[UNKNO5]) exhibited excellent growth over a wide range of conditions. All these strains had a 

growth rate greater than one doubling•d-1 between 8 and 60 mmho•cm-1 and between 20° and 

35°C. UNKNO4 had growth rates higher than 1.5 doublings•d-1 between 15 and 60 mmho•cm-1

and between 20° and 35°C. 

A visual assessment of the lipid contents of the most rapidly growing strains was conducted by 

staining the cells with Nile Red, the stained cells were examined using fluorescence microscopy. 

 Based on this assessment (which was not carried out with nutrient-starved cells), TETRA5 and 

UNKNO4 had the highest estimated lipid contents. TETRA4, HYMEN2, PYRAM2, and 

UNKNO5 also appeared to accumulate substantial quantities of lipid. 

Publications: 

Rhyther, J.H.; Carlson, R.D.; Pendoley, P.D.; Jensen, P.R. (1987) “Collection and 

characterization of saline microalgae from South Florida.” FY 1986 Aquatic Species Program 

Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 122-

136. 
National Renewable Energy Laboratory 

50 A Look Back at the Aquatic Species Program—Technical Review 

II.A.2.i. Collection and Selection of High Energy Thermophilic Strains of Microalgae 

Subcontractor: Montana State University 

Principal Investigator: Keith E. Cooksey 

Period of Performance: 3/86 - 4/87 

Subcontract Number: XK-4-04136-04 

The goal of this research was to develop a technique for rapidly screening microalgae for high 

lipid content, and to use this method to select microalgae with potential for liquid fuel 

production. Dr. Cooksey’s laboratory initiated the development of the Nile Red lipid staining 

procedure, which is fully described in Section II.A.1.f. The Nile Red staining procedure was used 

to screen for high lipid strains of microalgae, first using cultures collected mainly from Florida 

and maintained at Montana State University, and in cultures containing diatoms freshly isolated 

from hot springs in Yellowstone National Park. Because algae to be used in outdoor mass 

culture in the desert southwest would be subject to high temperatures, the Florida strains, isolated 

at 28°C, were first tested for growth and lipid production at 35°C. Although some strains 

produced fairly high levels of lipid, most grew poorly. Some diatom strains were then isolated 

from the hot springs, based on the premise that they would be more likely to tolerate extremes of 

temperature and pH variation. In these cultures, Nile Red was used to screen the initial sample 

for lipid-producing strains. These cells were then cultured, made unialgal and axenic, and tested 

for growth rate and lipid production. The strains tested showed growth rates of 0.5 to 2 

doublings/d and lipid contents of 9%-54%, similar to the properties of oil-producing algae 

isolated by other methods. 

Publications: 

Berglund, D.; Cooksey, B.; Cooksey, K.E.; Priscu, L.R. (1987) “Collection and screening of 

microalgae for lipid production: Possible use of a flow cytometer for lipid analysis.” FY 1986 

Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3071, pp. 41-52. 

Cooksey, K.E. (1987) “Collection and screening of microalgae for lipid production.” Final 

Subcontract Report to the Solar Energy Research Institute, Solar Energy Research Institute, 

Golden, Colorado, May 1987, 42 pp. 

Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R. (1987) “Fluorometric determination of 

the neutral lipid content of microalgal cells using Nile Red,” Journal of Microbiological Methods

6:333-345. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 51 

II.A.3. The SERI Microalgae Culture Collection

II.A.3.a. History of SERI Microalgae Culture Collection 

The SERI Microalgae Culture Collection was first established in 1984 by Dr. Bill Barclay to 

provide a central repository for strains that were believed to have potential as biomass fuel 

production organisms. The intent was to provide documented and partially characterized 

microalgal strains to researchers interested in conducting biofuels research or in developing algal 

mass culture technologies. The publicly available collection was described in a series of Culture 

Collection Catalogs published between 1984 and 1987. It was initially limited to strains that had 

been characterized quite extensively with respect to growth properties and chemical composition, 

and that were believed to hold the most promise. These catalogs contain a wealth of information 

for many of these strains, often including photomicrographs, proximate chemical compositions, 

lipid contents of cells grown under various environmental conditions, growth characteristics in 

different media types and different temperatures, and the results of small-scale outdoor 

production pond trials. Furthermore, media compositions are provided in these catalogs. 

The original 1984-1985 Microalgae Culture Collection Catalog listed the following criteria for 

selection of strains to be placed in the cataloged public collection (in descending order of 

importance): 

• Energy yield (growth rate x energy content) 

• Type of fuel products available from biomass (hydrocarbon, diesel, alcohol, 

methanol) 

• Environmental tolerance range (temperature, salinity, pH) 

• Performance in mass culture (highly competitive, predator resistant) 

• Media supplementation requirements (addition of vitamins, trace minerals) 

• Amount of culture and composition data available on the clone or strain 

• Budget for the culture collection 

Although conceptually sound, these criteria carried with them the requirement to characterize the 

strains fairly extensively before a decision could be made as to whether they should be included 

in the collection. This detailed characterization became increasingly difficult as the number of 

strains available increased. As a consequence, many strains were maintained that were not 

officially documented in the catalogs. 

From the inception of the culture collection until the late 1980s, strains in the collection were 

provided free of charge to anyone who requested them, with the hope that the research conducted 

(and published) using these strains would increase the overall understanding of these organisms. 
National Renewable Energy Laboratory 

52 A Look Back at the Aquatic Species Program—Technical Review 

Many laboratories took advantage of this. In the first year after the publication of the SERI 

Microalgae Culture Collection Catalog, more than 100 cultures were shipped to various groups 

studying biofuels production, natural product discovery, aquaculture, and the general physiology, 

biochemistry, and molecular biology of microalgae. In the ensuing years, hundreds of additional 

cultures were provided to researchers free of charge. Then, in the early 1990s, concerns were 

raised by the SERI Legal Department that the cultures should be considered as valuable 

intellectual property, and a moratorium was placed on providing cultures to outside parties; this 

moratorium persisted until the ASP were eliminated in late 1995. 

The SERI Microalgae Culture Collection consists almost exclusively of eukaryotic, single-celled 

microalgae. Included in the collection are members of several algal classes, with a predominance 

of chlorophytes (green algae) and diatoms. These two groups tended to dominate under the high 

temperature/high light screening and selection regimes used to identify good production strain 

candidates. Of the strains present in the final culture catalog produced (published in 1987, 

including an addendum), 26% were chlorophytes, 60% were diatoms, 8% were chrysophytes, and 

6% were eustigmatophytes. The following pages list the strains described in each of the three 

culture collection catalogs published during the course of the ASP. 

The first culture collection catalog (1984-1985) listed 11 strains, which included five 

chlorophytes, four diatoms, one chrysophyte, and one eustigmatophyte. Some of these strains 

were obtained from other culture collections, including the University of Texas algal culture 

collection. The strains listed in the original 1984-1985catalog were as follows: 

Table II.A.6. Microalgal strains listed in the first SERI Culture Collection Catalog, 

1984-1985. 

Species Original SERI 

Strain 

Designation 

Final SERI 

Strain 

Designation 

Strain Alias Collector 

Ankistrodesmus falcatus S/ANKIS-1 ANKIS1 Pyramid Lake 91- W. Thomas 

Botryococcus braunii S/BOTRY-1 BOTRY1 UTEX #572 --- 

Chaetoceros gracilis S/CHAET-1 CHAET1 CHGRA R. York 

Chlorella sp. S/CHLOR-1 CHLOR1 S01 S. Lien 

Isochrysis galbana S/ISOCH-1 ISOCH1 Tahitian T-ISO J.-L. 

Nannochlropsis salina S/NANNO-1 NANNP1 GSBSTICHO J. Rhyther 

Nitzschia sp. S/NITZS-1 NITZS1 Mono Lake D. 

Oocystis pusilla S/OOCYS-1 OOCYS1 Walker Lake W. Thomas 

Phaeodactylum S/PHAEO-1 PHAEO1 TFX-1 --- 

Phaeodactylum S/PHAEO-2 PHAEO2 BB W. Thomas 

Platymonas sp. 

(later Tetraselmis suecia) 

S/PLATY-1 TETRA1 --- E. Laws 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 53 

The next edition of the SERI Microalgae Culture Collection Catalog (1985-1986) included the 

following additional strains: 

Table II.A.7. Microalgal strains added to the SERI Culture Collection Catalog, 1985-

1986. 

Species Original SERI 

Strain 

Designation 

Final SERI 

Strain 

Designation 

Strain Alias Collector 

Amphora coffeiformis S/AMPHO-1 AMPHO1 --- W. Barclay 

Boekelovia hooglandii S/BOEKE-1 BOEKE1 --- W. Barclay 

Chaetoceros sp. S/CHAET-2 CHAET14 SS-14 W. Thomas 

Chlorella ellipsoidea S/CHLOR-2 CHLOR2 BL-6 W. Thomas 

Chlorella sp. S/CHLOR-3 CHLOR3 SC-2 W. Thomas 

Cyclotella cryptica S/CYCLO-1 CYCLO1 DI-35 M. Tadros 

Monoraphidium sp. S/MONOR-1 MONOR1 Mom’s Ranch W. Barclay 

Monoraphidium sp. S/MONOR-2 MONOR2 --- W. Barclay 

Nannochloropsis sp. S/NANNO-2 NANNP2 Nanno-Q R. Lewin 

Nitzschia dissipata S/NITZS-2 NITZS2 DI-160 M. Tadros

The 1986-1987 SERI Microalgae Culture Collection Catalog (including an addendum) added 29 

more strains, bringing the total number of strains in the collection to 50. The 1986-1987 catalog 

included the following additional strains: 

Table II.A.8. Microalgal strains added to the SERI Culture Collection Catalog, 1986-

1987. 

Species Final SERI 

Strain 

Designation 

Strain Alias Collector 

Amphiprora hyalina ENTOM3 BB-333 M. Tadros 

Amphora sp. AMPHO27 MLS-1, ASU 0032 M. Sommerfeld 

Amphora sp. AMPHO28 GR-2, ASU 3001 M. Sommerfeld 

Chaetoceros muelleri CHAET6 NM-6 W. Barclay 

Chaetoceros muelleri CHAET9 UT-147 S. Rushforth 

Chaetoceros muelleri CHAET10 S/CHAET-4, UT-27 S. Rushforth 
National Renewable Energy Laboratory 

54 A Look Back at the Aquatic Species Program—Technical Review 

Chaetoceros muelleri CHAET15 49-1A W. Thomas 

Chaetoceros muelleri CHAET38 SH9-1 R. York 

Chaetoceros muelleri CHAET58 --- J. Johansen 

Chaetoceros muelleri CHAET61 --- S. Rushforth 

Chaetoceros muelleri CHAET63 --- S. Rushforth 

Cyclotella cryptica CYCLO2 F-1 P. Roessler 

Cyclotella cryptica CYCLO4 UT-65 W. Barclay 

Ellipsoidon sp. ELLIP1 70-01 R. Lewin 

Franceia sp. FRANC1 LCC-1, ASU 0146 M. Sommerfeld 

Nannochloris sp. NANNO2 HB44 R. Carlson 

Nannochloris sp. NANNO12 120-01 R. Lewin 

Navicula saprophila NAVIC1 F-2 P. Roessler 

Navicula acceptata NAVIC6 SB-264 M. Tadros 

Navicula sp. NAVIC7 BB 260 M. Tadros 

Navicula acceptata NAVIC8 SB-304 M. Tadros 

Navicula saprophila NAVIC24 --- J. Johansen 

Nitzschia dissipata NITZS13 SB-307 M. Tadros 

Nitschia communis NITZS28 --- J. Johansen 

Pleurochrysis carterae PLEUR1 --- W. Barclay 

Tetraselmis sp. TETRA4 HB47 R. Carlson 

Thalassiosira weissflogii THALA2 CO-F15 W. Barclay 

Thalassiosira weissflogii THALA6 SH14-89 R. York 

Unidentified Prasinophyte GREEN3 --- J. Johansen
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 55 

II.A.3.b. Current status of the SERI/NREL Microalgae Culture Collection 

Of the strains included in the most recent Culture Collection Catalog (the 1986-1987 edition, 

including the addendum), 37 are still viable. In addition, approximately 260 additional strains 

are part of the collection, but were never characterized well enough to be included in the catalog. 

All of these strains are in the process of being transferred to the University of Hawaii, where they 

will be maintained within the Center for Marine Biotechnology. The university intends to again 

make these strains available to the research community. A complete list of the strains still being 

maintained is included below: 

Table II.A.9. Current list of microalgal strains in the SERI Culture Collection 

Strain Species Class 

ACHNA1 Achnanthes orientalis Bacillariophyceae 

ACHNA2 Achnanthes orientalis Bacillariophyceae 

AMPHO1 Amphora coffeiformis Bacillariophyceae 

AMPHO2 Amphora coffeiformis Bacillariophyceae 

AMPHO3 Amphora coffeiformis Bacillariophyceae 

AMPHO5 Amphora coffeiformis Bacillariophyceae 

AMPHO6 Amphora coffeiformis Bacillariophyceae 

AMPHO7 Amphora delicatissima capitata Bacillariophyceae 

AMPHO8 Amphora coffeiformis punctata Bacillariophyceae 

AMPHO10 Amphora delicatissima Bacillariophyceae 

AMPHO11 Amphora coffeiformis Bacillariophyceae 

AMPHO12 Amphora coffeiformis punctata Bacillariophyceae 

AMPHO13 Amphora coffeiformis punctata Bacillariophyceae 

AMPHO14 Amphora coffeiformis Bacillariophyceae 

AMPHO18 Amphora coffeiformis Bacillariophyceae 

AMPHO21 Amphora coffeiformis linea Bacillariophyceae 

AMPHO22 Amphora coffeiformis Bacillariophyceae 

AMPHO23 Amphora delicatissima Bacillariophyceae 

AMPHO24 Amphora coffeiformis Bacillariophyceae 

AMPHO25 Amphora sp. Bacillariophyceae 

AMPHO26 Amphora coffeiformis tenuis Bacillariophyceae 

AMPHO28 Amphora coffeiformis Bacillariophyceae 

AMPHO29 Amphora coffeiformis Bacillariophyceae 

AMPHO30 Amphora coffeiformis Bacillariophyceae 
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56 A Look Back at the Aquatic Species Program—Technical Review 

Strain Species Class 

AMPHO31 Amphora coffeiformis Bacillariophyceae 

AMPHO32 Amphora coffeiformis Bacillariophyceae 

AMPHO33 Amphora delicatissima Bacillariophyceae 

AMPHO34 Amphora coffeiformis Bacillariophyceae 

AMPHO35 Amphora coffeiformis taylori Bacillariophyceae 

AMPHO36 Amphora coffeiformis taylori Bacillariophyceae 

AMPHO37 Amphora coffeiformis linea Bacillariophyceae 

AMPHO38 Amphora delicatissima Bacillariophyceae 

AMPHO40 Amphora delicatissima Bacillariophyceae 

AMPHO46 Amphora sp. Bacillariophyceae 

ANKIS1 Ankistrodesmus falcatus Chlorophyceae 

BOROD2 Borodinella sp. Chlorophyceae 

BOTRY1 Botryococcus braunii Chlorophyceae 

CARB --- --- 

CHAET1 Chaetoceros muelleri Bacillariophyceae 

CHAET3 Chaetoceros gracilis Bacillariophyceae 

CHAET5 Chaetoceros muelleri Bacillariophyceae 

CHAET6 Chaetoceros muelleri Bacillariophyceae 

CHAET7 Chaetoceros muelleri Bacillariophyceae 

CHAET9 Chaetoceros muelleri Bacillariophyceae 

CHAET10 Chaetoceros muelleri Bacillariophyceae 

CHAET11 Chaetoceros muelleri Bacillariophyceae 

CHAET14 Chaetoceros muelleri Bacillariophyceae 

CHAET15 Chaetoceros muelleri Bacillariophyceae 

CHAET17 Chaetoceros muelleri Bacillariophyceae 

CHAET18 Chaetoceros muelleri Bacillariophyceae 

CHAET19 Chaetoceros muelleri Bacillariophyceae 

CHAET20 Chaetoceros muelleri Bacillariophyceae 

CHAET21 Chaetoceros muelleri Bacillariophyceae 

CHAET22 Chaetoceros muelleri Bacillariophyceae 

CHAET23 Chaetoceros muelleri Bacillariophyceae 

CHAET24 Chaetoceros muelleri Bacillariophyceae 

CHAET30 Chaetoceros sp. Bacillariophyceae 

CHAET39 Chaetoceros muelleri Bacillariophyceae 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 57 

Strain Species Class 

CHAET40 Chaetoceros muelleri Bacillariophyceae 

CHAET41 Chaetoceros muelleri Bacillariophyceae 

CHAET43 Chaetoceros muelleri Bacillariophyceae 

CHAET44 Chaetoceros muelleri Bacillariophyceae 

CHAET45 Chaetoceros muelleri Bacillariophyceae 

CHAET46 Chaetoceros muelleri Bacillariophyceae 

CHAET47 Chaetoceros muelleri Bacillariophyceae 

CHAET48 Chaetoceros muelleri Bacillariophyceae 

CHAET50 Chaetoceros muelleri Bacillariophyceae 

CHAET51 Chaetoceros muelleri Bacillariophyceae 

CHAET54 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET55 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET57 Chaetoceros muelleri-trans Bacillariophyceae 

CHAET58 Chaetoceros muelleri muelleri Bacillariophyceae 

CHAET59 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET60 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET62 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET64 Chaetoceros muelleri subsalsum Bacillariophyceae 

CHAET66 Chaetoceros muelleri Bacillariophyceae 

CHAET67 Chaetoceros sp. Bacillariophyceae 

CHAET68 Chaetoceros sp. Bacillariophyceae 

CHAET69 Chaetoceros sp. Bacillariophyceae 

CHAET73 Chaetoceros sp. Bacillariophyceae 

CHAET75 Chaetoceros sp. Bacillariophyceae 

CHAET76 Chaetoceros sp. Bacillariophyceae 

CHAET78 Chaetoceros sp. Bacillariophyceae 

CHLOC1 Chlorococcum sp. Chlorophyceae 

CHLOC2 Chlorococcum sp. Chlorophyceae 

CHLOC3 Chlorococcum sp. Chlorophyceae 

CHLOC6 Chlorococcum sp. Chlorophyceae 

CHLOC7 Chlorococcum sp. Chlorophyceae 

CHLOC8 Chlorococcum sp. Chlorophyceae 

CHLOC10 Chlorococcum sp. Chlorophyceae 

CHLOC11 Chlorococcum sp. Chlorophyceae 
National Renewable Energy Laboratory 

58 A Look Back at the Aquatic Species Program—Technical Review 

Strain Species Class 

CHLOR1 Chlorella sp. Chlorophyceae 

CHLOR2 Chlorella ellipsoidea Chlorophyceae 

CHLOR3 Chlorella sp. Chlorophyceae 

CHLOR5 Chlorella sp. Chlorophyceae 

CHLOR6 Chlorella sp. Chlorophyceae 

CHLOR8 Chlorella sp. Chlorophyceae 

CHLOR9 Chlorella sp. Chlorophyceae 

CHLOR10 Chlorella sp. Chlorophyceae 

CHLOR11 Chlorella sp. Chlorophyceae 

CHLOR12 Chlorella sp. Chlorophyceae 

CHLOR13 Chlorella sp. Chlorophyceae 

CHLOR14 Chlorella salina Chlorophyceae 

CHLOR15 Chlorella salina Chlorophyceae 

CHLOR16 Chlorella sp. Chlorophyceae 

CHLOR17 Chlorella sp. Chlorophyceae 

CHLOR18 Chlorella sp. Chlorophyceae 

CHLOR20 Chlorella sp. Chlorophyceae 

CHLOR24 Chlorella sp. Chlorophyceae 

CHLOR27 Chlorella sp. Chlorophyceae 

CHOOC3 (Chlorococcales) Chlamydophyceae 

CHOOC4 (Chlorococcales) Chlamydophyceae 

CHOOC5 (Chlorococcales) Chlamydophyceae 

CHOOC7 (Chlorococcales) Chlamydophyceae 

CHOOC8 (Chlorococcales) Chlamydophyceae 

CHOOC10 (Chlorococcales) Chlamydophyceae 

CHOOC13 (Chlorococcales) Chlamydophyceae 

CHOOC14 (Chlorococcales) Chlamydophyceae 

CHOOC16 (Chlorococcales) Chlamydophyceae 

CHROO1 Chroomonas sp. Bacillariophyceae 

CHRYS1 Chrysosphaera sp. Bacillariophyceae 

CHRYS2 Sarcinoid chrysophyte Bacillariophyceae 

CRICO1 Cricosphaera sp. Bacillariophyceae 

CRYPT1 Cryptomonas sp. Bacillariophyceae 

CYCLO1 Cyclotella cryptica Bacillariophyceae 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 59 

Strain Species Class 

CYCLO6 Cyclotella cryptica Bacillariophyceae 

CYCLO8 Cyclotella meneghiniana Bacillariophyceae 

CYCLO9 Cyclotella meneghiniana Bacillariophyceae 

CYCLO10 Cyclotella sp. Bacillariophyceae 

CYCLO11 Cyclotella cryptica Bacillariophyceae 

DIATO1 Navicula sp. nov Bacillariophyceae 

DUNAL1 Dunaliella sp. Bacillariophyceae 

DUNAL2 Dunaliella sp. Bacillariophyceae 

ELLIP1 Ellipsoidon sp. Eustigmatophyceae 

ENTOM1 Amphiprora hyalina Bacillariophyceae 

EUSTI1 (Eustigmatophyte) Eustigmatophyceae 

EUSTI2 (Eustigmatophyte) Eustigmatophyceae 

EUSTI3 (Eustigmatophyte) Eustigmatophyceae 

EUSTI5 (Eustigmatophyte) Eustigmatophyceae 

EUSTI6 (Eustigmatophyte) Eustigmatophyceae 

EUSTI7 (Eustigmatophyte) Eustigmatophyceae 

EUSTI8 (Eustigmatophyte) Eustigmatophyceae 

EUSTI9 (Eustigmatophyte) Eustigmatophyceae 

EUSTI10 (Eustigmatophyte) Eustigmatophyceae 

EUSTI11 (Eustigmatophyte) Eustigmatophyceae 

EUSTI12 (Eustigmatophyte) Eustigmatophyceae 

EUSTI13 (Eustigmatophyte) Eustigmatophyceae 

EUSTI14 (Eustigmatophyte) Eustigmatophyceae 

EUSTI15 (Eustigmatophyte) Eustigmatophyceae 

EUSTI16 (Eustigmatophyte) Eustigmatophyceae 

EUSTI17 (Eustigmatophyte) Eustigmatophyceae 

EUSTI18 (Eustigmatophyte) Eustigmatophyceae 

EUSTI19 (Eustigmatophyte) Eustigmatophyceae 

EUSTI20 (Eustigmatophyte) Eustigmatophyceae 

EUSTI21 (Eustigmatophyte) Eustigmatophyceae 

EUSTI22 (Eustigmatophyte) Eustigmatophyceae 

EUSTI23 (Eustigmatophyte) Eustigmatophyceae 

FLAGE1 Flagellate --- 

FLAGE2 Unknown flagellate --- 
National Renewable Energy Laboratory 

60 A Look Back at the Aquatic Species Program—Technical Review 

Strain Species Class 

FLAGE3 Pleurochrysis sp. --- 

FLAGE6 Green Flagellate --- 

FRANC1 Franceia sp. Chlorophyceae 

GLECA2 Gleocapsa sp. Cyanophyceae 

GLOEO1 Gloeothamnion sp. --- 

GREEN1 Green unicell --- 

GREEN2 Green unicell --- 

GREEN4 Green coccoid --- 

GREEN6 Green coccoid --- 

GREEN7 Green coccoid --- 

GREEN8 Unknown green coccoid --- 

GREEN9 Unknown green coccoid --- 

GREEN10 Unknown green coccoid --- 

GREEN11 Unknown green coccoid --- 

HYMEN2 Hymenomonas sp. Prymnesiophyceae 

ISOCH1 Isochrysis aff. galbana Prymnesiophyceae 

MONOR1 Monoraphidium sp. Chlorophyceae 

MONOR2 Monoraphidium sp. Chlorophyceae 

MONOR3 Monoraphidium sp. Chlorophyceae 

MONOR4 Monoraphidium minutum Chlorophyceae 

NANNO2 Nannochloris sp. Chlorophyceae 

NANNO3 Nannochloris sp. Chlorophyceae 

NANNO5 Nannochloris sp. Chlorophyceae 

NANNO7 Nannochloris sp. Chlorophyceae 

NANNO8 Nannochloris sp. Chlorophyceae 

NANNO9 Nannochloris sp. Chlorophyceae 

NANNO10 Nannochloris sp. Chlorophyceae 

NANNO12 Nannochloris sp. Chlorophyceae 

NANNO13 Nannochloris sp. Chlorophyceae 

NANNP1 Nannochloropsis salina Eustigmatophyceae 

NANNP2 Nannochloropsis sp. Eustigmatophyceae 

NAVIC1 Navicula saprophila Bacillariophyceae 

NAVIC2 Navicula saprophila Bacillariophyceae 

NAVIC3 Navicula saprophila Bacillariophyceae 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 61 

Strain Species Class 

NAVIC5 Navicula saprophila Bacillariophyceae 

NAVIC7 Navicula saprophila Bacillariophyceae 

NAVIC9 Navicula pseudotenelloides Bacillariophyceae 

NAVIC10 Navicula biskanterae Bacillariophyceae 

NAVIC12 Navicula acceptata Bacillariophyceae 

NAVIC13 Navicula saprophila Bacillariophyceae 

NAVIC14 Navicula pseudotenelloides Bacillariophyceae 

NAVIC15 Navicula pseudotenelloides Bacillariophyceae 

NAVIC16 Navicula pseudotenelloides Bacillariophyceae 

NAVIC17 Navicula pseudotenelloides Bacillariophyceae 

NAVIC20 Navicula pseudotenelloides Bacillariophyceae 

NAVIC21 Navicula pseudotenelloides Bacillariophyceae 

NAVIC22 Navicula saprophila Bacillariophyceae 

NAVIC23 Navicula saprophila Bacillariophyceae 

NAVIC24 Navicula saprophila Bacillariophyceae 

NAVIC26 Navicula saprophila Bacillariophyceae 

NAVIC28 Navicula saprophila Bacillariophyceae 

NAVIC31 Navicula acceptata Bacillariophyceae 

NAVIC32 Navicula acceptata Bacillariophyceae 

NAVIC33 Navicula pseudotenelloides Bacillariophyceae 

NAVIC35 Navicula acceptata Bacillariophyceae 

NEPHC1 Nephrochloris sp. --- 

NEPHR1 Nephroselmis sp. --- 

NITZS1 Nitzschia pusilla monoensis Bacillariophyceae 

NITZS3 Nitzschia pusilla elliptica Bacillariophyceae 

NITZS4 Nitzschia alexandrina Bacillariophyceae 

NITZS5 Nitzschia quadrangula Bacillariophyceae 

NITZS6 Nitzschia pusilla monoensis Bacillariophyceae 

NITZS7 Nitzschia quadrangula Bacillariophyceae 

NITZS9 Nitzschia inconspicua Bacillariophyceae 

NITZS10 Nitzschia microcephala Bacillariophyceae 

NITZS12 Nitzschia pusilla Bacillariophyceae 

NITZS13 Nitzschia dissipata Bacillariophyceae 

NITZS14 Nitzschia communis Bacillariophyceae 
National Renewable Energy Laboratory 

62 A Look Back at the Aquatic Species Program—Technical Review 

Strain Species Class 

NITZS17 Nitzschia hantzschiana Bacillariophyceae 

NITZS19 Nitzschia inconspicua Bacillariophyceae 

NITZS20 Nitzschia inconspicua Bacillariophyceae 

NITZS23 Nitzschia intermedia Bacillariophyceae 

NITZS24 Nitzschia hantzschiana Bacillariophyceae 

NITZS30 Nitzschia hantzschiana Bacillariophyceae 

NITZS35 Nitzschia inconspicua Bacillariophyceae 

NITZS37 Nitzschia pusilla Bacillariophyceae 

NITZS38 Nitzschia pusilla Bacillariophyceae 

NITZS39 Nitzschia pusilla Bacillariophyceae 

NITZS40 Nitzschia pusilla Bacillariophyceae 

NITZS43 Nitzschia pusilla Bacillariophyceae 

NITZS44 Nitzschia frustulum Bacillariophyceae 

NITZS45 Nitzschia inconspicua Bacillariophyceae 

NITZS49 Nitzschia sp. Bacillariophyceae 

OCHRO2 Ochromonas sp. Chrysophyceae 

OOCYS1 Oocystis pusilla Chlorophyceae 

OOCYS3 Oocystis parva Chlorophyceae 

OOCYS5 Oocystis sp. Chlorophyceae 

OOCYS9 Oocystis sp. Chlorophyceae 

OOCYS11 Oocystis sp. Chlorophyceae 

OOCYS14 Oocystis sp. Chlorophyceae 

OSCIL1 Oscillatoria limnetica Cyanophyceae 

OSCIL3 Oscillatoria subbrevis Cyanophyceae 

OSCIL8 Oscillatoria sp. Cyanophyceae 

OSCIL9 Oscillatoria sp. Cyanophyceae 

OSCIL10 Oscillatoria sp. Cyanophyceae 

OSCIL11 Oscillatoria sp. Cyanophyceae 

OSCIL12 Oscillatoria sp. Cyanophyceae 

OSCIL13 Oscillatoria sp. Cyanophyceae 

OSCIL14 Oscillatoria sp. Cyanophyceae 

OSCIL15 Oscillatoria sp. Cyanophyceae 

PAVLO2 Pavlova sp. Chrysophyceae 

PHAEO1 Phaeodactylum tricornutum Bacillariophyceae 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 63 

Strain Species Class 

PHAEO2 Phaeodactylum tricornutum Bacillariophyceae 

PLATY1 Platymonas sp. Chlorophyceae 

PLEUR1 Pleurochrysis dentata Prymnesiophyceae 

PLEUR4 Pleurochrysis dentata Prymnesiophyceae 

PLEUR5 Pleurochrysis sp. Prymnesiophyceae 

PLEUR6 Pleurochrysis sp. Prymnesiophyceae 

PRYMN2 (Prymnesiophyte) Prymnesiophyceae 

PSEUD1 Pseudoanabaena sp. Cyanophyceae 

PSEUD4 --- --- 

PYRAM2 Pyramimonas sp. Prasinophyceae 

STICH1 Stichococcus sp. Chlorophyceae 

STICH2 Stichococcus sp. Chlorophyceae 

SYNEC1 Synechococcus sp. Cyanophyceae 

SYNEC3 Synechococcus sp. Cyanophyceae 

SYNEC5 Synechococcus sp. Cyanophyceae 

TETRA1 Tetraselmis suecica Prasinophyceae 

TETRA2 Tetraselmis sp. Prasinophyceae 

TETRA3 Tetraselmis sp. Prasinophyceae 

TETRA4 Tetraselmis sp. Prasinophyceae 

TETRA5 Tetraselmis sp. Prasinophyceae 

TETRA6 Tetraselmis sp. Prasinophyceae 

TETRA7 Tetraselmis sp. Prasinophyceae 

TETRA8 Tetraselmis sp. Prasinophyceae 

TETRA9 Tetraselmis sp. Prasinophyceae 

TETRA11 Tetraselmis sp. Prasinophyceae 

THALA6 Thalassiosira weissflogii Bacillariophyceae 

THALA7 Thalassiosira weissflogii Bacillariophyceae 

THALA14 Thalassiosira weissflogii Bacillariophyceae 

THALA15 Thalassiosira weissflogii Bacillariophyceae 

THALA16 Thalassiosira weissflogii Bacillariophyceae 

UNKNO1 --- --- 

UNKNO5 Unknown olive-green unicell 

UNKNO6 Unknown coccolithophorid Prymnesiophyceae 

UNKNO8 Unknown coccolithophorid Prymnesiophyceae 
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64 A Look Back at the Aquatic Species Program—Technical Review 

Strain Species Class 

UNKNO10 Nitzschia sp. Bacillariophyceae 

UNKNO24 --- --- 

UNKNO36 --- --- 

UNKNO52 --- Cyanophyceae 

UNKNO58 --- --- 

VW291 --- --- 

Publications: 

Microalgae Culture Collection 1984-1985. Solar Energy Research Institute, SERI/SP-231-2486; 

59 pp. 

Microalgae Culture Collection 1985-1986. Solar Energy Research Institute, SERI/SP-232-2863, 

97 pp. 

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, R.; Lemke, P. (1986) “Microalgae 

Culture Collection 1986-1987.” Solar Energy Research Institute, SERI/SP-232-3079, 149 pp. 

Johansen, J.; Lemke, P.; Nagle, N.; Chelf, P.; Roessler, R.; Galloway, R.; Toon, S. (1987) 

“Addendum to Microalgae Culture Collection 1986-1987.” Solar Energy Research Institute, 

SERI/SP-232-3079a, 23 pp. 

II.A.4. Collection and Screening of Microalgae—Conclusions and 

Recommendations 

The collection, screening, and characterization of microalgal strains represent a major endeavor 

of ASP researchers during the 1980s. More than 3,000 algal strains were collected from sites 

within the continental U.S. (Figure II.A.5.) and Hawaii or obtained from other culture 

collections. This was the first major collection of microalgae that emphasized strains suitable for 

cultivation in saline waters at high (or variable) temperatures, and with the potential for oil 

production. The establishment of the SERI Culture Collection as a genetic resource was a major 

accomplishment of the ASP; unfortunately a large proportion of the collection was lost due to 

funding cutbacks. However, the approximately 300 strains remaining in the collection will be 

transferred to the University of Hawaii, and should be available to interested researchers. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 65 

Figure II.A.5. Microalgae Collection Sites Within the Continental U.S. 

The collection and screening effort resulted in a large number of strains that had many 

characteristics deemed important for a biodiesel production organism. In reviewing the many 

procedures used by ASP researchers, however, it is clear that a more consistent screening 

protocol might have yielded results that could be compared more meaningfully. Although these 

types of standard protocols were being developed near the end of the collection and screening 

effort, they were not consistently used. Furthermore, because an optimized microalgal-based 

biofuel production process was never fully developed, the screening protocols could not be based 

on an actual process. Therefore, whether the screening criteria used in the ASP were accurate 

predictors of good performance in a biodiesel production facility is not really known. For 

example, lipid productivity over a given amount of time is one of the most important factors in a 

production process. There were no clear guidelines as to whether lipid productivity in an outdoor 

pond was better in a continuous, steady-state process or in a multistage process involving 

substantial culture manipulation (e.g., nutrient level control, “ripening” tanks). This information 

is critical, and has a profound impact on the type of screening that should be conducted. 
National Renewable Energy Laboratory 

66 A Look Back at the Aquatic Species Program—Technical Review 

In addition to the need for a better understanding of the most economically feasible commercial 

biodiesel production process, additional information about the true constraints with regard to 

lipid properties (e.g., fatty acid chain length, degree of unsaturation, polar lipid constituents, etc.) 

there is a need for better information on the impact of lipid composition on fuel quality. The 

lipophilic dye Nile Red was used as a screening tool to rapidly assess the lipid contents of 

isolates, but in retrospect this technique probably does not provide the level of detail regarding 

lipid quality that may be necessary. Indeed, the variability in the ability of various strains to take 

up this dye is a major problem that must be recognized. With the rapid advances that have been 

made in recent years in automated high performance liquid chromatography and detection, this 

technique seems readily adaptable for use as a powerful screening tool. 

For future screening endeavors, we recommend that an effort be made to naturally select strains 

at the locations that would likely be commercial microalgal production sites. In this manner, the 

algae would be exposed to the prevailing environmental conditions, particularly the indigenous 

waters. In small open-air vessels, the medium would be “inoculated” with a variety of 

indigenous strains, and a process of natural selection would occur such that the most competitive 

strains would dominate the cultures after a short while. Of course, the disadvantage of this 

method is that the dominant strains may not be good lipid producers. For this reason, genetically 

manipulating the dominant strains by classical or recombinant means may be necessary, such that 

they remain competitive and yet make acceptable amounts of lipid. Whether such manipulations 

can be made awaits further experimentation. 

One thing that was clear from the collection and screening effort was that diatoms and green 

algae would most likely be well represented in a “natural selection” screen as described in the 

preceding paragraph. Therefore, future efforts should probably focus on developing 

sophisticated genetic engineering tools focused on these groups. Such tools could be transferable 

to many different species within these groups; such transfer would be facilitated by the fact that 

powerful methods for generating genetic sequence information are becoming routine. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 67 

II.B. Microalgal Strain Improvement

II.B.1. Physiology, Biochemistry, and Molecular Biology of Lipid 

Production: Work by SERI Subcontractors

II.B.1.a. Introduction 

Eukaryotic algae, like all photosynthetic organisms, efficiently convert solar energy into biomass. 

The algal research program at SERI was designed as a long-term basic research effort to adapt or 

use photosynthesis and related metabolic pathways to produce renewable fuels and chemicals. 

Research at SERI under the Aquatic Species Program (Biodiesel) has focused on ways to 

increase the yield of oil from microalgae for cost-effective liquid fuel production. Initially, a 

large component of the research performed both by subcontractors and by SERI researchers was 

the collection of microalgal strains from saline environments in the desert southwest of the 

United States (a region targeted as a feasible location for large-scale microalgal culture), marine 

environments, and established culture collections. These organisms were then screened and 

numerous species were identified as candidates for biodiesel production; this research was 

described in Sections II.A. and II.B. of this report. However, no one species was identified that 

displayed the ideal combination of rapid growth, environmental tolerance, and high lipid 

production. Subsequent research efforts were directed toward understanding the biochemistry 

and physiology of lipid production in oleaginous microalgal strains, with the idea of using strain 

improvement technologies (breeding, cell fusion, genetic engineering, mutagenesis and selection) 

to develop algal strains with optimized traits for biodiesel production. 

Early in the research program it became obvious the maximal lipid accumulation in the algae 

usually occurred in cells that were undergoing physiological stresses, such as nutrient deprivation 

or other conditions that inhibited cell division. Unfortunately, these conditions are the opposite of 

those that promote maximum biomass production. Thus, the conditions required for inexpensive 

biodiesel production, high productivity and high lipid content, appeared to be mutually exclusive. 

 To overcome this problem, research efforts were focused on understanding the biochemistry and 

physiology of lipid accumulation, with emphasis on understanding the “lipid trigger”, a 

mechanism that could induce production of large quantities of lipid under nutrient deprivation. 

In addition, research was directed toward understanding genetic variation within microalgal 

populations and to develop methods to screen for high-lipid subpopulations within algal cultures. 

 The knowledge of the biochemistry and physiology of lipid synthesis, combined with basic 

studies on microalgal molecular biology, was used in the later years of the project in attempts to 

use genetic engineering to develop microalgal strains with optimal properties of growth and lipid 

production. 

Part II.B.2. of this report describes work by ASP subcontractors to understand the biochemistry 

and physiology of lipid accumulation in microalgae, including ultrastructural studies, the 

development of methods for screening for high lipid strains, and attempts to understand the 

biochemical lipid trigger. The research performed by SERI/NREL subcontractors on the 

physiology, biochemistry, and molecular biology of lipid production in oleaginous microalgae 
National Renewable Energy Laboratory 

68 A Look Back at the Aquatic Species Program—Technical Review 

took place during the second half of the 1980s and is presented here, roughly chronologically, 

according to the work performed by the individual subcontractors. 

II.B.1.b. Chrysophycean Lipids: Effects of Induction Strategy in the Quantity and 

Types of Lipids 

Subcontractor: Selma University 

Principal Investigator: Shobha Sriharan 

Period of Performance: 9/85 - 5/88 

Subcontract Number: XK-5-05104-1 

The purpose of the research performed by Dr. Sriharan and coworkers was to study the effects of 

nutrient deprivation and temperature on growth and lipid production in microalgae with potential 

for liquid fuel production. All the reports generated by this laboratory during the subcontract 

describe a virtually identical set of experiments performed on several species of microalgae. The 

benefit of this approach is that the productivity data can be compared between the species. 

However, the flaws in experimental design and reporting were carried through all experiments 

and reports. 

The basic design for these experiments was to grow the algal cells in batch cultures in media that 

contained either “sufficient” or “deficient” levels of N or Si, and to test for algal growth rate, 

productivity, and lipid production. Cultures were grown at 20°C and at 30°C to test for the effect 

of temperature on growth and lipid induction. Exponentially growing cells were inoculated into 

fresh media containing high or low levels of Si or N. Growth was monitored by measuring the 

optical density of the culture, and the growth rate was reported as the number of cell doublings 

per day. The cells were harvested and processed to determine lipid content (reported as a 

percentage of the AFDW), and fatty acid composition under the various growth conditions. 

The organisms studied were all diatoms, except for the chlorophyte M. minutum (which the 

authors initially reported to be a diatom, but they corrected this error in a later report). The 

diatoms tested were Chaetoceros SS-14, C. muelleri var subsalsum, Navicula saprophila, 

obtained from SERI, Cyclotella DI-35, Cyclotella cryptica Reimann, Lewin, and Guillard, and 

Hantzchia DI-60, obtained from M. Tadros at Alabama A&M University. All the organisms 

tested grew more rapidly at 30°C (versus 20°C) and in nutrient-sufficient media. A decrease in 

the total AFDW was reported for all strains grown in nutrient-deficient media compared to 

nutrient-sufficient cultures, and this was accompanied by an increase in the percentage of the 

AFDW made up of lipids. 

The most dramatic increases in the lipid content of the cultures were seen under N-deficient 

conditions in cells grown at 30°C. In C. cryptica, the total lipids, as a percentage of AFDW, 

increased from 15% to 44%. In Hantzschia, lipids increased from 29% to 53%, and in Navicula 

saprophila, lipids increased from 26% to 44%. In all cases, the increase in total lipids was due to 

increases in both the neutral lipid and polar lipid fractions. In several cases, the ratio of neutral 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 69 

lipids to polar lipids increased significantly in the nutrient-stressed cells (i.e., in Hantzschia and 

C. muelleri grown in Si-deficient media, and in Navicula grown in N-deficient media). Dr. 

Sriharan also presented data comparing the fatty acid profiles of lipids from diatoms grown under 

nutrient-sufficient and nutrient-deficient conditions. Although the data was incomplete, it 

indicated that changes in the fatty acid composition (lipid quality) did occur in nutrient-stressed 

cells, suggesting that nutrient deprivation can affect the lipid biosynthetic pathways. 

In all cases nutrient-deficiency resulted in a decreased rate of cell growth and a decrease in total 

cell productivity. Therefore, an increase in lipid as a percentage of cell mass may not be 

economically advantageous for liquid fuel production from mass-cultured algae if the conditions 

that induce lipid accumulation also result in a significant drop in total biomass, and thus in total 

lipid produced. Although this was not discussed by Dr. Sriharan, the total effect of nutrient 

limitation on lipid content of the algal cultures could be estimated by multiplying the total 

biomass (AFDW) produced by the percentage of the AFDW attributable to lipid under nutrientsufficient or nutrient-deficient conditions. In general, these calculations demonstrated an 

increase in lipid content of the cultures induced by nutrient stress, in the range of a 20% to 30% 

increase in total lipid. 

The results reported here clearly suggest that algal productivity is increased under nutrientsufficient conditions and at elevated temperatures. However, it is difficult to determine the 

validity of the data presented regarding nutrient-deprivation as a lipid trigger. Growth curves 

were not presented for the organisms studied. Therefore, it cannot be determined if the low 

nutrient levels limited growth throughout the period of the experiment, or whether the nutrients 

became depleted and the lipid effects correlated with a decrease in cell division, as reported 

elsewhere. It is also not clear at what point in the cell cycle the cells were harvested for 

determination of AFDW and lipid content, and how to compare the data for nutrient-sufficient 

and nutrient-deficient cells. In several experiments, the authors reported that the cultures were 

only harvested when lipid droplets were seen in the cells, although this would seem to bias 

experiments designed to test nutrient effects on lipid production. All in all, it is difficult to 

determine whether the experiments were badly performed, or just poorly reported. 

In summary, the data presented by Dr. Sriharan was difficult to interpret for these reasons; 

however, several general conclusions can be made. Diatoms seem to be promising candidates for 

neutral lipid production. Many species produce constitutively high levels of lipid, and the level 

of lipid as a percentage of biomass can be increased by growing the cells under nutrient-limited 

conditions (the data presented here suggests that N-limitation may be more effective than Silimitation). In addition, Dr. Sriharan’s results suggest that nutrient-limitation may alter the lipid 

biosynthetic pathways in diatoms to increase lipid production and possibly affect lipid 

composition. 
National Renewable Energy Laboratory 

70 A Look Back at the Aquatic Species Program—Technical Review 

Publications: 

Sriharan, S.; Bagga, D. (1987a) “Effects of induction strategies on Chaetoceros (SS-14), growth 

with emphasis on lipids.” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 273-284. 

Sriharan, S.; Bagga, D. (1987b) “Influence of nitrogen and temperature on lipid production in 

microalgae (Hantzschia DI-60).” Energy from Biomass and Wastes X, Conference Proceedings, 

(Klas, D.L., ed.), Institute of Gas Technology, Chicago, pp. 1689-1190. 

Sriharan, S.; Bagga, D.; Sriharan, T.; Das, M. (1987) “Effect of nutrients and temperature on 

lipid production and fatty acid composition in Monoraphidium minutum and Cyclotella DI-35.” 

FY 1987 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3206, pp. 108-126. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1988) “Algal lipids: Effect of induction steategies on the 

quantity and types of lipids produced.” Final Technical Report, Solar Energy Research Institute, 

Golden, Colorado, 16 pp. 

Sriharan, S.; Sriharan, T.P.; Bagga, D. (1989) “Lipids and fatty acids of diatom Chaetoceros 

muelleri var subsalsum and the control of their production by environmental factors.” Energy 

from Biomass and Wastes XII, Conference Proceedings, (Klas, D.L., ed.), Institute of Gas 

Technology, Chicago, 10 pp. 

Sriharan, S.; Bagga, D.; Nawaz, M. (1991) “The effects of nutrients and temperature on biomass, 

growth, lipid production, and fatty acid composition of Cyclotella cryptica Reimann, Lewin, and 

Guillard.” Appl. Biochem. Biotech. 28/29:317-326. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1989) “Environmental control of lipids and fatty acid 

production in the diatom Navicula saprophila.” Appl. Biochem. Biotech. 20/21:281-291. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1990) “Effects of nutrients and temperature on lipid and 

fatty acid production in the diatom Hantzshia DI-60.” Appl. Biochem. Biotech. 24/25:309-316. 

II.B.1.c. Genetic Variation in High Energy Yielding Microalgae 

Subcontractor: City College of the City University of New York 

Principal Investigator: Jane C. Gallagher 

Period of Performance: 3/86 - 12/87 

Subcontract Numbers: ZK-4-04136-5; ZK 4-04-136-04 

The purpose of these studies was to investigate the intragenetic variability (e.g., between various 

isolates of a single species) in microalgae with potential for high lipid production. The rationale 

for this work with respect to the ASP is that variability within and between species of microalgae 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 71 

has implications for algal collection strategies, for strain selection for high lipid producers, and 

for genetic manipulation of microalgae by classical breeding or genetic engineering. 

Historically, microalgae have been classified based on morphological similarities. Previous 

studies by Dr. Gallagher and others (see Gallagher 1986) suggested significant physiological 

variability between isolates of a single species. In these studies, various isolates of a species 

grown under identical conditions (to control for environmentally induced changes in gene 

expression) often showed significant differences in characteristics such as nutrient uptake, 

growth rates, and pigment content. These results indicated that there may be inherent genetic 

differences between the individual strains. The studies by Dr. Gallagher compared 

electrophoretic banding patterns of specific proteins to obtain quantitative estimates of the 

genetic differences between isolates of two genera of oil-producing microalgae. 

The organisms studied were A. coffeiformis (class Bacilliarophyceae) and Nannochloropsis spp. 

(class Eustigmatophyceae). The basic approach was to streak the isolated algae onto agar plates, 

then to pick single colonies and restreak the cells to ensure that each strain was unialgal. The 

isolates were propagated under identical growth conditions to minimize differences caused by 

environmentally induced changes in gene expression. Each strain was examined using light 

microscopy (LM) and scanning electron microscopy (SEM) to look for morphological 

differences and to confirm species identity. Crude protein extracts from each strain were 

separated by polyacrylamide gel electrophoresis. The gel was then stained to detect several 

specific enzymes, including phosphoglucose isomerase, hypoxanthine dehydrogenase, αketoglutarate dehydrogenase, malate dehydrogenase, α-hydroxybutarate dehydrogenase, and 

tetrazolium oxidase. (Dr. Gallagher also tried unsuccessfully to stain for several other enzymes. 

Poor staining may be a consequence of the location of these enzymes within cellular membranes 

in microalgae.) An extract from the diatom Skeletonema costatum (clone SKEL) was run on 

each gel to serve as an internal standard, and the migration pattern for each enzyme was reported 

as the ratio of the migration distance for the unknown Amphora or Nannochloropsis enzyme to 

the migration of the known enzyme from Skeletonema. An example of this type of experiment is 

shown in Figure III.B.1. This method allowed for detection of very small differences in the 

migration patterns of the various forms of the enzymes. These differences could result from 

subtle variations in protein charge or conformation due to one or several amino acid changes. 

Isolates that showed two bands for a specific enzyme were assumed to be heterozygous at that 

allele. 

For the studies of Amphora, Dr. Gallagher obtained 47 isolates, 32 of which were isolated from a 

salt marsh in Woods Hole, Massachusetts, on the same day in August 1985. Another six strains 

had been isolated from the same site during the summers of 1979 or 1980 and maintained in 

culture, and five strains were obtained from laboratory cultures maintained by other investigators. 

It is unclear from Dr. Gallagher's reports how many of the 47 Amphora isolates were tested as 

described earlier. 

All strains that were subsequently analyzed for enzyme banding patterns were first examined by 

LM and SEM. Microscopy confirmed that all the strains were A. coffeiformis, although some 
National Renewable Energy Laboratory 

72 A Look Back at the Aquatic Species Program—Technical Review 

variation was observed in the morphology of the frustule between strains, for example, in the 

presence or absence of costae (rib-like protrusions) or in the shape or number of punctae (holes). 

These changes were assumed to be due to genetic differences between the strains, as unialgal 

clones maintained in culture for 6-7 years did not show variations in frustule morphology 

between individuals. Genetic similarity was calculated based on the electrophoretic banding 

patterns using the statistical methods of Nei (1972). The zymograms indicated significant 

variation between isolates of the same species, even between strains isolated from the same site 

on the same day. These differences were not correlated with the extent of morphological 

variation, and some morphologically identical strains showed differences in the protein banding 

patterns. 

For Nannochloropsis, 115 strains were obtained, all from culture collections. The electrophoretic 

banding patterns also indicated significant genetic diversity between strains, even between 

samples isolated from the same location. However, the zymogram data for Nannochloropsis was 

limited due to the high percentage of “null” alleles (no staining of some enzymes) in some 

isolates. It was unclear whether this was caused by undetermined genetic differences between the 

isolates (and between Amphora and Nannochloropsis), or due to difficulties in extraction of the 

proteins from Nannochloropsis. More data would be needed to fully analyze the genetic 

differences between isolates of this genus. 

What are the implications of this research for the Aquatic Species Program? The significant 

amount of genetic diversity between individuals of a species, even when isolated from very 

similar locations, suggests that researchers involved in collecting microalgal strains as potential 

lipid producers should obtain more than one isolate from each site. In fact, these results suggest 

that it may be adequate to sample fewer sites to obtain a sufficiently varied collection of 

microalgal strains. 

In a previous study (discussed in Gallagher 1985), Dr. Gallagher described experiments 

performed on isolates of the diatom S. costatum similar to those described here. The data 

suggested significantly less genetic variation between isolates of Skeletonema, even between 

strains isolated from different locations, than was seen between Amphora strains isolated from 

the same environment. This difference was attributed to the fact that Amphora is an attached, 

benthic organism that produces amoeboid gametes, whereas Skeletonema is planktonic, and 

produces swimming sperm. These “lifestyle” differences would result is lower potential for gene 

flow between Amphora populations, although the presence of heterozygotes indicates 

interbreeding among Amphora at localized sites. These observations suggest that benthic 

organisms may have greater genetic diversity than planktonic forms. 

Based on the data in this study, Dr. Gallagher also concluded that breeding or genetic 

engineering of microalgae may be more successful using morphologically similar phenotypes, as 

her results suggest less diversity at the protein level between identical morphotypes. However, 

genetic engineering research during the past 15 years in other organisms indicates that cells can 

often express genes from very different species, so these differences between strains probably 

will not affect the expression of genes transferred between these similar organisms. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 73 

While working under the SERI subcontract, Dr. Gallagher also participated in a study that 

provided evidence that the carotenoid violaxanthin functions as a major light harvesting pigment 

in Nannochloropsis (Owens et al. 1987). Carotenoids generally are considered accessory 

pigments in photosynthetic organisms, involved primarily in photoprotection, fluorescence 

quenching, and light harvesting. Nannochloropsis is a member of the class Eustigmatophyceae, 

which are unusual in that they can contain violaxanthin as up to 60% of their total pigments. 

These authors used room temperature fluorescence excitation and emission data to provide the 

first evidence that violaxanthin can function in photosynthetic light harvesting. 

Understanding the fundamental processes involved in microalgal photosynthesis is important to 

the ASP since light-driven photosynthesis results in the production of chemical reductants that 

drive the synthetic dark reactions; lipids are storage products that can be produced from excess 

photosynthate. One possible implication is that carotenoids absorb at different wavelengths than 

chlorophyll, absorbing green light that penetrates into the water column. This feature could be 

beneficial for mass culture of organisms, allowing denser cultures to grow in a deep raceway. 

Publications: 

Gallagher, J.C. (1986). “Population genetics of microalgae.” In Algal Biomass Technologies: An 

Interdisciplinary Perspective (Barclay, W.; McIntosh, R., eds.), Beihefte zur Nova Hedwigia, 

Heft 83, Gebrüder Borntraeger, Berlin-Stugart, pp. 6-14. 

Gallagher, J. C. (1987a). “Patterns of genetic diversity in three genera of oil-producing 

microalgae” (abstr.), FY 1987 Aquatic Species Program Annual Report, (Johnson, D.A.; Sprague, 

S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, p. 209. 

Gallagher, J.C. (1987b). “Genetic variation in oil-producing microalgae.” FY 1986 Aquatic 

Species Program Annual Report, (Johnson, D.A., ed.), Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, p.331-336. 

Owens, T.G.; Gallagher, J.C.; Alberte, R.S. (1987) “Photosynthetic light-harvesting function of 

violaxanthin in Nannochloropsis spp. (Eustigmatophyceae).” J. Phycol. 23:79-85. 

Additional References: 

Nei, M. (1972) Amer. Natur. 106:283. 
National Renewable Energy Laboratory 

74 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.B.1. Drawing of representative gels showing the banding patterns of various 

enzymes from clones of Amphora coffeiformis.

All bands are graphed as a ratio of the distance travelled by protein bands in Amphora to the 

distance travelled by stand bands in Skeletonema costatum. PGI: phosphoglucose isomerase; 

XDH: hypoxanthine dehydrogenase; TO: tetrazolium oxidase; ADH: analine dehydrogenase. 

(Source: Gallagher 1987). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 75 

II.B.1.d. Ultrastructure Evaluation of Lipid Producing Microalgae 

Subcontractor: Oak Ridge National Laboratory, Oak Ridge, Tennessee 

Principal Investigator: Jean A. Solomon 

Period of Performance: 10/84 - 11/86 

Subcontract Number: N/A 

The goal of this project was to gain further understanding of the physiology of lipid accumulation 

in microalgae by examination of the ultrastructure of cells containing high levels of storage 

lipids. The questions that Dr. Solomon addressed were: 

1. Where does the lipid accumulate within the cells; and 

2. What other ultrastructual changes are seen in microalgae induced to accumulate 

lipid? 

Three oleaginous microalgal strains were used in this study, Ankistrodesmus fulcatus (SERI 

strain ANKIS1; class Chlorophyceae), Isochrysis aff. glabana (ISOCH1, class 

Prymnesiophyceae), and Nannochloropsis salina (NANNO1, class Eustigmatophyceae). 

Ultrastructural changes were monitored by transmission electron microscopy (TEM). In this 

technique, cells are chemically fixed and embedded in a plastic resin. The resin is then cut into 

thin sections (70-100nm), stained with heavy metals, and viewed in an electron microscope. The 

first step was to develop adequate fixation and embedding techniques for the algal species to be 

studied. This is often problematic for microalgae, presumably due to the chemical and physical 

properties of the algal cell wall, which can act as barriers to penetration of the fixatives or resin. 

Dr. Solomon tested five fixation protocols (see Solomon 1985, p.74, Table 1), all variations of 

standard methods of fixation using glutaraldehyde and osmium tetroxide, dehydration with an 

organic solvent, embedding of the cells in an acrylic resin, and poststaining of the sections with 

uranyl acetate and lead citrate. Initially, Dr. Solomon reported that the best fixation of 

Ankistrodesmis and Isochrysis was achieved by exposing the cells briefly to glutaraldehyde and 

osmium simultaneously, followed by dehydration in acetone and embedding in Spurrs resin. 

However, a later report stated that Ankistrodesmis was better preserved by exposing the cells 

sequentially to glutaraldehyde and then osmium (in cacodylate buffer supplemented with sucrose 

as an osmoticum). Also, Araldite/Embed12 resin was used, as it appeared to provide better 

penetration into the cells. For Isochrysis, the initial protocol was also modified by adding 

sucrose. Fixation of Nannochloropsis was poor with any method used; the scaley cell wall of 

this organism seemed to provide a significant barrier to adequate penetration of fixatives and 

resins. 

Nitrogen deprivation was used to trigger the production of lipids in the cells. The cells were 

grown in N-replete medium, then collected by centrifugation and resuspended in growth medium 

without added N. Samples were fixed immediately and at regular intervals during the following 

13 days, and thin sections were cut and examined for ultrastructual changes by TEM. 
National Renewable Energy Laboratory 

76 A Look Back at the Aquatic Species Program—Technical Review 

As expected, N deprivation resulted in the accumulation of lipid within the cells of all three 

microalgal species. The lipid appeared primarily as droplets within the cytoplasm, not within the 

chloroplast or other cellular organelles. The lipid droplets often appeared adjacent to a 

mitochondrion. In Ankistrodesmus, N-deficiency also produced an increased number of starch 

granules within the chloroplasts, and resulted in the formation of unusual membrane structures 

consisting of packed, concentric layers of double membranes within the cytoplasm. Whether 

these unusual structures were the site of excess lipid accumulation, or were structural artifacts of 

the fixation process, was unclear. 

It is difficult to conclude much more about ultrastructural changes that might have been induced 

in these cells following N deprivation. The sample size was very small. One hundred-nm thick 

sections may represent less than 1/100th of the volume of a microalgal cell. In addition, only a 

few cells within a population can be examined easily by this technique. Finally, there is a high 

likelihood that the chemical fixation methods used in the study can create artifacts that are not 

related to actual cell structure. However, these studies supported the observation that significant 

levels of storage lipids can accumulate in the cytoplasm of microalgal cells exposed to N 

deficiency. Dr. Solomon's microscopic observations in Ankistrodesmus also suggested the 

presence of a discrete lipid trigger mechanism within each cell, as lipid did not appear to 

accumulate gradually within all cells of a population after N deprivation. Instead, individual 

cells appeared to accumulate large amounts of lipid during a 1-2 day period. This result was 

supported by the flow cytometric data also performed in Dr. Solomon's laboratory, which is 

described below. 

Publications: 

Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986b) “Ultrastructural and Flow Cytometric Analyses 

of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute, 

Golden, Colorado, SERI/STR-231-3089. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 77 

Figure II.B.2. Electron micrographs of nitrogen-sufficient (top) and nitrogen-deficient 

(bottom) cells of Nannochlorposis salina. 

Note the accumulation of large lipid droplets (L) in the cytoplasm in the nitrogen-deficient cells. 

The lipid often appeared adjacent to a mitochondrion (M). N: nucleus. C: chloroplast. Scale 

bars = 0.5 μm. The numbers in the lower left corner of each figure are from the original 

publication (Solomon et al.1986b). 
National Renewable Energy Laboratory 

78 A Look Back at the Aquatic Species Program—Technical Review 

II.B.1.e. Improvement of Microalgal Lipid Production by Flow Cytometry 

Subcontractor: Oak Ridge National Laboratory, Oak Ridge, Tennessee 

Principal Investigator: Jean A. Solomon 

Period of Performance: 12/86 - 11/87 

Subcontract Number: DK-4-04142-01 

The purpose of this project was to determine if flow cytometry could be used to select for 

subpopulations of high lipid-producing algae within an algal culture. Flow cytometry is a 

method that measures the light scattered or emitted by particles as they pass through a laser 

beam. Scattered light is believed to reflect the size, shape, and refractive properties of cells. Dr. 

Solomon initially used exponentially growing and nutrient-stressed cells of the chrysophycean 

alga Boekelovia to demonstrate that the extent of right-angle scatter, which indicates changes in 

internal cell morphology, can be correlated to the lipid content of microalgal cells. 

In subsequent studies, a lipid-specific fluorescent dye, Nile Red (see work by Dr. Cooksey, 

described in Section II.A.1.f.), was used to stain intracellular lipids. Nile Red is excited at a 

wavelength of 488 nm, and emits yellow-green light at 520-580 nm. In contrast, chlorophyll 

autofluorescence can be measured at wavelengths greater than 630 nm. Therefore, in contrast to 

the scattered light data mentioned above, flow cytometric analyses of cells stained with Nile Red 

would be more specific for changes in lipid content. Preliminary experiments in which cells of 

Boekelovia were stained with Nile Red demonstrated that increased yellow green fluorescence 

could be correlated with increased numbers of lipid droplets in the cells, suggesting that this 

method could work to screen for cells with high lipid contents. 

Of the three microalgal species analyzed by TEM (Section II.B.1.d.), only Isochrysis was found 

to be appropriate for flow cytometric analysis. The cells of this strain are small and spherical, the 

optimal shape for flow cytometric analysis, and take up Nile Red well. In contrast, 

Ankistrodesmus cells are long and thin (40 nm x 4 nm). Nannochloropsis did not take up the 

Nile Red dye, possibly because of cell wall properties that also prevented good chemical fixation 

for microsopy. 

In the initial experiments, cells were screened for lipid content based on Nile Red fluorescence 

alone. Several improvements to this procedure were implemented during the course of the study. 

 First, efforts were made to optimize the Nile Red staining protocol. The best staining was 

achieved using a concentration of 1 mg Nile Red in 1 mL of cell suspension. The solvent for the 

Nile Red stain was changed from heptane to acetone, due to interfering fluorescence from 

undissolved heptane droplets. Finally, the researchers found that the fluorescence signal from 

Nile Red is unstable and decays rapidly. However, the fluorescence level stabilizes after about 

45 minutes, so all readings were taken at least 45 minutes after staining the cells with Nile Red. 

Another important change was to measure the chlorophyll autofluorescence as well as Nile Red 

fluorescence. This ensured that only viable cells containing lipid and intact chloroplasts would 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 79 

be analyzed. In addition, the amount of chlorophyll is an indication of cell size. Cell sorting 

based on the ratio of chlorophyll fluorescence to Nile Red fluorescence would normalize the 

results to account for differences in cell size and age and allow detection of individual cells with 

unusually high lipid levels resulting from natural genetic variation. A decrease in the ratio of 

chlorophyll to Nile Red fluorescence would indicate lipid accumulation. 

In one set of experiments, Isochrysis cultures were stressed by transferring the cells into Ndeficient media, then screened for lipid content using flow cytometry, either by lipid content 

alone (Nile Red fluorescence) or by monitoring the chlorophyll to Nile Red fluorescence ratios. 

The daughter cells containing high or low levels of lipid were recultured in N-replete medium for 

1 week or 1 month, then subjected again to N deprivation, and resorted. The lipid content of the 

daughter population was compared to that of the parent cells. These experiments produced 

inconsistent results. In some cases, the population of daughter cells selected for their high lipid 

content showed a wider range of lipid contents than the parent cells; other sorts produced 

daughters without significant differences in lipid content from the parents. One interesting 

observation was the bimodal distribution of cells in all populations subjected to N stress. Cells 

fell into two classes with low or high chlorophyll-to-lipid ratios. This again supports the author's 

theory, discussed in the section on ultrastructural analysis of lipid accumulation, that cells 

respond as individuals to a lipid trigger, rather than gradually increasing the lipid content of the 

entire culture. 

These results suggested that flow cytometry might be used to select for populations of high lipid 

algae if more was understood about the relationship of the physiological state of parent cells to 

lipid accumulation. Analysis of the growth of Isochrysis in N-replete media showed several 

phases, including a period of exponential growth that declined to a stationary phase. After about 

2 weeks, the nutrients were depleted and the cells entered a stressed phase. A series of 

experiments was performed in which cells in various growth phases were stained with Nile Red 

and sorted based on lipid content alone (no chlorophyll measurements). The sorts on cells in 

exponential phase were usually not successful, but if the parent cells were in stationary phase or 

from very old cultures (stressed), the mean lipid content of the daughter population was about 

20% higher than that of the parental cells. These results suggested that successful screening for 

high-lipid cells using flow cytometry was related to the cell cycle. The exponential cultures 

contained cells in all stages of cell growth and division. Cells that were preparing to divide 

would be larger and would be selected as high-lipid, so that the set of high lipid cells selected 

would actually contain only the largest and oldest cells, rather than high lipid genetic variants. 

Stationary or stressed populations are not actively dividing, so the cells are more uniform in size 

and sorting of the cells for high lipid should be more likely to identify true high lipid variants 

within the population. 

The experiments using Isochrysis cultures in various growth stages as the parental population 

were repeated to test these assumptions. Cells were sorted based either on lipid levels alone (Nile 

Red fluorescence only) or using the ratio of chlorophyll to Nile Red fluorescence to control for 

cell size. The results presented in the available reports support the hypotheses. Sorting cells 

based solely on lipid content produced high-lipid daughter populations if the parent population 
National Renewable Energy Laboratory 

80 A Look Back at the Aquatic Species Program—Technical Review 

was in a stationary or stressed growth phase. Exponential cultures produced variable results. If 

the parental cells were sorted based on ratios of chlorophyll-to-lipid fluorescence, high-lipid 

populations could be produced from exponentially growing parent cultures. 

These conclusions were based on very limited data. Only a few experiments were performed. In 

addition, several daughter cultures did not grow up after the sorting process, and failure of a 

growth chamber resulted in the loss of some cultures. The data were written up in only two 

technical reports to SERI, and it was difficult to determine the exact protocol used for each 

sorting experiment. However, the results of these studies are intriquing, and suggest that flow 

cytometry might be a viable method for screening for high lipid genetic variants within (or 

between) strains of oleaginous microalgae. The procedure would be limited to strains in which 

the cells are small and spherical. Dr. Solomon's data suggest the best results would be achieved 

by using stationary or stressed cells as the parent population. In addition, cells should be selected 

based on a low chlorophyll-to-Nile Red fluorescence ratio, which would indicate high-lipid 

levels with respect to cell size. However, it is interesting that high-lipid daughter strains were not 

produced in the experiments in which exponentially growing cells were transferred directly to Ndeficient media; yet, cells allowed to gradually deplete their N supply to induce the stressed 

condition could be used successfully in a flow cytometric screen. This suggests either that lipid 

accumulation occurs by different mechanisms under these two conditions, or, more likely, that 

the stressed cells from very old cultures had all entered a similar metabolic state so that size and 

lipid contents would be more indicative of genetic differences. 

Publications: 

Solomon, J.A (1985) “Ultrastructure evaluation of lipid accumulation in microalgae.” Aquatic 

Species Program Review: Proceedings of the March 1985 Principal Investigators’ Meeting,

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 71-82. 

Solomon, J.A. (1987) “Flow cytometry techniques for species improvement.” FY 1986 Aquatic 

Species Program: Annual Report (abstr.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3071, p. 252. 

Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986a) “Ultrastructural and flow cytometric analyses of 

lipid accumulation in microalgae.” Annual Report, Oak Ridge National Laboratory, Oak Ridge, 

Tennessee, ORNL/M-258, 50 pp. 

Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986b) “Ultrastructural and Flow Cytometric Analyses 

of Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute, 

Golden, Colorado, SERI/STR-231-3089. 

Solomon, J.A.; Palumbo, A.V. (1987) “Improvement of microalgal strains for lipid production.” 

FY 1987 Aquatic Species Program: Annual report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3206. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 81 

II.B.1.f. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae 

Subcontractor: Montana State University 

Principal Investigator: Keith E. Cooksey 

Period of Performance: 1/86 - 10/87 

Subcontract Numbers: XK-5-05073-1; XK-6-05073-1 

The goal of this research was to understand the biochemistry of lipid accumulation in microalgae, 

in particular, to provide information on the biochemical triggers that induce lipid synthesis. The 

Nile Red fluorescence technique developed by Dr. Cooksey's laboratory and at SERI (described 

above in Sections II.A.1.f. and III.B.1.e.) was used to study lipid accumulation in microalgae in 

response to N or Si depletion. Nile Red fluorescence was used to monitor the lipid levels in 

batch cultures of Chlorella over time. As the N became depleted, lipids accumulated in the 

cultures, predominately as triglycerides. The triglyceride levels began to increase before N was 

totally depleted from the medium. Microscopic examination showed that individual cells within 

the population began to accumulate lipid at different times, similar to results obtained by Dr. 

Solomon in Isochrysis. Dr. Cooksey concluded that lipid accumulation begins as the cells enter 

stationary phase and cell division ceases; the timing of this event would be different for 

individual cells within a population. 

Dr. Cooksey's laboratory next performed a complex series of experiments designed to correlate 

media factors, (i.e., nitrate concentrations, pH, and carbon availability), with lipid accumulation 

in CHLOR-1. Cell growth and lipid accumulation were monitored in batch cultures, with cells 

grown in unbuffered Bold's medium, or media buffered at pH 7, 9, or 10. In unbuffered Bold's 

medium, the initial pH was 7, and increased to pH 8 by day 6, and up to pH 9.5 by day 9. The 

cells grew in all conditions, with the best growth at pH 9. Under all growth conditions, the level 

of nitrate in the media decreased, but never fell below 25% of the initial levels. 

Accumulation of neutral lipids was monitored by Nile Red fluorescence. There was no lipid 

accumulation in cultures maintained at or below pH 9. However, in buffered medium with a pH≥

10, or in unbuffered medium that experienced an increase in pH during the growth period, the 

cultures generally showed a significant increase in lipid levels that was accompanied by a 

decrease in cellular growth rates. 

Nutrient limitation, generally nitrate or silica, can trigger lipid accumulation in microalgae. 

Nutrient deprivation can cause a decrease in cell division, which presumably results in 

“targeting” of excess fixed carbon into storage lipids. The data obtained by Dr. Cooksey's 

laboratory suggested that a shift in pH, which has been correlated with decreased rates of cell 

division, could also trigger lipid accumulation. These data suggested that nutrient limitation 

might not directly affect biochemical pathways to enhance lipid synthesis; rather, lipid 

accumulation may be an indirect consequence of inhibition of a stage in the cell cycle. In other 

photosynthetic systems studied, the data indicated that cells synthesize triglycerides in the light 

and utilize these lipids as energy stores in the dark and during cell division. If division were 
National Renewable Energy Laboratory 

82 A Look Back at the Aquatic Species Program—Technical Review 

blocked, the rate of neutral lipid utilization would be slower than the rate of synthesis, so 

triglycerides would accumulate in the cells. To help test this hypothesis, Dr. Cooksey used light 

microscopy to examine cells grown in media with different pH ranges. Cultures grown at pH 7-9 

consisted almost entirely of small, single cells. However, at pH 10 and higher, a large proportion 

of the cells was in the form of autosporangial complexes, i.e., their nucleii had divided, but the 

autospores had not separated. The specific effect of increased pH on cell division is not clear, 

although some evidence suggests that increased pH can lead to increased flexibility of the 

autospore wall, preventing individual cells from breaking free. Alternatively, increased pH could 

affect precipitation of media components, indirectly affecting the cell cycle. 

Although the data presented here suggest that nutrient deprivation or increased pH may affect 

lipid levels simply as a consequence of decreased cell division, additional research by Dr. 

Cooksey's laboratory suggested that treatments that increase lipid accumulation may also affect 

the biochemistry of lipid biosynthesis. Analysis of the lipid classes present in the cells at the end 

of the 10-day growth period showed accumulation of triglycerides in cells at high pH, with a 

decrease in glycolipids and polar lipids. The nonpolar storage lipids predominantly contain 16- 

and 18-carbon saturated or monounsaturated fatty acids (16:0 and 18:1), which are considered 

“precursor” fatty acids in lipid biosynthesis. The polar lipids and glycolipids usually contain a 

higher proportion of polyunsaturated fatty acids. However, analysis of the fatty acid composition 

of the storage lipids showed that at higher pH, more of these precursor lipids were seen in the 

polar lipids and glycolipids. This suggests a switch in the lipid synthesis patterns that results in 

less desaturation of the fatty acids esterified to the polar lipids. 

In summary, the finding that an increase in pH can also lead to lipid accumulation in cells before 

N is depleted suggested a method to uncouple lipid accumulation from nutrient deprivation, and 

provided another method to study the biochemistry of lipid accumulation in microalgae. The 

data from Dr. Cooksey's laboratory supported the premise that lipid triggers such as nutrient 

deprivation or pH increase affect lipid accumulation in microalgae by similar mechanisms, i.e., 

inhibition of cell division, leading to decreased utilization of storage lipid while new synthesis of 

lipid continues. However, he also proposed that different stresses may affect different stages of 

the cell cycle. As there is evidence that the different lipid classes (neutral lipids versus polar 

lipids) may be synthesized at different times during the cell cycle, this could affect the quality 

and the quantity of the lipids synthesized. For example, pH stress appears to block release of 

autospores (after DNA replication); N deprivation could have multiple effects on the 

photosynthetic machinery or on a number of biochemical pathways in the cell that could directly 

or indirectly affect lipid synthesis. R. Thomas, a graduate student working with Dr. Cooksey, 

found that treating the cells with monofluoroacetate (MFA) also decreased cell growth and 

caused neutral lipid accumulation. MFA inhibits the TCA cycle, presumably preventing TCA 

oxidation of fatty acids and thus increasing the pool of acetyl CoA for synthesis of new fatty 

acids. (Thomas suggested that MFA could be used as a trigger for lipid accumulation in algal 

ponds, with the caveat that MFA is toxic to all living systems). 

Dr. Cooksey concluded that to understand the biochemistry of neutral lipid accumulation in 

microalgae, it would be necessary to understand cellular cycles of lipid synthesis and utilization 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 83 

that are coupled to cell growth and division. It will also be important to consider not only factors 

that affect synthesis of storage lipid, but also to understand the metabolic shifts that result in 

production of membrane lipids or storage lipids. 

Publications: 

Cooksey, K.E. (1987) “Collection and screening of microalgae for lipid production.” Final 

Subcontract Report, Solar Energy Research Institute, Golden, Colorado, May 1987, 42 pp. 

Cooksey, K.E.; Guckert, J.B.; Thomas, R. (1989) “Triglyceride accumulation and the cell cycle 

in microalgae.” Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, pp. 139-158. 

Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R.(1987) Fluorometric determination of 

the neutral lipid content of microalgal cells using Nile Red, J. of Microbiol. Methods 6:333-345. 

Cooksey, K.E.; Williams, S.A.; Collis, P.R. (1987) “Nile Red, a fluorophore useful in assessing 

the relative lipid content of single cells,” In The Metabolism, Structure and Function of Plant 

Lipids, (Stumpf, P.K; Williams, S.A.; Callis, R.P., eds.), Plenum Press, N.Y., pp. 645-647. 

Guckert, J.B.; Cooksey, K.E.; Jackson, L.L. (1987) “Lipid solvent systems are not equivalent for 

analysis of lipid classes in the microeukaryotic green alga, Chlorella.” Unpublished manuscript. 

Guckert, J.B.; Cooksey, K.E. (1990) “Triglyceride accumulation and fatty aid profile changes in 

Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition.” J. Phycol. 26:72-79. 

Thomas, R.M. (1990) “Triglyceride accumulation and the cell cycle in Chlorella.” Masters 

Thesis, Montana State University, July 1990. 

II.B.1.g. Biochemical Elucidation of Neutral Lipid Synthesis in Microalgae 

Subcontractor: University of Nebraska 

Principal Investigator: Steven D. Schwartzbach 

Period of Performance: 3/87 - 3/88 

Subcontract Number: XK-5-05073-3 

The goal of the research performed by Dr. Schwartzbach and coworkers was to understand the 

biochemistry and physiology of lipid accumulation in microalgae, in particular the biochemical 

responses to N deficiency as a trigger for lipid accumulation. Lipid biosynthesis is dependent on 

the availability of fixed carbon and the activity of enzymes involved in lipid synthesis. These 

experiments were directed at understanding how these processes are affected by N limitation in 

the algal cells. 
National Renewable Energy Laboratory 

84 A Look Back at the Aquatic Species Program—Technical Review 

The first set of experiments analyzed lipid synthesis in the eustigmatophytes Nannochloropsis 

salina and Nanno Q, two oleaginous strains from the SERI Culture Collection. Similar results 

were obtained for the two strains. The basic protocol was to innoculate the algal cells into media 

containing either non-limiting levels or low levels of nitrogen (0.1 mM NaNO3), and to monitor 

cell growth, chlorophyll content, and the lipid levels per cell and per culture volume. In cultures 

containing low N, cell division ceased after 50-60 hours, and the cells entered stationary phase as 

the N was depleted. In contrast, cells grown with sufficient N continued to divide. In the Nreplete cultures, the lipid content of the individual cells remained constant, and there was a 

steady increase in the amount of lipid per mL of culture as the cell number increased. In contrast, 

the N-deficient culture showed a significant increase in the level of lipid per cell. However, the 

lipid content of the culture per mL (or percentage of the AFDW composed of lipid) did not 

change. In N. salina, lipid made up 26%-32% of the AFDW, and in Nanno Q, lipid was 23%-

24% of the AFDW in cultures grown under N-replete and N-depleted conditions. These results 

indicate that N depletion causes the cells to stop dividing, while lipid synthesis continues. 

However, there is no net increase in lipid synthesis, and the trigger in these cells does not change 

the activity of enzymes involved in lipid biosynthesis. One caveat to these studies is that Dr. 

Schwartzbach measured only total lipid produced in the cells, including polar membrane lipids 

and nonpolar storage lipids; it is unclear from these studies and those described later whether N 

deficiency could differentially affect accumulation of the nonpolar lipids in these algae. 

Another result from the studies on Nannochloropsis was that the level of chlorophyll in the cells 

declined rapidly in N depleted cells. Thus, N depletion would also presumably decrease 

photosynthetic efficiency and the availability of fixed carbon. The next set of experiments was 

designed to separate the effects of reduced photosynthetic efficiency from direct effects of N 

limitation on biosynthetic enzyme activities. To accomplish this, a series of experiments was 

performed using the eukaryotic green alga Euglena gracilis var. bacillaris Cori. Euglena can 

grow heterotrophically using ethanol as the sole carbon source. The growth of cells in the 

presence of externally supplied carbon (ethanol) should not be limited by decreased 

photosynthesis, so the rate of lipid synthesis would be solely limited by lipid biosynthetic 

capabilities. 

Euglena is unique compared to most algae of interest to the ASP as potential producers of 

biodiesel. Euglena produces both lipid (primarily in form of the wax ester myristyl-miristate) 

and carbohydrate (the major product is paramylum, a β-1,3-glucan) as storage products. Using 

Euglena, a complicated series of experiments was conducted comparing the growth, lipid and 

carbohydrate content, and chlorophyll levels in algae under photosynthetic and heterotrophic 

growth conditions, as well as under aerobic and anaerobic conditions (Coleman et al. 1988b). 

Basically, cells were grown to N deficiency, then resuspended in fresh media containing either 

sufficient or limiting amounts of N. The new media also did, or did not, contain ethanol as a 

carbon source, and the cells were grown in the dark or in light. 

As was seen with the Nannochloropsis strains, cell growth under N deficient conditions caused 

an increase in the levels of storage products (in this case, lipid plus carbohydrate) per cell. 

However, there was no net increase in total lipid/carbohydrate when measured as a percentage of 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 85 

dry cell weight. This was true in cells grown autotrophically or heterotrophically. Nitrogen 

depletion caused the cells to stop dividing, but the storage products continued to accumulate in 

the cells at the same rate as in non-nitrogen limited cells. In addition, the proportion of 

carbohydrate and lipid was unchanged, thus there did not appear to be a N trigger effect, either 

directly or indirectly via carbon limitation, on the enzymes of the lipid or carbohydrate synthetic 

pathways. One caveat to this result was that in very old cultures, (i.e., 12 days after transfer of 

the cells to N-deficient media), the lipid as a percentage of the dry cell weight increased in all 

cultures. However, this was accompanied by a decrease in the total cell mass, and the lipids are 

apparently more stable than other cell components. 

Growth of Euglena under N-deficient conditions resulted in loss of chlorophyll, as seen for 

Nannochloropsis. Dr. Schwartzbach also used two-dimensional gel electrophoresis to monitor 

changes in the levels of chloroplast and mitochondrial proteins under N-deficient conditions 

(Coleman et al., 1988a). Under photosynthetic growth conditions (high light), exposure of the 

cells to N-deficient conditions resulted in a decrease in the levels of 37 proteins identified as 

components of the chloroplast. Under low light conditions, there was little change in the 

population of chloroplast proteins. The degradation of the chloroplasts under low N conditions 

was presumably due to photooxidation of chlorophyll, accompanied by degradation of newly 

synthesized photosynthetic membrane proteins that could not assemble properly into the unstable 

chloroplast. Synthesis of chlorophyll requires N to form δ-aminolevulinic acid, a chlorophyll 

precursor. Although photooxidation of chlorophyll occurs constantly in the light, synthesis of 

new chlorophyll molecules also occurs to replace the degraded molecules. However, if N levels 

are depleted, new chlorophyll cannot be produced, and photosynthetic efficiency decreases. This 

result is important with regard to biodiesel production. It suggests that there would be limitations 

on the amount of lipid that could be produced in outdoor ponds using N limitation as a trigger for 

lipid accumulation even if carbon was not limiting (i.e., for cells grown in outdoor ponds). 

One process that affected the biosynthetic pathways in Euglena and resulted in an increase in the 

total lipid in the cultures was cell growth under anaerobic conditions with ethanol as a carbon 

source (lipids increased from 5-10% to 45% of the AFDW). Growth via anaerobiosis caused the 

activation of the oxygen-sensitive pyruvate dehydrogenase in the mitochondria. This led to 

increased levels of acetyl CoA in the mitochondria, which activates the mitochondrial fatty acid 

synthesis pathways. However, the increased flow of carbon to lipid synthetic pathways was 

accompanied by degradation of non-lipid components under anaerobic condition, including 

paramylum, the main storage carbohydrate, which resulted in a decrease in total cell mass. Dr. 

Schwartzbach estimated that if the anaerobic cells had increased in cell mass to the same extent 

as cells grown aerobically, the lipids would only compose 15% of the dry weight. 

This observation that anaerobiosis could result in increased lipid yields by actually affecting the 

lipid biosynthetic pathway suggested that lipid synthesis could be increased by increasing the 

levels of the lipid precursors acetyl CoA and malonyl CoA. Little is known about lipid synthesis 

in algae, but data from other organisms suggested that pyruvate dehydrogenase and acetyl CoA 

carboxylase could function as regulatory enzymes in algal lipid synthesis. Understanding the 

biochemical factors that limit production of the lipid precursors could lead to biochemical or 
National Renewable Energy Laboratory 

86 A Look Back at the Aquatic Species Program—Technical Review 

genetic engineering strategies to increase the activity of these enzymes that could produce an 

organism with the ability to produce very high lipid levels. To this end, Dr. Schwartzbach 

initiated a project to isolate and characterize these enzymes from several algae, including 

Euglena, N. salina, Nanno Q, and Monoraphidium 2 (Smith and Schwartzbach 1988). They 

reported some very preliminary information on protein extraction techniques and assay 

techniques for these enzymes. This work was a precursor to a major effort at SERI/NREL in the 

late 1980s through the end of project in 1996 to identify key enzymes in the algal biosynthetic 

pathways and to increase lipid levels by manipulating these pathways through genetic 

engineering (see Sections II.B.2. and II.B.3.). 

In summary, although Euglena is not typical of the oleaginous microalgae targeted as potential 

biodiesel producers by the ASP, the data from Dr. Schwartzbach's laboratory point out the 

importanance of understanding the biochemical mechanisms by which algae accumulate lipids. 

For Euglena and the Nannochloropsis strains described here, N deprivation does not seem to 

function as an actual trigger to induce biosynthesis of lipid. Rather, it acts as a block to cell 

division. Lipid synthesis continues by normal pathways, and lipid levels increase per cell, with 

no net accumulation in the culture. This result confirms the conclusions of Cooksey and 

coworkers. In addition, N deficiency also affects other cell processes, such as photosynthetic 

efficiency, which could affect lipid accumulation as the availability of fixed carbon is decreased. 

Publications: 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987a) “Environmental control of lipid 

accumulation in Nannochloropsis salina, Nanno Q and Euglena.” FY 1987 Aquatic Species 

Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3206, pp. 190-206. 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987b) “Biochemistry of neutral lipid 

synthesis in microalgae.” FY 1986 Aquatic Species Program Annual Report (Johnson, D.A., ed.), 

Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 255. 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988a) “Preferential loss of chloroplast 

proteins in nitrogen deficient Euglena.” Plant Cell Physiol 29:1007-101. (Note: a preprint of this 

article was also submitted as a SERI Report, 47pp.) 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988b) “Environmental control of 

carbohydrate and lipid synthesis in Euglena.” Plant Cell Physiol. 29:423-432. 

Smith, C.W.; Schwartzbach, S.D. (1988) “Preliminary characterization of pyruvate 

dehydrogenase and acetyl-CoA synthetase.” Manuscript submitted as a report to SERI, 6 pp. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 87 

II.B.1.h. Transformation and Somatic Cell Genetics for the Improvement of Energy 

Production in Microalgae 

Subcontractor: University of Nebraska; Oregon State University 

Principal Investigator: Russel H. Meints 

Period of Performance: 1/86 - 12/89 

Subcontract Number: XK-5-05073-2 

By the mid-1980s, SERI researchers became convinced that optimal lipid production in 

microalgae could be achieved only by genetic manipulation of the cells to produce the desired 

traits. The overall goal of the research performed by Dr. Meints for SERI was to develop an 

algal virus system as a tool for the genetic manipulation of microalgae with potential for liquid 

fuel production. Viral vector systems had been used successfully to transfer DNA into other cell 

types. Dr. Meints' laboratory was preeminent in the field of algal viruses. The work performed 

from 1986 to 1989 was based on earlier studies from this laboratory on isolating and 

characterizing a unique group of viruses from symbiotic algae. Funding from SERI contributed 

to ongoing research on the algal viral system by Dr. Meints; much of this work was done in 

collaboration with Dr. James Van Etten, in the Department of Plant Pathology at the University 

of Nebraska. The list of publications at the end of this section includes articles produced before 

and after the period of the SERI subcontract. This information was included to give interested 

readers an overview of the work performed on this topic. 

Background: 

In the early 1980s, Dr. Meints and Dr. Van Etten were studying Chlorella-like green algae that 

live in a symbiotic relationship within cells of the protozoan Hydra viridis. They found that the 

algal cells could be excised from the hydra, which could exist free of the symbiont if given 

proper nutrients. However, it was not possible to culture the algae free of the hydra host. Further 

study demonstrated that when the algal cells were isolated from the host, virus particles rapidly 

began to multiply within the algae, resulting in lysis of the algal population within 24 hours. 

Ultrastructual and biochemical studies on this algal virus system produced the following results: 

• The virus consisted of a large (approximately 190-nm), polygonal particle, 

containing 30 to 40 polypeptides, the most abundant of which was a 46 kDa 

glycoprotein, presumably associated with the viral capsid. 

• The virus genome consists of about 130 kbp of double-stranded DNA. 

• New virus particles were assembled in the cytoplasm of the algal cells and 

released upon lysis of the algal cell wall. 

This virus, called HVCV (for Hydra viridis Chlorella virus), was one of the few viruses 

described in eukaryotic algae. HVCV might play a role in initiating or maintaining the symbiotic 
National Renewable Energy Laboratory 

88 A Look Back at the Aquatic Species Program—Technical Review 

relationship between the alga and its hydra host, possibly by altering the algal cell wall. 

Subsequent studies identified viruses in four other strains of Hydra obtained from commercial 

sources. The viruses fell into two classes, based on particle size, bouyant density, and DNA 

restriction patterns (HVCV-1, HVCV-2). In addition, a similar virus was isolated from symbiotic 

Chlorella from Paramecium bursaria (PBCV-1). 

To facilitate the study of these viruses, it was desirable to identify an algal strain that could be 

cultured free of the hydra or Paramecium host, which was susceptible to infection by the virus. 

This would allow production of large quantities of the virus and the study of viral replication and 

development. Sixteen strains of culturable Chlorella, which had been isolated from invertebrates 

such as Paramecium, Hydra, Stentor, and sponges, plus two free-living strains, were obtained. 

Attempts were made to infect these Chlorella strains with all the virus strains described earlier. 

None of the HVCV viruses (from Chlorella-Hydra hosts) were able to infect any Chlorella strain 

tested. However, two culturable Chlorella strains originally isolated from Paramecium

(Chlorella strains N1a and NC64) were infected with PBCV-1 (the P. bursaia Chlorella virus). 

The infection led to lysis of the algal cells and production of large amounts of infectious viral 

progeny. This result led to the development of a plaque assay system for the algal viruses, 

similar to a bacteriophage assay on bacterial lawns. The availability of this system, which caused 

synchronous infection of the algal cells and the production of large quantities of viral particles, 

allowed the researchers to characterize the virus biochemically. It also allowed researchers to 

study the regulation of viral gene expression and the effects of viral replication on algal 

physiology and gene expression. A large number of publications resulted from this research (see 

below). Several of the most interesting and possibly relevant findings are summarized here. 

• The virus particles attach at one vertex of their polygonal capsid to receptor sites 

on the algal cell wall. A lytic enzyme produced by the virus degrades the wall 

at this site, and the viral DNA is released into the cell. Living algal cells are not 

required for virus attachment and wall degradation (viruses can attach to and 

degrade isolated wall fragments), but living cells are necessary for release of the 

viral DNA. Complete viral capsids are assembled in viral assembly sites within 

the cytoplasm and subsequently filled with DNA. Virus particles are released 

through holes produced at discrete locations in the algal cell wall. 

• The plaque assay system was used to screen for other virues that infect algae. 

Viruses that infect Chlorella strains N1a or NC64A were found to be very 

common in nature. The viruses all had similar features, including a large, 

polygonal capsid and dsDNA; however, some viruses were distinguishable 

based on plaque size, reactivity to anti-PBCV-1 antisera, variations in the DNA 

restriction patterns and the extent of nucleotide modification. 

• The viruses are highly infectious and grow rapidly within the algal cells. Algal 

growth is inhibited rapidly following viral infection. Synthesis of host DNA 

and RNA is shut down, and the organellar and genomic DNA is degraded. Viral 

gene expression entails transcription of early and late genes, and may include 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 89 

expression of overlapping genes or transcription of genes from both DNA 

strands. Analysis of DNA from some viral isolates showed that the viral DNA 

is modified to varying extents, primarily in the methylation of adenine and 

cytosine residues. The data suggested that the virus produces a unique 

restriction enzyme that is specific for non-methylated sequences for degradation 

of the host DNA. The virus also produces a corresponding methyltransferase, 

which recognizes the same sequence as the restriction endonuclease. The 

methyltransferase methylates newly synthesized viral DNA, protecting it from 

degradation in the next round of infection. 

Dr. Meints received funding from SERI from 1986 through 1989. The overall goal of the SERIfunded research was to use the algal virus system to develop methods for genetically 

manipulating microalgae with potential for liquid fuel production. The research from Dr. Meints' 

laboratory is reviewed below with respect to the specific goals of the project. 

Isolation and characterization of natural hosts for the algal viruses. 

Water samples isolated from various sites were screened for the presence of algal viruses using 

the plaque assay. If viruses were not detected initially, an enrichment protocol was used in which 

a few algal cells were added to the water sample; after 48-72 hours the algal and cell debris were 

removed by centrifugation and the sample was reassayed for the presence of virus. Using these 

procedures, more than 50 individual algal viral isolates were identified. Although the viruses 

were all large polygonal particles with dsDNA genomes, analysis of the viral DNA by digestion 

with restriction endonucleases showed there were at least 15 different types of virus found. Sites 

that contained virus were further analyzed for the natural algal hosts; however, none were 

identified. It is unclear why the natural hosts could not be found. Dr. Meints proposed either 

that the viruses were propagated or maintained by some unknown mechanism, or, more likely, 

that the natural host was present in the sites tested at very low concentrations. This is possible in 

that each virus that infects an algal cell could produce 350 new virus particles, and that up to 100 

algal cells can exist within a single Paramecium. Based on the density of viral particles isolated 

from the various sites, a single Paramecium with algal symbionts could theoretically sustain a 

virus population in 350 liters of water. Because a natural alga host for the viruses was not found, 

the goal of characterizing the viral host was dropped, and further screening efforts were 

discontinued. 

In a separate, but related, series of experiments, Dr. Meints received 250 water samples collected 

by Dr. Ralph Lewin of the Scripps Institute in La Jolla, California. These were also screened for 

the presence of algal viruses, but viral particles were not found in any of Dr. Lewin's samples. 

Screening of the SERI algal collection for infection with the Chlorella viruses. 

Dr. Meints received a number of algal strains from Dr. Bill Barclay at SERI that were believed to 

have potential for biodiesel production. These included Chlorella 501 and A. falcatus (A record 
National Renewable Energy Laboratory 

90 A Look Back at the Aquatic Species Program—Technical Review 

of the precise number and identity of the strains sent to Dr. Meints could not be found). Growth 

conditions for these strains were optimized, and the algae were then tested for infection by the 

algal viruses. None of the SERI strains were susceptible to infection by any virus isolate tested. 

This project was discontinued in early 1987. 

Use of the lytic enzyme(s) produced by the virus for degradation of the host cell wall for the 

production of microalgal protoplasts. 

Infection of Chlorella N1a or NC64A by the algal viruses resulted in rapid lysis of the algal cells. 

Dr. Meints' laboratory developed methods to isolate the lytic enzymes and to use the lysin 

preparation to produce algal protoplasts (cells without cell walls). The protoplasts could be used 

in studies of somatic cell fusion (genetic improvement by fusion of two individuals with useful 

traits such as pH tolerance and high lipid production). Alternatively, the protoplasts could be 

used as targets in a genetic transformation system in which DNA plasmids are taken up directly 

into cells without walls. 

A crude lysin preparation was produced by infecting Chlorella N1a with PBCV-1. After several 

lytic cycles (approximately 24 hours), the sample was centrifuged to remove cell debris and 

virus. Initially, the supernatant from this preparation was used directly to produce protoplasts 

from Chlorella N1a cells. Alternatively, lysin activity was precipitated from the supernatant with 

65% ammonium sulfate. Cells were exposed to lysin in the presence of 1 M sorbitol as an 

osmoticum. (One interesting result from the protoplast studies is that algal strains exhibited 

significant differences in their sensitivities to osmotica commonly used for higher plant cells; i.e. 

mannitol, but not sorbitol, was toxic to Chlorella N1a. The sensitivity of individual algal strains 

to different osmotica will need to be determined empirically.) The cell wall of algal cells 

exposed to lysin was rapidly degraded over the entire cell surface, as judged by electron 

microscopy and staining of the cells with calcofluor white, which stains plant cell walls. The 

lysin preparation could be purified further by exposing the crude sample to an affinity matrix 

composed of algal cell wall fragments. Lysin activity was eluted by salt washes. Exposure of 

Chlorella N1a cells to this lysin sample resulted in degradation of the algal wall at specific sites; 

when the osmoticum was reduced to half strength, the alga protoplast was released through 

discrete holes in the wall. This result suggested the presence of more than one enzymatic activity 

in the crude lysin preparation. 

Somewhat surprisingly, the protoplasts did not lyse when transferred to water. However, 

exposure of lysin-treated cells, but not untreated cells, to low concentrations of detergent caused 

the release of chlorophyll. The amounts of chlorophyll released from lysin-treated cells was used 

as a measure of the extent of protoplast formation in a cell sample. The protoplasts were viable, 

as judged by staining with fluorescein diacetate. Unfortunately, although some regeneration of 

the cell wall occurred, the lysin-treated cells never formed new colonies. Attempts to use the 

viral-lysin to produce protoplasts from other microalgal strains met with little success. A 

manuscript was prepared and submitted to SERI that described the progress made on the use of 

lysin to produce algal protoplasts, but the article was never published in a technical journal. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 91 

Characterization of the chloroplast and mitochondrial genomes of microalgal strains. 

The goal of this research was to characterize the organellar genomes of Chlorella and other 

microalgae. As organellar DNA is thought to be highly conserved evolutionarily, the idea was to 

use similarities or differences between chloroplast or mitochondrial DNA as a measure of the 

taxomonic relatedness of algal strains. This information could be useful for experiments 

involving somatic cell fusion or gene transfer, as these procedures would likely have a higher 

chance of success between more closely related strains. Studies of the organellar genomes could 

also lead to the identification of promoters or replication origins that could be used to develop 

vectors for algal transformation. Due to the lack of significant progress on the first three goals, 

Dr. Meints concentrated the efforts of his laboratory on this project for the last 2 years of the 

subcontract. 

The first step was to develop methods for isolation of chloroplast and mitochondrial DNA from 

Chlorella N1a. Based on protocols used for higher plants, Dr. Meints exploited the differences in 

the C/G content between chloroplast DNA and nuclear DNA to separate the two genomes using 

density gradient centrifugation. Chloroplast DNA was identified by hybridization with 

heterologous chloroplast DNA markers. The chloroplast genome of Chlorella N1a was found to 

be circular, containing approximately 120 kbp of DNA. A restriction map of the chloroplast 

genome was produced and several genes were localized on the map by hybridization with 

chloroplast gene sequences from maize. Most chloroplast genomes contain two inverted repeats, 

each of which contains a copy of the 23S, 16S, and 5S ribosomal RNA genes. These repeats are 

flanked by a short and long single copy DNA region. Although Dr. Meints initially reported that 

Chlorella N1a chloroplast DNA contained this inverted repeat structure (Meints 1987), a 

subsequent article reported that the chloroplast genome of Chlorella N1a contains only a single 

copy of the ribosomal RNA gene region (Schuster et al. 1990b). This result was confirmed by 

Dr. Meints via a recent personal communication. Although most other green algae, including 

other chlorellans, contain chloroplast DNA similar to that commonly seen in most higher plants, 

i.e., containing two inverted repeats, this unusual chloroplast structure has been seen in two 

legumes (peas, broad beans), conifers, some red algae, and in at least one other green alga, 

Codium. 

Restriction analysis of chloroplast DNA from several exsymbiont and free-living strains of 

Chlorella showed variations between the strains that indicate genetic divergence and that suggest 

gene transfer and cell fusion between these species may be problematic. The results suggest that 

chloroplast DNA structure may be a useful taxonomic parameter, but more study is needed 

before definite conclusions on algal taxonomy or cell-cell compatability based on chloroplast 

DNA structure can be made. 

Isolating mitochondrial DNA from Chlorella N1a was technically problematic, and the 

mitochondrial genome isolated was first presumed to be a plasmid. Unlike some species in 

which mitochondrial DNA has a G/C content similar to that of nuclear DNA, in Chlorella N1a, 

the mitochondrial DNA had a low G/C content similar to that of chloroplast DNA, and the two 

genomes banded very closely on the density gradients. As with the chloroplast DNA, 
National Renewable Energy Laboratory 

92 A Look Back at the Aquatic Species Program—Technical Review 

heterologous probes were used to identify the mitochondrial DNA and to localize specific 

mitochondrial genes on the restriction map. The gene organization in the Chlorella N1a 

mitochondrial DNA was similar to that in higher plants, and distinct from the organization of 

mitochondrial genes in animals and fungi. It has been proposed that mitochondria in plants and 

green algae originated from a separate endosymbiotic event as compared to animals and fungi. 

This is supported by Dr. Meints’ results. 

Although not included in the original Statement of Work, Dr. Meints also reported under this task 

other related research efforts in his laboratory toward the development of a genetic 

transformation system for microalgae. Libraries were prepared from Chlorella N1a nuclear 

DNA and DNA from the algal virus. The goal of this project was to identify DNA sequences 

that could be used to develop transforming vectors, such as origins of replication, regulatory 

regions for gene expression, or algal genes to use in selectable marker systems. A library of the 

viral DNA was prepared in a lambda vector, which allowed for the sequencing of the viral 

genome and studies of viral gene stucture and expression. This work led to several significant 

discoveries that were published after SERI funding stopped, including the cloning of the major 

viral capsid protein (Graves and Meints, 1992), and the identification of a viral gene promoter 

that also functioned in higher plants (Mitra and Higgins, 1994). 

Dr. Meints’ laboratory also made several attempts to produce a library of Chlorella nuclear 

DNA, with little success. This appeared to be due to modification (probably methylation) of the 

algal DNA that resulted in degradation of the DNA by the bacterial host used for library 

construction. Several ways around this problem were proposed, including the use of a yeast 

cloning system or the use of a bacterial host that did not contain the enzymes for degradation of 

methylated DNA. A cDNA library was produced successfully before the end of the SERI-funded 

research efforts. 

Dr. Meints and his coworkers and collaborators produced a large quantity of data during the 4 

years of SERI-funded research and during the following years. They made significant 

contributions to the study of the biology, biochemistry, and molecular biology of a eukaryotic 

algal virus, and to the biology and molecular biology of the algal hosts, particularly with respect 

to the algal organellar genomes. Unfortunately, because of the specificity of the virus/algal 

interactions, the results obtained were not directly applicable to the development of a 

transformation system for the oleaginous algal strains of interest to NREL. The research also 

generated some valuable technical information, regarding toxicity of microalgae to common 

osmotica, construction of genomic DNA libraries, and organellar genome isolation, which could 

be useful for further studies of algal molecular biology and the development of genetic 

engineering techniques. The studies of the algal virus also resulted in the identification of a new 

restriction endonuclease (Jin et al. 1994) and a new adenine methyltransferase (Stefan et al. 

1991), as well as a viral promoter sequence that can function in plants (Mitra and Higgens 1994). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 93 

Publications: 

Anberg, S.M.; Meints, R.H. (1991) “Nucleotide sequence of the genes for ribulose-1,5-

bisphosphate carboxylase/oxygenase large subunit and ribosomal protein S145 from a Chlorellalike alga.” J. Phycol. 27:753-758. 

Graves, M.V.; Meints, R.H. (1992) “Characterization of the major capsid protein and cloning of 

its gene from algal virus PBCV-1.” Virology 188:198-207. 

Graves, M.V.; Meints, R.H. (1992) “Characterization of the gene encoding the most abundant in 

vitro translation product from a virus infected Chlorella-like alga.” Gene 113:149-155. 

Ivey, R.G.; Henry, E.C.; Meints, R.H. (1996) “A Feldmania algal virus has two genome size 

classes.” Virology 220:267-. 

Jin, A.; Zhang, Y.; Van Etten, J.L. (1994) “New restriction endonuclease CviRI cleaves DNA at 

TG/CA sequences.” Nuc. Acids. Res. 22:3928-. 

Krueger, S.K.; Ivey, R.G.; Meints, R.H. (1996) “A brown algal virus genome contains a ring zinc 

finger motif.” Virology 219:307-. 

Lee, A. M.; Ivey, R.G.; Meints, R.H. (1995) “Characterization of a repetitive DNA element in a 

brown algal virus.” Virology 212:474-. 

McCluskey, K.; Graves, M.V.; Mills, D.; Meints, R.H. (1992) “Replication of Chlorella virus 

PBCV-1 and host karyotype determination studies with pulse-field gel elecrtrophoresis.” J. 

Phycol. 28:846-850. 

Meints, R. (1987) “Studies of the chloroplast genome of exsymbiotic and free-living Chlorella.” 

FY 1987 Aquatic Species Program Annual Report, Solar Energy Research Institute, SERI/SP231-3206, pp. 210-228. 

Meints, R.H.; Burbank, D.E.; Van Etten, J.L.; Lamport, D.T.A. (1988) “Properties of the 

Chlorella receptor for the PBCV-1 virus.” Virology 164:15-21. 

Meints, R.H.; Lee, K.; Burbank, D.E.; Van Etten, J.L. (1984) “Infection of a Chlorella-like alga 

with the virus PBCV-1: Ultrastructural studies.” Virology 138:341-346. 

Meints, R.H.; Lee, K.; Van Etten, J.L. (1986) “Assembly site of the virus PBCV-1 in 

Chlorella-like alga: Ultrastructural studies.” Virology 154:240-245. 

Meints, R.H.; Kennedy, J.; Ziegelbein, M.; Amberg, S.; Van Etten, J.L. (1988) “Production of 

protoplasts from Chlorella-like algae.” Unpublished manuscript; submitted to SERI as a 

research report. 
National Renewable Energy Laboratory 

94 A Look Back at the Aquatic Species Program—Technical Review 

Meints, R.H.; Schuster, A.M.; Van Etten, J.L. (1985) “Chlorella viruses.” Plant Mol. Biol. 

Reports 3:180-187. 

Meints, R.H.; Van Etten, J.L. (1991) “Eukaryotic Algae.” In Microbioal Ecology: Principles, 

Methods and Applications, (Levin, M.A.; Seidler, R.J.; Rogul, M., eds.), McGraw-Hill, Inc. New 

York, pp. 883-888. 

Meints, R.H.; Van Etten, J.L.; Kuczmarski, D.; Ang, B. (1981) “Viral infection of the symbiotic 

Chlorella-like alga present in Hydra viridis.” Virology 113:698-703. 

Reisser, W.; Burbank, D.E.; Meints, S.M.; Meints, R.H.; Becker, B.; Van Etten, J.L. (1988) “A 

comparison of viruses infecting two different Chlorella-like green algae.” Virology 167:143-

149. 

Schuster, A.M.; Burbank, D.E.; Meister, B.; Skrdla, M.; Meints, R.H.; Hattman, S.; Swinton, D.; 

Van Etten, J.L. (1986) “Characterization of viruses infecting a eukayotic Chlorella-like green 

alga.” Virology 150:170-177. 

Schuster, J.A.; Graves, M.; Korth, K.; Ziegelbein, M.; Brumbaugh, J.; Grone, D.; Meints, R.H. 

(1990a) “Transcription and sequence studies of a 4.3 kbp fragment from a dsDNA eukaryotic 

alga virus.” Virology 176:515-523. 

Schuster, A.M.; Waddle, J.A.; Korth, K.; Meints, R.H. (1990b) “The chloroplast genome of an 

exsymbiotic Chlorella-like alga.” Plant Mol. Biol. 14:859-862. 

Skrdla, M.P.; Burbank, D.E.; Xia, T.; Meints, R.H.; Van Etten, J.L. (1984) “Structural proteins 

and lipids in a virus, PBCV-1, which replicates in a Chlorella-like virus.” Virology 135:308-315. 

Stefan, C.; Xia, Y.; Van Etten, J.L. (1991) “Molecular cloning and characterization of the gene 

encoding the adenine methyltransferase M.Cviri from Chlorella virus XZ-6E” Nuc. Acids Res.

19:307-. 

Van Etten, J.L.; Burbank, D.E.; Joshi, J.; Meints R.H. (1984) “DNA synthesis in a Chlorella-like 

alga following infection with the virus PBCV-1.” Virology 134:443-449. 

Van Etten, J.L.; Burbank, D.E.; Kuczmarski, D.; Meints, R.H. (1983) “Virus infection of a 

culturable Chlorella-like algae and development of a plaque assay.” Science 219:994-996. 

Van Etten, J.L.; Burbank, D.E.; Meints, R.H (1986) “Replication of PBCV-1 in ultra-violet 

irradiated Chlorella.” Intervirology 26:115-120. 

Van Etten, J.L.; Burbank, D.E.; Schuster, A.M.; Meints, R.H. (1985) “Lytic viruses infecting a 

Chlorella-like alga.” Virology 140:135-143. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 95 

Van Etten, J.L.; Burbank, D.E.; Xia, Y.; Meints, R.H. (1983) “Growth cycle of a virus, PBCV-1, 

that infects Chlorella-like algae.” Virology 126:117-125. 

Van Etten, J.L.; Meints, R.H. (1991) “Viruses and virus-like particles of eukaryotic algae.” 

Microbiological Reviews 55:586-620. 

Van Etten, J.L.; Lane, L.; Meints, R.H. (1991) “Unicellular plants also have large dsDNA 

viruses.” Seminars in Virology 2:71-77. 

Van Etten, J.L.; Meints, R.H.; Burbank, D.E.; Kuczmarski, D.; Cuppels, D.A.; Lane, L.C. (1981) 

“Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra 

viridis.” Virology 113:704-111. 

Van Etten, J.L.; Meints, R.H.; Kuczmarski, D.; Burbank, D.E.; Lee, K. (1982) “Viruses of 

symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis.” Proc. 

Nat'l. Acad. Sci. 79:3867. 

Van Etten, J.L.; Schuster, A.M.; Meints, R.H. (1988) “Viruses of eukaryotic Chlorella-like 

algae.” In Viruses of Fungi and Simple Eukaryotes, Mycology Series vol. 7 (Koltin, Y.; 

Leibowitz, M.J., eds.), Marcel Dekker, Inc., New York, pp. 411-428. 

Van Etten, J.L.; Xia, Y.; Meints, R.H. (1987) “Viruses of a Chlorella-like green alga.” In PlantMicrobe Interactions II, (Kosuge, T.; Nester, E.W., eds.), Macmillan Publishing Co., pp 307-325. 

Van Etten, J.L.; Xia, Y.; Narva, K.E.; Meints, R.H. (1987) “Chlorella Algal Viruses.” In 

Extrachromasomal Elements in Lower Eukaryotes (Fink, G.; et al., eds.), Plenum Press, New 

York, pp. 337-347. 

Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1989) “The mitochondrial genome of the 

exsymbiotic Chlorella-like green alga N1a.” Aquatic Species Program Annual Report,

September 1989, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 91-

118. 

Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1990) “The mitochondrial genome of an 

exsymbiotic Chlorella-like green alga.” Plant Mol. Biol. 14:187-195 

II.B.2. Physiology, Biochemistry, and Molecular Biology of Lipid 

Production: NREL In-House Researchers

II.B.2.a. Introduction 

During the first few years of the ASP, in-house research efforts in the area of lipid biosynthesis 

focused on understanding the lipid trigger and the effects of N starvation on lipid synthesis and 

photosynthetic efficiency. It was also shown that lipid accumulation can be induced in diatoms 

by Si starvation, a major component of the diatom cell wall. During the second half of the 1980s 
National Renewable Energy Laboratory 

96 A Look Back at the Aquatic Species Program—Technical Review 

and early 1990s, a major component of the research at SERI/NREL was the study of lipid 

biosynthesis in the oleaginous diatom C. cryptica. This was primarily the work of Paul Roessler, 

who identified a key enzyme involved in lipid accumulation and isolated and characterized both 

the protein and the gene for the enzyme acetyl-CoA carboxylase from Cyclotella. Other research 

efforts at NREL examined related biosynthetic pathways, including synthesis of chrysolaminarin, 

a storage carbohydrate, and lipid processing reactions such as fatty acid desaturation. The basic 

biochemistry and molecular biology research formed the basis of the efforts to manipulate 

microalgal lipids by genetic engineering, which will be described in Section II.B.3. 

One aspect of the algal research at SERI was the possibility of producing hydrogen by 

microalgae, for use as a gaseous fuel. During photosynthetic electron transport, electrons from 

reduced ferredoxin can be transferred to hydrogen ions to produce H2. This reaction is catalyzed 

by the enzyme hydrogenase. Unfortunately, hydrogenase is inhibited by molecular oxygen, a byproduct of the photosynthetic reaction, making the practical application of this process difficult. 

There was a significant research effort at SERI in the early 1980s, primarily by Dr. Steve Lien 

and Dr. Paul Roessler, to understand the biochemistry of hydrogen production by microalgae. 

The work on hydrogenase was funded by the Hydrogen Program at DOE, not the ASP, and will 

not be included in this report. However, studies on hydrogen production by microalgae are 

ongoing at NREL (SERI) in the Center for Basic Sciences, and interested readers should contact 

Dr. Michael Seibert for more information. 

II.B.2.b. Lipid Accumulation Induced by Nitrogen Limitation 

As a result of the algal screening efforts by SERI subcontractors and in-house researchers, 

several algal species were identified as good candidates for biodiesel production during the early 

1980s. This was facilitated by the development of a cytochemical staining technique for 

intracellular lipids that allowed researchers to visualize storage lipid droplets in algal cells (see 

Section II.A.1.f. and Lien 1981a). Two of the most promising candidates were the green alga N. 

oleoabundans, which showed a high lipid content and rapid growth, and a Chlorella strain 

(CHLS01) isolated from a local site. 

First, a sensitive method to monitor nitrate levels in liquid cultures using ion chromatography 

was developed to study the effects of N limitation on lipid accumulation in these organisms. 

Algal growth, lipid content, and chlorophyll a content were measured in batch cultures of N. 

oleoabundans and CHLS01. Cell division and chlorophyll accumulation occurred rapidly in the 

cultures as long as N was present. When N was depleted, cell division stopped, although 

biomass accumulation continued for several days. The major portion of the new biomass was 

composed of lipids and storage oils. N depletion resulted in a rapid decrease in the level of 

chlorophyll a in the cultures, suggesting that the cells might metabolize chlorophyll during 

periods of nitrogen stress. There was also an increase in the ratio of carotenoid to chlorophyll 

and a significant decrease in the complexity of the intracellular membranes in N-starved cells. 

These last three observations indicated that the photophysiology of the cells was affected, 

suggesting that the lipid trigger could also directly or indirectly alter photosynthetic efficiency in 

the treated cells (discussed in more detail below). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 97 

II.B.2.c. Studies on Photosynthetic Efficiency in Oleaginous Algae 

SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understand 

the processes affecting lipid accumulation (Lien and Roessler 1986). A recently published 

technical evaluation (Hill et al. 1984) identified two major requirements for economic feasibility 

of biodiesel production: 

1. Photosynthetic efficiency (which can simply be thought of as the percentage of 

incident radiation that is converted into biomass) needs to be 18%, and 

2. Algal biomass needs to consist of 60% lipid. 

Because very high lipid production is usually correlated with stress conditions (nutrient 

deprivation) that result in decreased photosynthetic efficiency and decreased growth, the two 

conditions of high lipid and high productivity seemed to be mutually exclusive. To overcome 

this technical hurdle, Lien and Roessler initiated a study to help understand the effects of 

nitrogen deprivation and lipid accumulation on photosynthetic efficiency. 

Three strains of oleaginous algae were used in this study: Chlorella CHLSO1, Ankistrodesmus

sp., and a newly isolated chrysophyte strain Chryso/F-1. The cells were grown in batch culture 

and monitored for nitrate concentration, light levels in the culture, chlorophyll concentration, and 

yield of cell mass and lipid (including total, neutral, and polar lipids). Maximum energy 

efficiency occurred as the culture approached N depletion. At this point, the culture showed a 

maximum density of photosynthetic pigments (before chlorophyll degradation and after N 

depletion), but the light energy reaching the cells was decreased due to the higher culture density. 

Thus, photosynthetic efficiency (biomass produced per light energy input) was maximized and 

the individual cells suffered less photooxidative damage due to lower light exposure. After the N 

in the culture was depleted, cell mass continued to increase for a time, eventually leveling off. 

All cultures experienced a two- to three-fold increase in total lipid, primarily as non-polar lipid. 

The photosynthetic efficiency decreased over the duration of the batch culture. However, in the 

early stages after the N was depleted, the cultures showed a decrease in energy efficiency with 

respect to total cell mass (AFDW) and with respect to the non lipid cell components, while 

photosynthetic efficiency remained constant or increased slightly with respect to lipid 

accumulation. In addition, N deprivation caused an increase in the efficiency of neutral, storage 

lipid production and suppressed the efficiency of polar structural lipid production. 

These studies provided interesting preliminary data on the energetics of cell mass and lipid 

accumulation in algae. Follow-up experiments were proposed, including investigations of the 

relationship between initial N concentration and photosynthetic efficiency and lipid production 

after N depletion, and studying the effects of N resupply after depletion to attempt to extend the 

period of lipid production. These experiments were not continued; however, the results 

described earlier suggest that understanding the timing or kinetics of lipid accumlation in 

microalgae will be essential to maximize lipid production in a mass culture facility. If N 

starvation is used to trigger lipid accumulation, the data suggest that maximal photosynthetic 
National Renewable Energy Laboratory 

98 A Look Back at the Aquatic Species Program—Technical Review 

efficiency with respect to lipid production (and probably the best time for harvesting lipidproducing cells), occurs just after the N is depleted from the cultures. 

Another set of experiments directed at optimizating photosynthetic efficiency in algal ponds was 

performed by SERI researcher Dr. Ken Terry. Previous studies had indicated that algal cells 

grown under high-intensity flashing light can use that light energy more efficiently than cells 

grown under the same intensity under constant illumination. The evidence suggests that an algal 

cell can integrate absorbed light energy such that the photosynthetic efficiency achieved under 

intermittent light conditions is similar to that attained under constant light of the same average 

intensity. This flashing light, or photomodulation, effect can be mimicked in vertically-mixed 

algal ponds, as cells circulate to the surface and back down to the lower levels in the pond where 

they receive minimal light. Thus, the photosynthetic efficiency of algal cells grown in ponds 

may be increased in high light by using mixing strategies that optimize this photomodulation 

effect. 

In order to better understand the effects of intermittent light on photosynthetic efficiency of 

microalgal cultures, Dr. Terry set up a system to measure photosynthetic rates and oxygen 

evolution in laboratory cultures of Chlorella pyrenoidosa and Phaeodactylum tricorutum under 

flashing light conditions. Intermittent light conditions were simulated by placing sectored disks 

in front of a light source, and using this to illuminate exponentially growing cultures that had 

been placed in an oxygen electrode chamber. Photosynthesis was then measured under varying 

light/dark ratios (generated by changing the configuration of the disk) and light intensities. The 

data generated were used to calculate the percent “integration” of the incident light by the algal 

cultures. More rapid flashing led to greater integration, although lower flash frequencies 

produced higher levels of integration as the percentage of time the cells spent in the light 

decreased. Although these data were preliminary, they supported the proposal that 

photosynthetic efficiency in microalgal ponds could be enhanced by optimized vertical mixing 

strategies. However, increased photosynthetic efficiency might be compromised by increased 

losses to respiration as the cells spend increased time away from the surface, and the energy costs 

to achieve optimal mixing could be prohibitive. 

Although Dr. Terry proposed follow-up studies using modulated light regimes that more closely 

mimic those seen in algal ponds, little further research on understanding photosynthetic 

efficiency in algal cultures was performed at SERI. Instead, the emphasis of the in-house 

research shifted to understanding the biochemistry and molecular biology of lipid accumulation. 

II.B.2.d. Lipid Accumulation in Silicon-Deficient Diatoms 

A note added to a chapter of the 1986 Annual Report (Lien and Roessler 1986) described 

preliminary data on the use of Si deficiency to trigger lipid accumulation in diatoms. Silicon is 

major component of diatom cell walls. Similar to the lipid trigger effect produced by Ndeficiency, Si depletion also results in a decrease in cell growth and often is accompanied by an 

accumulation of lipid within the cells. However, Si (unlike N) is not a component of other 

cellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 99 

apparatus. Therefore, any changes in cellular biochemistry and lipid accumulation induced by Si 

deficiency might be more easily interpreted than changes induced by N starvation. This work 

initiated a series of experiments by Paul Roessler during the late 1980s and early 1990s on the 

biochemistry and molecular biology of lipid accumulation in Si-deficient diatoms. 

The first set of experiments compared the effects of Si deficiency on lipid accumulation and cell 

physiology in several species of diatoms, including C. cryptica T13L, Thalassiosira pseudonana,

and Cylindrotheca fusiformis. Exponentially growing cultures were transferred to media that 

contained either excess Si or limited levels of Si so that the media became Si deficient while the 

cells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, and 

photosynthetic capacity were monitored under both conditions. In all three species, cell division 

decreased as soon as the Si was depleted in the media. However the species responded 

differently with respect to other physiological parameters. In C. cryptica, chlorophyll a synthesis 

was almost completely inhibited after 12 hrs in Si-depleted media; C. fusiformis showed little 

change in chlorophyll a synthesis after 72 hrs. T. pseudonana exhibited an intermediate effect, 

with some decrease in chlorophyll a synthesis noted after 36 hours without Si. The effect on 

photosynthetic capacity, measured as O2 evolution, also varied between the three species. In C. 

fusiformis and C. cryptica, photosynthetic capacity decreased 33% and 58%, respectively, after 

12 hours; T. pseudonana showed a steady decline in photosynthetic capability following Sidepletion. (However, photosynthetic capacity decreased in Si-replete cultures as well during the 

72 hours time course of the experiment, presumably due to the increased ratio of antenna 

chlorophyll molecules versus reaction center molecules in the self-shaded, dense cultures). 

The three species were also analyzed for accumulation of total biomass and lipid (Figure II.B.3.). 

 In C. fusiformis, biomass accumulation (measured as AFDW) for the duration of the experiment 

was similar in cultures with or without sufficient Si, although lipids made up a higher percentage 

of the AFDW in the Si-deficient cultures (26% versus 21% in Si-replete cells). In T. 

pseudonana, synthesis of cell mass and lipid was not affected until 36 hours after Si depletion. 

At this point, biomass and lipid accumulation rates decreased; however, there was little 

difference in the percentage of total lipid in the cells with or without Si at the end of the 72 hours 

experimental period. The situation with C. cryptica was very different. Twelve hours following 

Si depletion, there was a 38% decrease in the growth rate of these cells compared to the Sireplete culture. However, lipid synthesis continued at the same rate in the Si-deficient cells as in 

the Si-replete cells, resulting in a significant increase in the lipid content of the Si-starved cells. 

Interestingly, after these initial changes, the Si deficient cultures of C. cryptica showed little gain 

in total AFDW or lipid during the remaining 72 hours of the experiment. 

In order to determine if Si deprivation affected the composition of the lipids produced, the lipids 

were extracted and analyzed for the percentage of polar versus neutral lipids present. In all three 

species, the Si-deficient cultures showed a significant increase in the level of neutral lipids, 

primarily TAGs. For example, the percentage of neutral lipids in Si-deficient cultures of C. 

cryptica was 64%, compared to 32% in Si-replete cultures. In C. fusiformis, the percentage of 

neutral lipids increased from 17%-20% to 57% in Si-deprived cultures. 
National Renewable Energy Laboratory 

100 A Look Back at the Aquatic Species Program—Technical Review 

Based on these studies, C. cryptica was identified as the best candidate for further studies on the 

biochemistry of lipid accumulation. To determine the effects of Si deficiency on the synthesis of 

the cell components, the levels of protein, carbohydrate, and lipid were examined at various 

times after Si was depleted in the cultures. During the first 12 hours, protein and carbohydrate 

synthesis decreased. Lipid accumulation continued at a rate similar to that of the Si-replete 

cultures. This resulted in an increase in lipid content of the Si deficient cells from 19% to 27%. 

This observation was confirmed in subsequent studies that followed the incorporation of newly 

assimilated carbon (as H14CO3

-

) into the various cell components. Si depletion resulted in a net 

decrease in the rate of photosynthesis and carbon assimilation, but the individual cell fractions 

were affected differently. For example, the rate of 14C accumulation into lipids decreased by 

48% in the first 4 hours of Si-deprivation; the uptake of 14C into chrysolaminarin, the major 

carbohydrate storage product in diatoms, decreased 84%. Therefore, the increase in lipid content 

of Si-deficient cells was not due to an increase in the rate of lipid synthesis, but to a relative 

decrease in the rate of synthesis of protein and carbohydrate. 

Pulse-chase experiments were performed to test whether Si deficiency also caused the conversion 

of non-lipid cellular components into lipids. In these experiments, Si-replete cells were labeled 

with H14CO3

-

 for 1 hour, then transferred into Si-deficient media without labeled bicarbonate. 

The amount of labeled carbon in the lipid fraction was determined at various times following 

transfer to Si-free media. This experiment showed that carbon was slowly redistributed from the 

nonlipid components of the cells into lipid under Si-deficient conditions, but not under Si-replete 

conditions. Therefore, the accumulation of lipids in diatoms in response to Si-deficiency is 

apparently due to two factors: 

1. An increase in the proportion (but not the net amount) of newly assimilated 

carbon that is incorporated into lipids, resulting from a disproportionate 

decrease in the rate of lipid synthesis versus carbohydrate synthesis, and 

2. A slow conversion of nonlipid cell material into lipids.

Fractionation of the lipids produced demonstrated that Si deprivation resulted in an increase in 

the proportion of total lipid as neutral lipids, primarily TAGs, from 43% to 63% after only 4 

hours of Si deficiency. Analysis of the fatty acid composition of the accumulated lipids also 

showed changes induced by Si starvation. In Si-deficient cells, there was an increase in the 

proportions of mono- and unsaturated fatty acids (16:1, palmitoleic acid; 16:0, palmitic acid; and 

14:0, myristic acid), and a reduction in the proportions of the three major polyunsaturated fatty 

acids, (16:3, 20:5, and 22:6). These results are consistent with the finding that the predominant 

fatty acids found in triacylglycerol storage lipids in C. cryptica are 16:1, 16:0, and 14:1. These 

shorter, more highly saturated fatty acids are also the most desirable substrates for conversion 

into fatty acid methyl esters (biodiesel), as they would be less likely to polymerize during 

combustion and “gum up” an engine. 

Although Si depletion causes all diatoms tested to stop dividing, species responded differently 

with repsect to continued accumulation of biomass and lipid. C. cryptica showed a rapid 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 101 

response to Si-depletion, with a decrease in growth accompanied by a significant increase in the 

proportion of the biomass as lipid within 12 hours (the response to N starvation was usually 

much slower, as the cells could utilize internal N stores). This result again emphasizes the need 

to understand the kinetics of lipid accumulation in individual species under specific conditions 

for cost-effective lipid production in the ponds. 

Figure II.B.3. Changes in lipid mass, ash-free dry mass, and lipid content in Si-deficient 

cultures of three diatoms. 

A. C. fusiformis B. C. cryptica C. T. pseudonana. 

Symbols: () Si-deficient cultures; (z) Si-replete cultures. 
National Renewable Energy Laboratory 

102 A Look Back at the Aquatic Species Program—Technical Review 

II.B.2.e. Isolation and Characterization of Acetyl-CoA Carboxylase from C. cryptica 

To better understand the processes involved in lipid accumulation in microalgae, and to identify 

potential molecular targets for genetic manipulation, studies were initiated to examine the effects 

of Si deficiency on the enzymatic pathways involved in lipid and carbohydrate synthesis in C. 

cryptica. One possibility is that the increased levels of storage lipid in cells exposed to Si 

starvation could result from shifts in the relative activities of one or more enzymes in the lipid 

biosynthesis pathway. Acetyl-coenzyme A (acetyl-CoA) is known to be the immediate precursor 

of fatty acid synthesis, but the source of this compound varies in different organisms. For 

example, in mammalian cells, acetyl-CoA used in cytosolic fatty acid synthesis is produced from 

citrate via the action of ATP citrate lyase. In plants, acetyl-CoA can be produced in the 

chloroplasts from pyruvate, catalyzed by pyruvate dehydrogenase. Alternatively, acetyl-CoA 

could be produced by the mitochondrial pyruvate dehydrogenase. In this case, the acetyl-CoA 

(which cannot diffuse across the organellar membranes) would be broken down to acetate and 

free CoA by acetyl-CoA hydrolase. Acetate would diffuse to the chloroplast and become 

incorporated into acetyl-CoA by the action of acetyl-CoA synthetase. Once acetyl-CoA is 

produced, it is then used as a substrate by acetyl-CoA carboxylase (ACCase) to produce malonyl 

CoA. Malonyl-CoA is a substrate for fatty acid synthase and this reaction is considered to be the 

first committed step in fatty acid synthesis. 

These pathways had not previously been well-characterized in diatoms. To better understand the 

lipid synthesis pathways, Roessler first looked for the presence of these enzymes in extracts of C. 

cryptica, but found no citrate lyase activity. However, acetyl-CoA hydrolase, acetyl-CoA 

synthetase, and ACCase activity were all present. Enzyme activities were studied in Si-replete 

and Si-deficient cells (Figure II.B.4). The level of acetyl-CoA synthetase activity was similar 

under both conditions; however, the level of ACCase activity was two fold higher in Si-deficient 

cells after 4 hours, and four fold higher after 15 hours. Based on subsequent studies using 

protein synthesis inhibitors, the increased specific activity of the ACCase was believed to result 

from an increase in expression of the ACCase gene (Roessler 1988a; 1988c). 

ACCase is a biotin-containing enzyme that catalyses the carboxylation of acetyl-CoA to form 

malonyl-CoA. This reaction entails two partial reactions: the carboxylation of biotin, followed 

by the transfer of the carboxyl group from biotin to acetyl-CoA. In bacteria, the enzyme is 

composed of four non-identical subunits. However, in eukaryotes, biotin binding, biotin 

carboxylation, and carboxyl-transfer all occur on a single large multifunctional protein; the 

functional ACCase occurs as a multimer of this polypeptide. ACCase had previously been 

shown to play a key regulatory role in the rates of fatty acid synthesis in both animal and plant 

systems. A project was initiated to isolate and characterize ACCase from C. cryptica to clarify 

the role of this enzyme in lipid accumulation induced by Si starvation, and to compare the 

microalgal enzyme with those isolated from plants, animals, yeast, and bacteria. 

The enzyme was purified from C. cryptica by a combination of (NH4)2SO4 precipitation, gel 

filtration chromatography, and affinity chromatography based on the affinity of biotin to avidin. 

Consistent with ACCase enzymes isolated from other eukaryotes, C. cryptica ACCase was found 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 103 

to consist of a homo-tetramer of 185 kDa subunits. The activity of the enzyme was assayed by 

the incorporation of 14C bicarbonate into malonyl-CoA, and other factors were identified that 

affect the stability and activity of the enzyme. As seen for other ACCases, the enzyme required a 

slightly alkaline pH for optimum activity (pH 8.2), although the enzyme was most stable when 

stored at pH 6.5. The enzyme was also stabilized by sulfhydryl reductants (i.e., dithiothreitol), 

citrate, NaCl, and KCl; divalent metal cations (Mg2+ or Mn2+) were required for activity. A 

number of cellular metabolites were also tested for their affects on ACCase activity. The enzyme 

was inhibited by products of the ACCase reaction, including malonyl-CoA, ADP, and NaH2PO4, 

and also by palmitoyl-CoA, but it was not affected by various glycolytic or photosynthetic 

intermediates or by free CoA. Two herbicides that inhibit ACCases from monocot plants were 

also had little or no effect on C. cryptica ACCase. Thus, the ACCase from this diatom was 

found to be similar to higher plant ACCase enzymes in that it is composed of multiple, identical, 

multifunctional subunits. In addition, the Kms for the ACCase substrates (acetyl-CoA, MgATP, 

and bicarbonate) in C. cryptica were similar to those found in plant ACCase enzymes (Roessler 

1989; 1990). 
National Renewable Energy Laboratory 

104 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.B.4. Activity of various enzymes in Si-replete and Si-deficient C. cryptica. 

A. (Top) - Activities of several enzymes in Si-replete and Si-deficient C. cryptica cells. There is 

no significant difference in the activities of UDPglucose pyrophosphorylase, acetyl-CoA 

synthetase, or citrate synthase in the cells under the two conditions. However, there is a relative 

increase in acetyl-CoA carboxylase activity, and a decrease in chrysolaminarin synthase activity 

in Si-deficient cells. 

B (Bottom) - Graph showing the activity of ACCase in C. cryptica cells. Exponential-phase cells 

were transferred into Si-free media at 0 hr. At 6 hr, (arrow), the culture was split and 1.8 mM 

Na2SiO3 was added to one culture. (z) Si-deficient cells; () Si-replete cells. 

(Source: Roessler 1988a). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 105 

II.B.2.f. Cloning of the Acetyl-CoA Carboxylase Gene from C. cryptica 

Work on the key role of ACCase in lipid biosynthesis in other plant and animal systems, 

suggested that this enzyme might be a viable target for genetic manipulation in order to increase 

lipid production in microalgae. This notion was further supported by the work at SERI that 

showed changes in ACCase activity in Si-starved C. cryptica cells. 

The next step was to isolate the ACCase gene from a microalgal species. Although the ACCase 

gene had been isolated from yeast, rats, and the bacteria E. coli, the gene had not previously been 

isolated from any photosynthetic organism. In 1990 and 1991, Dr. Roessler took a sabbatical 

from SERI to work with Dr. John Ohlrogge at Michigan State University. Dr. Ohlrogge studies 

lipid biosynthetic pathways in higher plants. This work was partially funded by a Plant Biology 

Postdoctoral Fellowship to Paul Roessler from the National Science Foundation. The goal of this 

collaboration was to clone and characterize the ACCase gene from C. cryptica. To accomplish 

this task, the purified ACCase protein was first cleaved with cyanogen bromide (CNBr); the 

peptides generated were separated by SDS-PAGE, purified, and several of these peptides were 

analyzed to determine their amino acid sequence. (This work was done in collaboration with 

Calgene, a plant biotechnology company in Davis, California.) 

The amino acid sequences were used to design degenerate oligonucleotide primers that were used 

in a polymerase chain reaction (PCR) to amplify an ACCase gene fragment from C. cryptica’s 

total DNA. A 32P-labeled RNA transcript was produced from the ACCase DNA and used to 

screen a genomic library of C. cryptica DNA. A 14 kb cloned fragment that hybridized to the 

ACCase probe was cleaved into smaller fragments that were subcloned, sequenced, and analyzed 

for the presence of open reading frames (ORFs) and non coding intron sequences. This analysis 

showed that the ACCase gene from C. cryptica contains approximately 6.3 kbp of coding 

sequence, separated by a 447 bp intron close to the 5' end, and a 73 bp intron just upstream from 

the biotin binding site. The protein predicted by this nucleotide sequence would contain 2,089 

amino acids and have a molecular weight of 230 kDa. This is somewhat larger than the 

molecular weight of 185 kDa estimated by SDS-PAGE, discussed earlier. This discrepancy 

could be accounted for by inaccuracies inherent in using SDS-PAGE to estimate protein size, 

particularly for large proteins, and the probability of a signal sequence on the ACCase enzyme 

for targeting the protein to the chloroplast. Post-translational cleavage of the signal would result 

in a mature protein smaller than predicted from the primary DNA sequence. 

The deduced amino acid sequence of the ACCase from C. cryptica was compared with known 

sequences from yeast and rat (Figure II.B.5). The algal sequence showed approximately 50% 

identity with other sequences in the biotin carboxylase domain (at the amino terminus of the 

protein) and in the carboxyl transferase domain (at the carboxyl terminus of the sequence). 

However, the central portion of enzyme showed only about 30% identity with the yeast and rat 

enzymes, with most of the similarity in this region occurring in the biotin binding domain. This 

suggests that the central region of the protein probably functions primarily as a linker or spacer 

that moves the carboxylated biotin residue closer to the carboxyl transferase domain. The 

isolation of the ACCase gene from C. cryptica was an important step for the ASP; significantly 
National Renewable Energy Laboratory 

106 A Look Back at the Aquatic Species Program—Technical Review 

in that this was the first time a full-length sequence for an ACCase gene had been isolated from a 

photosynthetic organism. NREL was granted a patent on this gene in 1996, and there has been 

interest from at least one major plant biotechnology company in using this gene to manipulate 

oils and lipids in higher plants (Roessler and Ohlrogge 1993; Roessler et al. 1994). 

The availability of the purified ACCase protein and the cloned ACCase gene allowed NREL 

researchers to study the effects of Si deficiency on ACCase gene expression. Southern blots, in 

which a fragment of the cloned ACCase gene was used as a probe to analyze C. cryptica DNA, 

indicated that there is probably only a single copy of the ACCase gene in C. cryptica. ACCase 

gene fragments were used to monitor mRNA levels in Si-deficient cells using the ribonuclease 

protection assay (RPA). ACCase mRNA levels increased 2.5-fold between 2 and 6 hours after 

the beginning of Si-deprivation as compared to Si-replete cells, but then decreased to the control 

level after 23 hours. Thus, Si concentration appears to affect ACCase gene expression at the 

level of gene transcription, possibly as a result of increased promoter activity and/or by altering 

the rates of mRNA degradation. ACCase activity was also measured in cell lysates from Sistarved cultures; enzyme activity increased steadily over 23 hours to a final level 4.5-fold higher 

than that of Si-replete cultures. The increased level of ACCase activity was correlated with an 

increase in the amount of ACCase protein, as determined by Western blotting using anti-ACCase 

polyclonal antibodies. Although Si deficiency caused the levels of ACCase mRNA and protein 

to increase, the kinetics of the two processes were different. 

These results supported the hypothesis that diatoms could respond to Si deprivation by altering 

the activity of enzymes involved in lipid biosynthesis to partition more fixed carbon into storage 

lipids. If the activity of ACCase could be increased using mutation or genetic manipulation, it 

might be possible to produce a strain with constitutively high levels of TAG synthesis. This was 

a major premise of the genetic engineering experiments discussed in Section II.B.3. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 107 

Figure II.B.5. Schematic diagram indicating regions of similarity among the primary 

sequences of ACCase from C. cryptica, rat, and yeast. Regions (≥20 amino acids) that are 

present in all three sequences and that exhibit statistically significant homology (p<10-6) are 

indicated by thickened areas. 

(Source: Roessler and Ohlrogge 1993). 
National Renewable Energy Laboratory 

108 A Look Back at the Aquatic Species Program—Technical Review 

II.B.2.g. Biochemistry of Lipid Synthesis in Nannochloropsis 

From 1992 to 1995, Dr. Jane Schneider worked at NREL with Dr. Roessler on a project funded 

by the United States–Israel Binational Agricultural Research and Development Fund. The 

research was performed in collaboration with Dr. Assaf Sukenik and other scientists at the Israel 

Oceanographic and Limnological Institute in Haifa. The goal of the research was to understand 

the biochemistry of lipid synthesis in the eustigmatophyte Nannochloropsis sp., particularly with 

respect to fatty acid desaturation pathways. There has been a significant amount of research on 

lipid synthesis pathways in higher plants, and the pathways hve been assumed to be similar in 

lipogenic algae. However, unlike plants, nutrient deprivation produces major effects on the 

quantity and quality of lipids in algae; so there are likely to be significant differences in the 

biochemical pathways. In addition, like many algae, Nannochloropsis contains a high proportion 

of long fatty acids (i.e., C-20, C-22) with a high degree of unsaturation (20:5). These very long 

chain-polyunsaturated fatty acids (VLC-PUFAs) are important in aquaculture applications as 

they improve the nutritional quality of feed for fish and shellfish, and have nutritional and 

pharmaceutical applications for humans. Understanding the details of the biochemistry of lipid 

accumulation in microalgae could help researchers develop strategies for genetic manipulation of 

lipid synthesis pathways to affect not only the quantity but also the quality (chain length, degree 

of desaturation) of lipids produced for optimal biodiesel performance. 

In one set of experiments, pulse-chase radiolabeling was used to study de novo synthesis of lipids 

in Nannochloropsis. Exponentially growing cells under low light were fed 14C-bicarbonate or 

acetate for 1 hour. The cells were then washed and allowed to grow in unlabeled medium. At 

various time points, cells were removed and lipids extracted. The substrates resulted in a 

different distribution of labeled carbon in the lipids and fatty acids. The work demonstrated the 

probable existence of two pools of malonyl-CoA used as substrates for fatty acid synthesis, and 

resulted in a new model for the sites of desaturation of fatty acids and the identification of a new 

acyltransferase activity in this organism. In another set of experiments, Nannochloropsis cells 

were mutagenized using UV light and screened for unusual fatty acid profiles using gas 

chromatography. This work resulted in the isolation of a mutant deficient in 20:5 fatty acids, 

probably due to a mutation affecting a desaturase enzyme that utilizes 20:4 fatty acids as 

substrate. 

These experiments will not be described in detail here, primarily because the funding for this 

research did not come from DOE. In addition, it would require a lengthy discussion of the details 

of fatty acid synthesis and processing for the reader to understand the relevance of the findings. 

Readers interested in the details of this research are referred to the three publications that resulted 

from this research (Schneider and Roessler 1994; Schneider et al. 1995; Schneider and Roessler 

1995). 

II.B.2.h. Biochemistry and Molecular Biology of Chrysolaminarin Synthesis 

Another strategy that has been proposed to increase the proportion of lipid in algal cells is to 

limit the flow of newly assimilated carbon into other cellular pathways. Many diatoms, including 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 109 

C. cryptica, can produce a significant amount of a storage carbohydrate called chrysolaminarin, a 

β-(1→3)-linked glucan. Although some data were available on the chemistry of this compound, 

the biochemical pathways involved in the synthesis of chrysolaminarin were not known. The 

synthesis of most storage polysaccharides involves the condensation of nucleoside diphosphate 

sugars; for example, starch is formed in plants from ADPglucose, and UDPglucose is used to 

form sucrose in plants and glycogen in mammalian cells. These reactions are catalyzed by 

nucleoside diphosphate sugar pyrophosphorylases, such as UDPglucose pyrophosphorylase 

(UGPase), which catalyzes the following reaction: 

glucose-1-phosphate + UTP → UDPglucose + PPi

Roessler first looked for nucleoside diphosphate sugars pyrophosphorylases in cell-free extracts 

of C. cryptica, and identified significant amounts of UGPase activity. The enzyme activity was 

characterized to optimize in vitro assay conditions. The enzyme was activated in the presence of 

Mn2+ and Mg2+ but was not affected by 3-phosphoglycerate or inorganic phosphate; these 

chemicals are known to affect the activity of ADPglucose pyrophosphorylase in higher plants. 

Incubation of cell-free extracts with UDP[14C]glucose resulted in the incorporation of the labeled 

carbon into a β-(1→3)-glucan polymer, presumably chrysolaminarin, supporting the role of 

UGPase in chrysolaminarin synthesis in diatoms. Subsequent studies identified a second 

enzyme, UDPglucose:β-(1→3)-glucan-β-glucosyltransferase (also known as chrysolaminarin 

synthase), which catalyzes the synthesis of glucan using UDPglucose as substrate. The specific 

activity of both enzymes was examined in C. cryptica cells under Si-replete and Si-depleted 

conditions. The activity of UDPglucose pyrophosphorylase was similar under both conditions; 

however, the activity of chrysolaminarin synthase decreased by 31% in Si-deficient cells, 

suggesting that the partitioning of newly assimilated carbon into lipid may be partly due to 

decreased synthesis or inhibition of the chrysolaminarin synthase enzyme (Roessler 1987; 

1988a). 

Further research on UGPase in C. cryptica was put on hiatus for several years while the emphasis 

was on ACCase (discussed earlier) and on the development of genetic engineering protocols for 

microalgae (discussed in Section II.B.3.). However, the development of a successful genetic 

transformation system for C. cryptica, as well as advances in techniques that allow the downregulation of particular genes (i.e., antisense RNA, ribozymes) generated a renewed interest in 

UGPase. NREL researcher Eric Jarvis spent 6 months working at Ribozyme Pharmaceuticals, 

Inc., a biotechnology company in Boulder, Colorado, learning about these new methods. 

Antisense RNA is a method in which a cell is transformed with a synthetic gene that produces an 

RNA sequence complimentary to a specific messenger RNA (mRNA). Although the exact 

mechanism is not clear, the antisense RNA prevents translation from its complimentary mRNA, 

effectively lowering the level of that particular protein in the cell. Ribozymes are also RNA 

molecules produced by synthetic genes that can bind to, and cleave, very specific RNA 

sequences. Ribozymes can be designed to degrade specific mRNA molecules, effectively 

decreasing expression of a specific gene. 
National Renewable Energy Laboratory 

110 A Look Back at the Aquatic Species Program—Technical Review 

In C. cryptica, chrysolaminarin can make up 20%-30% of the cell dry weight, and thus 

chrysolaminarin synthesis pathways presumably compete for newly fixed carbon with the 

pathways for lipid biosynthesis. Dr. Jarvis and Dr. Roessler proposed that inhibiting 

chrysolaminarin production by inhibiting one or more genes in the carbohydrate synthesis 

pathway could result in the flow of more carbon into lipid production. Based on the earlier 

studies on chrysolaminarin synthesis, Dr. Jarvis initiated an effort to isolate the UGPase gene 

from C. cryptica DNA. A fragment of the C. cryptica UGPase gene was first produced by the 

PCR using degenerate oligonucleotide primers based on conserved sequences from known 

UGPase genes from potato, human, yeast, and Dictyostelium. This fragment was cloned and 

sequenced; the derived amino acid sequence showed 37% identity with the corresponding 

sequence from potato UGPase, confirming that a C. cryptica UGPase gene fragment had been 

cloned. The cloned PCR product was then used as a probe to isolate a genomic DNA clone 

containing the entire C. cryptica UGPase gene from a lambda library. One clone contained a 

DNA segment with a single long open reading frame, the 5' end of which showed homology to 

known UGPase genes. Surprisingly, the 3' end of this DNA showed homology to known genes 

coding for the enzyme phosphoglucomutase (PGMase). In chrysolaminarin synthesis, PGMase 

catalyzes the following reaction: 

glucose-6-phosphate → glucose-1-phosphate 

The glucose-1-P produced in this reaction is the substrate for UGPase in the production of 

UDPglucose, an immediate precursor of chrysolaminarin, as described earlier. Although 

PGMase and UGPase are thought to catalyze successive steps in the chrysolaminarin 

biosynthesis pathway, this was the first report of a naturally occurring fusion of these two genes 

in any organism. The C. cryptica UGPase/PGMase gene, designated upp1, contained 3,640 bps, 

including 3 introns, and coded for a protein composed of 1,056 amino acids, with a molecular 

weight of 114.4 kd. 

To confirm that the protein coded for by upp1 actually catalyzes both the UGPase and PGMase 

reactions, the protein was isolated from extracts of C. cryptica by sequential column 

chromatography (ion exchange, hydroxylapatite, and gel filtration). The two enzyme activities 

co-eluted throughout the purification procedure, and all fractions containing UGPase/PGMase 

activity contained a 114 kd protein as determined by SDS-PAGE. These results supported the 

presence of both enzyme activities in C. cryptica on a single multifunctional protein. A patent 

submitted by NREL on this unique gene was allowed in October 1996. The research at NREL 

involving attempts to manipulate upp1 gene expression to affect carbon partitioning in C. 

cryptica will be discussed in Section II.B.3. of this report. 

Publications: 

Bergeron, P.W., Corder, R.E., Hill, A.M., Lindsey, H.; Lowenstein, M.Z. (1983) SERI Biomass 

Program Annual Technical Report: 1982. Solar Energy Research Institute, Golden, Colorado, 

SERI/TR-231-1918, 83 pp. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 111 

Corder, R.E.; Hill, A.M.; Lindsey, H.; Lowenstein, M.Z.; McIntosh, R.P. (1984) SERI Biomass 

Program FY 1983 Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR231-2159, 107 pp. 

Hill, A.; Feinberg, A.; McIntosh, R.; Neenan, B.; Terry, K. (1984) “Fuels from microalgae: 

Technical status, potential, and research issues.” Solar Energy Research Institute Report,

SERI/SP-231-2550. 

Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. (1992) “DNA nucleoside composition and methylation 

in several species of microalgae.” J. Phycol. 28:356-362. 

Lien, S. (1981a) “Photobiological production of fuels by microalgae.” In Energy from Biomass: 

Proceedings of the First E.C. Conference (Palz, W.; Chartier, P.; Hall, D.O., eds.), Applied 

Science Publishers, London, pp. 697-702. 

Lien, S. (1981b) “Algal research at SERI: A basic research on photobiological production of 

fuels and chemicals in microalgae.” Proceedings of the July 1981 Subcontractors' Review 

Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-624-1228, pp. 59-65. 

Lien, S. (1982) “Studies on the production and accumulation of oil and lipids by microalgae.” In 

Proceedings of the SERI Biomass Program Principal Investigators' Review Meeting, Aquatic 

Species Program Reports, June 23-25, 1982, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-1808, pp. 45-54. 

Lien, S.; Roessler, P. (1986) “The energetics of biomass and lipid production by lipogenic 

microalgae under nitrogen deprivation.” Aquatic Species Program Review: Proceedings of the 

March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2700, pp. 100-117. 

Lien, S.; Spencer, K.G. (1983) “Algal oil production and lipid metabolism research.” Aquatic 

Species Program Review: Proceedings of the March 1983 Principal Investigators' Meeting, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 3-18. 

Ohlrogge, J.; Jaworski, J.; Post-Beittenmiller, D.; Roughan, G.; Roessler, P.; Nakahira, K. (1993) 

“Regulation of flux through the fatty acid biosynthesis pathway.” In Biochemistry and 

Molecular Biology of Membrane and Storage Lipids of Plants (Murata, N.; Somerville, C.R., 

eds.), American Society of Plant Physiologists, Rockville, Maryland, pp. 102-112. 

Raymond, L.P. (1983a) “Aquatic biomass as a source of fuels and chemicals.” Conference 

report: First U.S.-China Conference on Energy, Resources, and Environment. Beijing, People’s 

Republic of China, Nov. 7-12, 1982. Solar Energy Research Institute, Golden, Colorado, 

SERI/TP-231-1699, 8 pp. 
National Renewable Energy Laboratory 

112 A Look Back at the Aquatic Species Program—Technical Review 

Roessler, P.G. (1987a) “Biochemical aspects of lipid accumulation in silicon-deficient diatoms.” 

FY 1986 Aquatic Species program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071. 

Roessler, P.G. (1987c) “Lipid accumulation in silicon-deficient diatoms.” In The Metabolism, 

Structure, and Function of Plant Lipids, (Stumpf, P.K.; Mudd, M.B.; Nes, W.D., eds.), Plenum, 

Publishing Corp., pp. 649-651. 

Roessler, P.G. (1987d) “UDPglucose pyrophosphorylase activity in the diatom Cyclotella 

cryptica. Pathway of crysolaminarin biosynthesis.” J. Phycol. 23:494-498. 

Roessler, P.G. (1988a) “Changes in the activities of various lipid and carbohydrate biosynthetic 

enzymes in the diatom Cyclotella cryptica in response to silicon deficiency.” Arch. Biochem. 

Biophys. 267:521-528. 

Roessler, P.G. (1988b) “Characteristics of abrupt size reduction in Synedra ulna

(Bacillariophyceae).” Phycologia 27:294-297. 

Roessler, P.G. (1988c) “Effects of silicon deficiency on lipid composition and metabolism in the 

diatom Cyclotella cryptica.” J. Phycol. 24:394-400. 

Roessler, P.G. (1989) “Purification and characterization of acetyl-CoA carboxylase from the 

diatom Cyclotella cryptica.” Aquatic Species Program Annual Report, Solar Energy Research 

Institute, (Bollmeier, W.S.; Sprague, S., eds.), SERI/SP-231-3579, pp. 121-129. 

Roessler, P.G. (1990) “Purification and characterization of acetyl-CoA carboxylase from the 

diatom Cyclotella cryptica.” Plant. Physiol. 92:73-78. 

Roessler, P.G.; Bleibaum, J.L.; Thompson, G.A.; Ohlrogge, J.B. (1994) “Characteristics of the 

gene that encodes acetyl-CoA carboxylase in the diatom Cyclotella cryptica.” In Recombinant 

DNA Technology II (Bajpai, K.; Prokop, A., eds.), Ann. N.Y. Acad. Sci. 721:250-256. 

Roessler, P.G.; Ohlrogge, J.B. (1993) “Cloning and characterization of the gene that encodes 

acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica.” J. Biol. Chem. 268:19254-

19259. 

Schneider, J.C.; Roessler, P. (1994) “Radiolabeling studies of lipids and fatty acids in 

Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga.” J. Phycol. 30:594-598. 

Schneider, J.C.; Livine, A.; Sukenik, A.; Roessler P.G. (1995) “A mutant of Nannochloropsis

deficient in eicosapentaenoic acid production.” Phytochem. 40:807-814. 

Schneider, J.C.; Roessler, P.G. (1995) “A novel acyltransferase activity in an oleaginous alga.” In 

Plant Lipid Metabolism (Kader, L.C.; Mazliak, P., eds.), Kluwer Academic Publishers, the 

Netherlands, pp. 105-107. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 113 

Terry, K.L. (1984a) “Microalgal technology research at SERI: Modulated light photobiology.” 

Aquatic Species Program Review: Proceedings of the April 1984 Principal Investigators’ 

Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 160-169. 

Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogen 

starved green alga Neochloris oleoabundans.” Enzyme Microb. Technol. 5:435-440. 

Walsh, R.G. (1984) “Microalgal technology research at SERI: The biochemistry of lipid trigger 

mechanisms in microalgae—alteration of lipid metabolism during transition from rapid growth to 

rapid lipid accumulation.” Aquatic Species Program Review: Proceedings of the April 1984 

Principal Investigators Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP231-2341, pp. 170-183. 

Patents: 

Roessler, P.G; Ohlrogge, J.B., “Gene Encoding Acetyl-Coenzyme A Carboxylase”, U.S. Patent 

5,559,220, issued 9/24/96. 

Jarvis, E.E.; Roessler, P.G. “Isolated-Gene Encoding an Enzyme with UDP-Glucose 

Pyrophosphorylase Activities from Cyclotella cryptica.” Allowed 10/24/96.

II.B.3 Manipulation of Lipid Production in Microalgae via Genetic 

Engineering 

II.B.3.a. Introduction 

The overall goal of the ASP was to cost-effectively produce biodiesel fuel from microalgal lipids. 

The early laboratory efforts focused on the characterization of microalgae with regard to traits 

deemed desirable for mass culture and fuel production, i.e., rapid growth, tolerance to 

environmental fluxes, and high production of TAG storage lipids. Although numerous promising 

organisms were identified, no individual strain demonstrated rapid growth with constitutively 

high lipid production. Although high lipid levels could be induced in many strains by starving 

the cells for an essential nutrient such as N or Si, the increase in lipid was accompanied by a 

decrease in cell division and total productivity. 

During the late 1980s and 1990s, the direction of the laboratory research efforts at NREL shifted 

to the study of the biochemical pathways involved in lipid synthesis, with the goal of identifying 

targets for genetic manipulation. As discussed earlier, the desirable traits for biodiesel 

production (high productivity and high lipid content) were found to be mutually exclusive 

conditions in the organisms studied. Therefore, it was decided to use mutagenesis or genetic 

engineering to manipulate the algal biosynthetic pathways to produce algal strains with 

constitutively high lipid levels. Another possibility would be to engineer an organism in which 

lipid synthesis could be regulated by inducing or repressing key genes. Very little was known 

about the molecular biology of oleaginous microalgae and the genetic regulation of lipid 

biosynthesis pathways, so a concentrated research effort in this direction was deemed critical to 
National Renewable Energy Laboratory 

114 A Look Back at the Aquatic Species Program—Technical Review 

the success of the biodiesel project. Another reason for the shift to research on genetic 

manipulation of algae was more practical. Funding levels for ASP decreased during this period 

from the high funding levels in the mid-1980s that had allowed large numbers of subcontractors 

and the development of the Outdoor Test Facility in Roswell, New Mexico (Section II.B.5.). 

Laboratory experiments emphasizing biochemistry, molecular biology, and genetic engineering 

could be performed with a limited budget and few personnel. 

This section of the report will describe the in-house research efforts at NREL to develop high 

lipid algae by genetically manipulating selected oleaginous strains. Microalgae generally 

reproduce asexually by simple fission. Many strains can also produce sexually, but the 

conditions required to induce algal sexual reproduction in the laboratory are not known for most 

species. Thus, genetic manipulation by classical “breeding” was not an option for the algae. The 

approaches explored at NREL were (1) mutagenesis and selection; and (2) genetic engineering. 

The information summarized in this report was taken from annual reports, scientific publications 

and meeting reports. No annual reports were generated by the ASP after 1993, and no quarterly 

reports after September 1995, so some of the most recent information presented was derived 

from the personal experience of Terri Dunahay and discussions with former coworkers (Paul 

Roessler and Eric Jarvis). 

II.B.3.b. Mutagenesis and Selection 

Work by SERI/NREL subcontractors in the early 1980s supported the idea that there is 

significant genetic variation within algal populations (i.e., Gallagher, Section II.B.1.c.). 

Therefore, one possible method for producing high lipid algal strains would be selection of 

natural genetic variants with desired traits, such as high lipid levels or increased tolerance to high 

salinity or temperature. The limiting factor to this approach has always been the difficulty of 

selecting for individuals exhibiting a desired trait among a large population of cells. The use of 

lipophilic dyes such as Nile Blue or Nile Red, coupled with flow cytometry, showed some 

potential for isolation of high lipid strains of microalgae (see work by Solomon and Cooksey, 

Sections II.B.1.e and f). It was not clear, however, that the variations detected in subpopulations 

of cells were the result of genetic variations that would be passed on to progeny. 

An alternative approach is to induce genetic variation in a population of cells using mutagenesis. 

Again, the ability to select for the desired trait is a limiting factor, but the production of large 

numbers of mutants by artificial means is a proven method for generating organisms with 

heritable traits, often the result of a mutation within a single gene. As a prelude to the initiation 

of mutagenesis and selection experiments with oleaginous microalgae, NREL researcher Ruth 

Galloway performed a series of experiments designed to understand the factors required to 

produce mutants in microalgae. These included media requirements for growth, the ability to 

form colonies on agar plates, sensitivity to herbicides and other growth inhibitors, and the 

sensitivity of algal strains to mutagens such as UV light or fluorodeoxyuridine (Galloway 1990). 

 Nine algal strains from the SERI Culture Collection were tested, including organisms from three 

classes: the chlorophyceae (M. minutum MONOR1 and MONOR2), the eustigmatophyceae 

(Nannochloropsis (NANNP1 and NANNP2), and the bacilliarophyceae (C. cryptica T13L, C. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 115 

mulleri CHAET9, Amphora AMPHO17, Nitzschia pusilla NITSC12, and N. saprophila

NAVIC1). 

Growth of each strain was evaluated qualitatively after spotting the cultures onto media 

containing nitrogen or carbon sources, or after growing the cells under phototrophic, 

mixotrophic, or heterotrophic conditions. Cell growth was also evaluated in the presence of a 

large number of growth inhibitors including various antibiotics and herbicides. Although there 

was some variability between the algal strains, several generalizations could be made. Most 

strains could use either NO3

-

 or NH4

+

 as a nitrogen source. Mixotrophic growth on various 

carbon sources was more variable, and only AMPHO17, MONOR2, and CYCLOT13L were able 

to grow heterotrophically, using glucose as a carbon source. The ability to grow 

heterotrophically would be important for the isolation of photosynthetic mutants. 

Predictably, antibiotics that inhibit bacterial cell wall synthesis such as ampicillin and 

carbenicillin did not inhibit the growth of the algal strains. Antibiotics that inhibit bacterial 

protein synthesis by binding to the 30S ribosome showed variation in their effects on algal 

growth. For example, all strains tested grew well on kanamycin and neomycin and showed no 

growth on erythromycin; while the growth response differed for the strains on spectinomycin and 

streptomycin. Whether this result was due to differences in 30S (organellar) ribosomal structure 

between the algal strains, to differences in uptake of the antibiotics by the individual strains, or to 

other factors that affect sensitivity, is unclear. All strains showed sensitivity to photosynthesis 

inhibitors diuron, metronidazol, and atrazine, and to the herbicide glyphosate (“RoundUp”), 

which affects the shikimic acid pathway. However, sensitivity varied between the strains to 

compounds that affect the enzyme acetolactate synthase and to chemicals that inhibit microtubule 

synthesis. (The details of these growth experiments can be found in Tables 2, 3, 4, 5, and 7 of 

Galloway 1990). Many of the growth inhibitors used in this study affect specific proteins in the 

target organism, and many of these proteins have been well characterized in a number of 

systems. Isolating the corresponding gene from an algal mutant using heterologous gene probes 

to characterize the mutation and/or to use the mutant gene as a selectable marker for 

transformation studies should be relatively easy. 

Attempts were also made to generate mutants in the algal strains by exposing the cells to UV 

light or to fluorodeoxyuridine, followed by plating the cultures on toxic levels of various growth 

inhibitors. Using UV mutagenesis, streptomycin-resistant mutants were obtained in MONOR2, 

as well as glyphosate-resistant mutants in both strains of Nannochloropsis, sulfometuron methylresistant mutants in NANNP1, and atrazine-diuron-resistant mutants in NAVIC1. In addition, 

tunicamycin-resistant mutants of NAVIC1 were produced following treatment with 

fluorodeoxyuridine. Mutants were not obtained for the other diatoms, CYCLOT13L, CHAET9, 

or NITZS12, whether this was due to poor colony formation by these strains, inefficacy of the 

mutagen, or inappropriately high levels of the selective agent is not known. One interesting point 

was that the green algal strains, Monoraphidium and Nannochloropsis, produced mutants with 

traits thought to be due to recessive nuclear gene mutations (i.e., glyphosate resistance or 

photosynthesis mutants). On the other hand, in Navicula, the only diatom in which mutants were 

generated, the types of mutations produced were indicative of dominant mutations, i.e., 
National Renewable Energy Laboratory 

116 A Look Back at the Aquatic Species Program—Technical Review 

atrazine/diuron resistance (resulting from a chloroplast gene mutation) or resistance to 

tunicamycin, an inhibitor of n-glycosylation. These results indicate that Monoraphidium and 

Nannochloropsis are probably haploid; the diatoms are diploid. Design of strategies for 

generation of algal mutants will have to consider the ploidy of the target organism. For example, 

generating nitrate reductase-deficient mutants for use in a genetic transformation system using 

homologous selectable markers (described in detail later) should be relatively simple in haploid 

strains, but would be much more difficult in diploids. For the diatoms, a better approach would 

be to utilize a dominant gene as a selectable marker, such as a mutant form of the enzyme 

acetolactate synthase (discussed later), or a heterologous gene such as the neomycin 

phosphotransferase II (NPTII). The latter gene confers drug resistance by inactivating antibiotics 

such as kanamycin or geneticin (G418). 

In summary, the research performed by Dr. Galloway demonstrated the potential to produce algal 

mutants with a wide variety of phenotypes, particularly in the green algae, using simple 

mutagenesis and selection techniques. It would be important to first optimize and understand the 

growth conditions for the target strains. The conditions to be used for selection (inhibitor 

specificities and concentrations) should be determined for each strain. However, the generation 

of mutants will probably be more useful as a tool in developing selectable marker systems, rather 

than as a method to directly produce high lipid algal strains, primarily because there is no simple 

way to screen for high-lipid phenotypes. The use of mutagenesis to develop of homologous 

selectable marker systems for algal transformation will be discussed in detail later. 

Mutagenesis and selection was used successfully in another study at NREL to generate mutants 

in one aspect of lipid synthesis, fatty acid desaturation (Schneider et al. 1995, described in 

Section II.B.2.g.). In this experiment, UV mutagenized cells of Nannochloropsis were allowed 

to form colonies, then grown in small-scale liquid cultures. Lipids were extracted from each 

sample and analyzed by gas chromatography for any significant alteration in the proportion of 

fatty acids. This project resulted in the identification of a mutant lacking in 20:5 fatty acids, 

apparently due to a mutation in a 20:4 desaturase. In this case, a simple screen was used to look 

for changes in a quantitative trait. This result suggests that, with the right method to screen for 

mutants with the desired properties, mutagenesis could result in microalgae with altered lipid 

compositions. However, this project was very labor intensive, with hundreds of colonies 

screened to identify a single mutant. 

II.B.3.c. Development of a Genetic Transformation System for Microalgae 

Introduction: 

During the past 2 decades, manipulation of organisms via genetic engineering has become 

routine in a number of animal, bacterial, fungal, and plant systems. However, before the research 

was done at NREL, very little work in this area had been done with microalgae. In fact, the only 

species for which there was a reproducible transformation system was the single-celled, 

flagellated green alga C. reinhardtii, which is studied extensively in laboratories as a model 

photosynthetic cell. The focus of the research in the ASP during the early 1990s was to develop 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 117 

genetic transformation methods for microalgae with potential for biodiesel production. Based on 

the collection and screening efforts of the 1980s, this approach was considered to have the 

highest potential to produce organisms with high constitutive lipid levels, and to use genetic 

manipulation to understand the molecular regulation of lipid synthesis in the oleaginous algae. 

Studies on the biochemistry and molecular biology of lipid production in C. cryptica had 

identified acetyl-CoA carboxylase as a key regulatory enzyme in lipid synthesis (Section 

II.B.2.e.). One initial goal was to introduce additional copies of this gene into C. cryptica with 

the hope of increasing the activity of the enzyme and the flux of fixed carbon into lipid. 

Several projects will be discussed in the following section of this report that were directed 

towards the development and use of genetic transformation systems in oleaginous microalgae. 

The initial approach was to use use available promoters and marker genes that were reported to 

function in other eukaryotic systems. Various methods were also tried to get DNA into the cell, 

initially focusing on enzymatically removing the cell wall or perturbating the cell membrane 

using electroporation. Unsuccessful experiments represented a “Catch 22” scenario, as negative 

results could mean either the DNA was not getting into the cells, or the DNA entered but could 

not be expressed at detectable levels. Subsequent experiments were designed to increase the 

understanding of the processes involved in DNA uptake and expression and to increase the 

probability of obtaining transformants by developing methods for detecting rare transformation 

events within a population of cells. 

The projects that will be discussed here include a basic study on the DNA composition of 

microalgal strains, with implications for the choice of reporter or marker genes used to monitor 

gene expression in transgenic algae. Other aspects of the research that will be discussed include: 

• the use of the luciferase gene to monitor DNA uptake and expression in 

Chlorella protoplasts, 

• attempts to develop heterologous and homologous genetic markers for algal 

transformation, 

• the development of methods to introduce DNA into algal cells through the cell 

wall, and 

• the successful development of a stable genetic transformation system for 

diatoms. 

Once the methods were available to obtain genetic transformants, efforts were made to use the 

transformation system to manipulate lipid content in the algae by overexpressing or 

downregulating key genes. In addition, the transformation system was used to introduce a 

reporter gene under the control of various regulatory sequences, to better understand the 

regulation of gene expression in microalgae. 
National Renewable Energy Laboratory 

118 A Look Back at the Aquatic Species Program—Technical Review 

Analysis of Microalgal DNA Composition: 

Several oleaginous microalgal strains had been identified as potential candidates for biodiesel 

fuel production. These organisms became the target of genetic engineering efforts to manipulate 

the lipid biosynthetic pathways. Before the work on genetic transformation of algae at NREL, 

very little information was available on the molecular biology of these organisms. One of the 

first steps was to develop techniques to isolate and purify DNA from these organisms. A 

desirable protocol would disrupt the cell wall using methods gentle enough to prevent shearing of 

the genomic DNA. This was not trivial for some species, such as Monoraphidium, which has a 

very resistant wall that contains sporopollenin. A method that worked for most species tested 

(described in Jarvis et al. 1992) was developed based on a protocol used to isolate yeast DNA 

(Hoffman and Winston 1987). The cells were suspended in buffer that contained 2% Triton X100 and 1% SDS, then added to a tube that contained glass beads and an equal volume of 

phenol:cholorform:isoamyl alcohol (PCI). The cells were agitated for 1 minute using a vortex 

mixer. The DNA in the aqueous phase was purified by re-extraction with PCI, ethanol 

precipitation, and treated with RNase A. For some species, the DNA had to be purified further 

by using precipitation with hexadecyltrimethylammonium bromide (CTAB; Murray and 

Thompson 1980) to remove contaminating carbohydrates or by purifying the DNA on CsCl 

gradients. This procedure produced DNA that digested well with many common restriction 

endonucleases, but even highly purified DNA would not digest well with all restriction enzymes. 

NREL researcher Eric Jarvis theorized that poor digestion of the DNA by some enzymes could 

be attributable to characteristics of the DNA. All DNA is composed of four nucleosides; 

deoxycytidine, deoxyguanosine, deoxythymidine, and deoxyadenosine, (abbreviated dC, dG, dT, 

dA); in double stranded DNA, dC is always paired with dG, and dT with dA. The percentage of 

each nucleoside (often represented as %GC) is variable between species. Restriction enzymes 

cut DNA at specific nucleotide sequences, generally recognizing 4-6 bp motifs. Therefore, the 

frequency of cutting by a particular enzyme will be affected by the total nucleotide composition 

of the DNA (i.e., an enzyme that recognizes CCGG would cut infrequently in an organism with a 

low %GC). The GC content is also reflected in the codon usage by each organism, as DNA with 

a high GC content would show a bias toward codons ending with G or C in the variable third 

position. DNA can also contain unusual modified nucleosides, including 5-

hydroxymethyldeoxycytidine (hm5

dC) and 5-hydroxymethyldeoxyuridine (hm5dU), although the 

biological significance is unclear. Another common modification is the presence of methylated 

nucleosides, in particular 5-methyldeoxycytidine (m5

dC) and 6-methyldeoxyadenosine (m6dA). 

The degree of methylation has been associated with levels of gene expression. In addition, some 

microorganisms use DNA methylation as a defense mechanism, in that methylated DNA 

sequences are often not recognized by endonucleases from invading pathogens. Although the 

presence of methylated nucleosides is characteristic for some species, the degree of methylation 

can vary on a short time scale with changing environmental conditions. In contrast, the %GC 

and presence of modified nucleosides are characteristic for a particular organism. These 

characteristics only on an evolutionary time scale. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 119 

DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleoside 

composition was analyzed by reverse-phase HPLC and by digestion with restriction 

endonucleases. The results of the HPLC analysis are summarized in Table I.B.4-1. Although the 

diatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of the 

green algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium

DNA contains 71% GC. The table also shows the presence of m5

dC in the algal DNA. All 

species tested contained some level of this modified base, although once again Monoraphidium

stands out with approximately 11% m5

dC. The only other unusual feature was the presence of 

12% hm5

dU in the dinoflagellate C. cohnii (data not shown); dinoflagellates were not considered 

to be good candidates for biodiesel fuel production, so this observation was not explored further. 

These data provided a good background for developing genetic transformation systems for these 

organisms. As mentioned above, the GC content of an organism can be reflected in the codon 

usage, suggesting that an organism with a high GC content such as Monoraphidium may not 

successfully express heterologous marker genes. This was found to be true for the green alga 

Chlamydomonas; successful transformation of this organism was achieved only by the use of 

homologous selectable markers (discussed in more detail later). Also, GC content should be 

considered when designing synthetic DNA probes based on protein sequences, i.e., for isolation 

of algal genes by PCR. In addition, DNA methylation can affect the ability to construct DNA 

libraries and to clone algal DNA, and may require the use of bacterial host strains that are 

insensitive to DNA methylation. 

Table II.B.1. DNA Nucleoside Composition of Several Microalgal Strains (Modified 

from Dunahay, et al, 1992, p .333 and Jarvis et al, 1992) 

Algal species M5

dC 5GC 

Chlorophyceae 

Chlamydomonas reinhardtii 0.16 61.6 

Chlorella ellipsoidea 1.48 51.6 

Monoraphidium minutum 11.2 70.9 

Bacillariophyceae 

Cyclotella cryptica 1.95 43.2 

Navicula saprophila 0.20 46.2 

Nitzschia pusilla 0.78 45.4 
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120 A Look Back at the Aquatic Species Program—Technical Review 

Phaeodactylum tricornutum 0.14 48.0 

Charophyceae 

Stichococcus sp. 0.30 44.8 

Prasinophyceae 

Tetraselmis suecica 3.32 57.5 

Dinophyceae 

Crypthecodinium cohnii 1.54 43.7 

Transient Expression of Luciferase in Chlorella ellipsoidea: 

The first step in transformating any organism is getting the foreign DNA inside the cell. For 

organisms with a cell wall, methods must be devised to either remove or permeabilize the wall, 

or to get DNA into the cell through the intact wall. Bacterial cell walls do not seem to represent 

a significant barrier to DNA uptake, and can be induced to take up foreign DNA simply by being 

washed in low osmotic medium and glycerol, followed by a brief heat shock. Cell walls can be 

removed enzymatically from yeast cells to form spheroplasts, or from plant cells to form 

protoplasts. These wall-less cells can be induced to take up DNA by chemically permeabilizing 

the cell membrane with polyethylene glycol and/or calcium. Alternatively, DNA can enter yeast 

spheroplasts or plant protoplasts via electroporation, a method in which a rapid, high voltage 

electric pulse is used to produce transient pores in a cell membrane. 

Based on their research backgrounds, NREL researchers tended to view microalgae as either 

single cell plants, or pigmented yeasts. In either case, the initial tendency was to try to produce 

wall-less algal cells as targets for transformation. There had previously been some reports of 

protoplast production in green microalgae of the genus Chlorella (Braun and Aach 1975; 

Berliner 1977). NREL researcher Eric Jarvis decided to attempt to introduce foreign DNA into 

Chlorella protoplasts, with the eventual goal of adapting these protocols for other algal strains 

with biodiesel production potential. 

The production of a stably transformed line of cells involves several steps, including introducing 

the foreign DNA into the target cell, expressing the foreign gene, stabilizating (replicating) the 

new DNA by the host cell, and survival and proliferation of the genetically altered cells. 

Transient expression assays can be used to monitor and optimize just the first two of these 

processes, i.e., DNA entry and expressing a foreign gene in a population of cells, and thus can be 

useful intermediate steps in developing genetic transformation systems. Transient assays usually 

involve the introduction of a gene that codes for an enzyme detectable by a simple biochemical 

assay (often referred to as a reporter gene). Dr. Jarvis decided to use one such gene, the firefly 

luciferase gene, to monitor the entry and expression of foreign DNA into Chlorella protoplasts. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 121 

The alga used for these studies was C. ellipsoidea (strain CCAP 211/1a, obtained from the 

Culture Collection of Algae and Protozoa, Freshwater Biological Association, United Kingdom). 

Protoplasts were produced using a protocol adapted from Global and Aach (1985). The cells 

were grown to early stationary phase, then incubated overnight in 10 mg/mL Cellulysin, a crude 

commercial preparation of the cellulose-degrading enzyme cellulase. Protoplast production was 

monitored by sonication of the treated cells in water; generally about 80% of the cells were 

disrupted by this treatment and were considered to be protoplasts. A plasmid containing the 

luciferase gene driven by plant regulatory sequences was introduced in the protoplasts by mixing 

the cells with the plasmid DNA for 30 minutes in the presence of 50 mM CaCl2 and 13% 

polyethylene glycol (mw 4000). The cells were washed and incubated in a regeneration medium 

overnight. The cells were then harvested and luciferase activity was monitored in crude protein 

extracts. Luciferase catalyzes the oxidation of luciferin with the production of a photon of light 

via the following reaction: 

luciferase, Mg2+, O2

LUCIFERIN + ATP -----------------------> OXYLUCIFERIN + AMP + CO2 + hv 

The light produced can be monitored using a scintillation counter or a luminometer. 

The results of these experiments are shown in Figure II.B.6. Luciferase activity was detectable in 

protoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed to 

plasmid or to polyethylene glycol. Intact cells did not take up the DNA. There was a significant 

decrease in luciferase expression when carrier DNA was left out of the transformation reaction 

(“carrier DNA” is usually sheared genomic DNA from calf thymus or salmon sperm that is added 

to reduce the effects of cellular nucleases on the added plasmid DNA). Monitoring of the 

luciferase activity over time showed that the activity was maximal at about 24 hours after 

exposing the protoplasts to the plasmid; expression decreased over time and was virtually 

undetectable after 80-100 hours. Unfortunately, attempts to regenerate the protoplasts into viable 

walled cells were unsuccessful. 

These results were important as they demonstrated the first successful steps in developing a 

genetic transformation system for microalga, including the production of viable protoplasts, the 

introduction of DNA into the protoplasts, and the expression of a foreign gene by the algal cells. 

This last point was very significant, as homologous genes were required to achieve 

transformation in another green alga (Chlamydomonas). The dogma in the field was that 

heterologous gene expression in green algae would likely be unsuccessful due to codon biases 

resulting from high GC contents. The work resulted in a publication (Jarvis and Brown 1991), 

and was the basis for later studies in which the luciferase gene was used to monitor promoter 

activities in Cyclotella (discussed later). However, attempts to adapt this procedure to algal 

strains with significance to the biodiesel project were unsuccessful. The composition of 

microalgal cell walls is highly variable between species and even between isolates of the same 

species. Some unsuccessful efforts were made to determine the enzymatic conditions for wall 

degradation for several oleaginous algal strains. However, the conclusion, in the words of the 
National Renewable Energy Laboratory 

122 A Look Back at the Aquatic Species Program—Technical Review 

project manager at the time, was that this was “an endless pit of fruitless endeavor”, and the 

decision was made to explore other methods of introducing DNA into microalgal cells. In 

addition, although low levels of luciferase expression were acheived in Chlorella, the decision 

was made to pursue the development of selectable marker systems that would allow the isolation 

of very rare individual transformants within a population of microalgal cells. This will be 

discussed in the following section. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 123 

Figure II.B.6. Transient expression of firefly luciferase in Chlorella ellipsoidea.

A. (Top) - Histogram showing luciferase expression in protoplasts of C. ellipsoidea. 

Expression of the luciferase gene is expressed in relative light units (RLU), which are the net 

photons counted during a 5-min period. See text for explanation. 

B. (Bottom) - Kinetics of luciferase expression in C. ellipsoidea protoplasts. Each symbol 

represents the result of a single assay. Control cultures were grown in the dark (▲) or light 

(∆). Duplicate cultures of plasmid-treated protoplasts were also grown in either the dark (■) 

or light (□). 

(Source: Jarvis and Brown 1991). 
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124 A Look Back at the Aquatic Species Program—Technical Review 

Development of Homologous Selectable Markers for Monoraphidium and Cyclotella: 

Transient expression assays can be useful for the rapid assessment of DNA uptake and 

expression by cells as demonstrated by the expression of luciferase in Chlorella protoplasts, 

described earlier. However, attempts to produce similar results in other algal strains were 

unsuccessful. The problem with an experiment that produces no signal is that it is impossible to 

know if this is because the DNA did not get into the cell, or if the DNA entered the cell but was 

not expressed at detectable levels. In the latter case, poor expression could result from 

degradation of the foreign DNA, inappropriate regulatory signals, or differences in the codon 

usage. 

One of the most promising organisms with regard to high lipid production and tolerance to 

environmental fluxes was the green alga M. minutum (strain MONOR2). However, MONOR2 

DNA was shown to be highly unusual in GC content and degree of methylation. As mentioned 

elsewhere in this report, successful transformation of the green alga C. reinhardtii, which also 

has an elevated GC content, required the use of homologous selectable markers. The literature 

suggested that this unusual GC content would inhibit the expression of foreign genes, such as 

bacterial antibiotic resistance genes that had been used successfully as transformation markers in 

plant and mammalian systems. Based on this information, it was decided to attempt to develop 

homologous selectable markers for transforming MONOR2 and other strains with programmatic 

importance. Use of a selectable marker, in contrast to a transient expression assay, would allow 

the identification of very rare transformation events. Under the appropriate selection conditions, 

one transformed cell can be detected in a very large population of nontransformed cells, whereas 

in transient assays, a significant number of cells in a population must be expressing the foreign 

gene in order to detect the new enzymatic activity. The use of a homologous gene as a marker 

would greatly increase the chance for successful expression of the introduced gene, as there 

would be no problems associated with codon bias or foreign regulatory sequences. Although 

some success was achieved toward the development of a homologous selectable marker system, 

the emphasis of the research at NREL was shifted after the successful development of a 

transformation system for diatoms that used a chimeric selectable marker. A significant effort 

was put into the development of homologous markers, particularly for non-diatom species, from 

1989 to 1994, so it is relevant here to summarize the progress made in this area. 

The general protocol for developing a homologous selectable transformation system involves 

several steps. First, a mutation is created or identified in a specific gene. The gene should be 

essential for growth under “normal” conditions; however, the mutated strains will grow under 

modified growth conditions. This will allow for positive selection of transformed cells. Then the 

corresponding wild-type gene is isolated and inserted into a plasmid vector. The wild-type gene 

is introduced into the mutant cells, and transformants are detected by the ability to grow under 

the normal, defined growth conditions. In contrast to the transient assay described earlier, use of 

a selectable marker involves not only DNA entry and expression, but also stabilization of the new 

DNA in the cell and viability and growth of the newly transformed cells. Genes with good 

potential for use as selectable markers should not only code for a protein essential for growth 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 125 

under defined conditions, but should also produce a protein that can be detected by a simple 

enzymatic assay. In addition, the use of a gene that has been well characterized in other systems 

will help isolate the gene from the species of interest and simplify the development of enzyme 

assays and growth conditions for isolating mutants and transformed cells. 

Two genes that meet these criteria were targeted for the development of homologous selectable 

markers for MONOR2 and for C. cryptica T13L. One codes for the enzyme nitrate reductase 

(NR). NR had been used successfully to transform Chlamydomonas (Kindle et al. 1989) and 

several species of fungi (Daboussi et al. 1989) and methods were available to isolate NR mutants 

and selection of transformed strains. In addition, there was some interest at NREL in the role of 

nitrogen uptake and utilization in lipid accumulation, and isolating the wild-type NR gene would 

permit further investigation of these questions. 

NR mutants can be isolated based on their resistance to chlorate. Cells with functional NR will 

take up chlorate along with nitrate and reduce the chlorate to the toxic compound chlorite. 

Therefore, cells with a mutation in the NR gene will be unable to grow using nitrate as the sole N 

source, but will be able to grow in the presence of chlorate, as long as urea or ammonium is 

added as an alternative N source. Using this scheme, several putative NR mutants grew from 

non-mutagenized cells of MONOR2 and C. cryptica T13L. Biochemical assays suggested that at 

least two of the MONOR2 mutants contained defects within the NR structural gene. 

The next step was to isolate the wild-type gene from MONOR2 for complementation of the NRminus mutants. A partial cDNA clone of NR from Chlorella vulgaris was obtained from Dr. 

Andrew Cannons (University of Southern Florida). Southern blot analysis indicated that the 

Chlorella DNA sequence showed significant homology to a sequence in MONOR2 genomic 

DNA. Degenerate primers for use in the PCR were designed based on conserved regions in the 

NR genes from three green algal species and several higher plants. A 700-bp PCR product was 

generated using MONOR2 genomic DNA as a template and confirmed to represent a fragment of 

the NR gene by sequence analysis. A MONOR2 genomic DNA library was constructed in a 

lambda phage vector. Although the library appeared to be representative of the algal genome in 

that it contained approximately 300,000 separate clones of about 20,000 bp each, repeated 

screening of the library with the NR gene fragment failed to produce any positive results. Two 

additional libraries were constructed, but again, screening with the MONOR2 NR sequence did 

not result in the isolation of a genomic NR sequence. It was concluded that the libraries were 

probably incomplete; i.e., they did not contain DNA representative of the total algal genome, 

possibly because of problems associated with the unusual composition of the MONOR2 DNA. 

This project was put on hold when successful transformation was achieved in C. cryptica, and 

had not been pursued further when the project was terminated in 1996. 

A gene that encodes the enzyme orotidine-5'-phosphate decarboxylase (OPDase) was also 

targeted for use as a selectable transformation marker. OPDase is a key enzyme in the synthesis 

of pyrimidines. Organisms with defects in the OPDase gene will only grow if pyrimidines such 

as uracil are added to the growth medium. OPDase mutants can be selected by growing cells in 

the presence of the drug 5-fluoroorotic acid (FOA); OPDase converts FOA into a compound that 
National Renewable Energy Laboratory 

126 A Look Back at the Aquatic Species Program—Technical Review 

is toxic to the cells. Therefore, OPDase mutants would grow in the presence of FOA and require 

uracil; wild-type cells (or mutants transformed with the wild-type OPDase gene) would be 

susceptible to FOA and would require added uracil in the growth media. NREL researcher Eric 

Jarvis attempted to develop the OPDase system as a selectable marker for MONOR2. Cells were 

mutagenized by exposure to UV light, then grown in the presence of uracil and FOA. Putative 

OPDase mutants were identified as FOA-resistant colonies. Based on growth studies and 

spectrophotometric measurements of OPDase activity, one isolate of MONOR2 (3180a-1) was 

identified as a probable OPDase mutant for use as a host strain in the transformation system. 

The next step, as for NR, was isolate the wild-type OPDase gene from MONOR2. OPDase had 

previously been isolated from several species and demonstrated significant sequence 

conservation between genes from different organisms. Dr. Jarvis made a number of attempts to 

isolate the OPDase gene from MONOR2 via PCR, using degenerate primers based on conserved 

OPDase gene sequences. Several PCR products were generated using this approach, but 

sequence analysis of the cloned DNA fragments resulted in no clones with homology to the 

OPDase gene. Why this approach did not work for OPDase is unclear, as this same PCR 

technique had been used to isolate a fragment of NR. A second approach, in which a MONOR2 

genomic DNA library was screened for OPDase sequences using heterologous probes, was also 

unsuccessful. 

By 1994, a transformation system had been developed for the diatoms using a chimeric gene as a 

selectable marker (discussed in the following section); however, there was still interest in 

producing a selectable marker system that would work for high lipid (although genetically 

recalcitrant) green algal strains, such as MONOR2. Work began on developing a new selectable 

marker system that used a mutated version of the acetolactate synthase (ALS) gene as a 

selectable marker. ALS is an enzyme involved in the synthesis of branched-chain amino acid 

such as leucine and valine. In plants, this enzyme is inhibited by sulfonylurea and imidazolinone 

herbicides. Previous work at NREL by Galloway (1990) showed that many microalgae are also 

sensitive to these herbicides. Eric Jarvis repeated these experiments for MONOR2 and 

demonstrated that these cells are sensitive to low levels of the sulfonylurea herbicides 

chlorsulfuron and sulfometural methyl. The approach was to isolate the wild-type gene for ALS 

from MONOR2, and then to produce a gene that encodes a herbicide-resistant form of the 

enzyme by site-directed mutagenesis. Degenerate primers were produced based on known ALS 

sequences and used, this time successfully, to isolate an ALS gene fragment from MONOR2 

DNA. This sequence was used to screen the MONOR2 DNA libraries for a full-length ALS 

sequence, but once again, the screening efforts were unsuccessful. 

The feeling among the NREL researchers was that the use of a homologous selectable marker 

system would still be the best approach for developing genetic transformation systems for some 

organisms, in particular, those with unusual DNA compositions, and for haploid organisms for 

which generation of mutants should be relatively straightforward. Despite the promise of M. 

minutum as a high lipid producer, it may have not been the best organism for these studies 

because of its highly unusual DNA properties and “tough” cell wall that complicated biochemical 

extractions and assays. Some of the cloning problems seen with this organism might have been 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 127 

solved if time had permitted the generation of a cDNA library, or a new genomic DNA library 

using bacterial host strains optimized for use with highly modified or high GC DNA. 

Transformation of Chlamydomonas reinhardtii Using Silicon Carbide Whiskers: 

Based on the frustrating efforts to produce viable protoplasts from microalgae discussed earlier, 

efforts were initated to develop other methods for introducing DNA into microalgal cells through 

the intact algal cell walls. At the time this research was going on, the only microalga for which 

there was a reproducible transformation system was C. reinhardtii. Early efforts to transform 

this organism were facilitated by the availability of wall-less cells, either genetic mutants (cw15), or cells whose walls were degraded using autolysin, a species-specific cell wall-degrading 

enzyme produced during mating by C. reinhardtii gametes. High-frequency nuclear 

transformation was accomplished by agitating these wall-less cells in the presence of plasmid 

DNA, glass beads, and polyethylene glycol (Kindle 1990). This method was reported to work for 

walled cells, but at a very low frequency. DNA could also be introduced into walled cells of 

Chlamydomonas and into higher plant cells using microprojectile bombardment, or biolistics; 

however, this technique requires very expensive, specialized equipment. (This technique will be 

described in detail “Development of a Genetic Transformation System for the Diatoms Cyclotella

and Navicula.”) 

During the early 1990s, several reports demonstrated the feasibility of using silicon carbide 

whiskers (SiC) to mediate the entry of DNA into intact plant cells (Kaeppler et al. 1990; Asano et 

al. 1991). NREL researcher Terri Dunahay decided to try this approach to introduce DNA into 

intact algal cells. As reliable selectable markers were not yet available for any oleaginous 

microalgal strain, she decided to use Chlamydomonas as a model system. A strain of C 

reinhardtii that contains a defect in the gene for nitrate reductase (CC2453 nit1-305 mt-

) was 

obtained from the Chlamydomonas Genetics Center at Duke University, Durham, North 

Carolina. These cells cannot use nitrate as a N source, but grow well in the presence of ammonia 

or urea. Kindle (1990) had shown previously that NR-deficient cells could be transformed with 

the Chlamydomonas wild-type gene for NR; transformed cells expressing the added DNA could 

be detected by their ability to grow on nitrate as the sole N source. A plasmid containing the 

wild-type NR gene from Chlamydomonas was obtained from Dr. P. Lefebvre at the University of 

Minnesota, St. Paul, Minnesota. A protocol for SiC-mediated transformation was developed 

based on the glass bead transformation protocol of Kindle (1990). Exponentially growing cells 

were washed once in NH4

+

-free medium, then suspended in the same medium with plasmid 

DNA, sterilzed SiC whiskers, and polyethylene glycol (mw 8,000) to a final concentration of 

4.5%-5.0% w/v. The samples were agitated using a vortex mixer for periods from 30 seconds to 

10 minutes, then diluted into NH4

+

-free medium containing 0.6% agar (top agar) and plated onto 

agar plates that contained the same medium. Transformed colonies (containing a funtional NR 

gene) appeared in 1-2 weeks. 

Attempts to transform walled cells of Chlamydomonas using SiC were made in parallel with 

glass bead-mediated transformation to compare the two procedures. The results of a typical 

experiment are shown in Figure II.B.7. The number of transformants obtained using SiC varied 
National Renewable Energy Laboratory 

128 A Look Back at the Aquatic Species Program—Technical Review 

between experiments, but generally were in the range of 10-100 per 107 cells, comparable to 

transformation efficiency obtained with glass beads. Probably the most significant finding was 

the difference in cell viability after being agitated with either glass beads or SiC fibers. The 

viability of the cultures was greater than 80% even after agitation with SiC fibers for 10 minutes; 

only 10% of the cells survived agitation with glass beads for 60 seconds. The fact that SiCmediated transformation appears to be a more “gentle” protocol than glass bead treatment may be 

important when adapting the transformation procedure to other species that may have different 

wall properties. This work resulted in two publications (Dunahay 1993; Dunahay et al. 1997). 

The second paper was a collaboration with Dr. Jonathan Jarvik at Carnegie Mellon University. 

Dr. Jarvik's laboratory adopted and refined the SiC protocol and now uses it routinely to generate 

transformants in Chlamydomonas strains with intact walls. After the initial development of the 

SiC protocol, there was some work at NREL to adapt this procedure for other algal strains of 

interest to the biodiesel project. Initially, no genetic markers for these strains were available; 

however, the viability of Monoraphidium and Cyclotella were tested following agitation with 

SiC; both strains showed high survival rates after extended agitation with SiC. However, the 

successful development of a transformation system for Cyclotella using biolistics (discussed 

later) precluded further work on SiC-mediated algal transformation. A few attempts were made 

to generate transformants of Cyclotella or Navicula using SiC once a selectable marker system 

was developed. Only one transformant was generated in one experiment. The silica frustule of 

the diatoms likely acts as a significant barrier to penetration by SiC fibers. SiC would probably 

work better for introducting DNA into non-diatom cells such as Monoraphidium; these cells are 

very small and may not be a good target for biolistics, but might be readily pierced by SiC fibers. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 129 

Figure II.B.7. Cell survival and transformation efficiency of intact C. reinhardtii following 

vortex mixing with SiC fibers or glass beads. (Source: Dunahay 1993.) 
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130 A Look Back at the Aquatic Species Program—Technical Review 

Development of a Genetic Transformation System for the Diatoms Cyclotella and Navicula: 

Successful genetic transformation of microalgal strains with demonstrated potential for biodiesel 

fuel production was finally accomplished in 1994. Two factors that were critical in the 

development of the transformation system were: 

• the cloning of the acetyl-CoA carboxylase gene from C. cryptica, and thus the 

availability of ACCase regulatory sequences to drive expression of a foreign 

gene in the diatoms, and 

• the purchase by NREL of a microprojectile accelerator (also known as a particle 

gun) that can efficiently deliver DNA-coated gold or tungsten beads into walled 

cells. 

Except for the transient expression of luciferase in Chlorella protoplasts, all previous attempts at 

NREL to transform microalgae had been unsuccessful. Whether the problem was the inability to 

deliver foreign DNA into the cells through the algal cell wall, or inefficient expression of the 

foreign gene, is not clear. 

As discussed in the previous section of this report, a significant amount of work went into 

developing homologous selectable markers for microalgae, primarily for Monoraphidium. 

However, there were some attempts, mainly with diatoms, to use a heterologous antibiotic 

resistance gene as a selectable marker. The GC content of bacteria and diatoms are relatively 

similar; thus, codon bias should not prevent expression of a bacterial gene in the diatoms. The 

antibiotic kanamycin and its more potent analog G418, have been used extensively for genetic 

transformation in higher plants. These antibiotics function by binding to 30S ribosomes and 

inhibiting protein synthesis. Resistance to kanamycin or G418 can be induced in cells by 

expressing the bacterial gene neomycin phosphotransferase (nptII). This enzyme phosphorylates 

the antibiotic, preventing binding to the ribosome. Previous work at NREL (Galloway 1990) 

demonstrated that some algal strains are sensitive to kanamycin, suggesting that the kanamycinG418/nptII system might be the basis of a successful transformation system for microalgae. 

Further testing showed that most of the algal strains were sensitive to low concentrations of 

G418; however, the conditions for complete inhibition of cell growth had to be determined 

empirically for each strain. The required concentration of the antibiotic depended both on the 

osmoticum of the plating medium and on the plating density of the cells. For example, C. 

cryptica T13L grows well on both 10% and 50% ASW. When 2 x 106 cells of T13L were plated 

on 50% ASW agar plates, the cells were resistant to 50 μg⋅mL-1 G418. The same number of cells 

plated onto 10% ASW plus 50 μg⋅mL-1 G418 showed no growth, yet 3 x 107

 cells produced a 

confluent lawn of colonies under the same conditions. 

Early attempts to use the nptII gene as a selectable marker used a plasmid construct that had been 

used successfully for transformation in higher plants. This plasmid, pCaMVNeo, was obtained 

from Dr. Michael Fromm at the USDA Plant Gene Expression Center, Albany California. 
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A Look Back at the Aquatic Species Program—Technical Review 131 

pCaMVNeo contains the nptII gene driven by the cauliflower mosaic virus 35S ribosomal gene 

promoter (CaMV35S). Attempts were made to introduce pCaMVNeo into C. cryptica CYCLO1 

by electroporation, and later into C. ellipsoidea or CYCLO1 by agitating the cells with glass 

beads or SiC fibers. No G418-resistant colonies were generated by these methods. 

After the acetyl-CoA carboxylase (acc1) gene was cloned from C. cryptica T13L, NREL 

researcher Paul Roessler decided to try to use the 5'- and 3'-regulatory regions from this gene to 

drive expression of nptII in T13L. A plasmid (pACCNPT10) was constructed that contained a 

chimeric gene consisting of the coding region of the nptII gene flanked by 445 bp of the acc1 5' 

region (the putative promoter) and 275 bp of acc1 coding region following the nptII stop codon, 

followed by the acc1 3' noncoding regions (the putative transcriptional terminator). To increase 

the chance of encompassing the entire acc1 promoter, a second plasmid, pACCNPT5.1, was 

constructed that contained 819 bp of upstream sequence. In addition, all but 13 bp of the acc1 

coding region was removed from the 5' end of chimeric gene. Details of the plasmid 

constructions can be found in Dunahay et al. (1995), and plasmid maps are shown in Figure 

II.B.8. 

DNA entry into the algal cells was accomplished using the DuPont/Bio-Rad PDS/1000He 

microprojectile accelerator. The process, called biolistics, had been used successfully for 

introducting DNA into walled cells of higher plants, fungi, bacteria, and Chlamydomonas. In this 

procedure, plasmid DNA is precipitated onto small tungsten or gold particles and accelerated into 

cells using a burst of helium pressure. Early versions of this device used a gun powder charge to 

accelerate the particles. Because of prohibitive costs and restrictive licensing agreements, a 

homemade version of the particle gun was designed and built at NREL. No transformants were 

generated using this device, but as these experiments were performed before the acc1-nptII 

chimeric plasmids where constructed, whether the device actually functioned as planned is 

unclear. Ultimately, a commerical microprojectile accelerator was purchased. This device was 

optimized for very simple operation and used helium pressure to propel the DNA-coated 

particles. These properties resulted in greater reproducibility between shots and decreased 

toxicity caused by gases generated during the explosive charge. 

There was some initial skepticism on the part of at least one NREL researcher as to whether 

microprojectile bombardment would work to introduce DNA into diatoms through the Si 

frustule. However, the diatoms were transformed using the particle gun and the chimeric vectors 

in the first try. This turned out to be a simple and reproducible procedure (Figure II.B.9.). For 

each transformation, algal cells were harvested and spread in an approximate monolayer in the 

center of an agar plate containing growth medium and 50 μg⋅mL-1 ampicillin to inhibit bacterial 

growth. The plates were allowed to dry for 2 hours before bombardment. Just before 

bombardment, plasmid DNA was precipitated onto 0.5-1.0 μm tungsten particles, which were 

then propelled into the cells using the microprojectile accelerator. The exact parameters used are 

described in Dunahay et al. (1995). The cells were incubated for 2 days under nonselective 

conditions to allow the cells to recover and express the nptII gene. The cells were then washed 

from the orignal plates and replated onto agar that contained the appropriate concentration of 

G418. G418-resistant colonies appeared in 7-10 days. These putative transformants were picked 
National Renewable Energy Laboratory 

132 A Look Back at the Aquatic Species Program—Technical Review 

from the plates and tested for continued resistance to G418. The presence of the foreign gene was 

tested by hybridizing the algal DNA with an nptII gene probe (Southern analysis). The cells were 

tested for the presence of the and for the NPTII protein by probing with an NPTII-specific 

antibody (Western blotting), Figures II.B.10 and II.B.11. 

Both the pACCNPT10 and pACCNPT5.1 plasmids worked well to generate transformants in two 

strains of C. cryptica (T13L and CYCLO1), as well as in the diatom N. saprophila (NAVIC1). 

These two species belong to different orders (C. cryptica is a centric diatom, Order Centrales; N. 

saprophila is a pennate diatom, Order Pennales). Southern analysis indicated that the plasmid 

DNA was not replicating independently in the cells but had integrated into the host genome, 

presumably into the nuclear DNA. The chimeric gene integrated into one or more independent 

sites, often in form of tandem repeats. The nptII DNA remained stably integrated into the host 

genome for more than 1 year, even when the cells were grown under nonselective conditions. 

The successful development of a genetic transformation system for the diatoms was a major 

achievement for the ASP. This was the first report of genetic transformation of any diatom 

species, and one of the few reports in which a heterologous gene was used as a selectable marker 

for stable nuclear transformation of an alga. The use of algal regulatory sequences to drive 

expression of the bacterial gene in diatoms apparently was a key factor in the successful 

development of a transformation protocol for these organisms. When the pCaMVNeo plasmid 

was introduced into diatoms via particle bombardment, no G418-resistant transformants were 

generated. However, when another plasmid that contains the CaMV35S promoter and the firefly 

luciferase gene were introduced into the diatoms by cotransformation with pACCNPT5.1, a 

number of transformants selected based on their resistance to G418 also expressed significant 

luciferase activity. This result suggests that even though microalgae can in some cases recognize 

and use foreign promoter sequences, homologous promoters may be necessary to drive 

expression of foreign selectable markers at levels high enough to overcome the selective 

pressure. The research that resulted in the development of a genetic tranformation system for 

diatoms resulted in a publication (Dunahay et al. 1995) that was a finalist for the Provasoli 

Award for best publication in the Journal of Phycology for that year. In addition, a patent 

describing this technology was applied for and issued in August 1997. Diatoms represent a very 

large proportion of the world's biomass, and are responsible for nearly one-fourth of the net 

primary production. However, little is known about the biochemistry and molecular biology of 

these organisms. The availability of a genetic transformation system for diatoms could have a 

major impact on increasing the understanding of the basic biology of these organisms and should 

promote their use in biotechnological applications in addition to the intended goal of lipid 

production. The following section will describe the initial attempts to use the genetic 

transformation protocol to manipulate levels of storage lipids in C. cryptica. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 133 

Figure II.B.8. Plasmid maps of pACCNPT10, pACCNPT5.1, pACC1 

A. (Top) - Maps of plasmids containing the neomycin phosphotranferase gene (nptII) flanked 

by regulatory regions from the acetyl-CoA carboxylase gene from C. cryptica. Both plasmids 

worked well as expression vectors in the diatoms C. cryptica and Navicula saprophila.

B. (Bottom) - Map of plasmid pACC1, containing the full-length genomic sequence of the 

acetyl-CoA carboxylase gene (acc1) from C. cryptica. 

 - nptII gene sequence;  - acc1 coding sequence;  - acc1 regulatory sequences 

(Source: Dunahay et al. 1995; Roessler and Ohlrogge 1993). 
National Renewable Energy Laboratory 

134 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.B.9. Simplified schematic showing the protocol for transformation of diatoms by 

microprojectile bombardment (“gene gun”). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 135 

Figure II.B.10. Southern blot showing the presence of the nptII gene in transformed cells of 

C. cryptica T13L. 

Cells of C. cryptica T13L were transformed with pACCNPT5.1 via particle bombardment as 

described in the text. DNA from wild-type cells (wt) or G418-resistant strains (lanes a-f) were 

digested with HindIII and hybridized to a digoxigenin-labeled nptII sequence. The lane 

designated “5.1” contains HindIII-digested pACCNPT5.1as a control. The sizes of DNA 

fragments included as markers are indicated to the right. (Source: Dunahay et al. 1995). 
National Renewable Energy Laboratory 

136 A Look Back at the Aquatic Species Program—Technical Review 

Figure II.B.11. Western blot, showing the presence of the 30-kDa NPTII protein in 

transformed cells of C. cryptica T13L and N. saprophila. 

In this example, C. cryptica and N. saprophila were transformed with pACCNPT10 and 

pACCNPT5.1, respectively. Crude cell extracts were separated on SDS-polyacrylamide gels, 

blotted onto a nitrocellulose filter, and NPTII protein was detected using anti-NPTII primary 

antibodies and alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibodies. The 

polyclonal antiNPTII antibody also recognizes a band of approximately 80 kDa in C. cryptica;

however, the 30 kDa NPTII protein is seen only in the G418-resistant transformants. (Source: 

Dunahay et al. 1995). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 137 

II.B.3.d. Attempts to Manipulate Microalgal Lipid Composition via Genetic 

Engineering 

The overall goal of the studies on the biochemistry and molecular biology of lipid synthesis in 

microalgae was to increase the understanding of the lipid biosynthetic pathways and to identify 

enzymes that influence the rate of lipid accumulation and lipid quality. This information would 

be used to genetically manipulate the biosynthetic pathways for improvement in lipid production 

rates and to manipulate the nature of the lipids produced (i.e., the degree of fatty acid saturation 

and chain length) to optimize the production of biodiesel. 

The development of a genetic transformation system for diatoms allowed NREL researchers to 

begin testing ways to manipulate microalgal biochemical pathways. The first target enzyme was 

ACCase. Previous studies at NREL had shown that increased lipid production in diatoms 

induced by Si starvation was accompanied by an increase in the activity of the ACCase enzyme. 

Therefore, it was logical to ask whether the activity of the enzyme could be increased in the cells 

by adding additional copies of the gene encoding ACCase (acc1), and, if so, would increased 

activity of the protein stimulate the production of lipids in the algal cells? 

A full-length copy of the C. cryptica acc1 gene had been cloned and characterized at NREL (see 

Section II.B.2.f). The plasmid containing this sequence was designated pACC1 (Figure II.B.8). 

Before the algal transformation system, attempts were made to express the algal gene in a 

bacterial system to ensure that the cloned gene encoded a functional ACCase enzyme and to test 

for the effects of overexpression. For expression of the C. cryptica acc1 gene in E. coli, the 

introns were removed and the 5' terminus was replaced with the 5' end of the E. coli βgalactosidase gene, which included the inducible promoter region. This fusion gene was 

introduced into E. coli. The transformed cells were analyzed for the production of algal ACCase 

protein by probing blots of (Sodium Dodecyl Sulfate, SDS) polyacrylamide gels with an antiACCase antibody or with avidin conjugated to alkaline phosphatase. (Avidin binds to the biotin 

moity in the functional ACCase protein.) The bacterial cells produced full-length algal ACCase, 

as well as a large number of shorter polypeptides recognized by the anti-ACCase antibody. 

Introducting the gene into other E. coli strains deficient in protease activity also produced these 

shortened peptides; therefore, they were presumed to be the result of truncated transcription or 

translation. The full-length ACCase protein was properly biotinylated in the transformed 

bacteria, but not as efficiently as in the E. coli native biotin-binding ACCase subunit. No effects 

were observed on lipid biosynthesis in the transformed E. coli strain. Attempts were also made 

to introduce the C. cryptica acc1 gene into yeast, as expression in a eukaryotic system would 

more likely mimic the effects in algae, but these experiments were unsuccessful. 

The next step was to introduce additional copies of the acc1 gene in diatoms, with the goal of 

increasing the activity of the ACCase enzyme and then assaying the effects of ACCase 

overexpression on lipid accumulation. The plasmid containing the full-length acc1 gene 

(pACC1) does not contain a selectable marker for transformation. Studies in other laboratories 

showed that nonselectable plasmids can be introduced into cells via cotransformation with a 

plasmid containing a selectable marker gene such as nptII. Although the exact mechanism for 
National Renewable Energy Laboratory 

138 A Look Back at the Aquatic Species Program—Technical Review 

this phenomenon is not clear, it is believed that during a given transformation procedure, a 

particular subpopulation of the cells becomes “transformation competent”. These cells may then 

take up multiple copies of DNA molecules present in the reaction. Introduction of pACC1 into 

the diatoms was mediated by microprojectile bombardment as described in a previous section, 

but with pACCNPT5.1 and pACC1 precipitated onto the tungsten beads in equimolar amounts. 

Transformed cells were selected based on their induced resistance to G418 and then screened for 

additional copies of the acc1 wild-type gene using PCR and Southern analysis. Between 20% 

and 80% of the G418-resistant colonies contained acc1 sequences in the cotransformation 

experiments. To facilitate the selection of transformants containing extra copies of the acc1 

gene, a plasmid was also constructed that contained both acc1 and nptII, designated pACCNPT4; 

transformants generated using pACCNPT4 and selected for G418-resistance almost always 

contained the acc1 gene as well. 

Transformed cells containing additional C. cryptica acc1 gene sequences were isolated in C. 

cryptica T13L, C. cryptica CYCLO1, and N. saprophila NAVIC1. Southern analysis indicated 

that the foreign DNA inserted into host genome, often in one or more random sites, and often in 

the form of tandem repeats. Several strains that contained one or more full-length sequences of 

the inserted acc1 gene were analyzed further to test for ACCase overexpression. The CYCLO 

T13L transformants showed two to three fold higher ACCase activity than wild-type cells, and 

there was a corresponding increase in acc1 gene transcript (mRNA) levels. However, 

preliminary analyses of the lipid composition of the cultures overexpressing acc1 did not indicate 

a detectable increase in lipid levels. These results suggest that the lipid biosynthesis pathways 

may be subject to feedback inhibition, so that increased activity of the ACCase enzyme is 

compensated for by other pathways within the cells. It was hoped that expression of C. cryptica

T13L acc1 gene in other algal strains might overcome this inhibition. Numerous N. saprophila

transformants were generated that contained full-length copies of the C. cryptica acc1 gene; 

although acc1 mRNA was detected using the RPA, the recombinant ACCase protein was not 

detected in any of the N. saprophila strains tested. Whether this result was due to inefficient 

translation of the mRNA, or degradation of the foreign protein due to improper biotinylation or 

targeting, is not known. Transformants were also generated in a second strain of C. cryptica, 

CYCLO1, but the program was discontinued before these strains could be analyzed fully. 

NREL researcher Eric Jarvis took another approach to genetically manipulating algal pathways 

for increased lipid production. Previous research had resulted in the cloning and characterization 

of the upp1 gene from C. cryptica (described in Section II.B.2.h.). This gene codes for a fusion 

protein containing the activities for UDPglucose pyrophosphorylase and phosphoglucomutase, 

two key enzymes in the production of chrysolaminarin. It was postulated that decreasing 

expression of the upp1 gene could result in a decrease in the proportion of newly assimilated 

carbon into the carbohydrate synthesis pathways, and consequently increase the flow of carbon to 

lipids. 

Two techniques that are becoming widely used for gene inactivation are ribozymes and antisense 

RNA. Dr. Jarvis spent 6 months working at Ribozyme Pharmaceuticals, Inc., a biotechnology 

company in Boulder, Colorado, learning about these new methods. Antisense RNA is a method 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 139 

in which a cell is transformed with a synthetic gene that produces an RNA sequence 

complimentary to a specific mRNA. Although the exact mechanism is not clear, the antisense 

RNA prevents translation from its complimentary mRNA, effectively lowering the level of that 

particular protein in the cell. Ribozymes are also RNA molecules produced by synthetic genes 

that can bind to, and cleave, very specific RNA sequences. Ribozymes can be designed to 

degrade specific mRNA molecules, effectively decreasing expression of a specific gene. 

Several ribozymes sequences designed to cleave upp1 RNA were constructed based on computer 

predictions of the secondary structure of the target RNA. The ribozyme constructs were shown 

to specifically cleave the target RNA in vitro. The ribozyme sequences were then inserted into 

the pACCNPT10 vector in the untranslated acc1 sequence between the nptII stop codon and the 

acc1 termination sequence (see Figure II.B.8). C. cryptica T13L was transformed with these 

vectors as described earlier and transformants were selected based on acquired resistance to 

G418. Extracts were made of the transformed strains and analyzed for UGPase activity. 

Unfortunately, insertion of the ribozyme sequences did not result in detectable decreases in 

UGPase expression. Although these initial experiments were unsuccessful, gene inactivation 

technologies acquired during this project seemed a promising approach for manipulation of algal 

lipid synthesis pathways. At the time project funding was terminated, work was in progress to 

continue with the ribozyme experiments and to test antisense RNA constructs as an additional 

method for inactivating algal pathways. 

II.B.3.e. The Effect of Different Promoters on Expression of Luciferase in Cyclotella 

Little is known about the regulation of gene expression in diatoms, partly because genetic 

transformation was not possible in this group of algae before NREL’s transformation system was 

developed. The availability of this transformation system now allows the study of the roles of 

regulatory DNA sequences in gene expression. As a first step toward a better understanding of 

gene transcription in diatoms, NREL researchers Paul Roessler and Steve Milstrey used the 

firefly luciferase reporter gene (discussed earlier), to study the level of gene expression as 

controlled by various DNA regulatory sequences from the diatom C. cryptica and other 

organisms. They also used this system to try to define the regions of the ACCase gene promoter 

involved in the Si-depletion response. 

Various plasmids were constructed in which different combinations of 5’ regulatory DNA 

sequences (promoters) and 3’ regulatory DNA sequences (terminators) were linked to the firefly 

luciferase gene (luc). The regulatory sequences used in this study included both the ACCase 

promoter and the UDP-glucose pyrophosphorylase/phosphoglucomutase promoters from C. 

cryptica. Also tested were the simian virus 40 (SV40) promoter, which drives high levels of 

gene expression in mammalian cells, and the cauliflower mosaic virus 35S RNA promoter 

(CaMV35S), which is a strong constitutive promoter in plants. These plasmids were introduced 

into C. cryptica via cotransformation with the selectable marker plasmid pACCNPT5.1 as 

described earlier. Approximately half of the transformed strains produced in this manner 

contained the luc gene, as determined by PCR analysis. Based on past results, it is expected that 

the plasmid DNA was integrated into the genome of the cells. Luciferase activities in randomly 
National Renewable Energy Laboratory 

140 A Look Back at the Aquatic Species Program—Technical Review 

chosen transformants (eight from each plasmid type) were determined by the use of a 

luminometer. 

As expected, the promoter regions of both C. cryptica genes drove luciferase expression in the 

transformed C. cryptica cell at high levels. Less predictable was the finding that the SV40 

mammalian promoter also drove luciferase expression in C. cryptica at relatively high levels 

(although lower than seen using the homologous promoters), but the CaMV35S promoter was 

much less effective. In most of the constructs used in this study, the 3' terminator regulatory 

region was from the C. cryptica aac1 gene. Replacement of this sequence with the SV40 

terminator did not affect the levels of luciferase expression driven by the acc1 promoter, 

indicating that the source of the terminator sequence used may not be a critical determinant of 

gene transcription efficiency. 

Previous results at NREL indicated that Si deficiency may affect the expression of the acc1 gene 

in C. cryptica (see Section II.B.2.d.). To try to identify regions of the acc1 promoter that might 

be responsive to Si levels, three plasmids that contained varying lengths of the acc1 promoter 

region (900, 445, and 396 bp, respectively) fused to the luc gene were used to transform C. 

cryptica. Under Si replete conditions, the average luciferase activities of transformants 

containing these plasmids were very similar. Furthermore, the luciferase activity increased to the 

same extent (approximately twofold) 6 hours after transfer into Si-free medium. This suggests 

that the Si-responsive elements are either within the shortest (396-bp) promoter region tested or 

in a separate area of the genome. 

These results indicate that the firefly luciferase gene can be expressed in recombinant C. cryptica

cells, to provide a sensitive reporter system for analyzing gene expression and promoter function 

in diatoms. This and similar systems will likely be extremely useful for gaining a better 

understanding of the molecular biology of this important group of organisms. 

Publications: 

Brown, L.M.; Dunahay, T.G.; Jarvis, E.E. (1990) Applications of genetics to microalgae 

production. Dev. Industrial Microbiology, vol. 31 (J. Industrial Microbiol., Suppl. No.5), pp. 

271-274. 

Dunahay, T.G. (1993) “Transformation of Chlamydomonas reinhardtii with silicon carbide 

whiskers.” Biotechniques 15:452-460. 

Dunahay, T.G.; Adler, S.A.; Jarvik, J.W. (1997) “Transformation of microalgae using silicon 

carbide whiskers.” In Methods in Molecular Biology, vol. 62: Recombinant Gene Expression 

Protocols (Tuan, R., ed.), Humana Press, Totowa, New Jersey, pp. 503-509. 

Dunahay, T.G.; Jarvis, E.E.; Dais, S.S.; Roessler, P.G. (1996) “Manipulation of microalgal lipid 

production using genetic engineering.” Appl. Biochem. Biotechol. 57/58:223-231. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 141 

Dunahay, T.G.; Jarvis, E.E.; Roessler, P.G. (1995) “Genetic transformation of the diatoms

Cyclotella cryptica and Navicula saprophila.” J. Phycol. 31:1004-1012. 

Dunahay, T.G.; Jarvis, E.E.; Zeiler, K.G.; Roessler, P.G.; Brown, L.M. (1992) “Genetic 

engineering of microalgae for fuel production.” Appl. Biochem. Biotechnol. 34/35:331-339. 

Galloway, R.E. (1990) “Selective conditions and isolation of mutants in salt-tolerant, lipidproducing microalgae.” J. Phycol. 26:752-760. 

Jarvis, E.E. (1995) “Antisense RNA and ribozymes as tools for metabolic engineering.” Final 

Report DDF #07422041, National Renewable Energy Laboratory, 13 pp. 

Jarvis, E.E.; Brown, L.M. (1991) “Transient expression of firefly luciferase in protoplasts of the 

green alga Chlorella ellipsoidea.” Curr. Genet. 19:317-321. 

Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. (1992) “DNA nucleoside composition and methylation 

in several species of microalgae.” J. Phycol. 28:356-362. 

Roessler, P.G. (1995) “Expression of an algal acetyl-CoA carboxylase gene in E. coli.” In Plant 

Lipid Metabolism (Kader, J.-C.; Mazliak, P., eds.), Klewer Academic Publishers, pp. 46-48. 

Roessler, P.G.; Brown, L.M.; Dunahay, T.G.; Heacox, D.A.; Jarvis, E.E.; Schneider, J.C.; Talbot, 

S.G.; Zeiler, K.G. (1994) “Genetic engineering approaches for enhanced production of biodiesel 

fuel from microalgae.” In Enzymatic Conversion of Biomass for Fuels Production, (Himmel, 

M.E.; Baker, J.; Overend, R.P., eds.), American Chemical Society, pp. 256-270. 

Roessler, P.G.; Ohlrogge, J.B. (1993) “Cloning and characterization of the gene that encodes 

acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica.” J. Biol. Chem. 268:19254-

19259. 

Additional References: 

Asano, Y.; Otsuki, Y.; Ugaki, M. (1991), “Electroporation-mediated and silicon carbide fibermediated DNA delivery in Agrostis alba L. (Redtop).” Plant Sci. 79:247-252. 

Berliner, M.D. (1977) “Protoplast induction in Chlorella vulgaris.” Plant Sci. Lett. 9:201-204. 

Braun, E.; Aach, H.G (1975) “Enzymatic degradation of the cell wall of Chlorella.” Planta

126:181-185. 

Daboussi, M.J.; Djeballi, A.; Gerlinger, C.; Blaiseau, P.L.; Bouvier, I.; Cassan, M.; Lebrun, 

M.H.; Parisot, D.; Brygoo, Y. (1989) “Transformation of seven species of filamentous fungi 

using the nitrate reductase gene of Aspergillus nidulans.” Curr. Genet. 15:453 
National Renewable Energy Laboratory 

142 A Look Back at the Aquatic Species Program—Technical Review 

Global, E.; Aach, H.G. (1985) “Studies of Chlorella protoplasts. II. Isolation and fusion of 

protoplasts from Chlorella saccharophila (Krüger) nadson strain 211-1a.” Plant Sci. 39:213-218. 

Hoffman, C.S.; Winston, F. (1987) “A ten-minute DNA preparation from yeast efficiently 

releases autonomous plasmids for transformation of Escherichia coli.” Gene 57:267-272. 

Kaeppler, H.F.; Gu, W.; Somers, D.A.; Rines, H.W.; Cockburn, A.F. (1990) “Silicon carbide 

fiber-mediated DNA delivery into plant cells.” Plant Cell Rep. 9:415-418. 

Kindle, K. (1990) “High frequency nuclear transformation of Chlamydomonas reinhardtii.” 

Proc. Nat. Acad. Sci. USA 87:1228-1232. 

Kindle, K.L.; Schnell, R.A.; Fernández, E.; Lefebvre, P.A. (1989) “Stable nuclear transformation 

of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.” J. Cell Biol

109:2589-2601. 

Murray, H.G.; Thompson, W.F. (1980), “Rapid isolation of high molecular weight plant DNA.” 

Nuc. Acids Res. 8:4321-4325. 

Patents: 

Dunahay, T.; Roessler, P.G.; Jarvis, E.E. “Method to Transform Algae, Materials Therefor and 

Products Produced Thereby”, U.S. Patent 5,661,017, issued 8/26/97. 

II.B.4. Microalgal Strain Improvement – Conclusions and Recommendations 

Previous to the research performed by researchers in the ASP at NREL, very little work had been 

done in the area of microalgal strain improvement, particularly with a goal of developing a 

commercial organism. Although much remains to be done, significant progress was made in the 

understanding of environmental and genetic factors that affect lipid accumulation in microalgae, 

and in the ability to manipulate these factors to produce strains with desired traits. 

The evidence for a specific lipid trigger is not overwhelming. Interpreting exactly what is 

happening in the nutrient-deprived cells is difficult, particularly when cells are starved for N, as 

the lack of an important nutrient is likely to produce multiple and complex reactions in a cell. 

However, lipid accumulation in some algal species can be induced by nutrient limitation. Cell 

division slows or stops, and the cells begin to accumulate lipid as cytoplasmic droplets, formed 

primarily of neutral TAGs. The trigger hypothesis is supported by microscopic and flow 

cytometric evidence that showed that the lipid droplets do not form gradually within all cells in a 

population; rather, individual cells seem to sense the trigger and lipid accumulation occurs 

rapidly within the individual cells. However, lipid accumulation is always correlated with the 

cessation of cell division. Other factors that inhibit cell division, such as a pH shift, can also 

induce lipid accumulation in some strains. The evidence suggests that the rate of synthesis of all 

cell components, including lipids, proteins, and carbohydrates, is decreased in nutrient-stressed 

cells. However, the rate of lipid synthesis remains, at least for some strains of diatoms, higher 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 143 

than the rate of protein or carbohydrate synthesis, resulting in a net accumulation of lipid in 

nutrient-starved cells. Another hypothesis is that cessation of cell division in nutrient-limited 

cells leads to decreased utilization of storage lipid while new synthesis of lipid continues, causing 

a net accumulation of lipid in the cells. 

One of the most important findings from the studies on lipid accumulation in the microalgae is 

that, although nutrient stress causes lipid to increase in many strains as a percentage of the total 

biomass, this increase is generally accompanied by a decrease in total cell and lipid productivity. 

As discussed elsewhere in this report, economically viable production of algal lipids for fuel 

production will require optimization of productivity as well as a clear understanding of the 

kinetics of lipid accumulation, in order to harvest the cells at a time when lipid production is 

maximal. 

In addition to the effect on total lipid production, nutrient deprivation seems to have other effects 

on the lipid biosynthetic pathways. Several laboratories reported that nutrient limitation also 

resulted in a change in the types of lipids seen in the algal cells, specifically, an increase in the 

ratio of neutral lipids (storage TAGs that are important in the production of biodiesel) as 

compared to the polar membrane lipids. It will be important to characterize this phenomenon 

further in any algal strain targeted for biomass production to maximize the desired lipid product. 

The progress made by the ASP in the understanding of the biochemistry and molecular biology 

of lipid biosynthesis in algae, and the success in the area of algal genetic engineering are more 

clear cut. An enzyme that appears to play a key regulatory role in the synthesis of lipids in 

plants, and likely in algae, ACCase, was purified from the diatom C. cryptica. The gene that 

codes for this enzyme was then cloned; this was the first report of cloning a full-length ACCase 

sequence from any photosynthetic organism. Obtaining this gene was advantageous for two 

reasons: 

1. The regulatory sequences from this gene were used to develop a genetic 

transformation system for diatoms, and 

2. Having this gene (in combination with the transformation system) allowed 

researchers to test for the effects of overexpression of this enzyme on lipid 

accumulation. 

The development of the transformation system for an oleaginous microalgal strain was a major 

goal of the ASP, and a significant effort was put into this project during the early 1990s. This 

was the first successful transformation of any non-green alga. The method was simple and 

reproducible, and should work for a variety of diatom strains, as long as the cells can form 

colonies on solid medium and are sensitive to one of the known selectable agents, such as G418 

or kanamycin. Although little work is currently being done on the development of genetically 

engineered algal strains for commercial applications, the ability to transform these algae should 

have positive ramifications for the algal biotechnology community. 
National Renewable Energy Laboratory 

144 A Look Back at the Aquatic Species Program—Technical Review 

Preliminary experiments were also performed within the ASP to use this genetic transformation 

system to introduce genes into the algal cells, with the goal of manipulating lipid biosynthesis. 

Additional copies of the ACCase gene were introduced into cells of C. cryptica and N. 

saprophila. Although ACCase activity was increased in these cells, there was no detectable 

increase in lipid accumulation. The project was terminated before these experiments, and similar 

experiments designed to down-regulate genes involved in carbohydrate synthesis could be 

pursued further. This could be an interesting and possibly rewarding path for future research, if 

only to help in understanding the biochemical and molecular biological factors that affect lipid 

accumulation in these cells. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 145 

 III. Outdoor Studies and Systems Analysis 

III.A. Projects Funded by ERDA/DOE 1976-1979

III.A.1. Introduction 

The concept of microalgae biomass production for conversion to fuels (biogas) was first 

suggested in the early 1950s (Meier 1955). Shortly thereafter, Golueke and coworkers at the 

University of California–Berkeley demonstrated, at the laboratory scale, the concept of using 

microalgae as a substrate for anaerobic digestion, and the reuse of the digester effluent as a 

source of nutrients (Golueke et al. 1957; Golueke and Oswald 1959). 

Oswald and Golueke (1960) presented a conceptual analysis of this process, in which large (40-

ha) ponds would be used to grow microalgae. The algae would be digested to methane gas used 

to produce electricity. The residues of the digestions and the flue gas from the power plant 

would be recycled to the ponds to grow additional batches of algal biomass. Wastewaters would 

provide makeup water and nutrients. The authors predicted that microalgae biomass production 

of electricity could be cost-competitive with nuclear energy. 

This concept was revived in the early 1970s with the start of the energy crisis. The National 

Science Foundation–Research Applied to National Needs Program (NSF-RANN) supported a 

laboratory study of microalgae fermentations to methane gas (Uziel et al.1975). Using both fresh 

and dried biomass of six algal species, roughly 60% of algal biomass energy content converted to 

methane gas. 

With the establishment of ERDA, the NSF-RANN activities were transferred to this new agency, 

which initiated a program in biomass fuels production. The Fuels from Biomass Program at 

ERDA funded a new project at Berkeley to develop a microalgae wastewater treatment and fuel 

production process. This project, started in 1976, was carried out at the Richmond Field Station 

of the University of California–Berkeley, and continued for about 4 years in parallel with several 

related projects. These projects included an ERDA-funded biophotolysis project (reviewed in 

Benemann et al. 1980), a NSF-RANN project on N-fixing blue-green algae (cyanobacteria) for 

fertilizer production (Benemann et al. 1977), and an EPA-funded project on algal bioflocculation 

in oxidation ponds (Koopman et al.1978; 1980). 

The initial objective of the ERDA microalgae fuels project was to develop methods by which 

particular species of microalgae could be maintained in open ponds used for wastewater 

treatment. There are many large (>100 ha) and many hundreds of smaller wastewater treatment 

pond systems in California and elsewhere in the United States. The problem addressed by this 

project was the removal (harvesting) of the algal biomass from the effluents. Not only did the 

algal biomass represent a potential resource for the production of biogas, but the algal solids 

discharged from the ponds were pollutants that resulted in eutrophication and dissolved O2

reduction in the receiving bodies of waters. Thus, there was considerable interest in lower-cost 

and less energy-intensive microalgae harvesting technologies and wastewater treatment processes 
National Renewable Energy Laboratory 

146 A Look Back at the Aquatic Species Program—Technical Review 

in general. In the absence of cost-effective microalgae harvesting technologies, microalgae pond 

systems, although widely used, could not meet the increasingly stringent wastewater treatment 

plant discharge standards, specifically in regards to suspended solids (mainly algal cells). A 

process that could reduce algal solids in pond effluents would have a ready market and potential 

near-term applications. 

This was the justification for the initial emphasis on wastewater treatment processes from 

microalgae production. However, the ERDA/DOE Fuels from Biomass Program soon shifted its 

emphasis towards large-scale biomass production systems, having multi-quad (quad = 1015 Btu) 

impacts. This accounted, in part, for the early emphasis by this program on large-scale biomass 

“energy plantations” and even immense ocean energy farms, which some thought would provide 

solutions for the perceived U.S. energy crisis (Benemann 1980). 

Large effects on U.S. energy supplies would probably not be plausible with wastewater treatment 

systems, which in aggregate represent a maximum potential of perhaps only one- or two-tenths of 

a quad of fuels (Benemann et al.1998, in preparation), a fraction of 1% of U.S. energy needs. 

However, wastewater systems can arguably serve as an initial step in the long-term development 

of larger, stand-alone systems. Although this argument was controversial at the time, the 

Univeristy of California–Berkeley project continued to emphasize wastewater treatment systems 

in its R&D. However, the supporting economic analyses carried out by the ASP (see Section 

III.D.), started to focus on very large-scale, stand-alone systems. Section III.A. of this report 

reviews the algal mass-culture projects that were supported by ERDA and DOE before the ASP 

was initiated. 

Publications: 

Benemann, J.R. (1980) “Biomass energy economics.” Intl. Energy Journal 1:107-131. 

Benemann, J.R.; Hallenbeck, P.C.; Weissman, J.C.; Murry, M.; Oswald, W.J. (1977) “ Fertilizer 

production with nitrogen-fixing heterocystous blue-green algae.” Final Report to the National 

Science Foundation, Sanitary Eng. Res. Lab., Univ. Calif. Berkeley. 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Goebel, R. P. (1980) 

“Development of microalgae harvesting and high rate pond technology.” In Algal Biomass, 

(Shelef, G.; Soeder, C.J., eds.), Elsevier, pp. 457-499. 

Benemann, J.R.; Koopman, B. L.; Weissman, J.C.; Oswald, W.J. (1976) “Biomass production 

and waste recycling with blue-geen algae.” In Microbial Energy Conversion, (Schlegel, H.G.; 

Barnea, J., eds.), Gottingen, pp. 413-426. 

Benemann, J.R.; Miyamoto, K.; Hallenbeck, P.C. (1980) “Bioengineering aspects of 

biophotolysis.” Enzyme and Microbial Technology 2:103-111. 

Benemann, J.R.; Weissman, J.C.; Koopman, B.L.; Oswald, W.J. (1977) “Energy production with 

microalgae.” Nature 268:19-23. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 147 

Golueke, C.G.; Oswald, W.J. (1959) “Biological Conversion of light energy to the chemical 

energy of methane.” App. Microbiol. 7:219-241. 

Golueke, C.G.; Oswald, W.J.; Gotaas, H.B. (1957) “Anaerobic digestion of algae.” App. 

Microbiol. 5:47-55. 

Koopman, B.L.; Benemann, J.R.; Oswald, W.J. (1980) “Pond isolation and phase isolation for 

control of suspended solids concentration in sewage oxidation pond effluents.” In: Algal Biomass

(Shelef, G.; Soeder, C., eds.), Elsevier, pp. 135-162. 

Koopman, B.L.; Thomson, R.; Yacksian, R.; Benemann, J.R.; Oswald, W.J. (1978) 

“Investigation of the pond isolation process for microalgae separation from woodland’s pond 

effluents.” Report to the Univ. of Calif., Berkeley, SERL Report No 78-5. 

Meier, R.L. (1955) “Biological cycles in the transformation of solar energy into useful fuels.” In: 

Solar Energy Research, (Daniels, F.; Duffie, J.A., eds.), Madison University Wisconsin Press, 

pp. 179-183. 

Oswald, W.J.; Golueke, C.G. (1960) “Biological transformation of solar energy.” Adv. Appl. 

Microbiol. 11:223-242. 

Uziel, M.; Oswald, W.J.; Golueke, C.G. (1975) “Solar energy and conversion with algal bacterial 

systems.” Univ. of California Berkeley, SERL Report to NSF/RANN/SE/GI-39216/Fr/75/5. 

III.A.2. Species Control in Large-Scale Algal Biomass Production 

This project addressed the problem of the uncontrolled nature of the algal populations in 

wastewater treatment ponds. Because the dominant algal species found in a pond could range 

from small unicellular to large colonial or filamentous species, harvesting of the algae for 

biomass conversion would require a universally applicable harvesting technology, such as 

centrifugation or chemical flocculation, to enable the recovery of any algal type. However, these 

processes are very expensive (Golueke and Oswald 1965; see also Shelef 1984 for an ASP 

supported assessment, and Benemann and Oswald, 1996 for a recent review). If, however, algal 

species could be controlled in the ponds, then filamentous microalgae species might be grown 

that would be easier and cheaper to harvest using microstrainers. Microstrainers, which are 

rotating screens (typically 25 to 50 μm openings) with a backwash, are already widely used for 

removing filamentous algae, mainly filamentous cyanobacteria (blue-green algae) from potable 

water supplies. Thus, the first objective of this project, initiated in 1976, was to investigate how 

to selectively cultivate filamentous microalgal species in waste treatment ponds (Benemann et al. 

1977). 

The first issue was that conventional waste treatment ponds are generally deep (2 m), and 

unmixed. Such ponds do not maximize algal productivity, nor do they provide a uniform 

hydraulic flow or physical-chemical environment. Thus, this project focused on the use of 

shallow, mixed, raceway-type ponds, the “high rate pond” of Oswald (1963), for microalgae 
National Renewable Energy Laboratory 

148 A Look Back at the Aquatic Species Program—Technical Review 

production and demonstration of algal species control in wastewater treatment. Initially four 

small (approximately 3 cm2

) and, four larger (12 m2) rectangular, paddle wheel-mixed ponds 

were built and used. The Richmond Field station also had a large pilot-scale (0.25 ha) shallow 

high rate pond that was fed settled (primary effluent) municipal wastewater, available for this 

research. 

A fundamental theory of species control was developed based on selective recycle of harvestable 

biomass (Figure III.A.1.). The concept is that harvesting filamentous algae and recycling part of 

the biomass back to the ponds (similar to the process of activated sludge), would favor the 

slower-growing filamentous algae over the faster-growing unicellular types (which would thus 

get washed out of the system). This theory was both mathematically proven and experimentally 

demonstrated in the laboratory in competition experiments with mixed cultures of Chlorella (a 

unicellular green alga) and Spirulina (a filamentous blue-green alga). Without biomass recycle 

Chlorella always out-competed Spirulina; with biomass recycle Spirulina could be made to 

dominate the culture (see also Weissman and Benemann 1978). 

This process was also tested in outdoor ponds. Experiments with Spirulina grown on wastewater 

were not encouraging, as this species dominates only at high salinities and alkalinities. Thus, the 

first issue was the source of algal species for the experiments. A pond in the city of Woodland 

was found to have a dominant culture of the filamentous cyanobacterium Oscillatoria. It was 

decided to use this alga in the initial tests. First the alga was isolated from a small pond sample 

and grown in the laboratory. It was then inoculated into a small circular pond fed with settled 

Richmond algae. The results of the first experiment are shown in Figure III.A.2. The 

Oscillatoria culture grew, but the unicellular organisms grew faster. Only by completely 

harvesting the entire culture and recycling all of the biomass retained in the microstrainer (almost 

pure Oscillatoria), could the culture be maintained. 

It was thought that perhaps the laboratory cultivation stage had selected for a laboratory-adapted 

strain that did not do well outdoors. A small microstrainer was taken to Woodland, and enough 

Oscillatoria biomass was collected and returned to the Richmond Field Station to inoculate one 

of the small circular ponds. The pond was diluted about one-third per day with wastewater, with 

about 40% of the algae harvest recycled. However, after a couple of weeks, a new algal species 

became dominant. This species, Micractinium, a colonial organism covered with large spines 

(thought to be a protection against zooplankton grazers), also was captured by the (26-μm 

opening) fabric of the microstrainers and recycled. This gave a Micractinium competitive 

advantage over the unicellular forms, but also over the Oscillatoria. Several more inoculations 

of Oscillatoria gave similar results, with Micractinium dominating. 

However, even Micractinium could not be stably cultured with recycling, as during apparently 

unfavorable conditions it washed out (probably due to loss of spines) and then was replaced by 

Scenedesmus. On the other hand, when conditions were favorable for Micractinium, biomass 

recycle helped the culture dominate the ponds faster, but, regardless of recycling, this alga 

replaced Scenedesmus. Thus, the theory worked in principle, but in practice selective biomass 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 149 

recycle could be only one, and not a sufficient, tool for controlling microalgal species in such 

ponds. 

Biomass productivities equivalent to a total solar energy conversion efficiency of about 2% 

(about 15 g/m2

/d) were achieved for about 1-week periods. Effluents from the more harvestable 

cultures were below the EPA wastewater treatment system discharge limits of 30 ppm suspended 

solids (SS), with reductions of over 80% for ammonia and 50% for total organic C. A 

preliminary economic analysis was also presented (Benemann et al. 1977). 

Publications: 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Oswald, W.J. (1977b) 

“Species control in large scale microalgae biomass production. Report to Univ. Calif. Berkeley,

SERL 77-5, SAN/740-77/1 (April 1977, revised November 1997). 

Benemann, J.R.; Oswald, W.J. (1966) “Systems and economic analysis of microalgae ponds for 

conversion of CO2 to biomass.” Final Report to the Pittsburgh Energy Technology Center, Grant 

No. DE-FG22-93PC93204. 

Golueke, C.G.; Oswald, W.J. (1965) “Harvesting and processing sewage-grown planktonic 

algae.” J. Wat. Poll. Cont. Fe. 37:471-498. 

Oswald, W.J. (1963) “The high-rate pond in waste disposal.” Dev. Ind. Microbiol. 4:112-125. 

Shelef, G.A; Sukenik, A.; Green, M. (1984) “Microalgae harvesting and processing: A literature 

review.” Report, Solar Energy Research Institute, Golden Colorado, SERI/STR-231-2396. 

Weissman, J.C.; Benemann, J.R. (1978) “Biomass recycling and species control in continuous 

cultures.” Bioeng. Biotech. 21:627-648. 
National Renewable Energy Laboratory 

150 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.A.1. Selection process through biomass recycle. 

A theoretical model of selective cell harvesting and recycling resulting in dominance of the 

slower-growing over the faster-growing species. (Source: Benemann et al. 1978.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 151 

Figure III. A.2. Initial experiment demonstrating the maintenance of Oscillatoria in outdoor 

ponds. 

Maintenance of a culture of Napa Oscillatoria using selective recycle. Bars indicate the relative 

volume, concentration and filament length distribution of Oscillatoria (white bar height 

indicates filaments shorter than 150 μm; vertical stripes indicate filaments between 150 and 375 

μm; slashes indicate filaments longer than 375 μm). Volumes of single algal cells were 

calculated on an equivalent concentration basis and are shown as triangles (shaded area, top). 

Vertical arrows marked R indicate when the culture was harvested with a microstrainer (30-μm 

opening fabric) and the captured algae (almost all Oscillatoria) returned to the pond. 

Experiments were carried out in a 3-m2

 circular pond mixed continuously at 3 cm/s. Settled raw 

sewage, supplemented with 100 ppm of bicarbonate was used as substrate. (Source: Benemann 

et al. 1977). 
National Renewable Energy Laboratory 

152 A Look Back at the Aquatic Species Program—Technical Review 

III.A.3 An Integrated System for the Conversion of Solar Energy with 

Sewage-Grown Microalgae 

During FY1976-1977, the project described in Section III.A.2. continued with the same overall 

objective: to determine what pond operating factors could allow control over algal species, and 

thus permit cultivation of algal types that allow low-cost harvesting by microstrainers. The 

biomass recycling process described earlier continued to be studied, using the 12-m2

 rectangular 

ponds and various pond operating strategies (mixing speeds, retention times, biomass recycle 

fraction), to test for their influence on microalgae species composition and productivity. Mainly 

the four 12-m2

 ponds were used, with some initial experiments with the large 0.25-ha pilot pond. 

An extensive series of experiments was carried out in the small ponds, with daily analysis of 

suspended solids (SS) concentrations (the best measure of algal biomass, although some, 10%-

15%, contributions from wastewater and bacterial solids could not be avoided). Other 

parameters measured, less frequently, were chlorophyll concentrations, BOD5, P, ammonia, and, 

microscopic algal counts, including cell dimensions. Experimental pond operating parameters 

tested included retention time (hydraulic dilution) and depth (though this was typically 25 cm), 

mixing speed, and biomass recycle ratios. 

Both at short and long retention times the algal cultures invariably became unharvestable with 

microstrainers. Intermediate hydraulic retention times selected for larger colonial algal species 

that were more readily harvestable. However, long retention times also resulted in low 

productivities. There was an optimum residence time, which varied with depth of the culture and 

climatic variables that selected for harvestable cultures. However, biomass recycling was only 

marginally effective in improving biomass harvestability by microstraining. Mixing speeds >15 

cm/s also improved harvestability by microstraining. Mixing speeds of 15 to 30 cm/s tended to 

induce algal flocculation. 

Problems were encountered with zooplankton grazing of the algal cultures. Coarse (150-μm) 

screens did not effectively remove the grazers. Shorter retention times reduced grazer pressures, 

but also made the cultures less harvestable by microstrainers. In all the ponds, Scenedesmus

dominated in the winter and spring, and then was replaced with Microactinium. Loss of 

dominance correlated with the breakup of the colonies, which may have been related to 

zooplankton grazing. The best productivity was 13.4 g/m2

/d, during a 10-month period, 

irrespective of harvest efficiency. For the most harvestable pond, productivity was only 8.5 

g/m2

/d (of which only 7.2 g/m2/d was harvested by the microstrainers). Clearly, optimizing for 

productivity and harvestability required quite different operating conditions. It was concluded 

that the use of microstrainer harvesting and biomass recycling was unlikely to lead to both a high 

algal productivity and effective harvesting process. 

After growing and harvesting an algal culture on sewage, enough nutrients remain to grow a 

second crop of microalgae. Such a second crop would then deplete available N. Due to excess 

inorganic and organic phosphates in sewage, sufficient P remains after harvest of the second 

algal crop to allow cultivation of additional batches of N-fixing microalgae. Of course, due to C 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 153 

limitation, CO2 must be supplied to these cultures. Such a process, which would achieve 

so-called tertiary wastewater treatment (nutrient removal) is shown schematically in Figure 

III.A.3., and was demonstrated during this project. Considerable problems were encountered 

with the secondary ponds (shown as a small box labeled “green algae” in Figure III.A.3.), due to 

culture instabilities, lack of sufficient algal removal in the first stage, grazers, etc. Figure III.A.4. 

shows a composite of the general results obtained, with a 7-day batch cultivation, at which point 

the culture settled quite well and, from its yellowish color was apparently N limited. The final, 

N-fixing, stage in this process was demonstrated under the following year's project and a 

NSF-RANN funded project (Benemann et al. 1977). 

In May 1977, cultures were started in the 0.25-ha high rate pond. That pond, mixed (poorly) 

with centrifugal pumps located in a 2.5-m deep sump, exhibited rather poor hydraulics. Sewage 

supply limitations resulted in longer retention times than desired. Despite these and other 

operational problems, the results were “reasonably consistent with the smaller 12-m2

 ponds, both 

in productivity and harvestability responses to detention time” (Benemann et al. 1978). 

Productivities of 19 g/m2

/d were observed over an 18-day campaign in summer, with a retention 

time of 3.5 days. Interestingly, zooplankton grazing was not as big a problem as with the smaller 

ponds. 

These 0.25 ha pond algal effluents were also tested for settling, in a “pond isolation” experiment, 

in which cultures were removed from the pond and held for as long as 3 weeks in a settling pond. 

In one experiment, when the culture had been grown at a dilution rate at 0.22 day-1, it settled 

more than 90% in one day, while a culture grown at a dilution rate of 0.5 day-1 required 3 weeks 

to settle. This suggested another approach to algal harvesting, which became the focus of the 

project described in Section III.A.4. 

Publications: 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Oswald, W.J. (1978) “An 

integrated system for the conversion of solar energy with sewage-grown microalgae.” Report, 

Contract D(0-3)-34, U.S. Dept. of Energy, SAN-003-4-2, June 1978. 
National Renewable Energy Laboratory 

154 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.A.3. Process schematic for tertiary wastewater treatment with microalgae. 

This schematic for an advanced wastewater treatment process uses a multi-stage pond system for 

complete organic waste degradation and nutrient removal. The initial wastewater treatment 

ponds are shown, followed by a smaller intermediate “green algae” pond for N depletion and a 

final pond for cultivating N-fixing blue-green algae and removing residual phosphates. CO2

supplementation would be required in the last two ponds, and could increase productivity in the 

initial pond. (Source: Benemann et al. 1978.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 155 

Figure III.A.4. Growth of microalgae on pond effluents. 

Graph showing typical changes in density, chloropyll a content and ammonia concentrations 

during batch growth of green microalgae on effluents from wastewater treatment ponds after 

settling of the primary culture. As the culture grew from the residual concentrations of about 20 

mg to about 170 mg/L, ammonia concentrations and chlorophyll a levels decreased. A 4- or 5-

day culture period appears to be optimal. (Source: Benemann et al. 1978.) 
National Renewable Energy Laboratory 

156 A Look Back at the Aquatic Species Program—Technical Review 

III.A.4. Large-Scale Freshwater Microalgal Biomass Production for Fuel and 

Fertilizer 

This 2-year project (Benemann et al. 1979; Eisenberg et al. 1980) extended from the end of 1977 

to Winter 1979. Initially the approach to establish microstrainable cultures using the 12-m2

ponds, described in Section III.A.3., continued to be investigated. Essentially the same results as 

before were obtained: detention time was found to be the key environmental variable determining 

algal colony size (but not necessarily species composition) and a negative correlation was found 

between numbers of algal grazers and the large colonial algal types easy to harvest with 

microstrainers. Apparently the grazers preferentially consumed the smaller algae. Overall, the 

harvestability results with the microstrainers continued to be poor, so this line of research was 

abandoned during the initial period of this project. 

Simultaneously with these studies, another project was being carried out under EPA sponsorship 

to study the settling of algae in the City of Woodland waste treatment ponds. This project used a 

“phase isolation” process, in which the algal cells were allowed to spontaneously settle when 

sewage inflow was stopped (Koopman et al. 1978, 1980). Although generally long times were 

required for this settling process (2-3 weeks), it was decided to investigate this general 

phenomenon of “bioflocculation” in high rate ponds. The process involved removing the algae 

from the paddle wheel-mixed ponds and placing them in a quiescent container, where they would 

spontaneously flocculate and rapidly settle. There are several apparently distinct mechanisms by 

which algae flocculate and then settle, including “autoflocculation”, which is induced by high pH 

in the presence of phosphate and divalent cations (Mg2+ and Ca2+), and flocculation induced by N 

limitation. Bioflocculation refers to the tendency of normally repulsive microalgae to aggregate 

in large flocs, that then exhibit a rather high sedimentation velocity. The mechanisms of 

bioflocculation involve extracellular polymers excreted by the algae, but the details remain to be 

investigated. 

Settling tests were carried out with the cultures from the 12-m2

 ponds. As with microstrainer 

harvesting, detention time and mixing velocity were the most important variables in promoting a 

bioflocculating culture. The rather rapid settling of many of the cultures was very encouraging. 

Also, the initial experiments with the 0.25-ha pond demonstrated a fairly rapid (<24 h) 

bioflocculation process. 

At this time (mid 1978), the 0.25-ha pond had been divided into two 0.1-ha high rate ponds 

mixed with paddle wheels, and the bioflocculation-settling process using these pilot ponds 

became the focus of further research. Two 32,000 L settling ponds, with concave bottoms as 

deep as 2.5 m, were constructed to test bioflocculation-settling with the effluents of these ponds 

at the pilot scale. In initial experiments the two ponds were operated in parallel with two smaller 

ponds, at similar dilution rates (and with the only difference that one set of ponds was screened to 

remove larger grazers). The results (summarized in Table III.A.1.) were reasonably reproducible 

between the ponds. Algal solids removal through bioflocculation, as measured by 24-h settling 

in an “Imhoff Cone,” was high, about 90%. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 157 

Additional experiments, with the ponds operated in parallel at 2.5- and 5-day retention times 

(during September 1978) again resulted in removal efficiencies of over 90% for both ponds, 

including the 32,000 L settling tanks, based on measurements of suspended solids and 

chlorophyll. However, the shorter retention-time pond had more than twice the productivity of 

the longer-retention time pond (15 versus 7 g/m2

/d). Bioflocculation was established as the 

method of choice for algal harvesting, as it seemed to be achievable even with high productivity 

cultures. Culture settleability was routinely determined during all the experiments with the high 

rate ponds. Table III.A.2. summarizes productivity and settleability for more than 1 year for the 

two 0.1-ha ponds. The 24-h laboratory settling tests correlated well with settling in the large (32-

m3

) settling ponds. Overall, a settling efficiency of greater than 85% was achieved, if the better 

of the two ponds was selected, without compromise in productivity. Annual gross productivity 

averaged almost 20 g/m2

/d, estimated at about 90% algal solids. In many cases bioflocculation 

removals were higher than 95% on a chlorophyll a basis, indicating almost complete 

sedimentation of the algal biomass. 

One factor that had a major effect on bioflocculation was mixing speed. At very low mixing 

velocities, the cultures settled very poorly. This is not unexpected, as any settling algae would 

have dropped out of the photic zone and been replaced by suspended cells in the absence of 

continuous mixing. However, a more fundamental investigation of the bioflocculation 

phenomenon (well reported in the ecological and some physiological literature) remains to be 

carried out. 

Another activity carried out during this project was the cultivation of N-fixing microalgae using 

the supernatant of secondary growth ponds (see Figure III.A.3.). One problem was that culture 

collection strains of N-fixing cyanobacteria inoculated into the N-deficient ponds quickly 

succumbed. However, when the N-deficient wastewater samples were incubated in sunlight for a 

few weeks, N-fixing strains, which were indeed culturable, appeared spontaneously. One strain, 

an Anabaenopsis sp., was successfully cultured over long periods. However, the productivity of 

N-fixing cultures was only about half of what was observed with green algae under similar 

conditions. That is understandable because of the high energy requirement of N-fixation, which 

is not desirable in biomass fuel production. However, use of such algae for phosphate removal 

from wastewaters is of interest. 

This project also investigated the anaerobic digestion of algal biomass using both a large (1,400-

L) and several smaller (20-L) digesters. Algae provided a good substrate for anaerobic digestion, 

although not as good as conventional sewage sludge based on the conversion of organic C to 

methane. However, attempts to regrow algae on the effluent of the digesters resulted in poor 

productivities, as some unidentified factor appeared to limit growth on the regenerated nutrients. 

This was not investigated further, but clearly requires some attention in the future. 

As part of this project, the energy requirements for high-rate pond operations, in particular 

mixing, were studied. The shaft power input required for mixing followed closely the predicted 

cube power law, demonstrating the need to keep mixing velocities below 30 cm/s to avoid 

excessive power inputs. At 15 cm/s, power inputs were only about 1 kWh/d (for the 0.1-ha 
National Renewable Energy Laboratory 

158 A Look Back at the Aquatic Species Program—Technical Review 

pond), increasing to about 10 kWh/d at 30 cm/s. Motor efficiency at the higher velocity (67%) 

was twice that at the lowest velocities, not a deciding factor. Clearly, mixing speeds higher than 

30 cm/s are impractical, at least for energy production systems. 

An energy analysis of such a microalgae wastewater treatment process was quite promising, with 

the wastewater system being at least energy self-sufficient based on the requirements for sewage 

and algal pond effluent lift requirements, as well as algal pond mixing and other requirements. 

Such systems should be able to generate a net positive energy output if operated to maximize 

algal biomass through the addition of CO2 (Figure III.A.3.). 

Overall this project marked a major advance in this technology, as it demonstrated at the pilot 

plant scale a relatively low-cost and reasonably reliable microalgae harvesting process that does 

not unduly interfere with other objectives, in particular microalgae productivity. (See Benemann 

et al. [1980] and Eisenberg et al. [1980].) 

Publications: 

Benemann, J.R.; Weissman, J.C.; Eisenberg, D.M.; Goebel, R.P.; Oswald, W.J. (1979) “Large 

scale freshwater microalgae production for fuel and fertilizer.” Report to the Univ. of Calif. 

Berkeley, SERL 79-3 (1979), Contract EY-76-S-03-0034. 

Eisenberg, D.M.; Benemann, J.R.; Tiburzi, T.; Weissman, J.C.; Oswald, W.J. (1980) “Large 

scale freshwater microalgae production for fuel and fertilizer.” Report to the Univ. of Calif., 

Contract EY-76-S-03-0034. 

Eisenberg, D.M.; Koopman, B.; Benemann, J.R.; Oswald, W.J. (1981) “Algal bioflocculation and 

energy conservation in microalgae sewage ponds.” Biotech. Bioeng. Symp., No. 11, pp. 429-449. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 159 

Table III.A.1. Microalgae harvesting by bioflocculation: Small and large pond results. 

Comparison of performance between two 1,000-m2

 and two 12-m2 ponds, operated under 

otherwise similar conditions except that one pond was screened to remove zooplankton and the 

other one was not so treated. Data averaged for July 1978. Note agreement in terms of 

productivity (grams VSS/m2

/d, essentially some 90% AFDW algal biomass). (Source: 

Benemann et al. 1979.) 
National Renewable Energy Laboratory 

160 A Look Back at the Aquatic Species Program—Technical Review 

Table III.A.2. Productivity and settleability of microalgae in the 0.1-ha high-rate ponds. 

Productivity (AFDW of suspended solids) averaged monthly. Varying hydraulic dilution rates 

and depths account for differences in productivities and harvestabilities between ponds. Algal 

biomass were estimated by microscopy at about 85% to more than 90% of AFDW productivity, 

the rest being waste derived solids. (Source: Benemann et al. 1979.) 

* Imhoff Cone removals are 24-h laboratory settling tests, indicating percentage of algal biomass 

that spontaneously flocculate and settle (“bioflocculation”) under quiescent conditions after the 

culture is removed from the mixed ponds.
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 161 

III.A.5. Other Microalgae Projects During the ERDA/DOE Period 

During the 1975-1979 period, several other projects on microalgae fuels production were funded 

by ERDA/DOE, including the biophotolysis projects using heterocystous cyanobacteria, 

discussed earlier (see review by Benemann et al. 1980). Another biophotolysis project tested an 

optical fiber system for diffusing solar light into algal cultures, thereby overcoming the light 

saturation limitation to photosynthetic efficiencies (Manley 1979). This was shown to be 

impractical and was abandoned after only some very initial work. However, optical fiber 

photobioreactors are today the centerpiece of the 10-year, very large Japanese R&D program for 

microalgae CO2 utilization (Section IV.B.1.c.). 

In 1976, Lawrence Livermore National Laboratory (LLNL) established a well funded in-house 

project that was very similar to the University of California-Berkeley project, including the use 

of microstrainers for harvesting filamentous microalgae, biomass recycle, and even a 

biophotolysis component using heterocystous cyanobacteria (Jeffries et al. 1977; Timourian et al. 

1997). This project failed to receive support from ERDA, and was disbanded in 1977. 

Professor Harry Gregor at Columbia University was funded for 2 years to develop membrane 

systems for cross-flow filtration harvesting of microalgae. However, the membranes available at 

the time, the pressure drops required, and the fouling problems encountered made this approach 

impractical (Gregor and Gregor 1978). 

At Woods Hole Oceanographic Institutions, Drs. John Ryther and Joel Goldman carried out 

extensive research on microalgae cultivation in outdoor ponds on mixtures of seawater-secondary 

sewage effluent. When Dr. Ryther relocated to the Harbor Branch Oceanographic Foundation in 

Florida in the late 1970s, he was supported by DOE and later the ASP for the production of 

freshwater plants (water hyacinths, etc.) and seaweeds (Ryther 1981; 1982; 1983), as well as for 

microalgae culture collection work (See Part II.A.2.). Dr. Goldman also wrote a review on the 

theoretical and practical aspects of microalgae cultivation under contract with DOE (Goldman 

1979a,b). One conclusion was that the productivity of microalgae systems would be limited, 

because of the light saturation effect and other factors, to below 50 mt/ha/yr. Although the 

analysis was correct, it was a very conservative conclusion, making no allowance for 

productivity improvements caused by fundamental and applied R&D advances, as discussed in 

the remainder of this report. 

Publications: 

Benemann, J.R.; Miyamoto, K.; Hallenbeck, P.C. (1980) “Bioengineering aspects of 

biophotolysis.” Enzyme and Microbial Technology 2:103-111. 

Goldman, J.R. (1979a) “Fuels from solar energy photosynthetic systems—state of the art and 

potential for energy production.” Report to the U.S. Dept. of Energy COOO-4141-241. 

Goldman, J.R. (1979b) “Outdoor algal mass cultures. I. Applications. II. Theoretical Aspects.” 

Water Research 13:1-19. 
National Renewable Energy Laboratory 

162 A Look Back at the Aquatic Species Program—Technical Review 

Gregor, H.P.; Gregor, C.D. (1978) “Synthetic membrane technology.” Sci. Amer. 239:112-128. 

Jeffries, T.W.; Mouthrop, P.H.; Timurian, H.; Ward, R.L.; Berger, B.J. (1977) “Biosolar 

production of fuels from algae.” UCRL 52 177, November 16, 1976. 

Manley, S. (1979) Unpublished Report to U.S. Department of Energy, for work in the laboratory 

of G. Pelofsky. 

Ryther, J.H. (1981) “Cultivation of macroscopic marine algae.” Proceedings of the July 1981 

Subcontractors’ Review Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden Colorado, SERI/CP-624-1228, pp. 111-118. 

Ryther, J.H. (1982) “Cultivation of macroscopic marine algae.” Proceedings of the June 1982 

SERI Biomass Program Principal Investigators' Review Meeting, Aquatic Species Program 

Reports, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1808, pp 85-102. 

Ryther, J.H. (1983) “Cultivation of macroscopic marine algae.” Proceedings of the March 1983 

Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden, Colorado, SERI/CP-231-1946, pp. 79-88. 

Timurian, R.; Ward, R.L.; Jeffries, T.W. (1977) “Growth of filamentous blue-green algae at high 

temperatures: a source of biomass for renewable fuels.” IZ Int. Seaweed Symp., Aug 20-27. 

III.B. The ASP Microalgal Mass Culture

III.B.1. Introduction 

The long-term objective of the ASP was to develop microalgae liquid fuel production processes. 

Since its inception, the ASP supported laboratory R&D projects (Section II) and algal mass 

culture projects. However, for the most part, the laboratory and outdoor projects were not 

integrated into a strongly unified program. This reflects in large part the difficulty of such 

integration. Also, during the early stages of the ASP, too close an integration would have been 

restrictive, as it was not yet clear at the time which research approaches, production systems or 

algal strains would be best. 

The extensive work on strain isolation, selection, characterization, etc., carried out by the ASP 

was used to a significant extent by the field projects, through the testing of a number of the 

isolates in algal mass cultures, specifically in the projects reviewed in this section. 

Unfortunately, the laboratory-level screening protocols had, in hindsight, relatively little 

predictive power for the ability of the strains to dominate and perform in outdoor ponds. 

Similarly, the laboratory work on the biochemistry, genetics and physiology of lipid biosynthesis, 

was difficult to apply to the goal of increasing lipid productivities in outdoor systems. Greater 

integration of laboratory and outdoor R&D is a challenge for any future microalgae R&D 

program. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 163 

The ASP initiated two outdoor projects in 1980, one in California using a paddle wheel-mixed 

raceway pond design (“high rate pond,” [HRP]), and another in Hawaii. The Hawaii project was 

to demonstrate a patented algal culture system, invented by then-ASP program manager, Dr. 

Larry Raymond (1981). This “Algal Raceway Production System” (ARPS) used very shallow 

flumes (<10 cm), rapid mixing by air lifts, covers with CuSO4 filters to screen out harmful 

infrared radiation, and harvesting of the biomass by foam fractionation, among many other 

claimed attributes (Figure III.B.1.). Very high productivities were claimed. But a review of the 

work (Raymond 1979), in which P. tricornutum was grown in a 0.5-m2

 system, revealed that this 

projection derived from a single batch culture, and in fact, the last data point showed biomass 

density actually decreasing. Benemann et al. (1982a,b, see Section III.D.5.), carried out a 

comparative analysis of the ARPS and the HRP designs, concluding that the ARPS would be too 

expensive and energy intensive, compared to the HRP design. These two projects in California 

and Hawaii, carried out for more than 6 years each, are reviewed in this section, followed by 

descriptions of the ASP projects in Israel, New Mexico, and related projects. 

Publications: 

Raymond, L. (1979) “Initial investigations of a shallow layer algal production system.” Am. Soc. 

Mech. Eng., New York. 

Raymond, L. (1981) “Mass algal culture systems.” U.S. Patent 4,253, 271. 
National Renewable Energy Laboratory 

164 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.1. The algal raceway production system. 

(Source: Raymond 1981.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 165 

III.B.2. The ARPS Project in Hawaii, 1980-1987 

III.B.2.a. Hawaii ARPS Project Initiation, 1980-1981 

As mentioned earlier, the concept for the ARPS project derived from the Raymond (1981) patent. 

The project was initiated in early 1980s, with construction of a single, 48-m2

 raceway system 

completed in early 1981 (Laws 1981). During this first year, chemostat experiments using two 

strains of P. tricornutum were carried out. The tests revealed large differences in protein and 

lipid productivity between the strains. This laboratory work also investigated cell harvesting by 

“foam fractionation” in which the foam formed by the aeration of the cultures via air lift mixing 

was collected and found to contain some 10-40 times the cell concentration of the liquid. 

However, the harvesting efficiency was not reported. 

One difficulty noted in the laboratory experiments was the low cell densities achieved, compared 

with the original reports by Raymond for the ARPS system. Researchers tried to increase cell 

density by increasing the pond depth to 0.6 m, rather than 0.1 m as proposed by Raymond 

(1979). This resulted in other problems (low cell density, shading-see below), and the depth was 

again reduced to 30 cm. The laboratory experiments were extrapolated to predict an outdoor 

productivity of almost 130 mt/ha/yr, at least for the best P. tricornutum strain, similar to the 

Raymond prediction. However, this extrapolation was based on the invalid assumption that such 

laboratory growth rates can be used to predict outdoor productivities. 

The initial work also studied the effect of CuSO4 filters, concluding that although any 

productivity increases would be minor, installation of CuSO4 filters is advisable in the APRS, “as 

it would help manage overheating of the culture and store a potentially valuable by-product, 

heat.” Finally, the “flashing light” effect was investigated. The time constants (1 s light: 1 s 

dark), and low light intensities used were quite different from the classic flashing light effect of 

Kok (1953), which uses approx. 1-5 μsec high intensity light flashes, followed by about five 

times longer dark periods. Only small, “not particularly encouraging”, productivity increases 

were noted. Still, a 70% increase in productivity was predicted, though the basis for this was not 

stated. 

Laws (1981) also reported on initial results with the 48-m2

, 0.6-m deep, airlift-mixed flume 

system. Cell densities were much lower than predicted, likely because of the great depth of the 

culture, which was later reduced. The report concluded that, assuming $30,000/ha/yr production 

cost, a biomass production of 180 mt/ha/yr AFDW would allow oil production (with protein 

byproducts) competitive with fossil fuel. However, this productivity figure was extrapolated 

from the indoor chemostat work, and increased by one-third, as “effects of modulated blue light 

on the system will allow the extra production to be achieved,” so the reliability of this prediction 

is questionable. 
National Renewable Energy Laboratory 

166 A Look Back at the Aquatic Species Program—Technical Review 

III.B.2 b. Second Year of the Hawaii ARPS Project, 1981-1982 

The second year of this project emphasized the use of “flashing light to enhance algal mass 

culture production” (Laws, 1982; see also Laws et al. 1983). The basic idea was that a “foil 

array” in the pond culture would generate a vortex that would create organized mixing in the 

ponds, expected to result in exposure of the cells to regular dark-light cycles (Figure III.B.2.). 

Based on data in the literature, this effect would be predicted to increase overall productivity. 

These a priori arguments were not supported by the algal physiological literature (the flashing 

light productivity enhancements are observed at much shorter time constants), and neither were 

the hydraulic arguments plausible (organized mixing would be seen only in a small fraction of 

the pond volume). However, the key issue here is not the theory but the actual experimental 

results. 

From November 1981 to January 1982, an average productivity of only about 3.3 g/m2

/d was 

recorded for the 50-m2

-flume reactor, a very low value for Hawaii, even in winter. After 

installation of the foils, productivities, from February to March 1982, increased to about 11 

g/m2

/d. This increase was attributed to the effect of the foils, though lack of a control did not 

allow isolation of this variable from other effects. Five-day running mean average 

photosynthetic efficiencies (PAR) are shown in Figure III.B.3. The author stated that 

productivity could be doubled with semi-continuous operations. One observation was infestation 

of the culture by algal predators, which could have been one reason for the rather large variability 

in productivities observed during this operation (Figure III.B.3.). However, day-to-day 

variability in productivities is a fact of outdoor pond microalgae cultivation, even in the best of 

cases. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 167 

Figure III.B.2. Hawaii ARPs with mixing foils 

a. (Top). Schematic of the 48-m2

 flume, showing heat exchangers, lift box, drain box and airlift 

mixer. 

b. (Bottom) Schematic of mixing resulting from foils inserted in the shallow flumes. 

(Source: Laws 1982.) 
National Renewable Energy Laboratory 

168 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.3. Five-day running productivity averages for the Hawaii system. (Source: 

Laws 1982.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 169 

III.B.2.c. Third Year of the Hawaii ARPS Project, 1982-1983 

Laws and Terry (1983; see also Laws 1984a) reported on the further development of the ARPS. 

Four 9.2 m2

 experimental raceways were built to allow replication of experiments and testing of 

variables, again using P. tricornutum. These raceways were used in a multifactorial experimental 

design testing eight variables at two levels each: 

1. depth (5 and 10 cm), 

2. dilution rate (0.2 and 0.4 d-1), 

3. night time temperatures (15°or 20°C), 

4. flow rates (15 and 30 cm/s), 

5. CuSO4 filters (present and absence), 

6. pH (8 and 9), 

7. N source (ammonia and urea), and 

8. salinity (15 and 35 ppt). 

Sixteen runs (of 256 possible) in four sequential blocks were carried out, with the assumption 

apparently being that these variables are non-interacting and additive (probably a poor 

assumption for biological processes). No block-to-block controls were provided, which could 

have been affected by light intensity and other variables. 

The ponds had been equipped with six sets of mixing foils, but rather suprisingly the presence or 

absence of the foils was not a variable tested. A complex data evaluation, in terms of “factor 

effects”, was presented, but no actual productivity data for any of the experiments are available. 

The authors concluded that “by far the most significant factor affecting biomass production” was 

culture depth, arguing that the “self-shading effects were more than offset by higher areal 

standing crops.” This was a rather puzzling conclusion as it is contrary to both theory and 

experience, which assumes that, everything else being equal, depth should not affect 

productivity. Of course, depth can affect pH, temperature, pO2, and other variables, which if not 

held constant will indeed affect productivity. But these should have been second-order effects, 

not the overriding factor in determining productivity. 

Although no actual productivity data were reported, Laws (1984) stated that this factorial 

experiment demonstrated maximum productivities of about 25 g/m2

/d, corresponding to an 

approximately 5% light conversion efficiency. The author claimed that this was 50% to 100% 

better than achieved with “conventional culture techniques,” though the basis for such 

extrapolations or comparisons was unclear. 
National Renewable Energy Laboratory 

170 A Look Back at the Aquatic Species Program—Technical Review 

III.B.2.d. Fourth Year of the Hawaii ARPS Project, 1983-1984 

During this year a number of experiments were carried out to determine productivity of the 

cultures as a function of several variables, this time studied independently (Laws 1984b,c; see 

also Laws et al. 1985). One task was to screen candidate species in the laboratory for possible 

growth outdoors. However, as the author concludes, “the fact that a given species grows well in 

the laboratory is no guarantee that it will perform well in an outdoor culture system.” One reason 

the project switched to different algal species was that the P. tricornutum strain used in the 

experiments described above was quite sensitive to even moderate (above 25°C) temperatures, 

and required temperature control (cooling) of the reactors. A Platymonas sp. was thus tested 

without temperature control in the outdoor flumes, at several dilution rates and maximal pH 

levels of 7 to 8. This strain showed a maximum productivity of about 26 g/m2

/d, about the same 

as observed with P. tricornutum with temperature control. 

Another question raised was the reproducibility of the data obtained with the flumes. During this 

year all four flumes were operated under identical conditions for two periods, resulting in very 

similar productivities (5% and 10% standard deviations in the two separate experiments). The 

Platymonas sp. strain was then inoculated into the larger (48 m2

) flume which was operated at 

various depths. A depth of about 12-cm gave the best results, producing about 26 g/m2

/d during 

February 1984. 

One important experiment carried out, again with the Platymonas sp., was to determine the 

effects of N and P limitation on algal lipid content. Lipids increased from less than 20% to 

almost 40% of dry weight upon combined N and P limitation. However, no actual productivity 

data were reported for this 2-week experiment. 

The major experiment carried out this year (Laws 1984b,c), was the cultivation of Platymonas in 

the 48-m2 flume, over most of the year. One major variable was dilution, with the culture diluted 

every 2, 3, or 4 days. As seen in Figure III.B.4., maximal productivity (50 g/m2

/d) was achieved 

by diluting the culture every 3 days. Further investigation of this phenomenon suggested that this 

was not an artifact, but a reproducible effect. Indeed, such a culture diluted every third day had 

twice the productivity on the third as on the first 2 days. This was a most unexpected, and 

controversial, result, and a major focus for the following years of this project. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 171 

Figure III.B.4. Productivity as a function of dilution. (Source: Laws 1984.) 
National Renewable Energy Laboratory 

172 A Look Back at the Aquatic Species Program—Technical Review 

III.B.2.e. Fifth Year of the ARPS Project, 1984-1985 

In FY1984-85 (Laws 1985), the research was directed toward the study of more thermotolerant 

species. Algal strains collected by the ASP researchers in the southwestern United States were 

evaluated using the Type I and II waters (see Section II.A.1.for a description of SERI Type I and 

Type II media). Several species, including Platymonas sp. (used previously), Amphora sp., C. 

gracilis, and Boekelovia sp. were grown in the two water types, each at two salinities and at four 

temperatures (25° to 32°C), with the data reported as the number of doublings per day. One 

interesting, but unexplained, observation was that at higher temperatures there was a consistent 

shift, among all four algae, of maximum doubling rates to the higher salinity and Type II waters. 

The small outdoor flumes were used to test this cultivation strategy. The cultures were diluted 

each third day, to a concentration of 2 x 106

 cells. The results were “consistent with those of 

earlier studies,” with solar conversion (PAR) efficiencies close to 10% (5% of total solar). The 

C. gracilis species was also tested, though at a 2-day dilution rate (requiring a one per day 

doubling time), with somewhat lower efficiencies (8%), though still rather high productivities. 

Also, Tetraselmis suecica was cultivated in the ponds with good results. Over a 78-day cycle, in 

spring 1984 and summer 1985, productivity was 37+5 g/m2

/d, with a corresponding PAR 

efficiency of 9.1%. 

Removing half the arrays had no significant effect on productivity; removing all foils reduced 

light conversion efficiencies from 8% to 5.5%. However, a major variable in such systems is the 

pO2 in the ponds, which may well account for the difference observed. The higher mixing (e.g., 

power inputs) caused by the foils may have increased outgassing of O2 from the pond enough to 

increase productivity, rather than to any flashing light effect. The reports of increased 

productivities caused by 3-day batch dilutions and foils remained controversial, and continued to 

be the major focus of this project. 

III.B.2.f. Sixth Year of the Hawaii ARPS Project, 1985-1986 

Research during FY 1985-86 (Laws 1987a) elaborated on the two key findings mentioned earlier: 

effects of a 3-day dilution interval and of the foil arrays. The effects of foil arrays were tested 

over a 12-month period in the 48-m2

 flume with Cyclotella sp., a diatom, which, like

Chaetoceros, is a good lipid producer. The experiment involved alternatingly operating the pond 

with and without the foils for 2-week periods. The presence of foils increased productivity by 

almost a third, similar to the prior experiments. 

The dilution effect was investigated with T. suecica, also in the 48-m2

 flume, with similar results 

as before, in terms of both overall and maximal 3rd day productivity. However, solar conversion 

efficiencies were lower than observed in previous years, perhaps due to the approximately 3°C 

higher temperature during this year, compared to the previous one. The author speculated that 

this could have been close to the maximal permissible temperature for growth of T. suecica, and 

thus resulted in lower productivities. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 173 

However, the effect of dilution interval on production in the 48.4-m2 flume was somewhat 

puzzling (Laws 1986). These findings were a subject of considerable discussion and 

controversy. One possible explanation was the measurement of actual biomass density, which 

varied from about 27-28 g/m2

 after dilution, to 80, 140, and 160 g/m2 for the 2-, 3-, and 4-day 

dilutions periods, respectively. However, this was considered an “unlikely” explanation. Indeed, 

the highest productivity was observed on day 3, with a steep decline on day 4. However, 4-day 

cycle cells still had lower productivity on day 3. Some “lingering effect of exposure to 

supraoptimal density conditions” was speculated to account for this phenomenon. The classical 

technique for studying such phenomena is the P versus I curve. Such studies were carried out 

with T. suecica cultures grown in the smaller 9.2-m2

 flumes. However, as the author noted, the 

results were “somewhat discouraging” as there was no difference as a function of dilution 

intervals, and productivities were only about 24 g/m2

/d, much lower than reported with the larger 

flumes. Thus, this issue remained as a major focus of this project. 

III.B.2.g. Seventh Year of the Hawaii ARPS Project, 1986-1987 

During the final year of the Hawaii ARPS project (Laws 1987), the goal was to screen for 

additional algal species in the smaller flumes and to further study the effect of dilution intervals. 

Four species were tested in the 9.2-m2

 flumes: Navicula sp., C. cryptica, C. gracilis, and 

Synechococcus sp. From prior work (see earlier Section, and also Laws et al. 1986, 1987a), 

photosynthetic efficiencies of 9.1% were reported with T. suecica, during a 78-day period, and 

9.6% for 122 days with C. cryptica. With the three other organisms listed above, somewhat 

lower efficiencies were noted during shorter time periods: 7.8 % for Navicula sp., 8.5% for C. 

gracilis, and 8.6% for Synechococcus. Somewhat “surprisingly” (their characterization), they 

observed that in a 2-day batch growth mode, initial cell concentrations ranging from about 50 to 

400 mg/L (AFDW) had no major effect on productivity. For C. cryptica, at an initial 

concentration of 40 mg/L at a depth of 12 cm, this would give an areal cell density of about 5 

g/m2

. For an equal daily productivity of 30 g/m2/d, averaged over 2 days, this would require the 

cells to divide 2.5 times the first day, and once the second day. Not impossible, certainly, but 

somewhat problematic. There is indeed some likelihood that some systematic measurement error 

influenced their productivity measurements (John Ryther, private communications, circa. 1986). 

This report also described lipid induction by Si limitation by C. gracilis and C. cryptica. In both 

microalgae Si limitation greatly reduced overall productivities, and lipid productivities, even 

though lipid contents increased. Laws (1987) concluded that lipid productivities would be 

maximized by maximizing total biomass production. 

In the final paper, Laws et al. (1988), reported on long-term (13-month) production of C. cryptica

in the large flume, with a 9.6% solar conversion efficiency reported with the foils and 7.5% 

without the foils, similar to earlier results with T. suecica. For 122 days, at optimal dilution (2-

day batch cycle) productivity of about 30 g/m2

/d was measured. This is, indeed, a high sustained 

productivity. 
National Renewable Energy Laboratory 

174 A Look Back at the Aquatic Species Program—Technical Review 

III.B.2.h. Hawaii ARPS Project, Conclusions 

This project evolved from one that focused on a demonstration of the ARPS concept using a 

single flume, to the investigation of fundamental issues in algal mass culture, using several 

smaller ponds and a simplified system design. In particular, this project reported very high 

productivities achieved by two methods: organized mixing in ponds (e.g., the foils), and optimal 

batch dilution (2- or 3-day intervals, depending on species). However, the basis for these 

productivity enhancements was speculative, and it proved difficult to demonstrate the 

reproducibility of these effects. The effects of foils could be better ascribed to degassing of 

oxygen from the ponds with foils (e.g., higher mixing power inputs) and the results from the 3-

day dilution experiments to some uncontrolled factors, in addition to possible methodological 

problems (Laws et al. 1985; 1986a,b; 1987). 

Laws (1989; see also Laws and Berning 1990) continued this research with Electric Power 

Research Institute funding for 1 year, moving the system to Kona, Hawaii. No significantly 

different information was produced. However, Laws concluded that lack of land area, and high 

costs, would make such a process impractical for fuel production in Hawaii. 

Publications: 

Cheng, E.D.H.; Dugan, G.L. (1996) “Low-energy mixing in algal culture raceways.” J. Energy 

Engineering, December, pp. 100-107. 

Kok, B. (1953) “Experiments in photosynthesis by Chlorella in flashing light.” In Algal Culture: 

From Laboratory to Pilot Plant (Burlew, J.B., ed.), Carnegie Inst. of Washington, Publ. 600, pp. 

63-75. 

Laws, E.A. (1981) “Year one progress report on algal production raceway project.” Proceedings 

of the July 1981 Subcontractors’ Review Meeting, Aquatic Species Program, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-624-1228, pp.1-32. 

Laws, E.A. (1982) “Results from preliminary studies on the use of flashing light to enhance algal 

mass culture productivity.” Proceedings of the June 1982 SERI Biomass Program Principal 

Investigators' Review Meeting—Aquatic Species Program Reports, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1808, pp. 31-42. 

Laws, E.A. (1984a) “Optimization studies in shallow algal mass culture flumes.” Proceedings of 

the April 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2341, pp. 108-131. 

Laws, E.A. (1984b) “Research and development of shallow algal mass culture systems for the 

production of oils.” Final Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/STR-231-2496. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 175 

Laws, E.A. (1984c) “Research, development, and demonstration of algal production raceway 

(ARP) systems for the production of hydrocarbon resources.” Subcontractor Report to the Solar 

Energy Research Institute, Golden, Colorado, SERI/STR-231-2206. 

Laws, E.A. (1985) “Productivity optimization of saline microalgae grown in outdoor mass 

culture.” Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species 

Program Review, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 

162-178. 

Laws, E.A. (1987) “Continuous microalgae production in shallow raceways at high 

productivity.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, pp. 209-225. 

Laws, E.A. (1987) “Productivity optimization of saline microalgae grown in outdoor mass 

culture.” Final Report, Contract XK-4-4109-1, Solar Energy Research Institute, Golden, 

Colorado. 

Laws, E.A. (1989) Final Report to the Electric Power Research Institute. 

Laws, E.A; Berning, B.L. (1991) Biotech. Bioeng. 37:936. 

Laws, E.A.; Lee, Y.K.; Tan, H.-M.; Hew, C.-S. (1985) “The effect of growth temperatures on the 

bioenergetics of photosynthetic algal cultures.” Biotech. Bioeng. 27:555-561. 

Laws, E.A.; Taguchi, S.; Hirata, J.; Pang, L. (1986) “High algal production rates achieved in a 

shallow outdoor flume.” Biotech. Bioeng. 28:191-197. 

Laws, E.A.; Taguchi, S.; Hirata, J.; Pang, L. (1988) “Optimization of microalgal production in a 

shallow outdoor flume.” Biotech. Bioeng. 32:140-147. 

Laws, E.A.; Terry, K.L. (1983) “Shallow algal mass culture system.” Proceedings of the March 

1983 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research 

Institute, Golden Colorado, SERI/CP-231-1946, pp. 33-46. 

Laws, E.A.; Terry, K.L.; Wickman, J.; Chalup, M.S. (1983) “Preliminary results from a simple 

algal production system designed to utilize the flashing light effect.” Biotech. Bioeng.

25:2319-2336. 

Raymond, L. (1979) “Initial investigations of a shallow layer algal production system.” Am. Soc. 

Mech. Eng., New York. 

Terry, K.L. (1984b) “Modulated light photobiology.” Proceedings of the April 1984 Principal 

Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2341, pp.160-169. 
National Renewable Energy Laboratory 

176 A Look Back at the Aquatic Species Program—Technical Review 

Terry, K.L. (1986) “Photosynthesis in modulated light: quantitative dependence on 

photosynthetic enhancement on the flashing rate.” Biotech. Bioeng. 28:988. 

III.B.3. High Rate Pond (HRP) Operations in California, 1981-1986 

III.B.3.a. HRP Design and Construction Phase, 1981 

The project described in this section succeeded the projects reviewed in Section III.A. that took 

place at the University of California-Berkeley. That research group moved essentially intact (as 

Ecoenergetics, Inc., later renamed EnBio, Inc.) to Fairfield, California, some 30 miles north of 

Berkeley, with a pond system set up in nearby Vacaville. 

The ASP funded this project starting in fall 1980. The objective was to demonstrate the HRP 

system using agricultural irrigation waters and fertilizers. The HRP was defined as a paddle 

wheel-mixed (approximately 10-20 cm/s), moderate depth (approximately 15-30 cm), algal 

production system. The R&D goal was to develop production technology for microalgae 

biomass with a high content of lipids. A detailed literature review concluded that the best option 

would be to use N limited (but not starved) batch cultures of green microalgae. 

The plan view of the facility is shown in Figure III.B.5. The system consisted of four 200-m2

and three 100-m2

 ponds, along with three deep harvesting ponds and four water and effluent 

storage ponds. This system thus provided considerable flexibility for the testing of a large 

number of variables and algal species, at a scale that would allow some confidence in the 

scale-up of the results. The units were lined with 20 mil PVC, to allow complete mass balances. 

The report to the ASP describing this work (Benemann et al. 1981) provided considerable detail 

on the design of the system and the various considerations that went into selection of different 

design options and operating variables. For example, Table III.B.1. lists the calculations on 

which basis the carbonation requirements for the ponds were estimated. 

After the facility was only partially constructed, the project was terminated by the ASP, as the 

Hawaii ARPS system, reviewed earlier, was deemed to have already demonstrated its superiority 

to the HRP design, even before any operations of either. However, after a hiatus of about 1 year, 

and with changes in the ASP management, funding for the California HRP project was reinstated 

in August 1982, and actual pond operations were initiated. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 177 

Figure III.B.5. Pond system design in California. 

The schematic shows the four 200-m2

 and three 100-m2 raceway ponds, three deep square algae 

harvesting (settling) ponds, and the mounds for location of the water supply and media recycle 

tanks. (Source: Benemann et al. 1981.) 
National Renewable Energy Laboratory 

178 A Look Back at the Aquatic Species Program—Technical Review 

Table III.B.1. Microalgae pond carbonation requirements. (Source: Benemann et al. 

1981.)
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 179 

III.B.3.b. HRP Operations in California, Oct-Nov. 1982 

After this project was restarted, construction was completed and the first inoculation of algae into 

one of the 100-m2

 ponds was made on August 13, 1982, using a mixed MicractiniumScenedesmus culture obtained from the Richmond wastewater ponds (Section III.A.). However, 

these algae settled out due to lack of flow deflectors, and the culture was soon dominated by a 

Selenastrum sp. Both biomass concentration and productivity were quite low. Without flow 

deflectors at the far end of the ponds (away from the paddle wheel) the hydraulics were so poor 

that the ponds exhibited almost zero productivity. This was due to the formation of large 

countercurrent eddies resulting in “dead zones,” where algal cells settled. After flow deflectors 

were installed, the pond was inoculated on September 21 with an almost pure culture of 

Scenedesmus that had arisen spontaneously in one of the 12-m2 inoculum ponds. The culture 

remained well suspended and grew well (Benemann et al. 1983). 

However, a similar inoculation into a 200-m2 pond resulted in almost complete settling of the 

culture, caused by poor pond hydraulics, even with similar flow deflectors installed. This 

indicated that the hydraulics of the ponds are critical to the success of the process and further, 

that the hydraulics are not predictable from one scale to another, even within a factor of two. 

After two flow deflectors were installed around the bends in the 100-m2

 ponds, these ponds 

exhibited much improved hydraulics, with few eddies or settling of algal cells. 

In contrast, similar deflectors did not improve hydraulics perceptibly in the 200-m2 ponds. Only 

after two more flow deflectors were installed at the end nearest the paddle wheels were 

satisfactory hydraulics observed in these larger ponds. A quantitative study of flow velocities 

was undertaken using a flow meter. The results were counterintuitive: flow velocities are higher 

on the inside than the outside of the channels. Clearly, pond hydraulics must be customized for 

each pond size and design to obtain even mixing. 

As expected, productivities were rather low in the initial experiments carried out during October 

and November 1982. Maximum productivities (measured for 2 days) were only about 9 g/m2

/d 

and average productivities less than 5 g/m2

/d. These initial experiments included assessment of 

species dominance, N limitations, and mixing velocities. Pond operations ceased by the end of 

November 1982. 

III.B.3.c. Continuing California HRP Pond Operations, 1983-1984 

This project was a continuation of the project described in III.B.3.b. It was performed by a new 

company, Microbial Products, Inc. (EnBio was dissolved when John Benemann left in 1983 for 

the Georgia Institute of Technology). The pond system described earlier continued to be used for 

this project. The objective was to obtain long-term productivity data with a pilot-scale system 

and generally demonstrate the requirements of large-scale algal mass cultivation (Weissman 

1984; Weissman and Goebel 1985, 1986). 

The first challenge was to obtain microalgal species that could be grown on the fresh to slightly 

brackish water available at the site. The common experience is that either inoculated strains from 
National Renewable Energy Laboratory 

180 A Look Back at the Aquatic Species Program—Technical Review 

culture collections fail to grow in the outdoor ponds, or that they grow initially but become 

rapidly outcompeted by indigenous strains. A common practice is to make the best of a bad 

situation and cultivate the invading organisms. This was also the experience and approach of this 

project. 

After inoculation of Scenedesmus obliquus strain 1450 from the SERI Culture Collection, a 

strain of Scenedesmus quadricuada invaded. This turned out to be the most successful organism, 

cultivated for 13 months in fresh water and an additional 3 months in brackish. After an Oocystis

sp. (Walker Lake isolate) was inoculated, a Chlorella sp. became dominant and was maintained 

(or maintained itself) for 2 months under semi-continuous dilution. However, some strains 

provided by SERI researchers could be grown for at least a few months outdoors, including an 

Ankistrodesmus falcatus and a freshwater Scenedesmus sp. So2a. 

Productivity for S. quadricuada grown semi-continuously which is harvested every few days (a 

“sequential batch” growth mode), averaged about 15 g/m2

/d for the 8 month period of March 

through October, with monthly averaged solar conversion efficiency ranging from 1.2% to 2.2% 

(Figure III.B.6.). “Typical” real biomass density just before harvest ranged from 60 to 100 g/m2

, 

except for May, which recorded the highest standing biomass (160 g/m2

) and productivity (20 

g/m2

/d). The continuously diluted cultures (diluted during the entire light period) exhibited 

approximately 20% higher productivity. 

From a large number (39) of experiments, a correlation of Tmax, Tmin, and total insolation with 

productivity reduced the variance in the prediction of productivity by about 50% when using any 

single variable, but not in combination. This suggested that one of these three factors generally 

dominated (e.g., too high or too low a temperature or too little insolation). Similar experiments 

were carried out with the other microalgae in combination with the study of variables such as 

mixing speed, O2 outgassing, CO2 addition, and N limitation (for lipid induction). 

The main conclusions of the extensive experimental program were: 

1. Productivities of 15 to 25 g/m2

/d were routinely obtained during the 8-month 

growing season at this location. However, higher numbers were rarely seen. 

2. Continuous operations are about 20% more productive than semi-continuous 

cultures, but the latter densities are much higher, a factor in harvesting. 

3. Culture collection strains fare poorly in competition with wild types. 

4. Temperature effects are important in species selection and culture collapses, 

including grazer development. 

5. Nighttime productivity losses increased to 10% to 20 % in July, when grazers 

were present; nighttime respiratory losses were high only at high temperatures. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 181 

6. There is a significant decrease in productivity in the afternoons, compared to the 

mornings, in the algal ponds. 

7. Oxygen levels can increase as much as 40 mg/L, over 450% of saturation, and 

high oxygen levels limit productivity in some strains but not others. Oxygen 

inhibition was synergistic with other limiting factors (e.g., temperature). 

8. Increasing TDS from 0.4 to 4 ppt decreased productivity, depending on strains. 

9. Mixing power inputs were small at low mixing velocities (e.g., 15 cm/s) but 

increased exponentially. Productivity was independent of mixing speed. 

10. The strains investigated in this study did not exhibit high lipid contents even 

upon N limitation. 

11. The transfer of CO2 into the ponds was more than 60% efficient, even though 

the CO2 was transferred through only the 20-cm depth of the pond. 

12. Harvesting by sedimentation has promise, but was strain specific and was 

increased by N limitation. 

13. Initial experiments demonstrated that media recycle is feasible. 

14. Project end input operating costs for large-scale production (@ $50/mt CO2, 

70% use efficiency, etc.) was $130/mt of algae, of which half was for CO2 and 

one-third for other nutrients, with pumping and mixing power only about 

$10/mt. 

This project answered a number of issues that had been raised about this process. One initially 

controversial observation was the finding that mixing speed had no effect on productivity (Figure 

III.B.7.). However, this experiment used a strain of Chlorella that did not settle, and care was 

taken to keep other parameters identical (in particular pH and pO2 levels). Thus, the increased 

productivities seen in some experiments (e.g., those of Hawaii), could possibly be accounted for 

by differences other than those of mixing, such as changes in outgassing of O2. 

From the perspective of large-scale biomass production, one conclusion from this research was 

that mixing power inputs make any mixing speed much above about 30 cm/s impractical, as the 

energy consumed would rapidly exceed that produced. The rate of mixing should only be 

between about 15 and 25 cm/s, sufficient to keep cells in suspension and transfer the cultures to 

the CO2 supply stations in time to avoid C limitations in large-scale (>1-ha) ponds. 

For low-cost production higher productivities would reduce capital, labor, and some other costs, 

but nutrient (e.g., CO2) related costs would not change. This suggested the need for low-cost 

CO2, and other nutrients, as well as a high CO2 utilization efficiency. Efficient utilization of CO2

appeared feasible based on the results obtained with even this unoptimized system. 
National Renewable Energy Laboratory 

182 A Look Back at the Aquatic Species Program—Technical Review 

Another major conclusion was that competitive strains would be required to maintain 

monocultures. The need for feedback from the outdoor studies to development of laboratory 

screening protocols was a major recommendation. Specifically, the relatively controllable 

parameters of CO2, pH, and O2 were of interest in determining species survival and culture 

productivity. Also, harvesting was identified as a specific area for further research. Finally, lipid 

induction remained to be demonstrated. These were the general objectives during the final year 

of this project, described in Section III.B.3.d. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 183 

Figure III.B.6. Long-term productivity of S. quadricauda in freshwater (100-m2

 pond). 

(Source: Weissman and Goebel 1985.) 
National Renewable Energy Laboratory 

184 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.7. Chlorella mixing velocity experiments. 

Data from 3.5-m2

 ponds operated under constant pH and pO2 levels and compared to a control 

culture operated at 30 cm/s mixing velocity. Numbers above the data points indicate productivity 

of the control. Max/min temperatures 20/30 o

C (15/25 oC for the 60 cm/s data set). (Source: 

Weissman and Goebel 1986.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 185 

III.B.3.d. Completion of the California HRP Project, 1985-1986 

Numerous microalgal strains were obtained from the SERI Culture Collection and tested in 

small-scale, 1.4-m2

, ponds (Weissman and Goebel 1986; 1987). All strains could be grown quite 

successfully in these small units, although some, such as Amphora sp., did not survive more than 

2 or 3 weeks before they were displaced by other algae. Cyclotella displaced Amphora under all 

conditions tested, even though Amphora was the most productive strain, producing 45 to 50 

g/m2

/d in short-term experiments. The green algae, e.g. Chlorella or Nannochloropsis, also 

could not be grown consistently. Their productivities were among the lowest, about 15 g/m2

/d 

(similar to that in the prior year). Thus, one fundamental conclusion was that productivity is not 

necessarily correlated with dominance or persistence. However, these factors may be related to 

oxygen effects, as shown in later experiments. 

Table III.B.2. summarizes the results of various experiments for the summer with small (3.5-m2

) 

ponds and seven of the algal strains under different operating regimes, including controlling the 

oxygen tensions through degassing by air sparging. 

A significant factor in pond operations was the oxygen level reached in the ponds, which 

influenced productivity and species survival. Ponds were operated with air sparging (and 

antifoam) to reduce DO levels, from typically 400% to 500% of saturation without air sparing, to 

150% to 200% of saturation with sparging. Foaming, caused by air sparging, was still was a 

problem in some cases, as with the Cyclotella. However this alga exhibited approximately the 

same productivity with or without sparging despite the 20%-30% opaque foam cover, suggesting 

some positive effect of the lower pO2. For other algal species productivity differences of 10% to 

20% were noted, and for some (e.g., C. gracilis), no specific effect of high versus low DO was 

noted. 

These outdoor results were reproducible enough to detect differences of greater than about 10% 

between treatments. The major result of this project was that productivities were 50% to 100% 

higher than the previous year, with some species of diatoms producing 30 to 40 g/m2

/d (AFDW, 

efficiency about 6% to 9% of PAR, or 3% to 4.5% total solar). The green algae were, as 

mentioned earlier, less productive than the diatoms. 

A more detailed study of oxygen effects was also carried out in the laboratory, avoiding the 

confounding factors of CO2 supply, temperature, and light intensity. In general the diatoms were 

insensitive to high DO; most, but not all, of the green algal strains exhibited marked inhibition by 

high oxygen levels (Figure III.B.8.). None of the oxygen-sensitive algae could be grown 

outdoors, suggesting this as a major factor in species dominance and productivity. 

Laboratory studies were also carried out at both high light intensity and high DO, to determine 

the synergism between these factors. Both the apparent maximum growth rate and dense culture 

productivity were determined for comparisons. Higher levels of DO intensified the inhibitory 

effects of higher light observed in some cases. This was true in particular for productivity, with 

growth rates also affected. Of course, the actual density of the culture is a major factor 
National Renewable Energy Laboratory 

186 A Look Back at the Aquatic Species Program—Technical Review 

determining productivity, and dense cultures avoid most, if not all, the deleterious effects of high 

light intensity. High O2 and low CO2 are other factors influencing the response to high light, 

with O2 being more inhibitory at both low CO2 and high light levels. High oxygen also affects 

chlorophyll content, although this effect is most pronounced at low light intensities where 

chlorophyll levels are 50% higher compared to high light intensities. 

Outdoor experiments were carried out to determine the effect of low CO2 (25 μM) and high 

(9-10) pH, which would be experienced in algal mass cultures, at least temporarily. Compared to 

the control cultures, one strain was not inhibited even at pH 10, two not at pH 9, and two were 

inhibited by about 33% at this pH, compared to the control at pH 8. Lowering pCO2 also resulted 

in similar levels of inhibition for the other strains. A role for bicarbonate in growth at high pH 

was established from the data, with metabolic costs estimated at about one-third of productivity, 

a major factor. This requires further investigation. 

One strain, a Cyclotella species, exhibited an increase of lipid content of more than 40% of dry 

weight upon Si limitation. However, lipid productivity (9 g/m2

/d), was not significantly different 

between Si-deficient and the Si-sufficient controls, because of the high productivity of the Sisufficient culture. Optimizing for lipid productivity was considered possible, but requires more 

detailed study. 

Perhaps most important, the data and simulations also suggest that maximizing productivity at an 

acceptable CO2/pH combination from the perspective of outgassing and CO2 loss from the ponds 

is possible, with operations above pH 8.0 required (for an alkalinity of 32 meq/L, higher for 

higher alkalinities) to avoid wasting of CO2. 

Laboratory studies were also carried out during this project. These included a study of light 

conversion efficiencies that concluded that at low light intensities very high light conversion 

efficiencies can be achieved (near the theoretical maximum of about 10 photons/CO2 fixed). 

However, these and other laboratories studies carried out during this project would require a 

much longer review than possible here. 

Finally, this project investigated harvesting of microalgae cultures with both polymers and FeCl3

(to enhance algal settling and sludge compaction) and cross-flow filtration. Organic flocculants at 

about 2 to 6 g/kg and FeCl3 at about 15 to 200 g/kg of algal biomass (AFDW) were required to 

remove 90% or more of the algal cells. Because of the high cost of the organic flocculants, costs 

were comparable for both flocculants tested. The organic polymers were also deemed to have 

significant potential for improvement and optimization. Cross-flow filtration, though effective, 

was estimated to be too expensive. A cost analysis of such a harvesting process was also 

presented. 

In conclusion, this project significantly advanced the state-of-the art of low-cost microalgae 

biomass production, and provided the basis for the Outdoor Test Facility, discussed in Section 

III.B.5., following the review of the ASP-supported project in Israel. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 187 

Publications: 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C. (1981) “Production of lipid hydrocarbon fuels and 

chemicals from freshwater microalgae.” Final Report, Contract Xb-0-9253-1, Solar Energy 

Research Institute, Golden, Colorado. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C. (1983) “Production of lipid hydrocarbon fuels and 

chemicals from freshwater microalgae.” Final Report, Solar Energy Research Institute, Golden, 

Colorado (unpublished). 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1982a) “Microalgae as a 

source of liquid fuels.” Proceedings of the June 1982 SERI Biomass Program Principal 

Investigators' Review Meeting, Aquatic Species Program Reports, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1808, pp. 1-16. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1983) “Production of liquid 

fuels and chemicals by microalgae.” Proceedings of the March 1983 Principal Investigators 

Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-1946, pp. 19-32. 

Weissman, J.C. (1984) “Cultivation of microalgae for liquid fuels.” Proceedings of the April 

1984 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-2341, pp.132-149. 

Weissman, J.C.; Goebel, R.P. (1985) “Design, fabrication, and operation of innovative 

microalgae culture experiments using open ponds.” Final Report, Subcontract XK-3-03135, Solar 

Energy Research Institute, Golden, Colorado. 

Weissman, J.C.; Goebel, R.P. (1985) “Production of liquid fuels and chemicals by microalgae.” 

Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species Program, 

Solar Energy Research Institute, Golden Colorado, SERI/CP-231-2700, pp.141-161. 

Weissman, J.C.; Goebel, R.P. (1987) “Design and analysis of pond systems for the purpose of 

producing fuels.” Report to the Solar Energy Research Institute, Golden, Colorado 

SERI/STR-231-2840. 

Weissman, J.C.; Goebel, R.P. (1987) “Factors affecting the photosynthetic yield of microalgae.” 

FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, pp. 139-168. 

Weissman, J.C.; Goebel, R.P.; Benemann, J.R. (1988) “Photobioreactor design: comparison of 

open ponds and tubular reactors.” Bioeng. Biotech. 31:336-344. 
National Renewable Energy Laboratory 

188 A Look Back at the Aquatic Species Program—Technical Review 

Table III.B.2. Outdoor results summary for California pond operations. 

Data from 3.5-m2

ponds. (Source: Weissman and Goebel 1986.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 189 

Figure III.B.8. Effects of oxygen tensions on laboratory algal cultures. 

The numbers above bars are standard deviations of duplicates. 

(Source: Weissman and Goebel 1986.) 
National Renewable Energy Laboratory 

190 A Look Back at the Aquatic Species Program—Technical Review 

III.B.4. The Israeli Microalgae Biodiesel Production Project 

In the mid-1980s an algal mass culture project for biodiesel production was supported by the 

ASP in Israel (Arad 1984, 1985, 1986), as a cooperative project among the following groups: 

1. Israel Oceanographic and Limnological Research Institute, with Dr. Ben-Amotz, 

who investigated lipid production at the laboratory and micropond scale; 

2. Ben-Gurion University of the Negev, with Professor A. Richmond as Principal 

Investigator, investigating algal mass cultures with outdoor ponds, essentially of 

the HRP design. 

3. Technion University, with Professor Gedaliah Shelef in charge of developing 

suitable microalgae harvesting technology. 

During the first 2 years of the project Ben-Amotz (1984, 1985) screened laboratory cultures of 

unicellular algae isolated in Israel and elsewhere. Of a score of strains tested, Nanochloropsis 

salina and B. braunii were the highest lipid producers, with lipid content as high as 50% in semicontinuous nitrate-limited cultures. Other strains had lipid contents <20%. Lipid composition 

and chemical characteristics (e.g., hydrocarbon contents) were also determined for many 

cultures. Nannochloropsis sp., P. tricornutum and C. gracilis were studied in more detail in 0.5-

Liter, pH-controlled chemostats for effects of temperature, light intensities, nutrients (Fe and 

nitrate), salinity and other parameters. The author concluded that “nitrogen limitation does not 

induce the production and accumulation of lipids,” but the “algae attain a low 

protein-carbohydrate ratio.” Previous reports in the literature describing lipid accumulation in 

algae induced by N limitation were attributed to trace element limitations. Actually, the data is 

typical of chemostat results, in which growth rate imposed by culture dilution do not allow lipid 

induction as is observed in batch or semi-continous cultures. 

During the final year of this project, Ben-Amotz (1986), optimized the growth of two cultures, C. 

gracilis and Nannochloris atomus in laboratory chemostats and in 0.35-m2 outdoor 

“microponds.” The ponds were mixed by air sparging, which would reduce pO2 levels. Maximal 

productivities of 40 g/m2

/d were obtained with C. gracilis during June-August, and highest 

photosynthetic efficiency (9.5% PAR) was achieved in the fall (when productivity was 27.3 

g/m2

/d, AFDW). During the winter, productivity decreased by about half, but lipid contents in 

the N-sufficient algal cells increased almost as much, reproducing the low-temperature effect on 

lipid content seen in the laboratory cultures. Attempts were also made to increase lipid 

production by Si limitation, but this was unsuccessful due to rapid contamination with green 

algae. 

The work by Professor Richmond and colleagues (1984; Boussiba et al. 1985, 1986), started with 

laboratory growth and lipid content experiments with more than a dozen algal strains. Outdoor 

cultivation was carried out for 2 years with small (2.5-m2

, 12-cm deep) paddle wheel-mixed high 

rate ponds. Among other factors, the effects of culture density on productivity and lipid content 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 191 

were studied, with the expected result that maximal productivity depended on the culture density 

(actually, on the areal concentration), but that this does not have major effects on lipid content. 

At the optimal density of 350 mg/L (45 g/m2

), productivity in the summer was 24.5 g/m2/d and 

lipid content about 16% for N. salina, and somewhat higher (28.1 g/m2

/d and 22 %) for 

Isochrysis galbana (both SERI Culture Collection strains). However, experiments with varying 

pond depths but constant areal biomass densities resulted in productivity differences of up to 

twofold, contrary to theory and expectations. Other factors (pO2, etc.) likely accounted for this. 

However, mixing speed had no significant effect on productivity. The authors stated: “These 

data reflect the complexity of the process of optimizing outdoor biomass production....” 

Professor Shelef (1984a,b; Shelef et al. 1985) carried out experimental and engineering studies of 

algal harvesting. The major effort was on the use of chemical flocculants for affecting algal 

sedimentation. Much of the work focused on I. galbana, grown, as above, on seawater of various 

concentrations. As expected, the higher the ionic strength (salinity), the greater amounts of 

chemical flocculants (alum, ferric chloride, chitosan) were required to induce algal flocculation. 

Autoflocculation, achieved by interrupting the CO2 supply, was also very effective. Other 

processes investigated were sand bed filtration, microstrainers (a 21 μm polyester weave allowed 

some 80%-90 % harvest efficiency), dissolved air flotation (after chemical flocculation, the 

method of choice for most commercial installations), and again, chemical “enforced” flocculation 

(recycling some of the precipitate to reduce flocculant needs). An economic analysis suggested 

various “allowable” flocculant costs for assumed biomass values. Overall, however, chemical 

flocculants are too expensive for biodiesel production. 

During the final year of this project, a 100-m2 pond was operated with I. galbana for 1 month 

(Arad 1986). In batch culture it took 12 days for the culture to enter stationary phase, and a 

productivity of 23.6 g/m2

/d was measured for about 2 weeks after starting dilutions. The culture 

was harvested with FeCl3 and alum using a dissolved air flotation unit from Technion. The 

flocculated algae had rather low lipid contents, compared to centrifuged algae. In conclusion, the 

Israeli project provided another dimension to the ASP effort, generally supporting the 

conclusions and results obtained by the U.S. work. 

Publications: 

Arad, S. (1984) “Development of outdoor raceway capable of yielding oil-rich halotolerant 

microalgae.” Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species 

Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 184-185. 

Arad, S. (1985) “Development of outdoor raceway for producing oil-rich halotolerant 

microalgae: An introduction.” Proceedings of the March 1985 Principal Investigators’ Meeting, 

Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-

2700, pp. 228-229. 
National Renewable Energy Laboratory 

192 A Look Back at the Aquatic Species Program—Technical Review 

Arad, S. (1987) “Integrated field-scale production of oil-rich microalgae under desert 

conditions.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3071, pp. 169-183. 

Ben-Amotz, A. (1984) “Identification of oil rich strains.” Proceedings of the April 1984 

Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden, Colorado, SERI/CP-231-2341, pp. 186-194. 

Ben-Amotz, A. (1985) “Development of outdoor raceway capable of yielding oil-rich 

halotolerant microalgae.” Proceedings of the March 1985 Principal Investigators’ Meeting, 

Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-

2700, pp. 230-243. 

Ben-Amotz, A. (1987) “Development of outdoor raceway capable of yielding oil-rich 

halotolerant microalgae.” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 199-208. 

Boussiba, S.; Vonshak, A.; Cohen, Z.; Abeliovich, A.; Kaplan, D.; Richmond, A. (1985) 

“Development of outdoor system for production of lipid-rich halotolerant microalgae.”

Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species Program, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 271-290. 

Boussiba, S.; Vonshak, A.; Cohen, Z.; Richmond, A. (1987) “Development of outdoor system for 

production of lipid-rich halotolerant microalgae.” FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 184-198. 

Richmond, A. (1984) “Development of outdoor system for production of lipid-rich halotolerant 

microalgae.” Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species 

Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 184-185. 

Shelef, G. (1984b) “Marine microalgae separation and harvesting.” Proceedings of the April 

1984 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-2341, pp. 206-224. 

Shelef, G.A.; Sukenik, A.; Green, M. (1984a) “Microalgae harvesting and processing: A 

literature review.” Report to the Solar Energy Research Institute, Golden, Colorado, SERI/STR231-2396. 

Shelef, G.; Sukenik, A.; Sandbank, E. (1985) “Separation and harvesting of microalgae from 

saline media.” Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species 

Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 244-270. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 193 

III.B.5. Design and Operation of a Microalgae Outdoor Test Facility (OTF) in 

New Mexico 

III.B.5.a. Facility Design and Construction. 

After the above noted projects carried out in California, the ASP decided to hold a competition 

for the development of a larger process development outdoor test facility (OTF) located in the 

southwestern United States. Two independent designs and proposals were commissioned, one 

consisting of enclosed production units (Aquasearch, Inc., Dr. Mark Huntley, Principal 

Investigator); the other of open ponds, similar to the design tested in California (Microbial 

Products, Inc., J.C. Weissman, Principal Investigator). Microbial Products, Inc., won this 

competition, with a proposed facility consisting of two 1,000-m2

 ponds, one plastic lined and 

another unlined, as well as supporting R&D using six small, 3-m2

 ponds, continuing and 

extending the work carried out in the prior projects in California (Weissman and Goebel 1987. 

See Section III.D. for a discussion of this engineering /cost analysis. No report for the 

Aquasearch proposal is available.). 

Although the proposal recommended establishing this facility in Southern California, the ASP 

selected a site in Roswell, New Mexico to establish the OTF. The project was located at an 

abandoned water research facility. Roswell has high insolation, abundant available flatland and 

supplies of saline groundwaters. The primary limitation of this site was temperature, which, in 

retrospect, turned out to be too low for more than 5 months of the year for the more productive 

species identified during the prior project. 

The objective of the first year of the research at this new site was to initiate a species screening 

effort at this site with the small 3-m2 ponds, which were installed while designing and 

constructing the larger facility. A major objective of this project was to identify cold weatheradapted strains (Weissman et al. 1987). 

Building the large system required installation of two water pipelines of 1,300-m in length (15 

and 7.5 cm, for brackish and fresh waters). The ponds were about 14 x 77 m, with concrete 

block walls and a central wooden divider. The paddle wheels were approximately 5-m wide, 

with a nominal mixing speed of 20 cm/s, and a maximum of 40 cm/s. Carbonation was achieved 

with a sump that allowed counterflow injection of CO2, to achieve high (90%+) absorption of 

CO2. One pond was plastic lined; the other had a crushed rock layer. The walls were cinder 

block. A 50-m2 inoculum production pond was included. Figure III.B.9. shows an overview of 

the layout of the completed facility. 
National Renewable Energy Laboratory 

194 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.9. Schematic of the microalgae OTF in Roswell, New Mexico. 

(Source: Weissman and Goebel 1988.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 195 

III.B.5.b. First Year OTF Experiments 

During the first year of the project (Weissman et al. 1988), all experimental work was carried out 

using the small ponds, which allowed essentially fully automatic operation and continuous 

dilution, as well as heating if needed. The objectives were to determine long-term productivity 

and stability for this site with previously studied and new species. Five of the strains inoculated 

into the 3-m2

 ponds were successfully cultivated, including two that derived from local isolates 

(one of which had invaded these ponds). Three of the culture collection strains could not be 

cultivated stably in the small ponds. Reproducibility of the experiments was tested, with the 

conclusion that differences between the different treatments should be judged significant only if 

these approached 20%. 

Productivities in the summer month of August reached 30 g/m2

/d for C. cryptica CYCLO1, but 

decreased to about half this level in September and October. At this point, M. minutum

(MONOR2) was used, as this is a more cold-tolerant organism. By November productivity of 

MONOR2 fell to about 10 g/m2

/d, and was very low (3.5 g/m2/d) in December in unheated 

ponds. Remarkably, despite these ponds freezing over repeatedly, the culture survived and 

exhibited some productivity. During August and September, productivities for CYCLO1 and 

Amphora sp. exhibited short-term excursions above 40 g/m2

/d. Faulty data are not suspected. 

A physical model of the pond environment developed by David Tillett at the Georgia Institute of 

Technology, combining climatic, design, operation, physico-chemical and biological process 

characteristics, was validated with temperature data from ponds in Roswell (see Section III.B.6). 

The large-scale system was completed by the second year. Some problems were encountered: 

the spongy clay at the site did not compact well, resulting in an uneven pond bottom. This made 

it difficult to clean and drain the ponds, and resulted in settling and sedimentation of solids. 

Significant differences were noted between the lined (north) and unlined (south) ponds, in terms 

of mixing velocities, head losses, and roughness coefficients. In any case, power inputs at low 

mixing velocities (<30 cm/s) were relatively low (<0.1 w/m2

). The efficiency of CO2 injection 

into the ponds through the carbonation sumps (at approximately 0.6 to 0.9-m depth) was 

estimated at close to 90%. From the measured gas transfer coefficient, outgassing losses from 

the lined pond were estimated as approximately 10% depending on pH levels. Also, the unlined 

pond lost 0.3-0.4 cm/d more water due to percolation. 

III.B.3.c. Full OTF System Operations 

Operation of the two large ponds (Weissman et al. 1989; Weissman and Tillett 1990, 1992) was 

initiated in August 1988. Ponds were inoculated with T. suecica and operated at relatively high 

mixing velocities (30 cm/s in the lined pond, 22 cm/s in the unlined pond) to reduce 

sedimentation. Productivities were only 11 and 10 g/m2

/d, respectively, lower than in the small 

ponds, but with an unknown amount of algal biomass settling out. After loss of this alga, M. 

minutum was inoculated, and productivities were, again, somewhat lower in the larger than the 

smaller ponds. 
National Renewable Energy Laboratory 

196 A Look Back at the Aquatic Species Program—Technical Review 

Experiments were also carried out in the small pond, primarily to determine the best operating 

pH and pCO2 range to help minimize CO2 outgassing while maximizing productivity. At 

reduced CO2 levels (higher pH) a decrease of 10% to 15% in productivity was observed with 

three algal species tested. Another variable tested was a 2- versus 3-day dilution routine, which 

had no significant effect. In addition, six cultures were examined for productivity in Si- or Ndeficient media. Only one strain exhibited significantly higher total (AFDW) productivities with 

nutrient deficiency, but no lipid data were collected. 

The conclusions from this work were, in brief: 

1. Power for pond mixing is within the expected range, and quite low (<1 kW/ha). 

2. Pond mixing should be in the 15-25 cm/s range, and pond depth 15-25 cm. 

3. CO2 utilization efficiencies of near 90% overall should be achievable with little 

compromise of productivity, through operation at an optimal pH/pCO2 range. 

4. Only preliminary 1,000-m2 pond operations were carried out during this year, 

hampered by design and operational problems, which lowered expected 

productivities. 

5. Large-scale pond productivities of 70 mt/ha/yr are realistic goals for this 

process, though probably not at this site because of low seasonal productivities. 

6. Very high, 50 g/m2

/d, single day, productivities were observed on some 

occasions. 

7. The small-scale ponds can be used to screen strains and optimize conditions. 

The final report (Weissman and Tillett 1992) in this series on the New Mexico OTF operations, 

reported on the demonstration of productivity for the two large ponds for 1 full year, continuation 

of the small-scale pond operations, and improvements in mixing and carbonation. One major 

improvement in the system was an automated data recording and operations system. 

Mixing was improved by improving the flow deflectors and increasing operating depths from 15 

to 22.5 cm, which is probably a better depth for large-scale systems. Culture instability was a 

problem, particularly in spring because of greater temperature fluctuations, and resulted in low 

average productivity of only 7 g/m2

/d for March through May. In contrast, the average 

productivity was 18 g/m2

/d for June through October, decreasing to 5-10 g/m2/d in November 

(depending on onset of cold weather), and only about 3 g/m2

/d in the winter months. 

Overall productivity, including 10%-15% down-time for the ponds for repairs and modifications, 

was 10 g/m2

/d, only one-third of ASP goals (Table III.B.3.). Clearly the major limiting factor 

was temperature, as smaller systems in warm climates have achieved annual yields two to three 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 197 

times as high. A major conclusion from this work is that scale-up is not a limitation with such 

systems. Climatic factors are the primary ones that must be considered in their siting. 

A countercurrent flow injection system was installed in the sumps resulting in a carbonation 

system that was essentially 100% efficient in CO2 transfer. Overall CO2 utilization was higher 

than 90%. The unlined pond performed nearly as well as the lined pond, with minor decreases in 

productivity (10%-20%), CO2 utilization efficiency (5%-10%) and a small increase in mixing 

power. The unlined pond consumed only 0.04 w/m2

, allowing the entire 1,000-m2pond to be 

powered by the equivalent of a 40-w light bulb. Species stability in the lined and unlined pond 

exhibited no significant difference. This work clearly established the feasibility of using unlined 

ponds in microalgae cultivation. This was a critical issue, as plastic lining of ponds is not 

economically feasible for low-cost production. 

In the small 3-m2

 systems, two variables were investigated: Si supply and pH. Both are major 

cost factors in pond operation, due to sodium silicate costs and CO2 outgassing. They affect 

overall productivity as well as lipid production. For Cyclotella, for example, productivity was 

about 20 g/m2

/d at pH 7.2 or 8.3, but only 15 g/m2/d at pH 6.2. As the higher pH range is 

preferred, where CO2 outgassing is minimal, this demonstrates the feasibility of operating such 

cultures within the constraints of a large-scale production system. Si additions could be halved 

with only a modest decrease in productivity, suggesting that Si supply could be reduced, 

particularly if low Si-containing diatoms are cultivated. Also Si limitation can be used to induce 

lipid production, as was demonstrated during this project, with lipid biosynthesis increasing as 

soon as intracellular Si content dropped, with a 40% lipid content being achieved. However, 

overall, lipid productivity did not increase as CO2 fixation limitation also set in. This remains as 

a major issue for the future (See also Section III.B.5.d.). 

Table III.B.3. Long Term OTF Results from 0.1-Ha Raceways. 

(Source: Weissman and Tillett 1990.) 

Pond Liner CO2 use 

(std.m3/d) 

Dates Productivity 

(gm afdw/m2/d) 

Carbon Use 

Efficiency 

Water Loss 

YES 15.2 10/1/88-9/30/89 9.8 59 5.7 mm/d 

NO 13.4 10/1/88-9/30/89 8.3 50 6.2 mm/d 

NO 14.6 10/1/89-9/30/90 10.5 82 

YES 22.0 6/1/90-10/30/90 19 81 

NO 19.2 5/1/90-9/30/90 18 88 

Notes: std m3

/d: standard cubic meters per day. 

gm/afdw/m2

/d: grams of ash-free dry mass per square meter per day. 

Pond liner: YES indicates a plastic lined pond; NO indicates an unlined (dirt bottom) 

pond.
National Renewable Energy Laboratory 

198 A Look Back at the Aquatic Species Program—Technical Review 

III.B.5.d. Conclusions

The performance of the large-scale system improved considerably in all aspects during the 2 

years of operations. The parallel use of the smaller-scale ponds helped guide this research, in 

particular in selecting algal strains and identifying operating characteristics. The high CO2

utilization efficiency demonstrated in the small and large-scale ponds was another major 

accomplishment of this project. 

The major limitation of this project was the overall low productivity in the large-scale ponds. 

This was due in large part to the adverse climatic conditions at this location, and the initial 

suboptimal nature of the large-scale pond operations. Even so, productivities were lower than 

anticipated, with annual average productivities only about one-third the projected productivities 

by the ASP that would be required for minimal economics (see Section III.D.). This must be a 

major ongoing objective for future research, first in terms of overcoming the lower temperature 

limitations on productivity, and second by relocating this type of process development to more 

favorable climatic sites. (See Section III.B.6. for a discussion of temperature effects.) 

But perhaps the major limitation of this project was that it did not carry out a longer-term process 

development effort. Although 2 years of data were collected for the large-scale ponds, the rapid 

advances made suggested that further research would have allowed continued improvements in 

performance and increased understanding of the overall process in specific critical areas of 

culture maintenance. 

The engineering evaluation of the operation of the 0.1-ha raceway ponds showed these systems 

to be potentially very efficient in terms of energy, water, nutrient and CO2 utilization, and even 

basic construction cost inputs. Most important, the absence of liners did not significantly reduce 

pond performance (e.g., productivity). This was a major observation of this project, giving 

greater confidence in the engineering analysis and cost projections carried out by the ASP and 

DOE, discussed again in Section III.C. 

A major uncertainty in this project was the nature of the species control achieved. A review of 

the data would suggest considerable success with species control, with several species cultivated 

successfully for relatively long periods. However, considerably more research will be required 

on this subject, as the tools were not available to allow a closer study of possible population 

dynamics (e.g., strain selection and even replacement) within the ponds. Thus, the subject of 

species control still requires considerable effort, as discussed further in the following section. 

Publications: 

Weissman, J.C.; Goebel, R.P. (1987) “Design and analysis of pond systems for the purpose of 

producing fuels.” Report to the Solar Energy Research Institute, Golden, Colorado, 

SERI/STR-231-2840. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 199 

Weissman, J.C.; Tillett, D.T. (1989) “Design and operation of an outdoor microalgae test 

facility.” Aquatic Species Program, Annual Report (Bollmeier, W.S; Sprague, S., eds.), Solar 

Energy Research Institute Golden Colorado, SERI/SP-231-3579, pp. 41-57. 

Weissman, J.C.; Tillett, D.T. (1992) “Design and operation of an outdoor microalgae test facility: 

large-scale system results.” FY 1989-1990 Aquatic Species Project Report, National Renewable 

Energy Laboratory, Golden, Colorado, NREL/MP-232-4174, pp 32-56.

Weissman, J.C.; Tillett, T.D.; Goebel, R. (1987) “Design and operation of an outdoor microalgae 

test facility.” FY 1987 Aquatic Species Program, Annual Report (Johnson, D.A.; Sprague, S., 

eds.), Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3206, pp. 231-250. 

Weissman, J.C.; Tillett, D.T.; Goebel, R.P. (1989) “Design and operation of an outdoor 

microalgae test facility.” Final Report to the Solar Energy Research Institute, Subcontract 

XK-7-06113-1. 

III.B.6. The Effects of Environmental Fluctuation on Laboratory Cultures 

III.B.6.a. Species Control and Productivity 

In the time frame between the end of the California projects and before initiation of the Roswell 

project, John R. Benemann and Dr. David Tillett carried out a research project in support of these 

outdoor pond projects (Benemann and Tillett 1987, 1990). This project included studies of the 

effect of nutrient limitations, specifically N and Si, on lipid induction, the effects of fluctuating 

conditions (temperatures, O2, light) on culture dominance and productivity, and the development 

of a mathematical model of the algal pond environment. That “Algal Pond Model,” (APM) 

could be used in designing experimental protocols and predicting culture performance. 

A basic premise of this project was that the algal pond environment experienced by microalgal 

cells is characterized by relatively consistent and predictable fluctuations in a rather limited set of 

variables, specifically light intensity, temperature, pH/pCO2, and pO2. The first two are 

essentially uncontrolled, although somewhat predictable, variables. The second two factors are 

consequences of light and temperature, as well as the pond chemistry and hydraulics 

(outgassing), and algal productivity. Further, it was argued that some of these variables, such as 

the last three, could be well modeled, based on mass and heat balances and algal growth models. 

 One uncertainty is whether short-term (<0.1 h) light fluctuations caused by pond mixing, can be 

averaged over the time scales of interest (>1 h). With this assumption it would be possible to 

predict, and reproduce on a small-scale, the key environmental parameters of outdoor ponds to 

which microalgae likely would respond, determining productivity and culture stability. 
National Renewable Energy Laboratory 

200 A Look Back at the Aquatic Species Program—Technical Review 

Such “down-scaling” of parameters, as summarized in Table III.B.4., would allow more realistic 

modeling at the laboratory, or very small outdoor scale, of the conditions encountered by 

microalgae in large-scale ponds. And it would allow, in turn, more controlled and easily 

interpretable experiments on species productivity and even dominance. As one of its objectives, 

and in collaboration with the prior studies (Section III.B.5.; Weissman and Tillett 1989, 1992), 

this project resulted in the development of an APM. Many experiments were also conducted on 

species growth responses and competition under fluctuating environmental conditions. Finally, 

lipid productivity was investigated under conditions of nutrient limitation. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 201 

Table III.B.4. Scale down parameters for microalgae culture. 

Feasiblity of scaling pond environmental parameters to laboratory reactors. 

(Source: Benemann and Tillett 1987.) 
National Renewable Energy Laboratory 

202 A Look Back at the Aquatic Species Program—Technical Review 

III.B.6.b. The Algal Pond Growth Model. 

The APM (Benemann and Tillett 1987, 1990; Tillett 1989) describes a shallow, paddle wheelmixed pond system. It incorporated climatic, design, operational, physicochemical, and 

biological parameters and submodels to predict the pond, and algal culture, behavior. Simplying 

assumptions in the model include the following: 

• the pond exhibited no nutrient, biomass or temperature inhomogeneities (e.g., 

gradients), 

• mixing was essentially plug flow for large systems and mixed tank behavoir for 

smaller ones, 

• CO2 and O2 outgassing can be estimated from measured gas transfer 

coefficients, and 

• evaporation was 1.5 times pan A evaporation data, for example. 

Climate input parameters were obtained from local U.S. climate stations in machine-readable 

form, including daily data on air temperatures (diurnal), relative humidity, rainfall, wind speed 

and total solar radiation. These data were then averaged over several years to provide daily and 

monthly average data sets. The use of such climate data is critical for predicting pond conditions 

at a particular site and location. Design variables included pond area, depth, mixing velocity, and 

length-to-width ratios. Physical inputs were the alkalinity of the media, the starting and ending 

pH for the carbonation station, and the outgassing coefficient. Finally, for a biological parameter 

a productivity assumption (from 20-40 g/m2

/d, or a % maximum sunlight conversion efficiency) 

was used, along with C content, O2 yield, heat of combustion, and saturating light intensity for 

photosynthesis (assuming simple light saturation, and application of the Bush equation). 

The model included an energy balance (input sunlight, radiation, evaporation, air temperatures, 

etc.), which continuously predicted the pond temperature, based on pond depth, and ambient 

conditions. From the productivity assumption (on a diurnal basis) and a CO2 outgassing 

coefficient for the ponds, the total inorganic C balance can be calculated based on alkalinity and 

pH as the major determinants of inorganic C in ponds. Like CO2 (and pH, etc.), O2 is also 

dependent on productivity, and outgassing. The model was written in Fortran (Tillett 1989). 

The model was validated at the Roswell test site, with the 3-m2

 ponds, which were instrumented 

(for pH, DO, wind, air temperature, etc.) and a data acquisition system developed to obtain short- 

(< 1h) and long- (>1 d) term data. The model was also validated with the larger ponds. 

Measured and predicted pond temperatures agreed well, as seen in Figure III.B.10a for a diurnal 

data set for a heated and unheated small pond, and Figure III.B.10b for a single unheated pond 

for 2 weeks. 

Simulations were also run for a larger, 1,000-m2

 earthen pond as built at Roswell (an arid and 

cool climate), which were then compared with a site in West Palm Beach, Florida (a humid, 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 203 

warm climate), using monthly average climate data. The model was exercised for 4 

representative months of the year for both locations, and assuming ponds of 10, 20, 30 and 100-

cm depth. (This last point was to demonstrate the limited effect of managing pond temperature 

extremes by depth.) Minimum water temperatures do not rise above 10°C for shallow ponds for 

most of the year in Roswell; they never drop below this level in West Palm Beach. Maximum 

summer temperatures seem to be only modestly higher in Florida than in New Mexico. These 

results point to low temperatures as a major factor in Roswell operations. 

Only a limited attempt was made to verify the model in relation to productivity, by using a fitted 

Ik (saturating light intensity) parameter, as well as an assumed heat of combustion (5.7 Kcal/g) 

and biochemical conversion efficiency (from prior work by Weissman and Goebel 1987). 

Although the agreement between calculated and measured productivities was excellent (both 

gave about 15.3 g/m2

/d), this was probably fortuitous, as the Ik actually used in the model was 

well below what had been previously measured with the same organism (Monoraphidium sp.). 

This requires further investigation. 

One interesting use of the model was to predict CO2 utilization and outgassing from various 

assumed pH, pCO2 and alkalinity regimes. This is a central issue in the operation of algal pond 

systems, as these parameters must be used to optimize for productivity and overall CO2

utilization efficiencies. The higher the pH and the lower the alkalinity, the greater the utilization 

efficiency. However, for CO2 supply, pH must be decreased transiently. With moderate 

alkalinity (2.5-5 mM), and CO2 requirements, the pH transients can be relatively small, allowing 

minimal outgassing even for seawater systems, resulting in predicted CO2 use efficiencies of over 

85%. 

A final important parameter is dissolved O2, which was predicted to accumulate within 1 hour to 

a level of 210% of air saturation with a 30-cm deep culture and a productivity of 30 g/m2

/d. 

Much higher concentrations would build up during the day, and are actually observed in ponds. 

This could be a major factor in reduced productivities in ponds (see Section III.B.5.). 

This model is of sufficient detail and predictive value to minimally direct the laboratory and 

small-scale outdoor research in making these more representative of the outdoor pond 

environments. Laboratory applications are discussed in the following section. 
National Renewable Energy Laboratory 

204 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.10. Comparison of measures and predicted pond temperatures in Roswell, New 

Mexico. 

a.). Top: Comparison of measured and predicted diurnal temperature profiles for heated and 

unheated ponds. October 4, 1987. 

b.). Bottom: A comparison of predicted (line) versus measured (symbols) maximum-minimum 

temperatures in miniponds using simplified inputs for Roswell, N.M. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 205 

III.B.6.c. Microalgae Competition under Fluctuating Conditions in the Laboratory 

A major issue in microalgae mass cultures is the understanding of the factors that determine 

species dominance. A considerable theoretical background can be gleaned from the ecological 

and ecophysiological literature, where this problem, as it applies to lotic systems (ponds, lakes, 

rivers, oceans) is the subject of an enormous literature (briefly reviewed in Benemann and Tillett 

[1987]). Fundamental to this is Hutchins’ so-called “Paradox of the Plankton.” Hutchins pointed 

out the fact that only a limited number of nutrients could limit algal growth, and thus, applying 

Liebing's Law of the Minimum, only a limited number (one per nutrient) of algal species that 

should be able to compete in any environment. Rather, we find literally hundreds, if not 

thousands of species and uncountable strains in even the smallest and most uniform of 

environments. Indeed, even Hutchins did not realize the greatness of microbial biodiversity in 

nature, hinted at by earlier work in clonal variations (See Section II.B.), but only recently 

revealed in ever-increasing detail using modern tools of molecular phylogenetics. The solution 

to the Paradox is that natural environments are not steady-state systems, but are exposed to 

periodic and random fluctuations in physical-chemical (let alone biotic) environmental 

parameters. These fluctuations allow for additional niches, allowing for organisms specialized in 

the exploitation of particular temporal combination of limiting factors. Also, non-steady-state 

conditions would select for different species and strains. For example, in a continuous 

cultivation using natural samples for inoculum into enriched media, the first algal species to 

appear and dominate are soon replaced by other species, which are slower growing but better at 

exploiting a light-limited (dense culture) environment. 

Continuous algal cultures in 1-L vessels were set up in the laboratory, which allowed operations 

under fluctuating conditions of temperature, O2 concentrations, and pH. In these experiments, 

several algal strains were inoculated together, then productivity and species dominance were 

observed for as long as 3 weeks (over 10 dilution times). In initial experiments, Chlorella and 

Chaetoceros co-dominated; Cyclotella was lost or greatly diminished, possibly because of 

different light spectral use of the two types of algae (greens and diatoms). Many other 

experiments were carried out, with fluctuating pH, dilution rates, and light intensities and even 

gas sparger types on the dominance of these and one additional algal strain (Ankistrodesmus). All 

these factors tended to affect species dominance, even the gas sparger, and the results were not 

clearly interpretable in terms of major dominance factors. One conclusion from these initial 

experiments was that several factors, alone and in combination, can determine species 

dominance. 

III.B.6.d. Lipid Productivity of Microalgae 

The most fundamental assumption in microalgae biomass production for biodiesel fuels is that it 

will be possible to achieve near theoretical solar conversion efficiencies by overcoming the light 

saturation effect (see Section IV.A.2.c. for a brief discussion). The second most fundamental 

assumption is that it will be possible to achieve such very high productivities with microalgae 

cultures high in oils, approaching or even exceeding 50% of lipids by dry weight. This second 

assumption was tested by this project. 
National Renewable Energy Laboratory 

206 A Look Back at the Aquatic Species Program—Technical Review 

The concept of producing microalgae with a high oil content goes back almost 50 years, to work 

carried out, and even patented, by Sphoer and Milner (1949), who reported oil contents as much 

as 80% of the dry weight. Lipid content is affected by many parameters, but most particularly by 

N (and, for diatoms, Si) limitation, which can result in extraordinarily high lipid contents. 

However, it appeared from earlier work, and also from a survey of 30 species by Shiffrin and 

Chisholm (1981), that total productivity declined sharply upon nutrient limitation, resulting in a 

decline in total lipid productivity, although lipid content increased as a percentage of the cell 

mass. However, a re-analysis of the data suggested that the evidence for this was not clear-cut, 

as only rather widely spaced data points had been collected. In fact, an essential assumption in 

this field is that a “lipid trigger” activates lipid biosynthesis without necessarily reducing 

photosynthesis, at least for a transitory period (see Section II). 

The experimental approach was to first grow the algal cultures under nutrient sufficiency and 

then to induce deficiency during batch cultivation, using light (single versus two-sided 

illumination of the 1-L flasks) as a second variable. In continuous cultures, the growth rates and 

cellular N contents were dependent on illumination, and there was only a modest increase in lipid 

content with decreasing cellular N content, with lipid culture productivity maximal at about 5% 

N biomass. The key experiment was the up-shifting of the light received by the culture (e.g., 

from single- to double-sided illumination). The results are shown in Figure III.B.11., which 

demonstrate that cells shifted to a higher light intensity start growing (AFDW increases) at the 

rate of the higher light level cultures. However, lipid productivity shoots up to a much higher 

rate than with either of the steadily illuminated cultures. In practical terms, this could be 

exploited by diluting cultures for lipid induction. This experiment demonstrated the possibility 

of producing high lipids content by nutrient limitation while achieving a substantial increase in 

overall lipid productivity. Experiments with continuously diluted cultures, however, did not 

exhibit such responses, indicating the necessity to carefully control and modulate conditions to 

maximize lipid production. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 207 

Figure III.B.11. Maximizing lipid productivity light shift-up. 

A. Top. Biomass yield during light shift. 

B. Bottom. Lipid yield during light shift. 

(Source: Benemann and Tillett 1987.) 
National Renewable Energy Laboratory 

208 A Look Back at the Aquatic Species Program—Technical Review 

III.B.6.e. Competition Studies with Continuous and Semicontinuous Cultures 

A key underlying assumption in this work was that monoculture productivity is an indicator of 

competitiveness in mixed cultures. That is, the most productive culture will also be the most 

competitive. Another assumption is that productivity and competitiveness will be affected by 

fluctuations in environmental variables and cannot be predicted from single parameter variations 

or steady-state operations. Testing these hypotheses was a central objective of these 

experiments, which were carried out with continuous and then semicontinuous (once a day 

dilution) laboratory cultures (1-L bottles). 

The continuous cultures were used to test productivity of several strains under simulated outdoor 

conditions (approximately sinusoidal diurnal temperature changes from 15°-32°C overnight, a 

14-h constant light period, 0.6-0.7 d-1 dilution, and 6-h gassing with pure O2 rather than air). As 

in earlier studies, Chlorella and Monoraphidium exhibited higher productivities (250-300 

mg/L/d) than the other strains tested (Porphyridium, Ankistrodesmus, and Chaetaceros, with 

some 150-200 mg/L/d productivity). Under constant conditions, similar ranking and differences 

were observed between the three fastest-growing strains. Mixed culture experiments between 

Chlorella and Chaetoceros were then carried out, with the results comparing fairly well with the 

APM results (Section III.B.6.b.). Figure III.B.12. shows experiments in which a 90/10 

Chaetaceros/Chlorella mixed inoculum grew in continuous cultures, with cell species changing 

faster experimentally than in the model, which, however, predicts the overall trend. Overall 

results with these strains were in accord with prior experience (Weissman and Goebel 1985, 

1988). 

However, the continuous cultures proved logistically and experimentally very difficult, and the 

experimental design was switched to a simpler, once a day (before dawn), dilution. Fluctuating 

conditions were achieved primarily by timers and actuators (e.g., for O2 versus air supply, for 

temperature control in the water baths). This allowed greater reproducibility and simultaneous 

operation of many more cultures, as many as 16 in four water baths, allowing collection of an 

extensive data set. The effects of constant versus fluctuating temperature, pO2, and light, on 

culture productivities and dominance in mixed cultures were studied. In summary, the overall 

result was that fluctuating temperature and other conditions had major effects and were different 

from those at constant temperature. Also, growth responses for unialgal cultures can predict the 

outcome of species competition experiments. Still, much more experimental work is required on 

these problems. 

Publications: 

Benemann, J.R.; Tillett, D. (1987a) “Effects of fluctuating environments on the selection of high 

yielding microalgae.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, pp. 285-300. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 209 

Benemann, J.R.; Tillett, D.M. (1987b) “Microalgae lipid production.” In Energy from Biomass 

and Wastes XI, Conference Proceedings, (Klass, D.L., ed.), Institute of Gas Technology, 

Chicago. 

Benemann, J.R.; Tillett, D.M. (1987c) “Effects of fluctuating environments on the selection of 

high yielding microalgae.” Final Report, Subcontract XK-4-04136-06, Solar Energy Research 

Institute, Golden, Colorado. 

Benemann, J.R.; Tillett, D.M. (1987d), “Effects of fluctuating environments on the selection of 

high yielding microalgae.” FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3206, pp. 

77-91. 

Benemann, J.R.; Tillett, D.M. (1990) “Effects of fluctuating environments on the selection of 

high yielding microalgae.” Final Report, Subcontract XK 4-04136-06, Solar Energy Research 

Institute. Golden, Colorado. 

Benemann, J.R.; Tillett, D.M.; Weissman, J.C. (1987) “Microalgae biotechnology.” Trends in 

Biotechnology 5:47-53. 

Shifrin, N.S. (1984) “Oils from microalgae.” In Biotechnology for the Oils and Fats Industry

(Ratledge, C., et al., eds.), AOCS monograph 11. 

Shifrin, N.S.; Chisholm, S.W. (1981) “Phytoplankton lipids: interspecific differences and effects 

of nitrate, silicate and light-dark cycles.” J. Phycol. 17:374. 

Sphoer, H.A.; Milner, H.W. (1956) “Production of protein, lipids and carbohydrates by cultures 

of algae.” U.S. Patent 2,732,661. 

Spoehr, H.A.; Milner, H.W. (1949) “The chemical compostition of Chlorella; effect of 

environmental conditions.” Plant Physiol. 24:120. 
National Renewable Energy Laboratory 

210 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.B.12. Species competition between Chaetoceros and Chlorella in continuous 

cultures. 

Mixed culture results for a 90/10 cell density mixture of Chaetoceros and Chlorella. 

a.) Top: Culture productivity. 

b.) Bottom: Species cell concentration (solid line represents model predictions). 

(Source: Benemann and Tillett 1990.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 211 

III.C. Resource Analyses

III.C.1. Introduction 

One major concern of the ASP, as for any renewable energy option being developed by DOE, is 

the resource potential for the technology. How large an impact will it have on future U.S. energy 

supplies, or, in today's units, fossil CO2 or equivalent greenhouse gas reduction potential? This is 

required both by the mission of the DOE as well as the inherent need to justify budgetary 

decisions. However, potential contributions to fuel supplies should be only one, albeit an 

important, parameter in such an assessment. Other factors must also be considered, such as 

economics, time frame for implementation, and the possibility of success for the R&D effort. If 

large resource potential is the main criterion, this could result in focusing too many resources on 

a few technologies with a low probability of succeeding in practice. 

Microalgal biodiesel is one of many different biomass energy options, extending from co-firing 

of wood in power plants to energy farms and a myriad of fuel conversion options (Hughes and 

Benemann 1997). One important attribute of microalgae systems is that they need not compete 

with other biomass alternatives, but must be able to use water and land resources generally not 

considered for crop production. Also, microalgae fuel production systems could meet other 

objectives, from waste treatment to salinity management. Resource potentials are important, and 

have been studied extensively by the ASP. 

The ASP emphasized the production potential of microalgae in the southwestern United States. 

This choice was based in large part on the perspective that this area offered the best and largest 

resource potential for algal culture systems, including available saline water supplies, land area, 

sunlight, and CO2 sources. Each of these resources individually was projected to have a potential 

of many quads (1015 Btu) of energy, and overall potentials of several quads were projected (e.g., 

Chelf and Brown 1989). This certainly justified the emphasis by the ASP on this geographical 

area. In fact, a significant activity of the ASP was to document this resource potential. It was not 

a trivial task to assess these resource potentials. This required the development of suitable 

assessment techniques. This was a major accomplishment of the ASP, as reviewed in this 

section. However, estimating an actual resource potential is not a simple matter, as the 

juxtapositions of the requirements for microalgae production—water, land, CO2, and climate—

are very difficult to quantitate, and would greatly reduce the resource potential estimated from 

single factor analyses. 

Publications: 

Hughes, E.; Benemann, J.R. (1997) “Biological fossil CO2 mitigation.” In Proc. of the 3rd Int. 

Conf. on CO2 Capture, Disposal and Reuse Utilization, ICCDR-3, Mass. Inst. Tech., September 

1996 (in press, 1997). 
National Renewable Energy Laboratory 

212 A Look Back at the Aquatic Species Program—Technical Review 

III.C.2. The Battelle Columbus 1982 Resource Assessment Report 

This report (Vignon et al. 1982), comissioned by the ASP, was the first comprehensive 

discussion of the resource requirements for microalgae production. It covered the criteria that 

should and could be used to identify available water, land and other resources, to estimate their 

relative importance, and to evaluate various legal, institutional, and other resource constraints. 

These issues were discussed at some length, focusing on the southwestern United States, 

although most of the discussion was of a rather general nature. For example, land and water 

rights issues are addressed, which are certainly important, but are difficult to extrapolate over 

large areas. Similarly, permits for such facilities will be very important, but will also involve site 

specific considerations. 

The authors calculated various costs and energy inputs for water (seawater) pumping and 

transportation to arrive at permissible lift and distance criteria for water resources. Water lifting 

of some 75 m and pipeline distances of 6 km, with an approximately 1-m diameter pipeline, were 

estimated to cost about $31 million for a 400-ha system. This estimate for water supply is as 

high as later estimates for the total cost of building and operating an entire microalgae production 

system (see Section III.B.5.), which puts some perspective on the limits of lift and distance for 

water supplies. This report did not arrive at a prediction for the resource base, but was an 

important early introduction to the complexities of such resource assessments. 

Publications: 

Vigon, B.W.; Arthur, M.F.; Taft, L.G.; Wagner, C.K.; Lipinsky, E.S.; Litchfield, J.H.; 

McCandlish, C.D.; Clark, R. (1982) “Resource assessment for microalgal/emergent aquatic 

biomass in the arid southwest.” Battelle Columbus Laboratory Report, Solar Energy Research 

Institute, Golden, Colorado. 

III.C.3. The 1982 Argonne Study of CO2 Availability 

This report (Nelson 1982) was the first analysis of the availability of CO2 specifically for 

microalgal mass culture. There was, and still is, considerable interest in CO2 sources for EOR 

(enhanced oil recovery). Thus, a significant body of literature had developed in the preceding 5 

years, particularly for the southwestern United States, where several small pipelines were built to 

deliver CO2 for EOR. One study even considered the production of CO2 from a power plant flue 

gas for such a purpose. The Argonne report concluded that there would be little extra available 

CO2 for microalgae production until the time for EOR had passed, about the year 2020. Of 

course, since then declining oil prices, and increasing interest in CO2 mitigation, have changed 

this situation. The report also concluded that flue gas sources would be a poor source for CO2 for 

the microalgae ponds, as power plants were not generally located in a suitable area for 

microalgae cultivation. The authors also concluded that the delivery of pure CO2 would be 

expensive, even after CO2 became available after the EOR era. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 213 

Publications: 

Nelson. (1982) “An Investigation of the availability of carbon dioxide for the production of 

microalgae lipids in the southwest.” Report to the Argonne National Laboratory, unpublished. 

III.C.4. The 1985 SERI Resource Evaluation Report 

This study (Maxwell et al. 1985) assessed the availability and suitability of land, brackish water 

resources, and climate in the southwestern United States. The objective was to “stratify the 

Southwestern United States into zones of varying suitability for such [microalgal] systems.” The 

Battelle Columbus report discussed earlier (Vignon et al. 1982) was identified as a companion 

effort, although it was “not directly supportive of the stratification effort...” Climate, land 

resources, and water resources maps based on various process constraints and characteristics 

(e.g., freeze-free period, land slope, water depth) were overlayed to develop suitability maps. 

Because of insufficient water data, water supply was excluded from the final analysis. Available 

maps were also inadequate for land classification for slopes <10%. 

This report includes an excellent discussion of the sources of maps and information about water, 

water rights, land ownerships, slopes, topography, climate, etc. Of course, in the dozen (or more) 

years since this research was carried out, the computer revolution has made access to this data 

easier or at least different. The report provides a very comprehensive review of the problem, and 

supplies a large amount of specific information. One interesting point is that microalgae systems 

will likely encroach on pasture and non-irrigated agricultural land; much of this land has low 

productivity and value, but is generally accessible and already generally serviced by roads and 

power. 

A relative productivity map was developed for the southwestern United States, by combining 

frost-free days with insolation values. Many other such maps, including land suitability and 

water availability, were prepared, including a final overall suitability assessment (Figure 

III.C.1.). The authors proposed continuing such an assessment, pointing out the many limitations 

of the present study (such as water laws and rights issues) that required further studies. However, 

a point of diminishing returns is likely to be reached. It may be best to evaluate some very 

specific areas, even sites, for actual suitability for such a process. Indeed, any generic analysis 

may miss important details. For example, as indicated in Section III.B. from the experience at 

Roswell, New Mexico, that location is quite unsuitable for microalgae production, due to its short 

(approximately 200-day) growing season, although it appears in the overall suitability map 

(Figure III.C.1.). This was likely due to insufficiently restrictive temperature criteria (freeze-free 

days, etc.). 

Publications: 

Maxwell, E.L; Folger, A.G.; Hogg, S.E. (1985) “Resource evaluation and site selection for 

microalgae production systems.” Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/TR-215-2484. 
National Renewable Energy Laboratory 

214 A Look Back at the Aquatic Species Program—Technical Review 

Figure III.C.1. Overall suitability map for microalgae culture in the Southwestern United 

States. 

Zones of relative suitability for microalgae biomass production based on compositing of climate, 

land and water suitability maps. (Source: Maxwell et al. 1985.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 215 

III.C.5. The 1990 SERI Study on CO2 Sources 

The objective of this study (Feinberg and Karpurk 1990) was to examine CO2 resources for 

microalgae production in the year 2010 and beyond. This report was a very comprehensive and 

authoritative source of information on this subject, from merchant CO2 supplies and costs to 

potential competition from EOR for CO2 sources. CO2 recovery from existing processes was 

judged to be relatively low cost from ethanol and ammonia plants, and much more expensive 

from cement, refineries, or power plants. 

After a detailed review of the options, the authors estimated that the potential CO2 resource base 

was sufficient to support the annual production of roughly 2 to 7 quads of algal fuels. This 

corresponds to as much as 1.1 billion tons of CO2 per year, at prices ranging from about $9 to 

$90/t CO2. However, this analysis lacks the spatial resolution of the earlier study; thus, the actual 

CO2 availability (particularly of the low-cost supplies) was somewhat more speculative. 

Certainly CO2 resources will be a major limiting factor in microalgae production technology. 

However, as CO2 utilization has become a central objective of microalgae production systems, 

perhaps rather than looking at CO2 as a limitation it should be considered a site-specific 

opportunity, where the other requirements for microalgae production are met (e.g., land, climate, 

water, infrastructure). Table III.C.1. summarizes the conclusions of this report regarding CO2

costs and supplies. 

Publications: 

Feinberg, D.A.; Karpuk, M.E. (1990) “CO2 sources for microalgae based liquid fuel production.” 

Report, Solar Energy Research Institute, Golden, Colorado, SERI /TP-232-3820. 

Karpuk, M. (1987) “CO2 sources for fuels synthesis.” FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 269-275. 
National Renewable Energy Laboratory 

216 A Look Back at the Aquatic Species Program—Technical Review 

Table III.C.1. Summary of Availability and Cost of CO2 Sources 

(Source: Feinberg and Karpuk, 1990.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 217 

III.C.6. The 1990 SERI Study of Water Resources in New Mexico 

This study (Lansford et al. 1990) specifically addressed the saline groundwater water resources in 

New Mexico. The objective was to identify suitable areas where large (1,000-ha) microalgae 

facilities could be established based on land and water availability. This report did not consider 

CO2 availability. Selection criteria developed by SERI, such as water quality, land slope, and 

climatic conditions, were used in this analysis. The groundwater resource information was 

reviewed for potential suitability for microalgae culture. Total gross water reserves of some 20 

billion acre-feet were projected, of which about one-quarter was fresh water and the remainder of 

varying degrees of salinity. Freshwater sources would likely not be available in large quantities, 

as they would have higher value uses, would already be appropriated, or otherwise restricted. 

Thus, this report focused on saline groundwater sources. A first cut was by depth, likely well 

yields, size of reserves, and chemical composition. A detailed analysis of six groundwater basins 

of varying sizes and quality was then carried out. A qualitative analysis was carried out first, 

based on available data (Table III.C.2). Of these six, only two met all the criteria established by 

SERI for a microalgae facility, with the Tularosa Basin judged the best choice and Crows Flats 

next. For these regions, about 2.3 to 5 million acre-feet of useable water resources were 

identified as available. This report is an excellent example of the challenges of developing such 

a detailed resource analysis, to arrive at even an initial estimate for a single U.S. state, even after 

relaxing a key resource constraint (e.g., CO2). 

Publications: 

Lansford, R.; Hernandez, J.; Enis, P.J. (1987) “Evaluation of available saline water resources in 

New Mexico for the production of microalgae.” FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 227-248. 

Lansford, R.; Hernandez, J.; Enis, P.J.; Truby, D.; Mapel, C. (1990) “Evaluation of available 

saline water resources in New Mexico for the production of microalgae.” Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/TP 232-3597, 83 pp. 
National Renewable Energy Laboratory 

218 A Look Back at the Aquatic Species Program—Technical Review 

Table III.C.2. Suitable water resources in New Mexico. 

(Source: Landsford et al. 1990.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 219 

III.C.7. Conclusions 

The various ASP resources analyses indicated significant potential land, water, and CO2

resources, even within the limited geographic area (the southwestern United States) that was the 

focus of the ASP. Several quads (1015 Btu) of fuels were projected for the various available 

resources. Other areas, from Florida to California, could also be considered. Microalgae systems 

actually use fairly little water, compared to irrigated crop plants. In addition, many waste and 

saline water resources may be available and suitable for microalgae production. Many CO2

sources are available, and algal ponds could be purposefully co-located with CO2 sources, or 

even vice versa. This is already being done at a commercial microalgae facility in Hawaii. 

Finally, land is hardly a major limitation: two hundred thousand hectares, less than 0.1% of 

climatically suitable land areas in the United States, could, with maximal productivities, produce 

about 1 quad of fuels. Thus, although there are many practical limitations, which may make 

some earlier predictions optimistic, resource limitations should not be an argument against 

microalgae biodiesel systems. 

III.D. Engineering Systems and Cost Analyses 

III.D.1. Introduction 

One of the major accomplishments of the ASP was the development of detailed engineering/cost 

projections for large-scale microalgae biofuels production. These analyses generally supported 

the view that microalgae biomass production could be performed at sufficiently low cost as to 

plausibly become a renewable energy source, assuming however, that the rather ambitious R&D 

goals of the ASP could be met. A major conclusion from reviewing these studies is that most 

R&D goals for this technology are related to the algal cultures themselves (productivity, species 

control, and harvestability), rather than the engineering aspects, such as the ponds, CO2 transfer, 

or biomass processing. 

Historically, the first engineering and cost analysis for large-scale microalgae production of fuels 

was that of Oswald and Golueke (1960). These authors projected the costs of electricity 

generated from biogas (methane) obtained from the anaerobic fermentation of algal biomass. The 

algae were to be cultivated in very large (40-ha) raceway type ponds, mixed with pumps, and 

supplied with CO2 from a power plant. Other nutrients would come from the digesters. 

Municipal wastewaters would be used as make up for water and nutrients (C, N, P, etc.). The 

ponds were to be of earthen construction, with a depth of about 30-cm. Harvesting was assumed 

to be by simple settling. Electricity costs were projected to be competitive with nuclear power. 

Although few details were provided, the general concept outlined in this early publication has 

remained essentially unchanged. Perhaps the greatest change is that biomass productivities 

thought to be achievable at that time were less than 50 mt/ha/yr of biomass, while current 

projections are roughly two to five times higher. 

With the initiation of the ERDA/DOE funded projects at the University of California–Berkeley 

during the mid-1970s (Section III.A.), additional engineering and cost analyses were conducted 
National Renewable Energy Laboratory 

220 A Look Back at the Aquatic Species Program—Technical Review 

(Benemann et al. 1977). The early studies were based on large (8–20-ha) ponds, with multiple 

channels and mixing by recirculation pumps (the required deep concrete sumps and splash pads 

were a major cost factor). Both the settling pond for harvesting algae by sedimentation and a 

covered anaerobic lagoon were part of this initial design. Total systems costs were only about 

$10,000/ha (somewhat over twice that in current dollars). Based on a projected yield of about 

500 GJ/ha/y (10 GJ/t of algal biomass) of biogas, costs were projected at about $3/GJ. Although 

optimistic, this study served as a starting point for more detailed later studies. 

Publications: 

Oswald, W.J.; Golueke, C.G. (1960) “Biological transformation of solar energy.” Adv. Appl. 

Microbiol. 11:223-242. 

Benemann, J.R.; Baker, D.; Koopman, B.L.; Oswald, W.J. (1977) “A systems analysis of 

bioconversion with microalgae.” Proc. Symposium Clean Fuels from Biomass and Wastes,

(Klass, D., ed.) Institute of Gas Technology, Chicago, pp. 101-126. 

III.D.2. The Algal Pond Subsystem of the “Photosynthesis Energy Factory” 

A relatively detailed analysis of an algal wastewater treatment-energy production process was 

carried out by Benemann et al. (1977) as part of a larger study that examined a system integrating 

wastewater algal ponds with tree biomass production. The so-called “Photosynthetic Energy 

Factory” (InterTechnology Solar Corporation 1978) was to use the effluents of a waste treatment 

pond system to fertilize short-rotation trees for fuel farming. In turn, the power plant burning the 

woody biomass would provide CO2 for the algal ponds. 

A design of the algal pond subsystem was carried out by Benemann et al. (1978) for a typical 

municipal community of 50,000 people, generating approximately 18,000 m3

 of municipal 

wastewater per day. The assumption was that algal biomass would be grown up to the N growth 

potential of the wastewater, containing 65 mg/L of useable N (as organic N and ammonia). This 

required recycling about 5 to 7 tons of CO2 per day from the power plant to the algal ponds. A 

temperate site with an average insolation of about 15 GJ/m2

/d was assumed, with a solar 

conversion efficiency averaging only 2.7% of visible light (about 1.35% of total solar), somewhat 

higher in winter than summer. This is considerably lower than current assumptions. 

This study, for the first time, took into consideration monthly variations in temperature, 

insolation and other parameters. Algal harvesting was assumed to be with microstrainers (this 

analysis was carried out while this option was still being investigated, see Section III.A.3.). This 

report also carried out the first, though preliminary, analysis of the mixing power required for 

such large algal ponds and of the transfer requirements for CO2 to the algal culture. A 160-ha 

algal pond system was required to treat this wastewater flow year-round. This was about three 

times larger than a conventional oxidation pond system. Costs were projected to be competitive 

with conventional wastewater treatment systems. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 221 

Energy outputs were twice the energy inputs, based on digester gas production and requirements 

for pumping the wastewater, mixing the ponds, etc. The overall economics were very favorable 

because of the wastewater treatment credits. 

Although this concept appeared favorable, in practice the relatively small scale of the locally 

available municipal wastes could supply only a small fraction of fertilizer needs for the very 

large (>10,000 ha) energy plantations being projected. It does, however, point to the potential of 

this technology in wastewater treatment. 

Publications: 

Benemann, J.R.; Koopman, B.L.; Baker, D.; Goebel, R.; Oswald, W.J. (1977) “Preliminary 

design of the algae pond subsystem of the photosynthesis energy factory.” Final Report to 

Inter-Technology Solar Corp., Sanitary Eng. Res. Lab., Univ. of Calif.-Berkeley. 

InterTechnology/Solar Corp. (1978) “The photosynthesis energy factory: analysis, synthesis and 

demonstration.” U.S. DOE HCP/T3548-01. 

III.D.3. Cost Analysis of Microalgae Biomass Systems 

This report (Benemann et al. 1978) originated with a Request for Proposals (RFP) by ERDA for 

a “Cost Analysis of Algal Biomass Systems” which included both micro- and macroalgae. The 

contract was awarded to the Dyantech R/D Co., who subcontracted for the analysis of the 

microalgal work with CSO International, Inc. Although the RFP specified a minimum scale for 

such systems of “100 square miles,” a single large unit was not feasible, and the analysis was 

carried out for individual modules of 800-ha. This system was to be independent of wastewater 

treatment and nutrients, which were deemed too small to provide “meaningful” energy supplies. 

The first step in the analysis was to list 10 major sets of assumptions on which this process could 

be based (Table III.D.1.). These included 

• essentially effortless species control, 

• a yield of about 45 mt/ha/yr (20 t/ac/y), 

• 4% N and 0.4% P in the algal biomass, 

• 40 ha (100 ac) growth ponds with multiple channels, and 

• harvesting by bioflocculation. 

Month-by-month variations in biomass density, productivity, water and CO2 utilization, etc. were 

estimated based on a typical southwestern United States location, with productivities ranging 

from a minimum of 6 to a maximum of 18 g/m2

/d. 
National Renewable Energy Laboratory 

222 A Look Back at the Aquatic Species Program—Technical Review 

Based on these assumptions, designs of the various system components were carried out, and 

supporting calculations made for the subsystems, including earthworks, pumps to move and lift 

the water, the supply channels and piping required, transfer structures, settling ponds, ducting for 

CO2, etc. The algal biomass would be digested to methane gas, but this was not included in the 

analysis. Based on estimates for various components, total capital costs were estimated (in 1978 

dollars) at about $9,000/ha, without contingencies or engineering. Annualized costs, based on a 

15% per annum capital charge, plus $700/ha operating costs for labor and nutrients, assuming 

free CO2, were about $2,000/ha, or about $45/t biomass. As pointed out in the report, “the basis 

for choosing many of the design features was low cost, or, actually, the highest cost allowable.” 

Thus, this report was primarily useful in identifying the major design assumptions and cost 

centers for such a process. 

This report was the first truly detailed analysis of such systems, though it still was, in many 

aspects, highly conceptual. It was used by Regan (1980) for a similar analysis of a large-scale 

algal (B. braunii) hydrocarbon production process in Australia, and served as the basis for 

subsequent analysis by DOE and the ASP. 

Publications: 

Benemann, J.R.; Persoff, P.; Oswald, W.J. (1978) “Cost analysis of microalgae biomass 

systems.” Final Report prepared for the U.S. Dept. of Energy, HCP/t1-605-01 Under Contract 

EX-78-X-01-1 605. 

Regan, D.L. (1980) “Marine biotechnology and the use of arid zones.” Search 111:377-381. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 223 

Table III.D.1. Design assumptions for a microalgae production system. 

(Source: Benemann et al. 1978.) 
National Renewable Energy Laboratory 

224 A Look Back at the Aquatic Species Program—Technical Review 

III.D.4. Cost Analysis of Aquatic Biomass Systems 

In 1978, Dynatech R/D Company prepared a report that analyzed the feasibility of using both 

macro- and microalgae systems, as well as other aquatic plants, for fuel production (Dynatech 

R/D Company 1978a). A major emphasis of this report was seaweed systems (“Ocean Farms”), 

as these were a major focus of the ERDA/DOE Fuels from Biomass Program at the time. This 

report concluded that macroalgae systems, based on open ocean giant floating seaweed farms, 

were technically and economically infeasible. The report also addressed the land-based 

microalgae systems, based on the report by Benemann et al. (1978) discussed in the previous 

section. The authors concluded that CO2 supply from power plant stack gases required 

“prohibitively expensive” duct work, distribution and transfer systems. It recommended further 

development only of emergent higher aquatic plants, such as water hyachinths and marsh plants, 

which can use CO2 from the air. 

However, these conclusions were subjected to considerable critique. In response, a companion 

report was prepared that addressed “Reviewers’ Comments” (Dynatech R/D Co. 1978b). 

Although most of the comments related to the macroalgae systems, the conclusions regarding the 

microalgae process were also challenged, specifically with respect to CO2 transfer. It was 

pointed out that CO2 transport distances from the power plant to the ponds need not be longer 

than 10 km, as assumed in the Dynatech R/D Co. report and that CO2 transfer into the ponds 

could be both efficient and of low-cost. In other respects, including water and nutrients supply 

and use, the Dyantech R/D (1978a) report concluded that overall “it appears that there is a high 

probability that land-based aquatic biomass growth systems can be designed which are 

technically feasible and for which growth energetics are quite favorable.” This conclusion did 

“not necessarily imply economic feasibility.” 

Actually, the Dynatech R/D Co. (1978a) conclusions were more positive than the opinions of 

some participants in this project. For example, Goldman and Ryther (1977) had earlier rejected 

the concept of microalgae fuel production, because, among other arguments, the water and 

fertilizer resources for microalgae ponds would be prohibitive. However, Oswald and Benemann 

(1977) countered arguments, pointing out, for example, that such a simplistic analysis failed to 

consider water and nutrient recycling. In this connection, Goldman (1979a,b) also reviewed the 

fundamental and practical aspects of microalgae biomass production, including the productivity 

data with outdoor pond systems. As part of the Dynatech Report, DOE published a “Topical 

Analysis” of aquatic biomass systems (Goldman et al. 1977), a good review of the scientific basis 

at the time. 

Publications: 

Dynatech R/D Company, (1978a) “Cost analysis of aquatic biomass systems.” Report prepared 

for the U.S. Dept. of Energy, HCP/ET-4000-78-1, vol. 1. 

Dynatech R/D Company, (1978b) “Reviewers comments on cost analysis of aquatic biomass 

systems.” Report prepared for the U.S. Dept. of Energy, HCP/ET-4000-78-2, vol. 2. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 225 

Goldman, J.C.; Ryther, J.H. (1977) “Mass production of algae: bioengineering aspects.” In 

Biological Energy Conversion (Mitsui, A., et al., eds.), Academic Press, New York, pp. 367-378. 

Goldman, J.C.; Ryther, J.H.; Waaland, R.; Wilson, E.H. (1977) “Topical report on sources and 

systems for aquatic plant biomass as an energy source.” Report to the U.S. Dept. of Energy. 

Oswald, W.J.; Benemann, J.R. (1977) “A critical analysis of bioconversion with microalgae.” In 

Biological Energy Conversion (Mitsui, A., et al., eds.), Academic Press, New York, pp. 379-394. 

III.D.5. Microalgae as a Source of Liquid Fuels 

After the ASP was established at SERI in the late 1970s, the emphasis switched from methane 

production to algal oils as the fuel product. This was based on the known ability of some 

microalgae species to accumulate large amounts of algal lipids, in particular under conditions of 

nutrient (mainly N and Si) limitations (See Section II and Section III.B.5.d.). 

As discussed in Section III.A.1, initially the ASP set out to investigate both the HRP design 

described earlier and a patented, closed photobioreactor concept, the ARPS. In 1981, the DOE 

Office of Energy Research requested an in-depth engineering and cost analysis of both systems. 

However, by the time the final report was completed in 1982, the ARPS had already started to 

evolve toward a more standard design (Section II.B.2.), and the HRP project in California 

(Section III.B.3.) was re-instated. Thus, the comparative evaluation had become somewhat 

moot, and the final report (Benemann et al. 1982a) covered only the HRP system in detail, with 

the comparative HRP-ARPS analysis relegated to an unpublished appendix (Benemann et al. 

1982b). 

The HRP system followed quite closely on the earlier work of Benemann et al. (1978), but with 

some significant differences and much greater details for the engineering designs and cost 

estimates. As before, 40-ha earthwork ponds were used; however, this time with paddle wheel 

mixing. Productivities were now projected of 67.5 mt/ha/yr for an algal biomass containing 40% 

lipids (oils) by weight. This corresponded to about 90 mt/ha/yr for conventional algal biomass, 

yielding almost 160 barrels of crude oil/ha/yr. This was roughly twice as high as the prior study. 

Harvesting was again assumed to be by bioflocculation, followed by a centrifugation process to 

concentrate the biomass to a paste-like consistency. A solvent extraction process as used for 

soybean oil extraction was assumed, at three-time higher unit cost to account for the highmoisture in the paste. (However, as was pointed out, direct solvent extraction was unlikely to be 

feasible for such high moisture biomass.) As before, the residual biomass was to be 

anaerobically digested in covered ponds to produce methane gas, with the nutrients (and C) from 

the digester (and digester gas) recycled to the ponds. 

A major emphasis in this report was the development of engineering designs for the CO2 supply 

and transfer systems, a major point of criticism in the Dynatech R/D Co. (1978a) study. In fact, 

Mr. Don Augenstein, the author of the Dynatech R/D Co study, joined EnBio, Inc. and carried 

out the engineering designs and calculations for CO2 supply systems for the present report. A 
National Renewable Energy Laboratory 

226 A Look Back at the Aquatic Species Program—Technical Review 

key assumption was that the CO2 flue gas delivery pipe from the power plant was only 5 km 

long, at a cost of almost $3 million (1982$). The distribution piping system, including blowers 

and valves, was estimated at about $2 million for the 800-ha plant. A detailed analysis of power 

requirements and CO2 transfer issues was also carried out. 

Several cases and scenarios were analyzed, including operations at high (8.0-8.5) pH and low 

(7.0-7.5) pH, CO2 recycling from the methane produced, and the use of flue-gas and pure CO2. 

Flue gas CO2 required much higher capital and operating costs; pure CO2 required purchase of 

this nutrient. Various scenarios were analyzed for CO2 sources, including purchase and 

combustion of coal (e.g., co-siting a power plant). Another major variable analyzed was the 

capital and operating costs factors, such as labor costs and overhead, utilities and fuel costs, land 

costs, factors for buildings and power supplies, contingencies and contracting, architect and 

engineering fees, and capital-related cost factors (taxes, insurances, depreciations, maintenance, 

and returns on investment). These factors made a larger difference in final costs than most of the 

engineering design cost estimates (e.g., pond construction costs, paddle wheels). Somewhat 

surprisingly, there appears to be no standardization for such general cost factors. For the “base 

case” analysis, assuming no recyling of nutrients from the digester and a low pH of operation, 

costs were nearly identical, at almost $160/barrel oil, for pure CO2 and flue gas utilization cases. 

The higher cost of pure CO2 (@$45/mt) balanced by the higher capital and operating costs of the 

flue gas delivery system. 

The cost of oil at $160/barrel was excessive, even for the projected rising fossil fuel prices. By 

recycling nutrients and operating at a higher pH (reducing CO2 outgassing) and using lower cost 

($22/mt) CO2, overall costs could be reduced by about 20%-25%, to about $115 for the pure CO2

case, somewhat higher for the flue gas (coal) case (Table III.D.2.). This was a significant 

reduction, but still represented an excessive cost. This led to a reiteration of the entire 

engineering analysis, by using more optimistic engineering designs and estimates at each step, 

including, for example, a shorter flue gas delivery pipe. This resulted in a further capital costs 

reduction of about 25%. The capital cost related factors were also reduced, including power 

supply costs, building costs (to near $1,000/ha), and return on total capital (from 15% to 10% per 

annum). With these most optimistic assumptions, final costs were reduced to as low as 

$65/barrel of oil for a high pH flue gas case. Table III.D.2. summarizes these costs, for both for 

the conservative (base) case and optimistic cases. Updating these costs to roughly 1997 dollars 

would give, even under the optimistic case, costs for the oil of about $100/barrel. 

Other alternatives were also examined. Seawater systems were attractive if all the CO2 required 

for algal growth were to be supplied by the seawater. For the assumed productivities, this would 

require a hydraulic retention time of 1 day, and thus much larger settling ponds and settling 

velocity for the algae, but could be cost-effective. 

Another conclusion was that higher value byproducts were unlikely to significantly contribute to 

such systems, as their production (either in scale or objectives) would not be easily integrated 

with algal fuel production. The costs of methane and alcohol fuels from algal biomass (high in 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 227 

carbohydrates, rather than lipids) would likely be similar to that of algal oil production. Indeed 

the issues were not the final processing, but the primary production of the biomass. 

The authors identified four major research needs to achieve the objectives of high productivity in 

large-scale outdoor systems: 

1. Photosynthetic efficiency for light energy and high lipid production. 

2. Fundamentals of species selection and control in open pond systems. 

3. Mechanisms (and control) of algal bioflocculation. 

4. Effects of non-steady-state operating conditions on algal metabolism. 

The appendix to this report (Benemann et al. 1982b), analyzed the ARPS system as proposed by 

Raymond (1979, 1981) that was in development at the time at the University of Hawaii (Section 

III.B.1.). First, a detailed historical review of microalgae systems designs was presented, which 

traced the evolution of the two concepts. The main report carried out a detailed and updated 

review of all prior cost analyses. The specific claims made for the ARPS systems were analyzed 

in detail. For example, the CuSO4-filled cover was claimed to reduce harmful IR radiation, but 

this was not supported by the photosynthesis literature. Also, overheating would still be a major 

factor even with a CuSO4 cover, requiring a cooling process. In addition, the heated CuSO4

could not be plausibly used as a power source. Mixing power inputs would be prohibitive for 

this design. Increased productivities caused by a flashing light effect were not plausible. Most 

important, the costs for even the cover and liner for such a system would be prohibitive by 

themselves, without considering any other factors. 

This study clearly identified the major difficulties associated with microalgal mass culture for 

fuel production. Only a very low-cost system, based on open ponds without plastic liners, mixed 

at low velocities, and using a very simple harvesting process, could be considered in such a 

process. But even with these rather favorable, though plausible, assumptions, costs would still be 

well above those for current, or projected, oil prices. 

Publications: 

Benemann, J.R.; Augenstein, D.C.; Weissman, J.C. (1982a) “Microalgae as a source of liquid 

fuels, appendix: technical feasibility analysis.” Final Report, U.S. Department of Energy, 

unpublished, 126 pp. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1982b) “Microalgae as a 

source of liquid fuels.” Final Report, U.S. Department of Energy, 202 pp. 

Raymond, L. (1979) “Initial investigations of a shallow layer algal production system.” Am. Soc. 

Mech. Eng., New York. 
National Renewable Energy Laboratory 

228 A Look Back at the Aquatic Species Program—Technical Review 

Table III.D.2. Costs of microalgal biomass production. 

Productivity: 67.5 mt/ha/yr for 40% extractable lipid biomass (162 bbl oil/ha/yr). 

System Description: Twenty 40-ha growth ponds, harvesting by settling, with C recycle from the 

digesters and use of either pure CO2 delivered to the site for $22/mt, or generation of CO2 on site 

from coal at half this cost. 

Cost Estimates: Figures in parenthesis next to capital and operating cost items refer to the factors 

used for the conservative and optimistic cases, respectively. Maintenance, insurance, taxes, (6% 

and 4.8%, respectively), based on total capital costs, except for land and working capital, which 

are also not depreciated. ROI (return on total capital) based on total capital, before taxes. 

(Source: Benemann et al. 1982a.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 229 

III.D.6. Fuels from Microalgae Technology Status, Potential and Research 

Requirements 

In 1986, a report was generated by SERI ASP in-house researchers that analyzed factors and 

costs involved in microalgae biomass production systems (Neenan et al. 1986). The report 

reviewed the various system components and requirements for algal biomass production, and 

summarized and extended the available resource analyses for water, land, and most importantly, 

CO2 required for large-scale microalgal biomass production. It also reviewed the various fuel 

product alternatives from microalgae biomass, including ethanol, methane, PVO 

(“pseudovegetable oil”), biodiesel (methyl ester fuels), and even gasoline, (see also Feinberg 

1984). 

Although generally following the HRP system concept and design described earlier, the authors 

raised a number of issues and questions. For example, they concluded that paddle wheel mixing 

was not “the optimal design,” although paddle wheel mixing was used in their analysis. Some 

modifications were made in the engineering analyses, such as replacing channel dividers by 

plastic fences, and the use of clay liners in the ponds. For harvesting, microstrainers or belt 

filters were used as primary harvesters, followed by centrifugation. However, the overall design 

and cost estimates were essentially based on the Benemann et al. (1982) analysis, including scale 

(860-ha of pond surface). 

Using these inputs, an Algal Production and Economic Model was developed using various unit 

costs (costs of fertilizers, land, power, water, CO2, etc.) and design parameters (module size, 

depth, nutrient concentrations and losses, mixing velocities, etc.), financial factors, and operating 

parameters (retention times, algal biomass composition, growing seasons, efficiencies, etc.). As 

in the prior studies, the residues from the fuel extraction/processing would be converted to 

methane gas by anaerobic digestion. Schematics and process diagrams for the various processing 

options were developed. The reference case assumed a biomass with a 30% lipid content and a 

17 g/m2

/d average annual productivity (62.5 mt/ha/yr). The overall projected system costs for the 

reference case were $43,283/ha of ponds and $433/mt of algal biomass. This compares to 

$39,850/ha and $274/t in the “conservative” case of the Benemann et al. (1982) analysis (Table 

III.D.2., pure CO2 case). That was for a somewhat higher productivity (67.5 mg/ha/yr) and lipid 

content (40% versus 30% in this study). It is difficult to extract the specific cost differences 

between these analyses. However, the cost routines and parameters used had larger effects on 

costs than the engineering estimates. For one example, in Neenan et al. (1986) water costs were 

12% of overall costs, compared to less than 4% in Benemann et al. (1982). 

Compared to a biomass cost of $433/mt estimated for the reference case, the allowable feedstock 

costs for the various fuel options were calculated to be only somewhat above $100/mt (and even 

less for ethanol). Table III.D.3. provides a summary of this analysis, for a 36,000 mt/y (33,000 

t/y) microalgae biomass to fuels processing plant. The table provides capital and operating costs 

only for the fuel processing units, and derives an allowable algae cost, in $/t. There is a large 

discrepancy between these estimates and the projected biomass production costs. This led to the 

conclusion that such a process “is currently not commercially viable.” In fact, the main and 
National Renewable Energy Laboratory 

230 A Look Back at the Aquatic Species Program—Technical Review 

coproduct credits in this analysis were projections for the year 2010, when diesel or methane was 

expected to cost some four times current (1998) costs (even without inflation adjustment). This 

makes the economics of this process even less attractive. 

As in the prior analysis (Benemann et al. 1982a), a cost reduction and process improvement 

effort was undertaken and “attainability targets” developed. First, sensitivities were run for 13 

resources (such as power costs and evaporation), 15 facility design parameters (e.g., culture 

depth and mixing), three biological parameters (such as growing season) and eight financial 

parameters (cost escalations, etc.). Taken one at a time, most factors did not reduce costs 

significantly (except for growing seasons, culture depth and source water CO2 content). 

Although some of the results are difficult to interpret (for example, the large decrease in costs 

with increasing depth), the major conclusion was that no single parameter dominated costs 

sufficiently to achieve the goal of low-cost fuel production. Of course, several parameters in 

combination could do so. In particular, by increasing productivity to as high as 8% of total solar 

conversion efficiency and 50% lipids (50 g/m2

d), and by assuming a capital investment of 

$48,000/ha, an algal production cost of $211/mt was estimated. Using this cost, the microalgae 

biomass fuel processing costs were again estimated, allowing calculation of allowable fuel 

product costs, of $1.65/gal of biodiesel. 

The fuel processing cost estimates, the major contribution of this report, were very preliminary 

and based on many assumptions. For example, the costs of the transesterification plant were 

rather high, and might be reduced in the future. But, as a central conclusion, productivity was 

again the most important parameter: increasing production efficiencies by a factor of about four 

decreased production costs by almost half. The report concluded with a detailed analysis of the 

“attainable” process improvements, emphasizing the need for achieving multiple cost reductions, 

in addition to significantly increased photosynthetic efficiencies. The report concluded that 

“aggressive research is need to fulfill the performance requirements defined by this analysis”. 

Publications: 

Feinberg, D.A. (1984) “Fuel options from microalgae with representative chemical 

compositions.” Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR-231-2427. 

Neenan, B.; Feinberg, D.; Hill, A.; McIntosh, R.; Terry, K. (1986) “Fuels from microalgae: 

Technology status, potential, and research requirements.” Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-2550, 158 pp. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 231 

Table III.D.3. Costs of microalgal biofuels production: reference case. 

(Source: Neenan et al. 1986.) 

Summary of reference capital, operating, and allowable feedstock costs for microalgae fuel 

processing options. Of the five fuel options, the first four produce a high (30%) lipid biomass; 

the last (ethanol) fermentable carbohydrate, representing only 13% of biomass weight. 

Notes: 

a. Not including algae feedsock 

b. Process and cooling waters 

c. By-product prices: $0.07/m3

 for CO2 (captive use only), $7.40/MMBtu for 

methane, fuel gas, or LPG, and $13.30/MMBtu diesel fuel (exports) 

d. Main product prices: $7.40/MMBtu for methane, fuel gas, LPG, and $16.60 

($1.75/gal) for gasoline and diesel fuel, $1.20/gal for ethanol and $1.75/gal for 

biodiesel (methyl ester) or PVO 

e. The value ($/t) of the algal feestock in producing the main and coproduct mixes. 

For example, for the ester fuel case, the facility would produce 2.1 million 

gallons of biodiesel in addition to some 600,000 GJ of methane and 760 t of 

glycerol for export (plus 11 million m3

 CO2 and 2,400 tons N, recyled 

internally) at prices listed in c. and d. above. 
National Renewable Energy Laboratory 

232 A Look Back at the Aquatic Species Program—Technical Review 

Table III.D.4. Costs of microalgae biofuels production: attainability cases. 

(Source: Neenan et al. 1986.) 

Summary of Attainable Capital and Operating Costs for Fuel Processing Options. 

Notes: 

a. Includes microalgae biomass feedstock production costs of $211/mt 

b. Process and cooling waters 

c. By-product prices: $0.07/m3

 for CO2 (captive use only), $7.40/MMBtu for 

methane, fuel gas, or LPG, and $13.30/MMBtu diesel fuel (exports) 

d. Main product, million gallons/yr. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 233 

III.D.7. Design and Analysis of Microalgae Open Pond Systems 

This report (Weissman and Goebel 1987) originated from a competition held by the ASP for the 

development of a pilot plant (“test facility”) for microalgae production. As mentioned in Section 

III.A.4., two companies were selected to develop competing processes: Aquasearch, Inc., of San 

Diego, California, and Microbial Products, Inc., which had carried out the ASP pond project in 

California. The objectives were to arrive at cost projections for such a test facility, and also for 

scale-up costs of a future full-scale facility based on the selected process. Aquasearch, Inc., 

developed a concept for a closed system microalgae production process, using large plastic bag 

tubular reactors contained in a greenhouse. This system was not selected for further 

development, and no final report is available. Aquasearch, Inc. recently established in Hawaii a 

closed photobioreactor process for cultivating Haematococcus pluvialis, an alga high in 

astaxanthin. 

This study further developed the concept of the HRP system for large-scale microalgae 

production, providing considerable additional detail, and performing extensive sensitivity 

analysis of various design options. A site was identified near Brawley, California, in the Imperial 

Valley, for locating a pilot plant and a full-scale system. Ample groundwater resources were 

available at this site. There was also significant water supply available from the Salton Sea, 

which is as saline as ocean water, although of different ionic composition. As discussed in 

Benemann et al. (1978), the Salton Sea is a potential source of saline water and land for more 

than 10,000 ha of algal ponds, as such ponds could help manage the salinity of this inland sea, a 

major problem. The report proposed building two 0.4-ha ponds to validate the process, as well as 

several smaller ponds for process development and inoculum production. A covered anaerobic 

lagoon was to be included to test the digestion of the algae and the recycling of nutrients to the 

algal ponds. 

The design of these experimental and pilot plant-scale ponds was provided in great detail. A 

larger-scale, 400-ha, pond system was also designed and costed. This report presents the most 

detailed, comprehensive, and realistic cost estimates currently available for large-scale, low-cost 

microalgae biomass and fuels production. 

Several significant advances were made in this design and analysis. Perhaps most importantly, 

the report presented a fundamental analysis of CO2 supply and in-pond transfer issues, in 

combination with water chemistry and transit times between carbonation issues. The analysis 

concluded that CO2 utilization efficiencies can overall be very high, more than 95%, within 

parameters that would allow high microalgae productivity. Another innovation was the use of 

small amounts of high molecular weight polymers to improve the flocculation, settling 

characteristics and harvesting efficiency of the basic bioflocculation process. The polymers can 

be used in very small amounts, without contributing a major cost to the overall process. 

The base case (30 g/m2

/d) capital costs were estimated at almost $72,000/ha, without working 

capital, or almost twice as high as the prior effort (Benemann et al.1982). This was due to higher 

costs for many components, such as earthworks, which were several-fold higher. Among other 
National Renewable Energy Laboratory 

234 A Look Back at the Aquatic Species Program—Technical Review 

things, higher costs were assumed for rough and fine (laser) grading, which depends on the type 

of site assumed to be available. Also the 1987study estimated about $5,000/ha to provide a 3-5 

cm crushed rock layer, specified to reduce the suspension of silt from the pond bottom. There is, 

however, little evidence for a need for such erosion prevention, except perhaps for some areas 

around the paddle wheel and perhaps the turns. Further, the Weissman and Goebel (1987) study 

selected slipform poured concrete walls and dividers (baffles) as the design choice. For the 

curved portions of the walls and berms, the authors specified corrugated walls, with an average 

cost of about $25/m. This resulted in a cost of over $8,000/ha for the walls (perimeter central, 

etc.). Clearly, such design options and engineering specifications can result in very large 

differences in capital costs. For another example, in the present design, a power generation 

system was specified to produce electricity from the methane generated from the algal residues 

(at about 10% of total costs). This had not been included in the earlier study. Despite these 

higher costs, and perhaps because of them, this engineering design and cost analysis effort may 

be considered the most detailed and realistic one available. 

Table III.D.5. summarizes two design cases (from more than a dozen presented in the report), 

with 30 and 60 g/m2

/d average productivities. By using an annual capital charge of 25% 

(depreciation, return on investment, insurance, taxes), biomass costs of some $273/mt and 

$185/mt were estimated for the two productivity cases (Table III.D.5.). These costs were more 

than twice the cost derived from the “conservative” case in the earlier study (Benemann et 

al.1982), which used only the lower productivity. Note that this even included a significant 

credit for power production from the methane produced (most of which was used internally). 

Although this report is the most detailed and complete analysis of microalgae biomass production 

for fuels available, it can also be criticized for not attempting to examine cost reduction 

possibilities in the various design options, which would be required to make microalgae fuels 

production viable. Possible strategies for cost reduction were the objective of the study discussed 

in the following section. 

Publications: 

Weissman, J. C.; Goebel, R.P. (1987) “Design and analysis of pond systems for the purpose of 

producing fuels.” Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/STR-231-2840. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 235 

Table III.D.5. Capital and Operating Costs for an Open Pond System* 

(Source: Weissman and Goebel, 1987.) 

*Based on 400-ha system with nutrient and CO2 recycle from anaerobic digesters. 

A. CAPITAL COSTS
National Renewable Energy Laboratory 

236 A Look Back at the Aquatic Species Program—Technical Review 

Table III.D.5. Capital and Operating Costs for an Open Pond System* 

(Source: Weissman and Goebel, 1987.) 

*Based on 400-Ha system with nutrient and CO2 recycle from anaerobic digesters. 

B. OPERATING COSTS ($/ha/yr) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 237 

III.D.8. Systems and Economic Analysis of Microalgae Ponds for 

Conversion of CO2 to Biomass 

In recent years, there has been increasing interest in greenhouse gas mitigation technologies. As 

a consequence, there has been renewed interest in microalgae mass culture and fuels production 

from the perspective of CO2 utilization. This is not a new concept, as Oswald and Golueke 

(1960) had previously emphasized the potential for microalgae systems to reduce and avoid CO2

emissions and thus reduce the potential for global warming. Indeed, microalgae have a rather 

unique attribute: they can utilize concentrated CO2 for growth, rather than the air-levels of CO2

used by higher plants. This could allow the culture of microalgae on power plant flue gases, 

probably the only method for directly using such CO2 sources. Of course, once the microalgae 

biomass is converted to, and used as fuel, this CO2 is again released. However, an equivalent 

amount of fossil fuel is not burned and fossil CO2 released into the atmosphere, reducing overall 

CO2 emissions. 

In the early 1990s, some ASP work at NREL was supported by the Pittsburgh Energy 

Technology Center, PETC (now FETC, Federal Energy Technology Center, see Section III.D.9.). 

PETC also contracted with the University of California-Berkeley to analyze microalgae systems 

for power plant flue gas utilization and CO2 mitigation (Benemann and Oswald 1996). This 

report updated and extended the earlier cost analysis reviewed above. In particular it reanalyzed 

the assumptions on which these studies were based, and the costs for the various system 

components. For example, the costs of laser grading and earthworks were independently cost 

estimated through contacts with agricultural engineers with expertise in constructing rice paddies 

in northern California. Paddle wheel costs were based on the experience of the principal 

investigator (WJO) with large unit designs for waste treatment ponds. Pond walls and dividers 

were simple earthworks, much cheaper than the Weissman and Goebel (1987) design. Among 

the process innovations introduced was the use of a three-phase centrifuge to separate the algal 

lipids from the water and other biomass fractions. This provides a relatively straightforward 

method for lipid recovery (a major issue in prior studies) at only marginally higher costs than the 

centrifuge earlier specified for final concentration. However, overall this analysis was derivative 

of the prior studies. 

Table III.D.6. summarizes the costs projected by this analysis. Both 20 and 60 g/m2

/d 

productivities were assumed, with a high (40%) lipid biomass, equivalent to a 10% total solar 

conversion efficiency. Such very high productivities would clearly require a major R&D 

breakthrough. The theoretical approaches to such advances were reviewed. A reduction in light 

harvesting (“antenna”) pigments would increase the photosynthetic efficiencies at high light 

intensities. Microalgae with reduced antenna pigments would, however, not be very competitive 

in large-scale algal pond systems, and thus would be subject to contamination. However, 

nutrient limitation, required in any event to maximize lipid content in the algal cells, could be 

used as a strategy to limit such contaminants. 
National Renewable Energy Laboratory 

238 A Look Back at the Aquatic Species Program—Technical Review 

Both direct flue-gas utilization near the power plant and remote use of CO2 captured from flue 

gas and piped to the algal ponds were considered. With projected oil prices of $25/bbl and 

productivities of 5% solar conversion and 30 g/m2

/d assumed to be achievable in the near-term, 

projected cost were $77/mt to $100/mt CO2 avoided, similar to other direct flue gas mitigation 

options. With higher productivities (60 g/m2

/d) and oil prices ($35/bbl), CO2 avoidance with 

microalgae costs could drop below $10/mt CO2, a very competitive cost compared to other direct 

CO2 mitigation options. The estimates for CO2 mitigation are summarized in Table III.D.7. 

This report also estimated the costs of providing a significant inoculum for the culture systems, 

taking as the example the cultivation of B. braunii. This would involve producing small amounts 

of inoculum under highly controlled laboratory conditions, then amplifying the cultures using 

increasingly larger, but less controlled and less expensive photobioreactors. Such a process 

would add some 10% to 30% to overall costs, depending on the amount and control over 

inoculum production desired. The microalgae industry and harvesting technologies were also 

reviewed in some detail. 

A major emphasis in this report was the potential of microalgae CO2 utilization during 

wastewater treatment, recapitulating the work since the 1950s by this group (Section III.B.). 

Indeed, with CO2 mitigation being now the primary goal, rather than fuel production, what was 

before a cost could now add to the waste disposal credits of such systems. Microalgae 

wastewater treatment uses less energy, and thus fossil fuels, than conventional treatment 

processes, resulting in a reduction of greenhouse gas emissions. Wastewater treatment processes 

could provide a near-term pathway to developing large-scale microalgae production processes 

and could find applications around the world. With climate change a global problem, this now 

allows consideration of such international perspectives, even within a DOE-funded R&D 

program. 

The applications of microalgae to CO2 mitigation from power plants became a major focus of the 

ASP during the 1990s, as briefly reviewed in the following section. 

Publications: 

Benemann, J.R. (1993) “Alternative recommendation regarding biological CO2 utilization 

R&D.” In The Capture, Utilization and Disposal of Carbon Dioxide from Fossil Fuel Power 

Plants (Herzog, H., et al., eds.), U.S. Dept. of Energy, DOE/ER-30194/1, 56 pp. 

Benemann, J.R.; Oswald, W.J. (1966) “Systems and economic analysis of microalgae ponds for 

conversion of CO2 to biomass.” Final Report, Pittsburgh Energy Technology Center, Grant No. 

DE-FG22-93PC93204. 

Herzog, H., et al. (1993) “The capture, utilization and disposal of carbon dioxide from fossil fuel 

power plants.” Report, U.S. Dept. of Energy, DOE/ER-30194, vol. 1 and 2. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 239 

Table III.D.6. Summary of cost analysis of microalgae CO2 mitigation. 

400-ha system, @ $0.065/kWh for power sales. 

(Source: Benemann and Oswald 1996.) 
National Renewable Energy Laboratory 

240 A Look Back at the Aquatic Species Program—Technical Review 

Table III.D.7. Greenhouse gas balances and mitigation costs. 

400-ha system, @$0.065/kWh. 

(Source: Benemann and Oswald 1996.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 241 

III.D.9. NREL Studies of Flue Gas CO2 Utilization by Microalgae 

The last major engineering-related activity carried out by the ASP was a PETC-funded study, 

both laboratory and process design, for microalgae biodiesel production using power plant flue 

gases. The general arguments for use of microalgae in CO2 mitigation, from high productivity to 

relatively low water use were reviewed in several reports (Brown et al. 1991; Chelf et al. 1991). 

The authors concluded that the SOx and NOx impurities in flue gas would likely not have a major 

effect on algal cultures, and, indeed, that the NOx could not even provide the N requirements of 

the cultures. Prior work indicating an inhibitory effect of flue gases (for example Negoro et al. 

1991) was probably due to the acidification of the media and resulting pH drop, because of 

excessive flue gas transfer. 

To demonstrate that flue gases can be used for microalgae culture, NREL set up an experimental 

apparatus to supply controlled and measured amounts of such gases to the algal cultures. Figure 

III.D.1. shows a typical result, with no detectable difference between the simulated flue gas 

culture and the control gas (similar CO2 levels, but without SOx and NOx). With almost 1,000 

times more CO2 than SOx in flue gas, alkalinity neutralization would not be a major problem 

except where high water reuse and low alkalinities in the water coincide. In general, flue gas 

supply to algal cultures should not present a major problem. 

Aside from the experimental work, the ASP also carried out work on systems design and analysis 

for microalgae biodiesel production using power plant flue gases (Kadam 1994, 1995). The 

analysis was based on the production of essentially pure (liquified) CO2 from the flue gases of a 

500-MW power plant, using conventional amine scrubbing processes, and its supply to a 100-km 

remote microalgae production facility. Delivered costs were estimated at $40.5/mt CO2. The 

microalgae pond system design was based on the prior effort of Neenan et al. (1986; see Section 

III.D.6.) and included site selection criteria for specific power plants in New Mexico. The 

summary of the system model inputs and outputs are provided in Table III.D.8., which 

summarizes current and long-term projections for such a process. Although there are some 

significant differences between this and other cost analyses (prior sections), overall these results 

agree that through long-term productivity increases such processes could achieve CO2 mitigation 

costs competitive with other options. This places the focus on long-term efforts for productivity 

enhancements. 

Publications: 

Brown, L.M.; Zeiler, K.G. (1993) “Aquatic biomass and carbon dioxide trapping.” Energy 

Conv.Mgmt. 34:1005-1013. 

Chelf, P.; Brown, L.M.; Wyman, C.E. (1991) “Aquatic biomass resources and carbon dioxide 

trapping.” Biomass and Bioenergy 4:175-183. 

Kadam, K.L. (1994) “Bioutilization of coal combustion gases.” Draft Milestone Completion 

Report, Recovery & Delivery, National Renewable Energy Laboratory, Golden, Colorado. 
National Renewable Energy Laboratory 

242 A Look Back at the Aquatic Species Program—Technical Review 

Kadam, K.L. (1995) “Power plant flue gas as a source of CO2 for microalgae cultivation: 

technology and economics of CO2 recovery & delivery.” Draft Report, National Renewable 

Energy Laboratory, Golden, Colorado. 

Kadam, K.L. (1997) “Power plant flue gas as a source of CO2 for microalgae cultivation: 

economic impact of different process options.” Energ. Convers. Mgmt. 38:S505-S510. 

Zeiler, K.G.; Kadam, K.L. (1994) “Biological trapping of carbon dioxide.” Draft Milestone 

Completion Report, National Renewable Energy Laboratory, Golden, Colorado. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 243 

Figure III.D.1. Effects of flue gas components on microalgal growth. 

Growth of cells of Monoraphidium minutum exposed to 20 mL/min for 2 min 55 times during 

each 24-h period. (Source: Brown et al. 1994.) 
National Renewable Energy Laboratory 

244 A Look Back at the Aquatic Species Program—Technical Review 

Table III.D.8. Summary of costs for microalgae CO2 mitigation. 

Economic performance of processes with different maturities.1

(Source: Kadam 1995.) 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 245 

III.D.10. Conclusions. 

The cost analyses for large-scale microalgae production for fuels reviewed earlier evolved from 

the rather superficial analysis of the 1970s to the much more detailed and sophisticated studies 

during the 1980s, with some updates and advances during the present decade. The basic process 

did not change significantly from the conceptual designs first suggested by Oswald and Golueke 

(1960): very large open, shallow, unlined, mixed, raceway ponds. However, the design details 

have evolved significantly, and current engineering and cost analyses are much more realistic. 

There are, of course, still some major uncertainties with these engineering studies. For the 

fundamental raceway design these are the issues of scale and the need for some type of pond 

lining. Current commercial microalgae production ponds are typically 0.25-0.5-ha in size, and 

are lined with plastics to prevent percolation and silt suspension and to allow pond cleaning. 

However, there are also examples of much larger and unlined raceway ponds in a commercial 

production facility, specifically the Earthrise Farms Spirulina plant in southern California, where 

two large (approximately 5-ha) unlined ponds are currently operating. Similar systems are also 

used in wastewater treatment. The City of Hollister wastewater treatment plant includes a single 

7-ha raceway unlined pond mixed with an Archimedes screw. Even larger (>50 ha) unlined, 

unmixed ponds are also used for microalgae production in Australia for commercial production 

of Dunaliella, and in several countries in wastewater treatment. Thus, although some 

uncertainties remain (such as allowable channel width and wind fetch effects), in general the 

basic engineering designs and assumptions for the microalgae cultivation ponds appear well 

established. 

For the harvesting, fuel processing, and media/nutrient recycling subsystem designs the cost 

analyses are perhaps less robust, based on often untested assumptions. However, overall, none of 

these appear to provide a likely major show stopper. Still, most of these issues require more 

R&D. One area where little work has been done is in the extraction of the algal oils. Although 

in the most recent studies the use of large three-phase centrifuges was recommended (Benemann 

and Oswald 1996), this requires further study. 

Although no single design component or unit process in these engineering analyses has an 

overwhelming effect on costs, the cost projections are optimistic; therefore, there is relatively 

little scope for any further cost reductions. In most cases, engineering designs and specifications 

were based on the cheapest possible design and likely lowest costs. Also, the engineering design 

and system construction approaches were based on agricultural engineering practices, rather than 

those of chemical engineering, as agricultural materials and construction methods are more 

applicable, in addition to being of lower cost. 

A major conclusion from the cost analyses is that there is little prospect for any alternative 

designs for microalgae production systems that would be able to meet the requirements of 

microalgae production for fuels. This is particularly true of closed photobioreactors, in which the 

culture is entirely enclosed, in greenhouses, plastic tubes or bags, or other transparent enclosures. 

 The costs of even the simplest such system would likely be well above what is affordable for 
National Renewable Energy Laboratory 

246 A Look Back at the Aquatic Species Program—Technical Review 

fuel production processes. Even the simplest plastic sheeting cover over the ponds would much 

more than double total systems capital and operating costs. The simplest tubular 

photobioreactors are projected to have capital costs some ten times higher (e.g., $50/m2

) than 

open pond designs (Benemann 1998). And, despite many proponents of such closed 

photobioreactors, current commercial microalgae production systems use exclusively open pond 

cultures, even for very high-value microalgae products. The few attempts at large-scale (>1 t/yr) 

production of microalgae in closed systems have failed. 

Of course, closed photobioreactors could have benefits in areas such as better control over 

environmental conditions (pH, temperature) and biological contaminants, and higher cell 

concentrations, reducing liquid handling and harvesting costs. Thus, it would be theoretically 

possible to grow algal strains not able to dominate in open ponds, at higher productivities and 

reduced harvesting costs, thereby making up for the higher costs of closed photobioreactors 

(which proponents assume to be only marginally higher than open pond systems). Closed 

systems of various types may find important applications in the production of the “starter 

culture” or inoculum that will be required to initiate and maintain large-scale open pond 

operations. This could be particularly important when genetically improved or genetically 

engineered algal strains are used. 

At the other engineering design extreme are the very large (up to 100 ha) unmixed ponds used in 

the production of Dunaliella in Australia (and, until recently, also used for Spirulina production 

in Mexico). Such production processes are of even lower cost than the mixed raceway designs. 

However, due to hydraulic and CO2 supply limitations (among others), productivities are 

maximally only a few g/m2

/d, a small fraction of those required for microalgae fuels production. 

Thus, there seem to be few practical choices in the basic engineering design of a raceway pond 

system. Even the mixing options are restricted; paddle wheels are overall more economical, 

flexible, and suitable than the alternatives (e.g., Archimedes screws, recirculation pumps, or airlifts). 

However, the most important issues raised in these economic and engineering analyses are not 

the engineering designs, or even the cost estimates, but the biological assumptions on which such 

designs are based. These have changed dramatically during the past 2 decades in one major 

aspect: productivity. Productivity projections have escalated from less than 50 mt/ha/y in the 

initial studies (e.g., Benemann et al. 1977), to almost 300 mt/ha/y (on an equivalent heat of 

combustion basis) in the most recent extrapolations (Benemann et al. 1993). In terms of 

photosynthetic efficiency, these improvements are from about 2% total solar energy conversion 

to a near-theoretical 10% efficiency. This dramatic increase in projected productivities was 

based on two main factors: first the significant advances in the state-of-the-art during these 2 

decades, with significantly higher productivities than originally anticipated being measured in 

outdoor systems. And second, the clear necessity to achieve very high efficiencies for any 

sunlight-to-fuels process. Although there are theoretical, and practical, approaches to achieving 

such high efficiencies, they will without a doubt require relatively long-term R&D efforts (see 

Section IV.A.2.). 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 247 

Productivity, in terms of solar conversion efficiency, is only one of the objectives of future R&D 

in this field. A related issue is that much of this productivity must be in the form of algal lipids, 

suitable for utilization and upgrading to fuels. Although some progress was made in this area in 

the laboratory, through physiological and genetic means (see Section III.B.5.d.; also Section II), 

this still will require considerable research. Another area that will require significant research is 

the development of a low-cost harvesting process. Again, the engineering and economic realities 

constrict the choices to the lowest-cost option, which would appear to be a simple settling 

process, followed by further mechanical concentration and processing. 

The major conclusion of these analyses is that microalgae production for fuels is currently not 

limited by engineering designs, but by the many microalgae cultivation issues, from species 

control in large outdoor systems to harvesting and lipid accumulation to overall productivity. 

Future R&D must focus on these biological issues as a primary research objective, in the quest 

for low-cost production processes. 
National Renewable Energy Laboratory 

248 A Look Back at the Aquatic Species Program—Technical Review 

IV. Conclusions and Recommendations

IV.A. Microalgal Strain Improvement 

IV.A.1. Conclusions 

From the early 1980s through the mid-1990s, there was a major effort by ASP researchers and 

contractors to identify or develop microalgal strains that demonstrated properties conducive to 

cost-effective biomass and lipid production. The characteristics deemed desirable in these strains 

include high productivity, high lipid content, competitiveness in outdoor culture, and tolerance to 

fluctuations in temperature and salinity. Although a number of strains were identified as possible 

candidates, no one strain was found to possess the optimal characteristics. As discussed 

elsewhere in this report, perhaps the most significant observation is that the conditions that 

promote high productivity and rapid growth (nutrient sufficiency) and the conditions that induce 

lipid accumulation (nutrient limitation) are mutually exclusive. Further research will be needed 

to overcome this barrier, probably in the area of genetic manipulation of algal strains to increase 

photosynthetic efficiency or to increase constitutive levels of lipid synthesis in algal strains. 

The collection and screening efforts produced a number of significant findings. The SERI/NREL 

Microalgae Culture Collection was established as a valuable genetic resource and was the first 

microalgal collection that focused on organisms from brackish or saline environments. The 

organisms remaining in this collection (see Section II.A.3.) are being transferred to the 

University of Hawaii and should be available to interested researchers. 

Although a number of algal strains were investigated for growth and lipid production properties, 

the best candidates were found in two classes, the Chlorophyceae (green algae) and the 

Bacilliarophyceae (diatoms). Organisms were identified in both classes that showed high 

productivity, ability to grow in large-scale culture, and lipid accumulation upon nutrient stress. 

However, in some ways the diatoms may turn out to be better candidate organisms for biofuels 

production. The highest lipid levels (40%-60% of the AFDW) were found in diatoms. Limiting 

the availability of Si, a major component of the diatom cell wall, can induce lipid accumulation 

in diatoms. In green algae, lipid accumulation is induced by N starvation. N is a component of 

many cellular molecules, and N limitation would induce a complex response, affecting 

photosynthesis, protein and nucleic acid synthesis, and other biochemical processes. In contrast, 

Si is not involved in most intracellular processes, so the response to Si limitation should be 

simpler to interpret and control. The disadvantage to diatom cultivation is the added cost of Si 

supplementation in the medium for optimal growth, although this could be minimized by the use 

of strains with lightly silicified walls. Some green algal strains, for example M. minutum, are 

advantageous for mass culture applications in that they can survive temperature fluctuations that 

are lethal for diatoms. We found however, that the properties that make these organisms very 

hardy, such as a very tough cell wall, also make their biochemical and molecular studies 

problematic. Despite these generalizations, the ideal organism(s) for a biofuels production 

facility will likely be different for each location, particularly for growth in outdoor ponds. The 

best approach will likely be to screen for highly productive, oleaginous strains at selected sites, 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 249 

optimize growth conditions for large-scale culture, and optimize productivity and lipid 

production through genetic manipulation or biochemical manipulation of the timing of lipid 

accumulation in the selected strains. It is also likely that more than one strain will be used at a 

site, to maximize productivity at different times of the year. 

Significant progress was made during the last 15 years in the understanding of lipid accumulation 

in the microalgae, although there is still much to be learned. Clearly microalgal cells can be 

induced to accumulate significant quantities of lipid when the medium is limited for an essential 

nutrient. However, the actual mechanism that triggers the accumulation is unclear. Lipid 

accumulation is correlated with the cessation of cell division. A simple explanation is that lipid 

synthesis continues in the non-dividing cells, but since no new membranes are being synthesized, 

the lipid is shunted into storage lipids. Alternatively, non-dividing cells are not utilizing cellular 

energy reserves as rapidly as dividing cells, so lipid accumulates as synthesis occurs more rapidly 

than utilization. Nutrient deprivation affects specific biochemical pathways, as lipid 

accumulation is accompanied by an increase in the proportion of storage lipids (TAGs) to polar 

membrane lipids, and Si deprivation in diatoms increases the expression of at least one gene 

involved in lipid synthesis, acetyl-CoA carboxylase. In general, nutrient deprivation induces 

lipid accumulation in cells and is accompanied by a decrease in total (and total lipid) 

productivity. However, studies of lipid accumulation suggest that an understanding of the 

kinetics of the process could be critical and could allow the identification of a stage where 

biomass productivity and lipid levels are optimal for maximal lipid accumulation. 

Significant progress was also made in understanding the molecular biology of microalgae. Many 

of the green algae were found to contain DNA with unusually high GC ratios, and often with 

unusual modifications that would make these organisms more difficult as targets for genetic 

engineering. In contrast, the DNA content of diatoms is more typical of other eukaryotes. Work 

at NREL by ASP researchers resulted in the cloning and characterization of several genes 

involved in lipid and carbohydrate accumulation in diatoms, including the ACCase gene and a 

“fused” gene encoding the enzymes UDPglucose pyrophosphorylase and phosphoglucomutase. 

Isolation of these genes facilitated the development of a genetic transformation system for the 

diatoms. These genes were used in preliminary attempts to manipulate lipid production in these 

organisms. The successful development of the transformation system led to an increased 

understanding of the factors involved in introducing and expressing foreign genes in these 

organisms, and should facilitate the development of similar methods for other algal strains. 

There is still much to be done in the area of microalgal strain development for lipid or biofuels 

production. A number of suggestions for possible research areas will be discussed in the 

following sections. 
National Renewable Energy Laboratory 

250 A Look Back at the Aquatic Species Program—Technical Review 

IV.A.2. R & D Recommendations 

IV.A.2.a. General Considerations 

The conclusions presented elsewhere in this report focus attention on the fundamental issue of 

how to maximize the overall productivities of microalgae systems and suggest that there still is 

considerable scope for improvements. The issue of productivity, in its various guises and 

aspects, from species control to lipid (oil) yield and harvesting, is therefore recommended as a 

central subject for any future U.S. R&D program in microalgae biodiesel production. 

Essentially, the focus would be on developing the microbial catalysts that can convert solar 

energy to a liquid fuel at high overall efficiency. This effort will require a relatively long-term 

R&D effort, which would, at least initially, be focused on the fundamental and early-stage 

applied research required for such a biocatalyst development effort. 

This recommendation implies that engineering design, cost and resource analyses, and even 

outdoor pond operations, discussed in Section III, would be relatively minor parts of such a 

projected R&D program, at least initially. The argument is that most of the variables of 

large-scale microalgal culture can be scaled-down to very small-scale, even laboratory systems. 

This allows detailed investigation of the key parameters in maximizing productivities. At present 

the central issues in scale-up are those of algal species dominance and grazer control (and other 

biological invasions). However, at this point these are secondary to the necessity and priority of 

establishing a high benchmark for productivity and lipid induction, under physicochemical and 

other environmental conditions that would allow extrapolation to large-scale outdoor systems at 

typical locations. 

Again, this will require a relatively long-term R&D effort to accomplish, although guideposts, 

such as efficiency goals, to the needed advances can be provided. One issue is how to select 

strains for genetic improvements. This is still a difficult choice, as a relatively large investment 

is required to develop any novel genetic system. On the other hand, selection of the best strains 

for such a targeted genetics development effort is still some time off. Thus, a parallel track is 

recommended: strain selection (screening and improvements) would be carried out alongside 

with genetic engineering studies to demonstrate productivity enhancements using microalgal 

strains with already well-developed genetic systems. Such improvements would be in the 

efficiency of photosynthesis, described next, and lipid productivities, extending the ASP research 

reviewed in Section II. 

IV.A.2.b. Maximum Efficiency of Photosynthesis 

Many environmental factors affect the performance of the complex photosynthetic machinery in 

microalgae, reducing its efficiency to well below the maximum at which photosynthesis can 

perform. That maximum is dictated by the underlying mechanisms, biophysical constraints, and 

physiological adaptations. One objective of applied microalgal R&D would be to develop strains 

and techniques that achieve productivities as close as possible to the maximum. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 251 

However, somewhat surprisingly, there is still argument about the maximum limit for 

photosynthetic efficiencies. The arguments boil down to the mechanisms assumed and the many 

possible loss factors that may or may not be considered. Most researchers agree that an absolute 

minimum of eight quanta (photons) of light absorbed are required by the two-photosystem 

mechanism (Z-scheme) of photosynthesis to reduce one molecule of CO2 (and closer to 10 to 12 

quanta if the energy needs for CO2 fixation and cell metabolism are considered). However, there 

have been many reports of higher efficiencies. For example, recently Greenbaum et al. (1995) 

reported that some algal mutants lacking one photosystem still fixed CO2 (and produced H2), 

suggesting less than 8 (and as few as 4) quanta per CO2 reduced. However, recent reports cast 

doubts on this interpretation, and the two-photosystem mechanism appears robust. 

The maximum efficiency can be estimated at about 10% of total solar (Bolton 1996). Such 

efficiencies have been used in the projections for microalgae biodiesel production (see Section 

III.D.). However, high sunlight conversions are observed ony at low light intensities. Under full 

sunlight, typically one-third or less of this maximal efficiency, biomass productivity is obtained, 

because of the light saturation effect. 

Light saturation is simply the fact that algae, like many plants, can use efficiently rather low 

levels of light, typically only 10% of full sunlight (and often even less). Above this level, light is 

wasted. In fact, full sunlight intensities can damage the photosynthetic apparatus, a phenomenon 

known as photoinhibition. Light saturation and photoinhibition result from several hundred 

chlorophyll molecules collaborating in light trapping, an arrangement ideally suited for dense 

algal cultures, where on average a cell receives little light. However, exposed to full sunlight, the 

photosynthetic apparatus cannot keep up with the high photon flux and most of the photons are 

wasted, as heat and fluorescence, and can damage the photosynthetic apparatus in the process. 

One possibility, suggested by Neidhardt et al. (1998), is that photosynthetic productivity and 

light utilization could be maximized in microalgae by reducing the size of the light-harvesting 

antenna through mutation or genetic engineering. This is an interesting idea that will be 

discussed further in the next section. 

Publications: 

Bolton, J.R. (1996) “Solar photoproduction of hydrogen.” Report to the Int. Energy Agency, 

under Agreement on the Production and Utilization of Hydrogen, IEA/H2/TR-96. 

Greenbaum, E.; Lee, J.W.; Tevault, C.V.; Blankinship, S.L.; Metz, L.J. (1995) “Carbon dioxide 

fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking 

photosystem I.” Nature, August 3rd, (1995). 

Kok, B. (1953) “Experiments in photosynthesis by Chlorella in flashing light.” In Algal Culture: 

From Laboratory to Pilot Plant (Burlew, J.B., ed.), Carnegie Inst. of Washington, Publ. 600, pp. 

63-75. 
National Renewable Energy Laboratory 

252 A Look Back at the Aquatic Species Program—Technical Review 

Kok, B. (1973) “Photosynthesis.” Proceedings of the Workshop on Bio Solar Hydrogen 

Conversion (Gibbs, M., et al., eds.), September 5-6, Bethesda, Maryland, pp. 22-30. 

Melis, A.; Neidhardt, J.; Bartoli, I.; Benemann, J.R. (1998) Proc. Biohydrogen '97.

Neidhardt, J.; Benemann, J.R.; Baroli, I.; Melis, A. (1998) “Maximizing photosynthetic 

productivity and light utilization in microalgae by minimizing the light-harvesting chlorophyll 

antenna size of the photosystems.” Photosynthesis Res., in press. 

IV.A.2.c. Overcoming Light Saturation, Photooxidation, and Other Limitations 

The problem of light saturation has been a subject of research in photosynthesis for almost 5 

decades, with the report by Kok (1953) that microalgae cultures exposed to short (milliseconds) 

flashes of bright light, followed by longer periods of darkness, exhibited the same light 

conversion efficiencies as cultures exposed to the same total photon flux averaged for the entire 

period. The interpretation was straightforward: only a limited number of photons can be used per 

unit time, and the millisecond light/dark periods allow averaging high photon fluxes. A large 

body of literature has developed on this subject, including laboratory work by the ASP (Terry 

1984, 1986; see also Section II). The mass culture work in Hawaii (Section III.B.2.), among 

many others, attempted to use this phenomenon to increase algal productivities. However, 

practical applications are not plausible because of the very short time periods involved. Another 

approach, central to the Japanese microalgae program (Section IV.B.1.c.), has been to diffuse 

light throughout the depth of the culture, using optical fibers, thus avoiding high a surface 

irradiance. But this approach is also not practical for biodiesel production because of the very 

high cost of the system. 

A potential practical solution to the light saturation problem, and also probably to 

photoinhibition, has been recognized for many years (e.g., Kok 1973): reduce the number of 

chlorophyll molecules cooperating in photosynthesis (the so-called “antenna” chlorophylls) from 

a few hundred to a few dozen. This would allow the photosynthetic apparatus to absorb only as 

much light as it can use. The benefits of reduced absorption are that it would: 

• reduce waste, 

• limit photooxidative damage to the photosynthetic reaction center, and 

• increase the overall productivity of an algal culture, by a factor of at least 3 (see 

Benemann and Oswald 1996 for a recent discussion). 

However, it has only recently become possible to consider achieving this objective, through the 

detailed understanding of photosynthesis at the molecular level, and the development of genetic 

engineering tools that could now allow us to redesign the photosynthetic apparatus. Recent work 

by Melis et al. (1998) and Neidhardt et al. (1998) demonstrated, at the physiological level, the 

feasibility of obtaining high efficiencies and high light saturation levels with algal cultures. 

Much more research is required, but the molecular and genetic tools are available to achieve the 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 253 

desired high photosynthetic efficiencies by algal mass cultures. Such tools can also be used to 

direct the flow of photosynthate to desired metabolic products, such as lipids (see Section II). 

Future R&D should demonstrate the feasibility of genetically engineering an improved 

photosynthesis system using algae for which such genetic systems are already well established. 

Once proven, these techniques can then be transferred to strains suitable for mass culture. 

IV.A.2.d. Microalgal Strains for Mass Culture: Source and Genetic Improvements 

The immediate issue arises of how to isolate, select, improve, and maintain the algal strains 

required for large-scale, low-cost microalgae cultivation. The ASP spent considerable effort in 

this area, with the isolation, screening, maintenance, laboratory studies, outdoor cultivation, and 

genetic improvement of microalgal strains (see Section II). In general, laboratory results were 

not predictive of outdoor performance. In addition, the strains most successfully maintained 

outdoors were those that spontaneously arose in and then dominated the ponds, often for 

considerable periods. Indeed, one conclusion from the outdoor culture work was that strains 

maintained in laboratory culture are, in general, not very competitive in open ponds. 

What was attempted in this context, the mass culture of specific, selected and productive algal 

strains in large open ponds for long periods of time, has only been accomplished in algal mass 

cultures in a few cases, and is still rare in most industrial or environmental microbiology 

applications. In the case of microalgae mass cultures, only a few strains, Spirulina, Dunaliella, 

Scenedesmus, and Chlorella, have been successfully mass cultured at a commercial or large 

(>0.1 ha) scale. In the most successful cases, Spirulina and Dunaliella are maintained in open 

ponds through the use of chemically selective media, containing high bicarbonate and high 

salinity, respectively. Scenedesmus was mass cultured at the pilot scale in Germany and 

Chekoslovakia, and other countries, with the cultures obtained from isolates that invaded and 

dominated the ponds. Commercial Chlorella production, using selected strains, has suffered 

from culture instabilities, requiring frequent inoculation and short production runs, greatly 

increasing the costs of the process. Thus, commercial-scale production of microalgae does not 

provide a good guide for this problem. 

In the case of industrial microbiology, only the traditional fermentations (e.g., ethanol, vinegar, 

cheese production) use selected strains that can be inoculated into and maintained in the 

production system, which must be relatively “clean” to avoid rapid contamination, but do not 

need to be sterilized. In environmental and agricultural microbiology it has not yet been possible 

to inoculate desired microbes (e.g., pollutant degraders, N fixers) into the open environments and 

demonstrate their survival and efficacy. 

Within this context, the demonstration of the ability to mass culture at least some algal strains on 

a relatively long-term and reliable basis by ASP-supported projects in California, Hawaii and 

New Mexico, must be considered a significant advance and accomplishment. These results 

provide a fundamental basis for future developments and improvements in this technology. 

However, a basic issue still to be resolved is the source of the microalgal strains to be used in 
National Renewable Energy Laboratory 

254 A Look Back at the Aquatic Species Program—Technical Review 

outdoor cultures. The results of the ASP Program suggest that one choice would be to allow the 

production system to self-select the organisms. Strains that naturally invade potential production 

sites could be screened for subtle combinations of fast growth, competitiveness in high densities, 

and adaptation to prevailing environmental conditions. In this context, most of the critical 

parameters—temperature, light intensity, pH fluctuations—can be modeled rather easily at a 

modest scale. Thus, it should be possible to select such strains in downscaled models that would 

allow much better control than possible in large ponds over the selective conditions desired. 

One factor essentially impossible to model or scale down is the biotic environment itself, that is, 

invasions by other microalgae, predation by grazers, infection by viruses, and other obvious or 

hidden biological effects that result in decreased productivities or even loss of culture. However, 

it appears from the experience with outdoor ponds, that these biotic effects are usually 

consequences of, not fundamental reasons for, loss of culture competitiveness. Further, some 

techniques have been developed to counteract such problems, for example rotifer grazing. In 

general, these problems will have to be dealt with when the technology has advanced to the point 

where large-scale culture efforts can be justified. That is, after high productivity cultures can be 

demonstrated at smaller scales, starting with laboratory simulations. 

It is thus recommended that small-scale systems, mimicking as much as possible the outdoor 

environment, be used as selection devices for microalgae strains suitable for outdoor algal mass 

cultures. Suitability for mass culture can be established at a relatively small scales (<200 m2

). 

Such selected “wild type” algal strains, would, of course, not necessarily exhibit the high 

biomass and lipid productivities required for the purposes of biodiesel production. Thus, 

considerable R&D will be required to genetically improve such strains. The techniques used to 

increase photosynthetic efficiencies or to optimize lipid quantity or quality, achieved with 

laboratory strains, must then be applied to the isolated strains suitable for algal mass culture. 

Thus the recommendation for future R&D in this field is for a parallel track effort: 

1. Demonstrate the feasibility to achieve with laboratory systems the high solar 

conversion efficiencies and lipid productivities required for biodiesel 

production. 

2. Isolate and study strains suitable for mass cultures, then apply the productivity 

enhancement techniques developed under laboratory conditions to these strains. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 255 

IV.B. Microalgal Mass Culture 

IV.B.1. Conclusions 

IV.B.1.a. Cost and Productivity Goals 

The overall conclusion from this review of 2 decades of DOE and ASP R&D in microalgal mass 

culture for biodiesel and other renewable fuels, is that this technology still requires relatively 

long-term R&D for practical realization. The initial, rather optimistic, cost and performance 

projections have not been met, or when met, the performance expectation (e.g., for productivity) 

have been raised. This was due, in large part, to the following factors: 

1. The expectations for the future costs of fossil fuels have declined. 

2. The value of by-product credits for waste treatment, greenhouse gas mitigation, 

or higher value coproducts are either uncertain or relatively low. 

3. The recent engineering designs and economic analyses have projected higher 

costs than earlier estimated, partly because of greater detail and realism, thus 

requiring higher productivities to achieve cost goals. 

4. The actual productivity results of the outdoor experimental work were well 

below the projections on which the economic analyses are based. 

In this concluding section, these issues are briefly addressed, followed by a discussion of future 

R&D needs and recommendations. 

The expectation for the economics of alternative fuels is a moving and uncertain target. Energy 

prices have been falling in real terms for more than 20 years, since the last oil-shock of the late 

1970s. Competing within current market realities is not plausible for most renewable energy 

technologies. Indeed, electric industry deregulation is removing price supports for such 

technologies as wood, wind, and geothermal power. The price of fossil fuels will probably start 

to reflect at least some of their externalities costs, including air pollution and greenhouse gases, 

and plausibly even a cost penalty to account for their non-sustainable nature. However, any 

projection of the future price or costs of fossil fuels, with which renewable fuels such as 

microalgae biodiesel would need to compete in the marketplace, is rather uncertain and arbitrary. 

For example, the use of a C-tax of some $50/t CO2 has been suggested, based on a current tax in 

Norway. However, if this were applied to all fossil fuels currently consumed, equivalent to some 

20 billion tons of CO2 world wide, it would increase the energy sector of the word economy by 

$1 trillion, more than tripling current expenditures on fossil fuels, a highly unlikely possibility. 

Perhaps a more modest tax of $50/tC (approximately $14/t CO2), would be a more appropriate 

upper bound for greenhouse gas mitigation penalties (e.g., credits for renewable energy sources). 

 At any rate, presently there is essentially no monetization of greenhouse gas mitigation, and any 

such figures are, at best, educated guesses. 
National Renewable Energy Laboratory 

256 A Look Back at the Aquatic Species Program—Technical Review 

However, greenhouse gas mitigation credits would likely be the overwhelming considerations in 

any future externalities cost accounting. Table III.D.7., summarized greenhouse gas credits 

required for microalgae systems, demonstrating the decisive effects of competitive fossil fuel 

costs on the necessary valuation of greenhouse gas mitigation. That table also demonstrates the 

major effect of productivity on the projected economics of such systems. 

Another potential enhancement of microalgae biodiesel economics is in wastewater treatment. 

Here the technology and economics would be dominated by the competitive costs with an 

activated sludge plant, or other wastewater treatment processes, including conventional 

microalgae pond systems. The latter, known also as facultative or stabilization pond systems, 

naturally treat municipal wastewaters (sewage), liquid animal manures, food processing wastes, 

and even some industrial effluents. In current technology, with very few exceptions (e.g., the 

City of Sunnyvale, California) the algal biomass is not harvested, and thus it is discharged to the 

nearest body of water (river, lake, etc.), used for irrigation, groundwater recharge, or it settles to 

the bottom of the ponds. Such systems are not designed for maximizing biomass production. 

However, through conversion to high rate ponds, they provide a possible entry for introducing 

and demonstrating of microalgae biomass fuel production and CO2 utilization. Of course, their 

economics would not be dictated, except marginally, by their waste treatment functions, and their 

impacts on U.S. greenhouse gas emissions and fuel resources would be modest, at most a fraction 

of 1% of U.S. energy consumption and greenhouse gas emissions. 

To expand the economic base and potential of such systems, other higher value coproducts or 

byproducts have been considered from such systems and processes. This is discussed in the 

following section. 

IV.B.1.b. Higher Value Byproducts and Coproducts 

The problem with integrating microalgae biodiesel production with any high value coproducts or 

byproducts, such as pigments, vitamins, or specialty chemicals, is that these would be produced 

in very large amounts, saturating any likely markets. And, of course, the requirements for 

producing such high value products are quite different from the needs for biomass fuels. One 

example for coproducts comes from fuel ethanol production from corn, which is economically 

dependent on animal feeds (distillers dried grains), byproducts for economic viability, in addition 

to the more than $1/gallon in subsidies. Indeed, only large byproduct markets, such as animal 

feeds, could be realistically considered in the context of biodiesel production. However, 

although it may be possible to coproduce proteins with algal lipids, such an optimization (e.g., 

for high protein feeds) is likely to be difficult. Another major problem, as in distillers dried 

grain, is the drying costs. Overall, higher-value feed coproducts cannot, and should not, be a 

major driving force in developing this technology. 

Of course, the likely route for the future development of practical and commercial large-scale 

microalgae culture technology will be through development of specialty foods and animal feeds 

coproduction. For example, Spirulina with two farms in the United States, comprising more than 

100-ha of ponds, is becoming a commodity product, with bulk prices declining by almost half 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 257 

(from the high 20s to the mid teens in dollars per kg) during the past 2 years. If this trend were to 

continue, to below $10/kg, this algal biomass could become a significant ingredient in 

aquaculture and other specialty animal feeds. Larger-scale systems for poultry feed production 

(microalgae high in xanthophylls, for example), or even cattle and hog feeds, could be foreseen, 

but require a decrease in costs (prices) to about $1,000/ton. 

But to be considered for fuel production, costs for microalgal biomass production would need to 

be reduced to the absolute minimum. This implies that productivities must significantly increase, 

and costs decrease from current levels. We argue that any future technology development effort 

for microalgae biodiesel production should exclude higher value byproducts or coproducts as a 

specific target. We believe that the needs of biodiesel production, specifically for high lipid 

productivities, must be the objectives of such a program. One exception is the combination of 

such a process with wastewater treatment, as there are few likely alternative uses for the biomass. 

 The other is the utility of such processes in greenhouse gas mitigation, also the objective of the 

Japanese R&D program in microalgae CO2 utilization, discussed briefly in the following section. 

IV.B.1.c. The Japanese R&D Program for Microalgae CO2 Utilization 

In Japan, the Research for Innovative Technology of the Earth program (RITE) has carried out an 

extensive progam for microalgal CO2 utilization. The Ministry of International Trade and 

Industry (MITI) funds this program, through the New Energy Development Organization 

(NEDO). The program was established in 1990 as a 10-year effort, carried out by approximately 

two dozen private companies, with some supporting work at various national laboratories and 

academic institutions. The budget is generally stated to be approximately $80 million (10 billion 

Yen) for 10 years; however, this is only for direct costs provided by RITE to the companies. If 

all indirect costs and supporting R&D at various institutes were to be included, this would easily 

double this budget. Also, microalgae-CO2 capture related R&D has been going on at the 

Japanese electric utilities, projects that are not part of the MITI-NEDO-RITE program. Thus, 

perhaps it is no exaggeration to estimate that during the 1990s the Japanese government and 

private companies will have invested more than $200 million (and perhaps closer to $250 

million) in this research. 

Although the Japanese microalgae R&D program is very large, it can be summarized rather 

briefly. Contrary to the U.S. approach, the Japanese effort has focused on closed 

photobioreactors, and on higher-value products. The argument made for closed photobioreactors 

was that these would require less land area than open ponds, because of much higher 

productivities. The higher productivities were assumed to be possible by using optical fibers to 

diffuse light into the reactors, and by greater control over environmental conditions (such as the 

ability to supply high CO2 levels to the cultures). Lower land requirements were also assumed to 

be possible with the optical fiber devices, although the land required for the concentrating 

mirrors was apparently not considered. The Japanese RITE program has yet to carry out, or at 

least report on, any technical, engineering, or economic analysis on such processes. A Japanese 

report (by researchers not part of the RITE program) to the IEA Greenhouse Gas R&D 
National Renewable Energy Laboratory 

258 A Look Back at the Aquatic Species Program—Technical Review 

Programme (IEA 1994) on microalgae “direct biofixation” of CO2, was based on prior U.S. 

engineering and cost analyses (Ikuta 1994), though they dried the algal biomass to replace coal. 

The Japanese RITE program has presented some results on their microalgae genetics program 

and photobioreactor development (Murakami and Ikenouchi 1997; Usui and Ikenouch 1975). But 

these add little detail to the development of this technology. 

One major emphasis of the Japanese program has been on developing high-value coproducts, 

from animal feeds to antibiotics to specialty chemicals. Some are rather esoteric, such as 

algae-based paper and concrete additives. In brief, the Japanese RITE Biological CO2 Fixation 

Program, and other Japanese R&D activities, perhaps in part by concentrating on such highervalue products, have not significantly advanced the technology for biofuels production or CO2

utilization, despite large investments. 

One exception is the work carried out by Mitsubishi Heavy Industries (MHI) and several electric 

utilities, in particular Tohoku Electric Co., near the northern city of Sendai, in the early 1990s. 

There, a small pond (approximately 3-m2

) project was carried out on the mass culture of diatoms 

and green algae. These studies initially used algal strains obtained from the NREL culture 

collection, and then with strains that spontaneously appeared and dominated the cultures at this 

site (Negoro et al. 1992, 1993). Productivity data were obtained and were generally in accord 

with the work at Roswell, New Mexico. (Two authors of this report, Benemann and Weissman, 

were consultants to this project.) 

Another interesting project was carried out by MHI and Tokyo Electric Power Co. (TEPCO), 

which demonstrated actual increased productivity in optical fiber bioreactors. However, the 

complication and costs of these devices resulted in this project shifting to more conventional, 

air-lift tubular reactors. Recently TEPCO-MHI released a publicity announcement of a major 

breakthrough in the production of ethanol from microalgae biomass. However, little specific 

technical information is available on this work. 

Publications: 

Ikuta, Y. (1994) “Design of a biological system for CO2 fixation, in carbon dioxide utilizationdirect biofixation.” Report to the Int. Energy Agency Greenhouse Gas R&D Programme. 

Murakami, M.; Ikenouchi, M. (1997) “The biological CO2 fixation and utilization project by 

RITE. 2. Screening and breeding of microalgae with high capability of fixing CO2.” Energy 

Conver. Mgmt. 38: Suppl. 493-498. 

Negoro, M.; Shioji, K.; Ikuta, Y.; Makita, T.; Utiumi, M. (1992) “Growth characteristics of 

microalgae in high-concentrations of CO2 gas: Effects of culture medium, trace components and 

impurities thereon.” Biochem Biotech. 34/35:681-692. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 259 

Negoro, M.; Hamasaki, K.A.; Ikuta, Y.; Makita, T.; Hirayama K.; Suzuki, S. (1993) “CO2

fixation by microalgae photosynthesis using actual flue gas discharged from a boiler.” Biochem 

Biotech. 39/40:643-653. 

Usui, N.; Ikenouchi, M. (1997) “The biological CO2 fixation and utilization project by RITE. 1. 

Highly effective photobioreactor system.” Energy Conver. Mgmt. 38: Suppl. 487-492. 

IV.B.1.d. Resource Projections and Microalgae Biodiesel R&D 

There are several arguments for and against a U.S. microalgae biodiesel R&D program. One of 

the more important, and perhaps contentious, issues, is the potential impact of such technologies 

on U.S. energy supplies, specifically liquid transportation fuels. The review in Section III.C. of 

the NREL resource analyses for microalgae biodiesel concluded that there is a potential for 

production of several quads (1015 Btu) of biodiesel fuels in the southwestern United States alone. 

However, as stated earlier, it will be difficult to find many locations where all the resources 

required for microalgae cultivation, flatland, brackish or waste waters, and low-cost CO2

supplies, are all available in juxtaposition. And, as also pointed out, the southwestern United 

States is not the ideal climatic location for such systems. For both these reasons, the resource 

potential estimated by these resource studies must be significantly discounted. 

In the case of utilization of power plant CO2, diurnal and seasonal factors would restrict direct 

CO2 (e.g., flue gas) utilization to about one-third of the power plant CO2. Even with CO2 capture 

and transportation (which greatly increases costs), only about half of the CO2 would be useable. 

With most coal-fired power plants located in the north, or in otherwise unfavorable climates, only 

a rather small fraction of power plant CO2 resources would likely be captured with microalgae 

systems in the United States. 

A conservative estimate is that microalgae systems would be able to mitigate, directly or 

indirectly, perhaps only about 1% of current power plant CO2 emissions, supplying an 

approximately equivalent amount of current transportation fuels. Herzog (1995) argued that such 

a potential, in fact, anything less than 10%, is not sufficient to justify a R&D effort, and that 

scarce resources should be devoted only to potentially high-impact technologies, such as the 

disposal of CO2 in the oceans or geological formations. However, Benemann (1995) countered 

that such a resource-only argument is too limited, as it ignores the issue of economics and 

technological risks. For example, the technical feasibility of ocean disposal is far from 

established, and the costs of such a process are not currently constrained by credible engineering 

and economic analyses. A balanced R&D portfolio would need to account for such factors. 

Also, it is inherently more attractive to use and recycle CO2, thus increasing economic activity, 

rather than to bury it or dump it into oceans. In addition, microalgae CO2 utilization could 

spin-off other technologies, as in the case of the ASP. Thus, although a decisive role for 

microalgae fuel production and greenhouse gas mitigation cannot be extrapolated, a modest R&D 

effort in this area is appropriate in the context of developing many such alternative technologies. 
National Renewable Energy Laboratory 

260 A Look Back at the Aquatic Species Program—Technical Review 

IV.B.1.e. Summary of Major Conclusions from the ASP Microalgal Mass Culture 

Work 

This report cannot do justice to the extensive and long-term R&D effort in applied microalgae 

mass culture carried out by DOE and the ASP over a 20-year period. Here only a very brief 

summary of the major conclusions is provided to put into context the recommendations for future 

R&D, which follow. 

Two major conclusions can be derived from the outdoor cultivation projects and 

engineering/economic analyses under the ASP, and can be briefly summarized: 

1. There appear to be no fundamental engineering and economic issues that would 

limit the technical feasibility of microalgae culture, either in terms of net energy 

inputs, nutrient (e.g., CO2) utilization, water requirements, harvesting 

technologies, or general system designs. 

2. Productivities, in terms of total biomass and algal lipids (oils) currently 

achieved during the ASP are substantially higher than those reported and even 

projected before the ASP, but still well below the theoretical potential, and the 

requirements for economical viability. 

The first conclusion should not imply that all these issues and problems have been solved. It 

does, however, suggest that the immediate R&D needs are not for engineering designs or cost 

analysis, or even in the operation of large, outdoor algal mass culture systems. Rather, from the 

second conclusion, the emphasis of any R&D effort must be on more fundamental and 

early-stage applied research issues faced in developing very high productivity algal strains. Ideal 

strains would dominate the pond cultures, achieve near-maximal productivities, efficiently 

biosynthesize large amounts of lipids, and be easy to harvest. 

Another conclusion from the DOE-ASP program is that the only plausible near- to mid-term 

application of microalgae biofuels production is integrated with wastewater treatment. In such 

cases the economic and resource constraints are relaxed, allowing for such processes to be 

considered with well below maximal productivities. 

IV.B.2. R & D Recomendations 

IV.B.2 a. Biodiesel Production and Algal Mass Culture for Wastewater Treatment 

The laboratory work outlined earlier will be a relatively longterm effort. Even after the 

demonstration at the laboratory and small-scale (e.g., 1 m2

) of the ability of genetically improved 

microalgae strains to exhibit high lipid productivities, many other factors and abilities are still 

required in algal mass culture. These, however, cannot be demonstrated in the laboratory. They 

include competitiveness, predation resistance, and harvestability. The issue thus arises of the 

need to carry out such algal mass culture research in parallel with the laboratory studies. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 261 

The outdoor projects of the ASP demonstrated the ability to mass culture microalgae under 

relatively unrestricted conditions (e.g., without a highly selective chemical environment), and to 

do so potentially at relatively low cost, as the inputs for power, nutrients, and water are rather 

modest. Considerable advances were also made in developing techniques for managing 

microalgae species in ponds, and demonstrating increasing biomass productivities. Of course, 

these subjects still require much more work. However, in the absence of improved microalgae 

strains, with the high total and lipid productivities required by the cost analyses, it would not be 

possible to make significant advances in this technology. Thus, a continuing emphasis on 

outdoor algal mass cultures, or on cost and resource analyses, is not recommended at this time. 

These accomplishments of the ASP now allow research to be focussed on the genetic work, as 

outlined above and in Section II, and to allow confident prediction of the ability to apply this 

research to mass culture systems. This is not to exclude some supporting outdoor studies, for 

example to verify the selection criteria used for the competitive strains for the genetic 

development. But, in general, the emphasis should be on genetics and strain improvements, not 

on outdoor culture technology development. 

However, a strictly laboratory-based R&D program, may rapidly loose touch with the realities of 

the eventual applications. Thus, some outdoor mass culture R&D is recommended, specifically 

for near-term development and demonstration of a combined microalgae wastewater treatment—

biodiesel production process. This recommendation is based on the potential for such systems to 

be developed and demonstrated rather quickly, and at relatively low cost. They would provide an 

early practical application of this technology, and justify the larger effort that would be required 

for the development of a significant microalgae-biodiesel industry. Benemann and Oswald 

(1996) present a detailed discussion of this approach. 

IV.B.3. Conclusions 

A microalgae biodiesel production system must be a solar conversion device, which operates at 

high efficiency and with minimal inputs at overall low cost. Cost constraints restrict 

consideration of such systems to the simplest possible devices, which are large unlined, open, 

mixed raceway ponds. Several decades of R&D in this field, in particular by the ASP, have 

revealed no plausible alternative to this basic design. Even some of the design details, such as 

the mixing devices (paddle wheels), depth (15-25 cm), mixing velocity (15-25 cm/s), CO2

transfer (countercurrent sumps), and others are fixed by the engineering and economic 

constraints. The commercial experience with open mass culture ponds suggests that such 

systems require relatively little further engineering development. Certainly, it would be of 

interest to determine the practical limits of such systems. Can single raceway pond scales be 

larger than 5 ha? What are the wind effects in such large systems? But overall, the engineering 

and hardware for the low-cost mass culture of microalgae cannot be considered a major R&D 

need in this field. 

Any effort toward the development of closed photobioreactors is probably too high risk in the 

present context. Although such devices could have a role in the buildup and production of 
National Renewable Energy Laboratory 

262 A Look Back at the Aquatic Species Program—Technical Review 

inoculum (starter cultures), they are not likely to be an essential or crucial component of 

large-scale, low-cost microalgae culture processes for energy production. 

Any future R&D program for microalgae CO2 capture and biofuels production must start with the 

development of the microalgae “biocatalysts.” The goal will be to construct strains via genetic 

engineering or other strain improvement methods that achieve very high solar conversion 

efficiencies and yield high lipid (oil) microalgal biomass, as required by the economic analyses. 

The central recommendation for a future R&D program is to emphasize such a biocatalyst 

development effort, building on the knowledge developed by the ASP. For a more near-term 

approach, there is a significant opportunity to develop and demonstrate microalgae biodiesel 

production as part of a wastewater treatment process; R&D in this area is also recommended. 

Finally, the international nature of the global warming problem now allows consideration of 

global impacts of such technologies. This could help justify a U.S., and an international, R&D 

effort, even if the impacts of microalgae biodiesel to future U.S. energy supplies were perceived 

to be modest.
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 263 

 V. BIBLIOGRAPHY 

V.A. SERI/NREL/DOE REPORTS AND PUBLICATIONS 

Anberg, S.M.; Meints, R.H. (1991) “Nucleotide sequence of the genes for ribulose-1,5-

bisphosphate carboxylase/oxygenase large subunit and ribosomal protein S145 from a Chlorellalike alga.” J. Phycol. 27:753-758. 

Anderson, D.B.; Molton, P.M.; Metting, B. (1981) “Assessment of blue-green algae in 

substantially reducing nitrogen fertilizer requirements for biomass fuel crops.” Proceedings of 

the July 1981, Subcontractors’ Review Meeting, Aquatic Species Program, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 69-73. 

Arad, S. (1984) “Development of outdoor raceway capable of yielding oil-rich halotolerant 

microalgae.” Aquatic Species Program Review: Proceedings of the April 1984 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, 

pp. 184-185. 

Arad, S. (1985) “Development of outdoor raceway for producing oil-rich halotolerant 

microalgae: An introduction.” Aquatic Species Program Review: Proceedings of the March 

1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-2700, pp. 228-229. 

Arad, S. (1987) “Integrated field-scale production of oil-rich microalgae under desert 

conditions.” FY 1986 Aquatic Species Program Annual Report; Solar Energy Research Institute, 

SERI/SP-231-3071; pp. 169-183. 

Barclay, W.R. (1984) “Microalgal technology and research at SERI: Species collection and 

characterization.” Aquatic Species Program Review: Proceedings of the April 1984 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, 

pp. 152-159. 

Barclay, W.; Chelf, P.; Nagle, N. (1987) “Environmental control of lipid production in diatom 

cultures.” Abstract, FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, p. 250. 

Barclay, W.; Johansen, J.; Chelf, P.; Nagle, N.; Roessler, P.; Lemke, P. (1986) “Microalgae 

culture collection, 1986-1987.” Solar Energy Research Institute, Golden, Colorado, SERI/SP232-3079, 149 pp. 

Barclay, B.; Nagle, N.; Terry, K. (1986) “Screening microalgae for biomass production potential: 

protocol modification and evaluation.” FY 1986 Aquatic Species Program Annual Report; Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 22-40. 
National Renewable Energy Laboratory 

264 A Look Back at the Aquatic Species Program—Technical Review 

Barclay, W.R.; Nagle, N.J.; Terry, K.L.; Ellingson, S.B.; Sommerfeld, M.R. (1988) 

“Characterization of saline groundwater resource quality for aquatic biomass production: a 

statistically-based approach.” Wat. Res. 22:373-379. 

Barclay, B.; Nagle, N.; Terry, K.; Roessler, P. (1985) “Collecting and screening microalgae from 

shallow, inland saline habitats.” Aquatic Species Program Review: Proceedings of the March 

1985 Principal Investigators’ Meeting, Solar Energy Research Institute, SERI/CP-231-2700, pp. 

52-68. 

Barclay, W.R.; Terry, K.L.; Nagle, N.J.; Weissman, J.C.; Goebel, R.P. (1987) “Potential of new 

strains of marine and inland saline-adapted microalgae for aquaculture.” J. World. Aquaculture 

Soc. 18:218-228. 

Ben-Amotz, A. (1984) “Development of outdoor raceway capable of yielding oil-rich 

halotolerant microalgae.” Aquatic Species Program Review: Proceedings of the April 1984 

Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP231-2341, pp. 186-194. 

Ben-Amotz, A. (1987) “Development of outdoor raceway capable of yielding oil-rich 

halotolerant microalgae” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, SERI/SP-231-3071, pp. 199-208. 

Ben-Amotz, A.; Tornabene, T.G. (1983) “Chemical profile of algae with emphasis on lipids of 

microalgae.” Aquatic Species Program Review: Proceedings of the March 1983 Principal 

Investigators’ Meeting; Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, 

pp. 123-133. 

Ben-Amotz, A.; Tornabene, T.G.; Thomas, W.H. (1985) “Chemical profiles of selected species 

of microalgae with emphasis on lipids.” J. Phycol. 21:72-81. 

Benemann, J.R. (1980) “Biomass energy economics.” Intl. Energy Journal 1:107-131. 

Benemann, J.R. (1993a) “Alternative recommendation regarding biological CO2 utilization 

R&D.” In The Capture, Utilization and Disposal of Carbon Dioxide from Fossil Fuel Power 

Plants (Herzog, H., et al., eds.), U.S. Dept. of Energy, DOE/ER-30194/1, 56 pp. 

Benemann, J.R. (1993b) “Utilization of carbon dioxide from fossil fuel-burning power plants 

with biological systems.” Energy Convers. Mgmt. 34:999-1004. 

Benemann, J.R. (1997) “CO2 mitigation with microalgae systems.” Energy Convers. Mgmt. vol. 

38, Suppl., pp. S475-S.479. 

Benemann, J.R.; Augenstein, D.C.; Weissman, J.C. (1982) “Microalgae as a source of liquid 

fuels, appendix: technical feasibility analysis.” Final Report to the U.S. Department of Energy, 

unpublished, 126 pp. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 265 

Benemann, J.R.; Baker, D.; Koopman, B.L.; Oswald, W.J. (1977) “A systems analysis of 

bioconversion with microalgae.” Proc. Symposium Clean Fuels from Biomass and Wastes

(Klass, D., ed.), Institute for Gas Technology, Chicago, pp. 101-126. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C. (1981) “Production of lipid hydrocarbon fuels and 

chemicals from freshwater microalgae.” Final Report, Contract Xb-0-9253-1, Solar Energy 

Research Institute, Golden, Colorado. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C. (1983) “Production of lipid hydrocarbon fuels and 

chemicals from freshwater microalgae.” Final Report, Solar Energy Research Institute, Golden, 

Colorado (unpublished). 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1982a) “Microalgae as a 

source of liquid fuels.” Proceedings of the June 1982 SERI Biomass Program Principal 

Investigators’ Review Meeting, Aquatic Species Program Reports, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1808, pp. 1-16. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1982b) “Microalgae as a 

source of liquid fuels.” Final Report, U.S. Department of Energy, 202 pp. 

Benemann, J.R.; Goebel, R.P.; Weissman, J.C.; Augenstein, D.C. (1983) “Production of liquid 

fuels and chemicals by microalgae.” Aquatic Species Program Review: Proceedings of the 

March 1983 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-1946, pp. 19-32. 

Benemann, J.R.; Koopman, B. L.; Weissman, J.C.; Oswald, W.J. (1976) “Biomass production 

and waste recycling with blue-green algae.” In Microbial Energy Conversion, (Schlegel, H.G.; 

Barnea, J., eds.), Gottingen, pp. 413-426. 

Benemann, J.R.; Hallenbeck, P.C.; Weissman, J.C.; Murry, M.; Oswald, W.J. (1977) “Fertilizer 

production with nitrogen-fixing heterocystous blue-green algae.” Final Report to the National 

Science Foundation, Sanitary Eng. Res. Lab., Univ. Calif. Berkeley. 

Benemann, J.R.; Koopman, B.L.; Baker, D.; Goebel, R.; Oswald, W.J. (1977) “Preliminary 

design of the algae pond subsystem of the photosynthesis energy factory.” Final Report to 

Inter-Technology Solar Corp., Sanitary Eng. Res. Lab., Univ. Calif. Berkeley. 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Oswald, W.J. (1977b) 

“Species control in large scale microalgae biomass production.” Report to Univ. Calif. Berkeley, 

SERL 77-5, SAN/740-77/1 (April 1977, revised November 1997.) 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Goebel, R. P. (1980) 

“Development of microalgae harvesting and high rate pond technology.” In Algal Biomass, 

(Shelef, G.; Soeder, C.J., eds.), Elsevier, pp. 457-499. 
National Renewable Energy Laboratory 

266 A Look Back at the Aquatic Species Program—Technical Review 

Benemann, J.R.; Koopman, B.L.; Weissman, J.C.; Eisenberg, D.M.; Oswald, W.J. (1978) “An 

integrated system for the conversion of solar energy with sewage-grown microalgae.” Report 

Contract D(0-3)-34, U.S. Dept. of Energy, SAN-003-4-2, June 1978. 

Benemann, J.R.; Miyamoto, K.; Hallenbeck, P.C. (1980) “Bioengineering aspects of 

biophotolysis.” Enzyme and Microbial Technology 2:103-111. 

Benemann, J.R.; Oswald, W.J. (1966) “Systems and economic analysis of microalgae ponds for 

conversion of CO2 to biomass.” Final Report, Grant No. DE-FG22-93PC93204, Pittsburgh 

Energy Technology Center. 

Benemann, J.R.; Persoff, P.; Oswald, W.J. (1978) “Cost analysis of microalgae biomass 

systems.” Final Report Contract EX-78-X-01-1, U.S. Dept. of Energy, HCP/t1-605-01. 

Benemann, J.R.; Tillett, D. (1987a) “Effects of fluctuating environments on the selection of high 

yielding microalgae.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071; pp. 285-300. 

Benemann, J.R.; Tillett, D.M. (1987b) “Microalgae lipid production.” In Energy from Biomass 

and Wastes XI, Conference Proceedings (Klass, D.L., ed.), Institute of Gas Technology, Chicago. 

Benemann, J.R.; Tillett, D.M. (1987c) “Effects of fluctuating environments on the selection of 

high yielding microalgae.” Final Report, Subcontract XK-4-04136-06, Solar Energy Research 

Institute, Golden, Colorado. 

Benemann, J.R.; Tillett, D.M. (1987d), “Effects of fluctuating environments on the selection of 

high yielding microalgae.” FY 1987 Aquatic Species Program, Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3206, pp. 

77-91. 

Benemann, J.R.; Tillett, D.M. (1990) “Effects of fluctuating environments on the selection of 

high yielding microalgae.” Final Report, Subcontract XK-4-04136-06, Solar Energy Research 

Institute, Golden, Colorado. 

Benemann, J.R.; Tillett, D.M.; Weissman, J.C. (1987) “Microalgae biotechnology.” Trends in 

Biotechnology 5:47-53. 

Benemann, J.R.; Weissman, J.C.; Eisenberg, D.M., Goebel, R.P.; Oswald, W.J. (1979) “Large 

scale freshwater microalgae production for fuel and fertilizer.” Report to the Univ. Calif. 

Berkeley, SERL 79-3 (1979) Contract EY-76-S-03-0034. 

Benemann, J.R.; Weissman, J.C.; Goebel, R.P. (1983) “Production of liquid fuels and chemicals 

by microalgae.” Aquatic Species Program Review: Proceedings of the March 1983 Principal 

Investigators' Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, 

pp. 19-32. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 267 

Benemann, J.R.; Weissman, J.C.; Koopman, B.L.; Oswald, W.J. (1977) “Energy production with 

microalgae.” Nature 268:19-23. 

Bergeron, P. (1989) “The economic impact of brine disposal on algal fuel facilities.” Aquatic 

Species Program Annual Report (Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3579, pp. 21-37. 

Bergeron, P.W.; Corder, R.E.; Hill, A.M.; Lindsey, H.; Lowenstein, M.Z. (1983). SERI Biomass 

Program Annual Technical Report: 1982. Solar Energy Research Institute, Golden, Colorado, 

SERI/TR-231-1918, 83 pp. 

Berglund, D.; Cooksey, B.; Cooksey, K.E.; Priscu, L.R. (1987) “Collection and screening of 

microalgae for lipid production: Possible use of a flow cytometer for lipid analysis.” FY 1986 

Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3071, pp. 41-52. 

Bird, K.T.; Hanisak, M.D.; Ryther, J.H. (1981) “Changes in agar and other chemical constituents 

of the seaweed Gracilaria tikvahiae when used as a substrate in methane digesters.” Resources 

and Conservation 6:321-327. 

Bollmeier, W. (1989) “SERI Program Overview.” Aquatic Species Program Annual Report

(Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3579, pp. 3-6. 

Boussiba, S.; Vonshak, A.; Cohen, Z.; Abeliovich, A.; Kaplan, D.; Richmond, A. (1985) 

“Development of outdoor system for production of lipid-rich halotolerant microalgae.” Aquatic 

Species Program Review: Proceedings of the March 1985 Principal Investigators’ Meeting, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp. 271-290. 

Boussiba, S.; Vonshak, A.; Cohen, Z.; Richmond, A. (1987) “Development of outdoor system 

for production of lipid-rich halotolerant microalgae.” FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 184-198. 

Brown, L.M. (1989) “Culture collection status.” Aquatic Species Program Annual Report

(Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3579, pp. 61-69. 

Brown, L.M. (1992) “Fuels from microalgae. Overview: Aquatic Species Project.” Aquatic 

Species Project Report, FY 1989-90, (Brown, L.M.; Sprague, S., eds.), National Renewable 

Energy Laboratory, Golden, Colorado, NREL/MP-232-4174, pp. 1-6. 

Brown, L.M.; Sprague, S. (1992) “Aquatic Species Project Report: FY 1991.” Report, April 

1992, National Renewable Energy Laboratory, Golden, Colorado, NREL/TP-422-4641, 30 pp. 
National Renewable Energy Laboratory 

268 A Look Back at the Aquatic Species Program—Technical Review 

Brown, L.M.; Sprague, S. (1993) “Biodiesel/Aquatic Species Project Report: FY 1992.” Report, 

May 1993, National Renewable Energy Laboratory, Golden, Colorado, NREL/TP-422-5259, 32 

pp. 

Brown, L.M.; Dunahay, T.G.; Jarvis, E.E. (1989) “Strategies for genetic improvement of 

microalgae with ability to grow in outdoor mass culture.” Aquatic Species Program Annual 

Report (Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3579, pp. 85-89. 

Brown, L.M.; Dunahay, T.G.; Jarvis, E.E. (1990). “Applications of genetics to microalgae 

production.” Dev. Industrial Microbiology, vol. 31 (J. Industrial Microbiol., Suppl. No.5), pp. 

271-274. 

Brown, L.M.; Sprague, S.; Jarvis, E.E.; Dunahay, T.G.; Roessler, R.G.; Zeiler, K.G. (1994) 

“Biodiesel from Aquatic Species Project Report: FY 1993.” Report, January 1994, National 

Renewable Energy Laboratory, Golden, Colorado, NREL/TP-422-5726, 32 pp. 

Brown, L.M.; Zeiler, K.G. (1993) “Aquatic biomass and carbon dioxide trapping.” Energy Conv. 

Mgmt. 34:1005-1013. 

Carlson, R.D.; Ryther, J.H.; Pendoley, P.D.; Jensen, P.R.; Blakeslee, M. (1986) “Collection and 

characterization of saline microalgae from south Florida.” Final Report, Solar Energy Research 

Institute, Golden, Colorado, 51 pp. 

Chelf, P. (1990) “Environmental control of lipid and biomass production in two diatom species.” 

J. Appl. Phycol. 2:121-129. 

Chelf, P.; Barclay, B.; Lemke, P. (1987) “Effect of environmental parameters on lipid production 

in Navicula saprophila.” FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, SERI/SP-231-3206, pp. 129-141. 

Chelf, P.; Brown, L.M. (1989) “Microalgae mass culture and the greenhouse effect: Resources 

update.” Aquatic Species Program Annual Report (Bollmeier, W.S.; Sprague, S., eds.), Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 9-15. 

Chelf, P.; Brown, L.M.; Wyman, C.E. (1991) “Aquatic biomass resources and carbon dioxide 

trapping.” Biomass and Bioenergy 4:175-183. 

Chelf, P.; Brown, L.M.; Wyman, C.E. (1992) “Aquatic biomass resources and carbon dioxide 

trapping.” Aquatic Species Project Report, FY 1989-90 (Brown, L.M.; Sprague, S., eds.), 

National Renewable Energy Laboratory, Golden, Colorado, NREL/MP-232-4174, pp. 7-31. 

Cheng, E.D.H.; Dugan, G.L. (1996) “Low-energy mixing in algal culture raceways.” J. Energy 

Engineering, December, pp. 100-107. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 269 

Cheng, L.; Lewin, R.A. (1987) “Lipogenic picopleuston.” Abstract, FY 1987 Aquatic Species 

Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3206; p. 75. 

Cohen, R.R.H. (1987) “Environmental effects on photorespiration in the diatom Chaetoceros 

muelleri.” Abstract, FY 1987 Aquatic Species Program Annual Report, (Johnson, D.A.; Sprague, 

S., eds.), Solar Energy Research Institute, SERI/SP-231-3206, p. 155. 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987a) “Environmental control of lipid 

accumulation in Nannochloropsis salina, Nanno Q and Euglena.” FY 1987 Aquatic Species 

Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3206, pp. 190-206. 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1987b) “Biochemistry of neutral lipid 

synthesis in microalgae.” Abstract, FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, SERI/SP-231-3071, p. 255. 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988a) “Preferential loss of chloroplast 

proteins in nitrogen deficient Euglena.” Plant Cell Physiol 29:1007-101. (Note: a preprint of this 

article was also submitted as a SERI Report, 47 pp.) 

Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988b) “Environmental control of 

carbohydrate and lipid synthesis in Euglena.” Plant Cell Physiol. 29:423-432. 

Cooksey, K.E. (1987) “Collection and screening of microalgae for lipid production.” Final 

Subcontract Report, Solar Energy Research Institute, Golden, Colorado, May 1987, 42 pp. 

Cooksey, K.E.; Guckert, J.B.; Thomas, R. (1989) “Triglyceride accumulation and the cell cycle 

in microalgae.” Aquatic Species Program Annual Report (Bollmeier, W.S.; Sprague, S., eds.), 

Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 135-154. 

Cooksey, K.E.; Guckert, J.B.; Williams, S.A.; Collis, P.R. (1987) “Fluorometric determination of 

the neutral lipid content of microalgal cells using Nile Red.” J. Microbiological Methods 6:333-

345. 

Cooksey, K.E.; Williams, S.A.; Callis, P.R. (1987) “Nile Red, a fluorophore useful in assessing 

the relative lipid content of single cells.” In The Metabolism, Structure and Function of Plant 

Lipids (Stumpf, P.K; Williams, S.A.; Callis, R.P., eds.), Plenum Press, NY, pp. 645-647. 

Corder, R.E.; Hill, A.M.; Lindsey, H.; Lowenstein, M.Z.; McIntosh, R.P. (1984) SERI Biomass 

Program FY 1983 Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR231-2159, 107 pp. 
National Renewable Energy Laboratory 

270 A Look Back at the Aquatic Species Program—Technical Review 

Dubbe, D.R.; Pratt, D.C. (1987) “Sustained production of cattail for energy applications.” 

Energy from Biomass and Wastes X, Conference Proceedings (Klass, D.L., ed.), Institute of Gas 

Technology, Chicago, pp. 363-383. 

Dunahay, T.G. (1993) “Transformation of Chlamydomonas reinhardtii with silicon carbide 

whiskers.” Biotechniques 15:452-460. 

Dunahay, T.G.; Adler, S.A.; Jarvik, J.W. (1997) “Transformation of microalgae using silicon 

carbide whiskers.” In Methods in Molecular Biology, vol. 62: Recombinant Gene Expression 

Protocols (Tuan, R., ed.), Humana Press, Totowa, New Jersey, pp. 503-509. 

Dunahay, T.G.; Jarvis, E.E.; Dais, S.S.; Roessler, P.G. (1996) “Manipulation of microalgal lipid 

production using genetic engineering.” Appl. Biochem. Biotechnol. 57/58:223-231. 

Dunahay, T.G.; Jarvis, E.E.; Roessler, P.G. (1995) “Genetic transformation of the diatoms 

Cyclotella cryptica and Navicula saprophila.” J. Phycol. 31:1004-1002. 

Dunahay, T.G.; Jarvis, E.E.; Zeiler, K.G.; Nagle, N.; Brown, L.M. (1992a) “Genetic engineering 

of microalgae for fuel production.” Aquatic Species Project Report, FY 1989-90 (Brown, L.M.; 

Sprague, S., eds.), National Renewable Energy Laboratory, Golden, Colorado, NREL/MP-232-

4174, pp. 57-119. 

Dunahay, T.G.; Jarvis, E.E.; Zeiler, K.G.; Roessler, P.G.; Brown, L.M. (1992b) “Genetic 

engineering of microalgae for fuel production.” Appl. Biochem. Biotechnol. 34/35:331-339. 

Dynatech R/D Company (1978) “Cost analysis of aquatic biomass systems.” Report, U.S. Dept. 

of Energy, HCP/ET-4000-78-1, vol. 1. 

Dynatech R/D Company (1978) “Reviewers comments on cost analysis of aquatic biomass 

systems.” Report, U.S. Dept. of Energy, HCP/ET-4000-78-2, vol. 2. 

Eisenberg, D.M.; Benemann, J.R.; Tiburzi, T.; Weissman, J.C.; Oswald, W.J. (1980) “Large 

scale freshwater microalgae production for fuel and fertilizer.” Report to the Univ. of Calif., 

Contract EY-76-S-03-0034. 

Eisenberg, D.M.; Koopman, B.; Benemann, J.R.; Oswald, W.J. (1981) “Algal bioflocculation and 

energy conservation in microalgae sewage ponds.” Biotech. Bioeng. Symp. No. 11., pp. 429-449. 

Ellingson, S.B.; Tyler, P.L.; Sommerfeld, M.R. (1989) “Characterization of growth and lipid 

yield in microalgae from the southwest using high salinity media.” Aquatic Species Program 

Annual Report (Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3579, pp. 71-81. 

Feinberg, D.A., (1984) “Fuel options from microalgae with representative chemical 

compositions.” Report, Golden, Colorado, SERI/TR-231-2427. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 271 

Feinberg, D.A.; Karpuk, M.E. (1989) “CO2 sources for microalgae based liquid fuel 

production.”Aquatic Species Program Annual Report (Bollmeier, W.S.; Sprague, S., eds.), Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 17-20. 

Feinberg, D.A.; Karpuk, M.E. (1990) “CO2 sources for microalgae based liquid fuel production.” 

Report, August 1990, Golden, Colorado, SERI/TP-232-3820, 35 pp. 

Gallagher, J.C. (1986) “Population genetics of microalgae.” In Algal Biomass Technologies: An 

Interdisciplinary Perspective (Barclay, W.; McIntosh, R., eds.), Beihefte zur Nova Hedwigia, 

Heft 83, Gebrüder Borntraeger, Berlin-Stutgart, pp. 6-14. 

Gallagher, J.C. (1987a) “Patterns of genetic diversity in three genera of oil-producing 

microalgae.” Abstract, FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, p. 

209. 

Gallagher, J.C. (1987b) “Genetic variation in oil-producing microalgae.” FY 1986 Aquatic 

Species Program Annual Report (Johnson, D.A., ed.), Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, pp. 321-336. 

Galloway, R.E. (1990) “Selective conditions and isolation of mutants in salt-tolerant, lipidproducing microalgae.” J. Phycol. 26:752-760. 

Goldman, J.R. (1979b) “Outdoor algal mass cultures. I. Applications; II. Theoretical Aspects.” 

Water Research 13:1-19. 

Goldman, J.R. (1979a) “Fuels from solar energy photosynthetic systems—state of the art and 

potential for energy production.” Report, U.S. Dept. of Energy, COOO-4141-241. 

Goldman, J.C.; Ryther, J.H. (1977) “Mass production of algae: bioengineering aspects.” In 

Biological Energy Conversion (Mitsui, A., et al., eds.), Academic Press, New York, pp. 367-378. 

Goldman, J.C.; Ryther, J.H.; Waaland, R.; Wilson, E.H. (1977) “Topical report on sources and 

systems for aquatic plant biomass as an energy source.” Report, U.S. Dept. of Energy. 

Golueke, C.G.; Oswald, W.J. (1965) “Harvesting and processing sewage-grown planktonic 

algae.” J. Wat. Poll. Cont. Fed. 37:471-498. 

Golueke, C.G.; Oswald, W.J. (1959) “Biological Conversion of light energy to the chemical 

energy of methane.” App. Microbiol. 7:219-241. 

Golueke, C.G.; Oswald, W.J.; Gotaas, H.B. (1957) “Anaerobic digestion of algae.” App. 

Microbiol. 5:47-55. 
National Renewable Energy Laboratory 

272 A Look Back at the Aquatic Species Program—Technical Review 

Graves, M.V.; Meints, R.H. (1992) “Characterization of the major capsid protein and cloning of 

its gene from algal virus PBCV-1.” Virology 188:198-207. 

Graves, M.V.; Meints, R.H. (1992) “Characterization of the gene encoding the most abundant in 

vitro translation product from a virus infected Chlorella-like alga.” Gene 113:149-155. 

Gregor, H.P.; Gregor, C.D (1978) “Synthetic membrane technology.” Sci. Amer. 239:112-128. 

Guckert, J.B.; Cooksey, K.E. (1987) “Biochemical elucidation of neutral lipid synthesis in 

microalgae.” Abstract, FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, p. 253. 

Guckert, J.B.; Cooksey, K.E. (1990) “Triglyceride accumulation and fatty aid profile changes in 

Chlorella (Chlorphyta) during high pH-induced cell cycle inhibition.” J. Phycol. 26:72-79. 

Guckert, J.B.; Cooksey, K.E.; Jackson, L.L. (1987a) “Biochemical elucidation of neutral lipid 

synthesis in microalgae: Report of a mechanism to induce neutral lipid accumulation and 

implications for increasing neutral lipid yields from microalgae.” FY 1987 Aquatic Species 

Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, 

SERI/SP-231-3206, pp. 174-189. 

Guckert, J.B.; Cooksey, K E.; Jackson, L.L. (1987b) “Lipid solvent systems are not equivalent 

for analysis of lipid classes in the microeukaryotic green alga, Chlorella.” J. Microbiol. Meth., 

submitted, never published. 

Habig, C.; DeBusk, T.A.; Ryther, J.H. (1984) “The effect of nitrogen content on methane 

production by the marine algae Gracilaria tikvahie and Ulva sp.” Biomass 4:239-251. 

Hanisak, M.D.; Ryther, J.H. (1986) “The experimental cultivation of the red seaweed Gracilaria 

tikvahie as an ‘energy crop’: An overview.” In Algal Biomass Technologies, An Interdisciplinary 

Perspective (Barclay, W.R.; McIntosh, R.P., eds.), Nova Hedwigia 83:212-217. 

Herzog, H., et al. (1993) “The capture, utilization and disposal of carbon dioxide from fossil fuel 

power plants.” Report to U.S. Dept. of Energy, DOE/ER-30194, vol. 1 and 2. 

Hill, A.M. (1982) “Cost budgeting for microalgae systems.” Proceedings of the June 1982 SERI 

Biomass Program Principal Investigators' Review Meeting, Aquatic Species Program Reports, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1808, pp. 17-30. 

Hill, A.M. (1984) “Analysis and evaluation of the economics of microalgal fuel production.” 

Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species Program, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 224-236. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 273 

Hill, A.; Feinberg, A.; McIntosh, R.; Neenan, B.; Terry, K. (1984) “Fuels from microalgae: 

Technical status, potential, and research issues.” Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-2550. 

Hon-Nami, K.; Hirano, A.; Kunito, S.; Suyuki Y.; Knoshita, K.; Ogushi, Y. (1997) “A new 

marine microalga cultivation in a tubular bioreactor and its utilization as an additive for paper 

surface improvement.” Energy Conv. Mgmt. 38: Suppl. pp. 481-486. 

Hubka, W.F.; Browne, S.H. (1983) “Microalgal systems production and processing simulation.” 

Aquatic Species Program Review: Proceedings of the March 1983 Principal Investigators' 

Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 171-172. 

Hughes, E.; Benemann, J.R. (1997) “Biological fossil CO2 mitigation.” Proc. of the 3rd Int. 

Conf. on CO2 Capture, Disposal and Reuse Utilization, ICCDR-3, Mass. Inst. Tech., September 

1996 (in press, 1997). 

Ikuta, Y. (1994) “Design of a biological system for CO2 fixation in carbon dioxide utilization—

direct biofixation.” Report to the Int. Energy Agency Greenhouse Gas R&D Programme. 

InterTechnology/Solar Corp. (1978) “The photosynthesis energy factory: analysis, synthesis and 

demonstration.” U.S. DOE HCP/T3548-01. 

Ivey, R.G.; Henry, E.C.; Meints, R.H. (1996) “A Feldmania algal virus has two genome size 

classes.” Virology 220:267. 

Jarvis, E.E. (1995) “Antisense RNA and ribozymes as tools for metabolic engineering.” Final 

Report DDF #07422041, National Renewable Energy Laboratory, Golden, Colorado, 13 pp. 

Jarvis, E.E.; Brown, L.M. (1991) “Transient expression of firefly luciferase in protoplasts of the 

green alga Chlorella ellipsoidea.” Curr. Genet. 19:317-321. 

Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. (1992) “DNA nucleoside composition and methylation 

in several species of microalgae.” J. Phycol. 28:356-362. 

Jeffries, T.W.; Mouthrop, P.H.; Timurian, H.; Ward, R.L.; Berger, B.J. (1977) “Biosolar 

production of fuels from algae.” UCRL 52 177, November 16, 1976. 

Jin, A.; Zhang, Y.; Van Etten, J.L. (1994) “New restriction endonuclease CviRI cleaves DNA at 

TG/CA sequences.” Nuc. Acids. Res. 22:3928-. 

Johansen, J.; Barclay, W. (1987) “Physiological variability within strains of Chaetoceros 

muelleri.” FY1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3071, pp. 137-168. 
National Renewable Energy Laboratory 

274 A Look Back at the Aquatic Species Program—Technical Review 

Johansen, J.; Barclay, W.; Nagle, N. (1990) “Physiological variability within ten strains of 

Chaetoceros muelleri (Bacillariophyceae).” J. Phycol. 26:271-287. 

Johansen, J.R.; Doucette, G.J.; Barclay, W.R.; Bull, J.D. (1988) “The morphology and ecology 

of Pleurochrysis carterae var dentata nov. (Prymnesiophyceae), a new coccolithophorid from an 

inland saline pond in New Mexico, USA.” Phycologica 27:78-88. 

Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987). “Collection, screening, and 

characterization of lipid producing microalgae: Progress during Fiscal Year 1987.” FY 1987 

Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3206; pp. 27-42. 

Johansen, J.R.; Theriot, E. (1987) “The relationship between valve diameter and number of 

central fultoportulae in Thalassiosira weissflogii (Bacillariophyceae).” J. Phycol. 23:663-665. 

Johnson, D.A.; Sprague, S. (1987) “FY 1987 Aquatic Species Program Overview.” Report, 

November 1987, Solar Energy Research Institute, Golden, Colorado, SERI/PR-231-3258, 22 pp. 

Johnson, D.A.; Weissman, J.; Globel, R. (1989) “An outdoor test facility for the large-scale 

production of microalgae.” Energy from Biomass and Wastes XII, Conference Proceedings

(Klass, D.L., ed.), Institute of Gas Technology, Chicago, pp. 277-287. 

Kadam, K.L. (1994) “Bioutilization of coal combustion gases.” Draft Milestone Completion 

Report, Recovery & Delivery, National Renewable Energy Laboratory, Golden, Colorado. 

Kadam, K.L. (1995) “Power plant flue gas as a source of CO2 for microaglae cultivation: 

technology and economics of CO2 recovery & delivery.” Draft Report to the National 

Renewable Energy Laboratory, Golden, Colorado. 

Kadam, K.L. (1997) “Power plant flue gas as a source of CO2 for microalgae cultivation: 

economic impact of different process options.” Energ. Convers. Mgmt. 38:S505-S510. 

Karpuk, M. (1987a) “CO2 sources for fuels synthesis.” FY 1986 Aquatic Species Program 

Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3206, p. 269. 

Karpuk, M. (1987b) “CO2 sources for fuels synthesis.” Abstract, FY 1986 Aquatic Species 

Program Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, 

pp. 269-275. 

Kok, B. (1953) “Experiments in photosynthesis by Chlorella in flashing light.” In Algal Culture: 

From Laboratory to Pilot Plant (Burlew, J.B., ed.), Carnegie Inst. of Washington, Publ. 600, pp. 

63-75. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 275 

Kok, B. (1973) “Photosynthesis.” Proceedings of the Workshop on Bio Solar Hydrogen 

Conversion (Gibbs, M., et al., eds.), September 5-6, Bethesda, Maryland, pp. 22-30. 

Koopman, B.L.; Benemann, J.R.; Oswald, W.J. (1980) “Pond isolation and phase isolation for 

control of suspended solids concentration in sewage oxidation pond effluents.” In Algal Biomass

(Shelef, G.; Soeder, C., eds.), Elsevier, pp. 135-162. 

Koopman, B.L.; Thomson, R.; Yacksian, R.; Benemann, J.R.; Oswald, W.J. (1978) 

“Investigation of the pond isolation process for microalgae separation from Woodland’s pond 

effluents.” Report to the Univ. of Calif., Berkeley, SERL Report No 78-5. 

Kresovich, S.; Wagner, C.K.; Scantland, D.A.; Groet, S.S.; Lawhon, W.T. (1982) “The 

utilization of emergent aquatic plants for biomass energy systems development.” Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/TR-98281-03, 103 pp. 

Krueger, S.K.; Ivey, R.G.; Meints, R.H. (1996) “A brown algal virus genome contains a ring zinc 

finger motif.” Virology 219:307-. 

Lansford, R.; Hernandez, J.; Enis, P.J. (1987) “Evaluation of available saline water resources in 

New Mexico for the production of microalgae.” FY 1986 Aquatic Species Program Annual 

Report; Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 227-248. 

Lansford, R.; Hernandez, J.; Enis, P.J.; Truby, D.; Mapel, C. (1990) “Evaluation of available 

saline water resources in New Mexico for the production of microalgae.” Report, August 1990, 

Solar Energy Research Institute, Golden, Colorado, SERI/TP 232-3597, 83 pp. 

Lee, A.M.; Ivey, R.G.; Meints, R.H. (1995) “Characterization of a repetitive DNA element in a 

brown algal virus.” Virology 212:474-. 

Laws, E.A. (1981) “Year one progress report on algal production raceway project.” Proceedings 

of the July 1981 Subcontractor's Review Meeting, Aquatic Species Program, Solar Energy 

Research Institute, Golden Colorado, SERI/CP-624-1228, pp. 1-32. 

Laws, E.A. (1982) “Results from preliminary studies on the use of flashing light to enhance algal 

mass culture productivity.” Proceedings of the June 1982 SERI Biomass Program Principal 

Investigators' Review Meeting, Aquatic Species Program Reports, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1808, pp. 31-42. 

Laws, E.A. (1984a) “Optimization studies in shallow algal mass culture flumes.” Proceedings of 

the April 1984 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2341, pp. 108-131. 

Laws, E.A. (1984b) “Research and development of shallow algal mass culture systems for the 

production of oils.” Final Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/STR-231-2496. 
National Renewable Energy Laboratory 

276 A Look Back at the Aquatic Species Program—Technical Review 

Laws, E.A. (1984c) “Research, development, and demonstration of algal production raceway 

(ARP) systems for the production of hydrocarbon resources.” Subcontractor Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/STR-231-2206. 

Laws, E.A. (1985) “Productivity optimization of saline microalgae grown in outdoor mass 

culture.” Aquatic Species Program Review: Proceedings of the March 1985 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, 

pp. 162-178. 

Laws, E.A. (1987) “Productivity optimization of saline microalgae grown in outdoor mass 

culture.” Final Report, Solar Energy Research Institute, Golden, Colorado, Contract 

XK-4-4109-1. 

Laws, E.A. (1987b) “Continuous microalgae production in shallow raceways at high 

productivity.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-307, pp. 209-226. 

Laws, E.A. (1989) Final Report to the Electric Power Research Institute. 

Laws, E.A; Berning, B.L. (1991) Biotech. Bioeng. 37:936. 

Laws, E.A.; Lee, Y.K.; Tan, H.-M.; Hew, C.-S. (1985) “The effect of growth temperatures on the 

bioenergetics of photosynthetic algal cultures.” Biotech. Bioeng. 27:555-561. 

Laws, E.A.; Taguchi, S.; Hirata, J.; Pang, L. (1986) “High algal production rates achieved in a 

shallow outdoor flume.” Biotech. Bioeng. 28:191-197. 

Laws, E.A; Taguchi, S.; Hirata, J.; Pang, L. (1988) “Optimization of microalgal production in a 

shallow outdoor flume.” Biotech. Bioeng. 32:140-147. 

Laws, E.A.; Terry, K.L. (1983) “Shallow algal mass culture system.” Proceedings of the March 

1983 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research 

Institute, Golden Colorado, SERI/CP-231-1946, pp. 33-47. 

Laws, E.A.; Terry, K.L.; Wickman, J.; Chalup, M.S. (1983) “Preliminary results from a simple 

algal production system designed to utilize the flashing light effect.” Biotech. Bioeng.

25:2319-2336. 

Lee, C.A.; McKim, T. (1981) “Water hyacinth wastewater treatment system.” Proceedings of 

the July, 1981, Subcontractors’ Review Meeting, Aquatic Species Program, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 75-109. 

Levine, L.S. (1982) “Biological and engineering parameters of algal mass culture.” Proceedings 

of the June 1982 SERI Biomass Program Principal Investigators' Review Meeting, Aquatic 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 277 

Species Program Reports, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-

1808, pp. 43-44. 

Lewin, R.A. (1980) “Tasks for the microbial geneticist in phycotechnology.” In Biotechnology 

for the Production of Chemicals and Fuels from Biomass, Proceedings of the International 

Workshop, Vail, CO, 9/29/80-10/20/80, pp. 105-111. 

Lewin, R.A. (1985) “Production of hydrocarbons by micro-algae; isolation and characterization 

of new and potentially useful algal strains.” Aquatic Species Program Review: Proceedings of the 

March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2700; pp. 43-51. 

Lewin, R.A.; Burrascano, C.; Cheng, L. (1987) “Some picopleuston algae from the Caribbean 

region.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3071, pp. 105-121. 

Lewin, R.A.; Cheng, L. (1988) “Some lipogenic, eukaryotic, picopleuston algae from the 

Caribbean region.” Report, Solar Energy Research Institute, Golden, Colorado. 

Lien, S. (1981a) “Photobiological production of fuels by microalgae.” In Energy from Biomass: 

Proceedings of the First E.C. Conference (Palz, W.; Chartier, P.; Hall, D.O., eds.), Applied 

Science Publishers, London, pp. 697-702. 

Lien, S. (1981b) “Algal research at SERI: A basic research on photobiological production of 

fuels and chemicals in microalgae.” Proceedings of the July 1981 Subcontractors' Review 

Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-624-1228, pp. 59-65. 

Lien, S. (1982) “Studies on the production and accumulation of oil and lipids by microalgae.” 

Proceedings of the SERI Biomass Program Principal Investigators' Review Meeting, Aquatic 

Species Program Reports, June 23-25, 1982, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-1808, pp. 45-54. 

Lien, S.; Roessler, P. (1986) “The energetics of biomass and lipid production by lipogenic 

microalgae under nitrogen deprivation.” Aquatic Species Program Review: Proceedings of the 

March 1985 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-2700, pp. 100-117. 

Lien, S.; Spencer, K.G. (1983) “Algal oil production and lipid metabolism research.” Aquatic 

Species Program Review: Proceedings of the March 1983 Principal Investigators' Meeting, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 3-18. 

Lien, S.; Spencer, K.G. (1984) “Physiology of oil-producing microalgae in response to stress 

conditions.” Aquatic Species Program Review: Proceedings of the April 1984 Principal 

Investigators' Meeting, Solar Energy Research Institute, SERI/CP-231-2341, pp. 79-107. 
National Renewable Energy Laboratory 

278 A Look Back at the Aquatic Species Program—Technical Review 

Lowenstein, M.Z. (1982) “FY 1982 Annual Report, Biomass Program Office.” November 1982 

Draft Report, Solar Energy Research Institute, Golden, Colorado, SERI/MR-231-1796. 

Mack, R.W. (1984) “Comparative studies on the carbohydrate composition of marine 

macroalgae.” Aquatic Species Program Review: Proceedings of the April 1984 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, 

pp. 2-7. 

Manley, S. (1979) Unpublished Report to U.S. Department of Energy for work in laboratory of 

G. Pelofsky. 

Maxwell, E.L.; Folger, A.G. (1982) “Resource assessment for saline aquatic biomass production 

systems.” Proceedings of the June 1982 SERI Biomass Program Principal Investigators' Review 

Meeting-Aquatic Species Program Reports, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-1808, pp. 55-67. 

Maxwell, E.L.; Folger, A.G.; Hogg, S.E. (1983) “Procedures for stratification of the U.S. 

Southwest for microalgae production systems based on climate, water, and land resources.” 

Aquatic Species Program Review: Proceedings of the March 1983 Principal Investigators' 

Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 135-154. 

Maxwell, E.L.; Folger, A.G.; Hogg, S.E. (1985) “Resource evaluation and site selection for 

microalgae production systems.” Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/TR-215-2484. 

McCluskey, K.; Graves, M.V.; Mills, D.; Meints, R.H. (1992) “Replication of Chlorella virus 

PBCV-1 and host karyotype determination studies with pulse-field gel electrophoresis.” J. 

Phycol. 28:846-850. 

McIntosh, R.P. (1985) “SERI Aquatic Species Program 1984 Annual Report.” March 1985 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/PR-231-2659, 53 pp. 

Meier, R.L. (1955) “Biological cycles in the transformation of solar energy into useful fuels.” In 

Solar Energy Research (Daniels, F.; Duffie, J.A., eds.), Madison University Wisconsin Press, pp. 

179-183. 

Meints, R. (1987) “Studies of the chloroplast genome of exsymbiotic and free-living Chlorella.” 

FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar 

Energy Research Institute, Golden, Colorado. SERI/SP-231-3206, pp. 210-228. 

Meints, R.H.; Burbank, D.E.; Van Etten, J.L.; Lamport, D.T.A. (1988) “Properties of the 

Chlorella receptor for the PBCV-1 virus.” Virology 164:15-21. 

Meints, R.H.; Lee, K.; Burbank, D.E.; Van Etten, J.L. (1984) “Infection of a Chlorella-like alga 

with the virus PBCV-1: Ultrastructural studies.” Virology 138:341-346. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 279 

Meints, R.H.; Lee, K.; Van Etten, J.L. (1986) “Assembly site of the virus PBCV-1 in Chlorellalike alga: Ultrastructural studies.” Virology 154:240-245. 

Meints, R.H.; Kennedy, J.; Ziegelbein, M.; Amberg, S.; Van Etten, J.L. (1988) “Production of 

protoplasts from Chlorella-like algae.” Unpublished manuscript; submitted to SERI as a 

research report. 

Meints, R.H.; Schuster, A.M.; Van Etten, J.L. (1985a) “Chlorella viruses.” Plant Mol Biol. 

Reports 3:180-187. 

Meints, R.H.; Schuster, A.M.; Van Etten, J.L. (1985b) “Characterization of viruses infecting 

Chlorella-like alga.” Abstract, FY1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 338. 

Meints, R.H.; Van Etten, J.L. (1991) “Eukaryotic Algae.” In Microbioal Ecology: Principles, 

Methods and Applications (Levin, M.A.; Seidler, R.J.; Rogul, M., eds.), McGraw-Hill, Inc., New 

York, pp. 883-888. 

Meints, R.H.; Van Etten, J.L.; Kuczmarski, D.; Ang, B. (1981) “Viral infection of the symbiotic 

Chlorella-like alga present in Hydra viridis.” Virology 113:698-703. 

Melis, A.; Neidhardt, J.; Bartoli, I.; Benemann, J.R. (1998) Proc. Biohydrogen '97. 

Milne, T.A.; Evans, R.J. (1987) “Catyalytic upgrading of whole algae and their components with 

shape-selective zeolites.” FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, SERI/SP-231-3206, pp. 264-265. 

Murakami, M.; Ikenouchi, M. (1997) “The biological CO2 fixation and utilization project by 

RITE (2)—screening and breeding of microalgae with high capability of fixing CO2.” Energy 

Conver. Mgmt. 38: Suppl. 493-498. 

Nagle, N.; Barclay, B. (1987) “The effects of light intensity on lipid production.” Abstract, FY 

1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, p. 251. 

Nagle, N.; Chelf, P.; Lemke, P.; Barclay, B. (1987) “Conversion of lipids through biological 

pretreatment.” FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., 

eds.), Solar Energy Research Institute, SERI/SP-231-3206, pp. 255-263. 

Nagle, N.; Chelf, P.; Lemke, P.; Barclay, B. (1989) “Conversion of lipids to liquid fuels by 

natural enzymatic pretreatment.” Energy from Biomass and Wastes XII, Conference Proceedings

(Klass, D.L., ed.), Institute of Gas Technology, Chicago, pp. 1107-1115. 
National Renewable Energy Laboratory 

280 A Look Back at the Aquatic Species Program—Technical Review 

Nagle, N.; Lemke, P. (1989) “Microalgal fuel production processes: Analysis of lipid extraction 

and conversion methods.” Aquatic Species Program Annual Report (Bollmeier, W.S.; Sprague, 

S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 157-164. 

Neenan, B. (1983) “Microalgae program element cost goals.” Aquatic Species Program Review: 

Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1946, pp. 69-77. 

Neenan, B.; Feinberg, D.; Hill, A.; McIntosh, R.; Terry, K. (1986) “Fuels from microalgae: 

Technology status, potential, and research requirements.” Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-2550, 158 pp. 

Negoro, M.; Hamasaki, K.A.; Ikuta, Y.; Makita, T.; Hirayama K.; Suzuki, S. (1993) “CO2

fixation by microalgae photosynthesis using actual flue gas discharged from a boiler.” Biochem 

Biotech. 39/40:643-653. 

Negoro, M.; Shioji, K.; Ikuta, Y.; Makita, T.; Utiumi, M. (1992) “Growth characteristics of 

microalgae in high-concentrations of CO2 gas. Effects of culture medium, trace components and 

impurities thereon.” Biochem Biotech. 34/35:681-692. 

Neidhardt, J.; Benemann, J.R.; Baroli, I.; Melis, A. (1998) “Maximizing photosynthetic 

productivity and light utilization in microalgae by minimizing the light-harvesting chlorophyll 

antenna size of the photosystems.” Photosynthesis Res., in press. 

Nelson. (1982) “An investigation of the availability of carbon dioxide for the production of 

microalgae lipids in the southwest.” Report to the Argonne National Laboratory, unpublished. 

Neushul, M. (1987) “Kelp genetics.” Abstract, FY 1986 Aquatic Species Program Annual 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, p. 337. 

Ohlrogge, J.; Jaworski, J.; Post-Beittenmiller, D.; Roughan, G.; Roessler, P.; Nakahira, K. (1993) 

“Regulation of flux through the fatty acid biosynthesis pathway.” In Biochemistry and Molecular 

Biology of Membrane and Storage Lipids of Plants (Murata, N.; Somerville, C.R., eds.), 

American Society of Plant Physiologists, Rockville, Maryland, pp. 102-112. 

Oswald, W.J. (1963) “The high-rate pond in waste disposal.” Dev. Ind. Microbiol. 4:112-125. 

Oswald, W.J.; Benemann, J.R. (1977) “A critical analysis of bioconversion with microalgae.” In 

Biological Energy Conversion ( Mitsui, A., et al., eds.), Academic Press, New York, pp. 

379-394. 

Oswald, W.J.; Golueke, C.G. (1960) “Biological transformation of solar energy.” Adv. Appl. 

Microbiol., 11:223-242. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 281 

Owens, T.G.; Gallagher, J.C.; Alberte, R.S. (1987) “Photosynthetic light-harvesting function of 

violaxanthin in Nannochloropsis sp. (Eustigmatophyceae).” J. Phycol. 23:79-85. 

Pratt, D.C. (1981) “Wetland biomass production.” Proceedings of the July 1981, Subcontractors’ 

Review Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-624-1228, pp. 119-126. 

Pratt, D.C. (1982) “Emergent aquatics: Stand establishment, management and species screening.” 

Proceedings of the June 1982 SERI Biomass Program Principal Investigators’ Review Meeting, 

Aquatic Species Program Reports, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-231-1808, pp. 69-84. 

Pratt, D.C.; Andrews, N.J.; Garver, E.G.; Penko, M.; Read, P.E.; Zimmerman, E.S. (1982) 

“Emergent aquatics: stand establishment, management, and species screening.” Subcontract 

Report, Contract No. XK-1-1087-1, Solar Energy Research Institute, Golden, Colorado, 

SERI/STR-231-1819, 56 pp. 

Pratt, D.C.; Dubbe, D.R. (1983) “Wetland biomass production.” Aquatic Species Program 

Review: Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 55-67. 

Pratt, D.C.; Dubbe, D.R.; Garver, E.G.; Linton, P.J. (1984) “Wetland biomass production: 

emergent aquatic management options and evaluations.” Final Subcontract Report, Contract No. 

XK-2-02094-01, Solar Energy Research Institute, Golden, Colorado, SERI/STR-231-2383. 

Radmer, R.; Behrens, P. (1987) “Temperature effects on microalgal photosynthetic efficiency.” 

FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, pp. 301-320. 

Raymond, L. (1979) “Initial investigations of a shallow layer algal production system.” Am. Soc. 

Mech. Eng., New York. 

Raymond, L. (1981) “Mass algal culture systems.” U.S. Patent 4,253,271. 

Raymond, L.P. (1983a) “Aquatic biomass as a source of fuels and chemicals.” Conference 

report: First U.S.-China Conference on Energy, Resources, and Environment. Beijing, People’s 

Republic of China, Nov. 7-12, 1982. Solar Energy Research Institute, Golden, Colorado, 

SERI/TP-231-1699, 8 pp. 

Raymond, L.P. (1983) “The SERI microalgae research group.” Aquatic Species Program 

Review: Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 89-98. 
National Renewable Energy Laboratory 

282 A Look Back at the Aquatic Species Program—Technical Review 

Raymond, L.P. (1984) “Microalgal technology research at SERI.” Proceedings of the April 1984 

Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden, Colorado, SERI/CP-231-2341, pp. 150-151. 

Regan, D.L. (1980) “Marine biotechnology and the use of arid zones.” Search 111:377-381. 

Reisser, W.; Burbank, D.E.; Meints, S.M.; Meints, R.H.; Becker, B.; Van Etten, J.L. (1988) “A 

comparison of viruses infecting two different Chlorella-like green algae.” Virology 167:143-149. 

Rhyne, C. (1987a) “Nutritional requirements for maximal growth of oil producing microalgae.” 

Abstract, FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, 

Golden, Colorado, SERI/SP-231-3071, p. 249. 

Rhyne, C. (1987b) “Nutritional requirements for maximal growth of oil producing microalgae.”

FY 1986 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, pp. 92-107. 

Richmond, A. (1984) “Development of outdoor system for production of lipid-rich halotolerant 

microalgae.” Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species 

Program, Solar Energy Research Institute, Golden Colorado, SERI/CP-231-2341, pp. 195-205. 

Ripin, M. (1983) “Systems overview of algal mass culture.” Aquatic Species Program Review: 

Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-1946, pp. 155-169. 

Roessler, P.G. (1987a) “Biochemical aspects of lipid accumulation in silicon-deficient diatoms.” 

FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/SP-231-3071, pp. 257-271. 

Roessler, P.G. (1987b) “The effects of silicon deficiency on several enzymes involved in lipid 

and carbohydrate biosynthesis in Cyclotella cryptica.” FY 1987 Aquatic Species Program Annual 

Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3206, pp. 159-173. 

Roessler, P.G. (1987c) “Lipid accumulation in silicon-deficient diatoms.” In The Metabolism, 

Structure, and Function of Plant Lipids (Stumpf, P.K.; Mudd, M.B.; Nes, W.D., eds.), Plenum, 

Publishing Corp., pp. 649-651. 

Roessler, P.G. (1987d) “UDPglucose pyrophosphorylase activity in the diatom Cyclotella 

cryptica: Pathway of crysolaminarin biosynthesis.” J. Phycol. 23:494-498. 

Roessler, P.G. (1988a) “Changes in the activities of various lipid and carbohydrate biosynthetic 

enzymes in the diatom Cyclotella cryptica in response to silicon deficiency.” Arch. Biochem. 

Biophys. 267:521-528. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 283 

Roessler, P.G. (1988b) “Characteristics of abrupt size reduction in Synedra ulna

(Bacillariophyceae).” Phycologia 27:294-297. 

Roessler, P.G. (1988c) “Effects of silicon deficiency on lipid composition and metabolism in the 

diatom Cyclotella cryptica.” J. Phycol. 24:394-400. 

Roessler, P.G. (1989) “Purification and characterization of acetyl-CoA carboxylase from the 

diatom Cyclotella cryptica.” Aquatic Species Program Annual Report (Bollmeier, W.S.; 

Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 

121-134. 

Roessler, P.G. (1990) “Purification and characterization of acetyl-CoA carboxylase from the 

diatom Cyclotella cryptica.” Plant. Physiol. 92:73-78. 

Roessler, P.G. (1995) “Expression of an algal acetyl-CoA carboxylase gene in E. coli.” In Plant 

Lipid Metabolism”, (Kader, J.-C.; Mazliak, P., eds.), Klewer Academic Publishers, pp. 46-48. 

Roessler, P.G.; Bleibaum, J.L.; Thompson, G.A.; Ohlrogge, J.B. (1994) “Characteristics of the 

gene that encodes acetyl-CoA carboxylase in the diatom Cyclotella cryptica.” In Recombinant 

DNA Technology II (Bajpai, K.; Prokop, A., eds.), Ann. N.Y. Acad. Sci. 721:250-256. 

Roessler, P.G.; Brown, L.M.; Dunahay, T.G.; Heacox, D.A.; Jarvis, E.E.; Schneider, J.C.; Talbot, 

S.G.; Zeiler, K.G. (1994) “Genetic engineering approaches for enhanced production of biodiesel 

fuel from microalgae.” In Enzymatic Conversion of Biomass for Fuels Production (Himmel, 

M.E.; Baker, J; Overend, R.P., eds.), American Chemical Society, pp. 256-270. 

Roessler, P.G.; Ohlrogge, J.B. (1993) “Cloning and characterization of the gene that encodes 

acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica.” J. Biol. Chem. 268:19254-

19259. 

Ryther, J.H. (1981) “Cultivation of macroscopic marine algae.” Proceedings of the July 1981 

Subcontractor’s Review Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden, Colorado, SERI/CP-624-1228, pp. 111-118. 

Ryther, J.H. (1982a) “Cultivation of macroscopic marine algae.” Proceedings of the June 1982 

SERI Biomass Program Principal Investigators’ Review Meeting, Aquatic Species Program 

Report, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1808, pp. 85-102. 

Ryther, J.H. (1982b) “Cultivation of macroscopic marine algae and freshwater aquatic weeds.” 

Subcontract Report, Solar Energy Research Institute, Golden, Colorado, SERI/TR-231-1820, 

35pp. 

Ryther, J.H. (1982c) “Cultivation of macroscopic marine algae.” Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/TR-98133-1A, 137 pp. 
National Renewable Energy Laboratory 

284 A Look Back at the Aquatic Species Program—Technical Review 

Ryther, J.H. (1982d) “Biomass production, anaerobic digestion, and nutrient recycling of small 

benthic or floating seaweeds.” Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/TR-98133-1B, 42 pp. 

Ryther, J.H. (1983) “Cultivation of macroscopic marine algae.” Proceedings of the March 1983 

Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, 

Golden, Colorado, SERI/CP-231-1946, pp. 79-88. 

Ryther, J.H. (1985) “Technology for the commercial production of macroalgae.” In Energy 

Applications of Biomass: Proceedings of the National Meeting on Biomass R&D for Energy 

Applications, Oct.1-3, 1984, Arlington, Virginia (Lowenstein, M.Z., ed.), Elsevier Applied 

Science Publishers, New York, pp. 177-188. 

Ryther, R.H.; Carlson, R.D.; Pendoley, P.D.; Jensen, P.R.(1987) “Collection and characterization 

of saline microalgae from South Florida.” FY 1986 Aquatic Species Program Annual Report, 

Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 122-136. 

Ryther, J.H.; DeBusk, T.A. (1982) “Significance of carbon dioxide and bicarbonate-carbon 

uptake in marine biomass production.” Energy from Biomass and Wastes VI, Conference 

Proceedings (Klass, D.L., ed.), Institute of Gas Technology, Chicago, pp. 221-233. 

Ryther, J.H.; DeBusk, T.A.; Andrews, D.A.; Habig, C.; LaBaff, L.P.; Williams, L.D.; Falkner, 

B.E. (1982) “Cultivation of macroscopic marine algae.” Proceedings of the June 1982 SERI 

Biomass Program Principal Investigators’ Review Meeting, Aquatic Species Program Reports, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-1808, pp. 85-101. 

Ryther, J.H.; DeBusk, T.A.; Blakeslee, M. (1984a) “Cultivation and conversion of marine 

macroalgae.” Final Subcontract Report, Solar Energy Research Institute, SERI/STR-231-2360, 

81 pp. 

Ryther, J.H.; DeBusk, T.A.; Peterson, J.E. (1984b) “Studies of marine macroalgae: Saline desert 

water cultivation and effects of environmental stress on proximate composition.” Final 

Subcontract Report, Solar Energy Research Institute, SERI/STR-231-2799, 64 pp. 

Schneider, J.C.; Roessler, P. (1994) “Radiolabeling studies of lipids and fatty acids in 

Nannochloropsis (Eustigmatophyceae), an oleaginous marine alga.” J. Phycol. 30:594-598. 

Schneider, J.C.; Livine, A.; Sukenik, A.; Roessler P.G. (1995) “A mutant of Nannochloropsis

deficient in eicosapentaenoic acid production.” Phytochem. 40:807-814. 

Schneider, J.C.; Roessler, P.G. (1995) “A novel acyltransferase activity in an oleaginous alga.” In 

Plant Lipid Metabolism (Kader, L.C.; Mazliak, P., eds.), Kluwer Academic Publishers, the 

Netherlands, pp. 105-107. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 285 

Schuster, A.M.; Burbank, D.E.; Meister, B.; Skrdla, M.; Meints, R.H.; Hattman, S.; Swinton, D.; 

Van Etten, J.L. (1986) “Characterization of viruses infecting a eukayotic Chlorella-like green 

alga.” Virology 150:170-177. 

Schuster, J.A.; Graves, M.; Korth, K.; Ziegelbein, M.; Brumbaugh, J.; Grone, D.; Meints, R.H. 

(1990a) “Transcription and sequence studies of a 4.3 kbp fragment from a dsDNA eukaryotic 

alga virus.” Virology 176:515-523. 

Schuster, A.M.; Waddle, J.A.; Korth, K.; Meints, R.H. (1990b) “The chloroplast genome of an 

exsymbiotic Chlorella-like alga.” Plant Mol. Biol. 14:859-862. 

Schwegler, B.R., Jr. (1983) “Water hyacinth production primary and advanced treatment of 

wastewater.” Final Subcontract Report, Solar Energy Research Institute, Golden, Colorado, 

Subcontract No. XK-0-9245-1, SERI/STR-231-1886, 39 pp. 

Schwegler, B.R.; McKim, T.W. (1982) “Water hyacinth wastewater treatment system.” 

Proceedings of the June 1982 SERI Biomass Program Principal Investigators’Review Meeting, 

Aquatic Species Program Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/CP-31-1808, pp. 103-113. 

Shelef, G.A.; Sukenik, A.; Green, M. (1984a) “Microalgae harvesting and processing: a literature 

review.” Report, Solar Energy Research Institute, Golden Colorado, SERI/STR-231-2396. 

Shelef, G.A; Sukenik, A.; Green, M. (1984b) “Marine microalgae separation and harvesting.”

Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species Program, Solar 

Energy Research Institute, Golden Colorado, SERI/CP-231-2341, pp. 206-223. 

Shelef, G.; Sukenik, A.; Sandbank, E. (1985) “Separation and harvesting of microalgae from 

saline media.” Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species 

Program, Solar Energy Research Institute, Golden Colorado, SERI/CP-231-2700, pp. 244-270. 

Shifrin, N.S. (1984) “Oils from microalgae.” In Biotechnology for the Oils and Fats Industry

(Ratledge, C., et al., eds.), AOCS monograph 11. 

Shifrin, N.S.; Chisholm, S.W. “Phytoplankton lipids: interspecific differences and effects of 

nitrate, silicate and light-dark cycles.” J. Phycol., 17:374. 

Skrdla, M.P.; Burbank, D.E.; Xia, T.; Meints, R.H.; Van Etten, J.L. (1984) “Structural proteins 

and lipids in a virus, PBCV-1, which replicates in a Chlorella-like virus.” Virology 135:308-315. 

Smith, C.W.; Schwartzbach, S.D. (1988) “Preliminary characterization of pyruvate 

dehydrogenase and acetyl-CoA synthetase.” Manuscript submitted as report to SERI, 6 pp. 
National Renewable Energy Laboratory 

286 A Look Back at the Aquatic Species Program—Technical Review 

Solomon, J.A (1985) “Ultrastructure evaluation of lipid accumulation in microalgae.” Aquatic 

Species Program Review: Proceedings of the March 1985 Principal Investigators’ Meeting,

Solar Energy Research Institute, SERI/CP-231-2700, pp. 71-82. 

Solomon, J.A. (1987) “Flow cytometry techniques for species improvement.” Abstract, FY 1986 

Aquatic Species Program: Annual Report, Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3071, p. 252. 

Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986a) “Ultrastructural and flow cytometric analyses of 

lipid accumulation in microalgae.” Annual Report, Oak Ridge National Laboratory, Oak Ridge, 

Tennessee, ORNL/M-258, 50 pp. 

Solomon, J.A.; Hand, R.E.; Mann, R.C. (1986b) “Ultrastructural and flow cytometric analyses of 

lipid accumulation in microalgae: A subcontract report.” Solar Energy Research Institute, 

Golden, Colorado, SERI/STR-231-3089, 50 pp. 

Solomon, J.A.; Palumbo, A.V. (1987) “Improvement of microalgal strains for lipid production.” 

FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar 

Energy Research Institute, SERI/SP-231-3206, pp. 142-154. 

Sommerfeld, M.R. (19xx) “Collection of high energy yeilding strains of saline microalgae from 

southwestern states.” Report, Solar Energy Research Institute, 52 pp. 

Sommerfeld, M.R.; Ellingson, S.B. (1987a) “Collection of high energy yielding strains of saline 

microalgae from southwestern states.” FY 1986 Aquatic Species Program Annual Report, Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 53-66. 

Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987b) “Screening microalgae isolated from the 

southwest for growth potential and lipid yield.” FY 1987 Aquatic Species Program Annual 

Report (Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3206; pp. 43-57. 

Sphoer, H.A.; Milner, H.W. (1956) Production of protein, lipids and carbohydrates by cultures of 

algae.” U.S. Patent 2,732,661. 

Spoehr, H.A.; Milner, H.W. (1949) “The chemical compostition of Chlorella; effect of 

environmental conditions.” Plant. Physiol. 24:120. 

Sriharan, S.; Bagga, D. (1987a) “Effects of induction strategies on Chaetoceros (SS-14), growth 

with emphasis on lipids.” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 273-284. 

Sriharan, S.; Bagga, D. (1987b) “Influence of nitrogen and temperature on lipid production in 

microalgae (Hantzschia DI-60).” Energy from Biomass and Wastes X, Conference Proceedings

(Klass, D.L., ed.), Institute of Gas Technology, Chicago, pp. 1689-1190. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 287 

Sriharan, S.; Bagga, D.; Sriharan, T.; Das, M. (1987c) “Effect of nutrients and temperature on 

lipid production and fatty acid composition in Monoraphidium mintum and Cyclotella DI-35.” 

FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar 

Energy Research Institute, SERI/SP-231-3206, pp. 108-126. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1988) “Algal lipids: Effect of induction strategies on the 

quantity and types of lipids produced.” Final Technical Report, Solar Energy Research Institute, 

Golden, Colorado, 16 pp. 

Sriharan, S.; Sriharan, T.P.; Bagga, D. (1989) “Lipids and fatty acids of diatom Chaetoceros 

muelleri var subsalsum and the control of their production by environmental factors.” Energy 

from Biomass and Wastes XII, Conference Proceedings (Klass, D.L., ed.), Institute of Gas 

Technology, Chicago, 10 pp. 

Sriharan, S.; Bagga, D.; Nawaz, M. (1991) “The effects of nutrients and temperature on biomass, 

growth, lipid production, and fatty acid composition of Cyclotella cryptica Reimann, Lewin, and 

Guillard.” Appl. Biochem. Biotech. 28/29:317-326. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1989) “Environmental control of lipids and fatty acid 

production in the diatom Navicula saprophila.” Appl. Biochem. Biotech. 20/21:281-291. 

Sriharan, S.; Bagga, D.; Sriharan, T.P. (1990) “Effects of nutrients and temperature on lipid and 

fatty acid production in the diatom Hantzshia DI-60.” Appl. Biochem. Biotech. 24/25:309-316. 

Stefan, C.; Xia, Y.; Van Etten, J.L. (1991) “Molecular cloning and characterization of the gene 

encoding the adenine methyltransferase M. CviRI from Chlorella virus XZ-6E.” Nuc. Acids Res.

19:307-. 

Suen, Y.; Hubbard, J.S.; Holzer, G.; Tornabene, T.G. (1987) “Total lipid production of the green 

alga Nannochloropsis sp. QII under different nitrogen regimes.” J. Phycol. 23:289-296. 

Tadros, M.G. (1985) “Screening and characterizing oleaginous microalgal species from the 

southeastern United States.” Aquatic Species Program Review: Proceedings of the March 1985 

Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP231-2700, pp. 28-42. 

Tadros, M.G. (1987a) “Screening and characterizing oleaginous microalgal species from the 

southeastern United States.” FY 1986 Aquatic Species Program Annual Report, Solar Energy 

Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 67-89. 

Tadros, M.G. (1987b) “Conclusion of the warm-water algae collection and screening efforts 

conducted in the southeastern United States.” FY 1987 Aquatic Species Program Annual Report

(Johnson, D.A.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP231-3206; pp. 58-74. 
National Renewable Energy Laboratory 

288 A Look Back at the Aquatic Species Program—Technical Review 

Tadros, M.G.; Johansen, J.R. (1988) “Physiological characterization of six lipid-producing 

diatoms from the southeastern United States.” J. Phycol. 24:445-452. 

Taylor, J.H. (1983) “The Middle Atlantic Consortium on Energy Research (MACER) Progress 

Report for December, January, and February.” Aquatic Species Program Review: Proceedings of 

the March 1983 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-1946, pp. 49-53. 

Terry, K.L. (1984a) “Microalgal technology research at SERI: Modulated light photobiology.” 

Aquatic Species Program Review: Proceedings of the April 1984 Principal Investigators’ 

Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 160-169. 

Terry, K.L. (1984b) “Modulated light photobiology.” Proceedings of the April 1984 Principal 

Investigators’ Meeting, Aquatic Species Program, Solar Energy Research Institute, Golden 

Colorado, SERI/CP-231-2341, pp.160-169. 

Terry, K.L. (1986) “Photosynthesis in modulated light: quantitative dependence on 

photosynthetic enhancement on the flashing rate.” Biotech. Bioeng. 28:988. 

Terry, K.L.; Raymond, L.P. (1985) “System design for the autotrophic production of 

microalgae.” Enzyme Microb. Technol. 7:474-487. 

Thomas, R.M. (1990) “Triglyceride accumulation and the cell cycle in Chlorella.” Masters 

Thesis, Montana State University, July 1990. 

Thomas, W.H. (1982) “Laboratory selection of microalgae for maximum lipid and protein 

production.” Proceedings of the June 1982 SERI Biomass Program Principal Investigators’ 

Review Meeting, Aquatic Species Program Reports, Solar Energy Research Institute, Golden, 

Colorado, SERI/CP-231-1808, pp. 115-137. 

Thomas, W.H. (1983b) “Microalgae from desert saline waters as potential biomass producers.” 

Progress in Solar Energy 6:143-145. 

Thomas, W.H.; Gaines, S.R. (1982) “Algae from the arid southwestern United States: an 

annotated bibliography.” Report for Subcontract XK-2-0270-01, to the Solar Energy Research 

Institute, Golden, Colorado, October 1982. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1983a) “Yields, photosynthetic 

efficiencies, and proximate chemical composition of dense cultures of marine microalgae: A 

subcontract report.” Solar Energy Research Institute, Golden, Colorado, SERI/STR-231-1896, 58 

pp. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1984a) “Cultural requirements, yields, 

and light utilization efficiencies of some desert saline microalgae.” Aquatic Species Program 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 289 

Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-2341; pp. 7-63. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1985) “Selection of desert saline 

microalgae for high yields at elevated temperatures and light intensities and in SERI Standard 

artificial media.” Aquatic Species Program Review: Proceedings of the March 1985 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700; 

pp. 5-27. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P. (1986) “Cultural requirements, yields 

and light utilization efficiencies of some desert saline microalgae.” Nova Hedwigia 83:60-69. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983b) 

“Selection of high-yielding microalgae from desert saline environments.” Aquatic Species 

Program Review: Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-231-1946, pp. 97-122. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Eldridge, P.; Neori, A.; Gaines, S. (1983c) 

“Microalgae from desert saline waters as potential biomass producers.” In Progress in Solar 

Energy, vol. 6, Biotechnology and Chemical Sciences Division, American Solar Energy Society, 

Boulder, Colorado, pp. 143-145. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A. (1981) “Effects of light intensity and 

nitrogen deficiency on yield, proximate composition, and photosynthetic efficiency of 

Phaeodactylum.” Proceedings of the Subcontractors’ Review Meeting, Aquatic Species Program, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-624-1228, pp. 33-58. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984b) “Yields, 

photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. I. 

Introduction and Phaeodactylum tricornutum experiments.” Biomass 5:181-209. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984c) “Yields, 

photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. II. 

Dunaliella primolecta and Tetraselmis suecica experiments.” Biomass 5:211-225. 

Thomas, W.H.; Seibert, D.L.R.; Alden, M.; Neori, A.; Eldridge, P. (1984d) “Yields, 

photosynthetic efficiency, and proximate composition of dense marine microalgal cultures. III. 

Isochrysis sp. and Monallantus salina experiments and comparative conclusions.” Biomass

5:299-316. 

Thomas, W.H.; Tornabene, T.G.; Weissman, J. (1984e) “Screening for lipid yielding microalgae: 

Activities for 1983.” Final Subcontract Report, Solar Energy Research Institute, Golden, 

Colorado, SERI/STR-231-2207. 
National Renewable Energy Laboratory 

290 A Look Back at the Aquatic Species Program—Technical Review 

Timurian, R.; Ward, R.L.; Jeffries, T.W. (1977) “Growth of filamentous blue-green algae at high 

temperatures: a source of biomass for renewable fuels.” IZ Int. Seaweed Symp. Aug 20-27. 

Tornabene, T.G. (1980) “Microbial production of hydrocarbons.” Proceedings, Bio-Energy ’80: 

World Congress and Exposition, Atlanta, GA, pp. 491-492. 

Tornabene, T.G. (1984) “Chemical profile of microalgae with emphasis on lipids.” Aquatic 

Species Program Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar 

Energy Research Institute, Golden, Colorado, SERI/CP-231-2341, pp. 64-78. 

Tornabene, T.G.; Ben-Amotz, A.; Hubbard, J.S. (1982) “Isolation, analysis and identification of 

lipids.” Report, Subcontract XK-2-02149-01, Solar Energy Research Institute, Golden, Colorado. 

Tornabene, T.G.; Benemann, J.R. (1985) “Chemical profiles on microalgae with emphasis on 

lipids.” Aquatic Species Program Review: Proceedings of the March 1985 Principal 

Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, 

pp. 83-99. 

Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogen 

starved green alga Neochloris oleoabundans.” Enzyme Microb. Technol. 5:435-440. 

Tornabene, T.G.; Holzer, G.; Peterson, S.L. (1980) “Lipid profile of the halophilic alga, 

Dunaliella salina.” Biochem. Biophys. Res. Comm. 96:1349-1356. 

Usui, N.; Ikenouchi, M. (1997) “The biological CO2 fixation and utilization project by RITE (1) 

highly effective photobioreactor system.” Energy Conver. Mgmt. 38: Suppl. 487-492. 

Uziel, M.; Oswald, W.J.; Golueke, C.G. (1975) “Solar energy and conversion with algal bacterial 

systems.” Univ. of California-Berkeley, SERL Report to NSF/RANN/SE/GI-39216/Fr/75/5. 

Van Etten, J.L.; Burbank, D.E.; Joshi, J.; Meints R.H. (1984) “DNA synthesis in a Chlorella-like 

alga following infection with the virus PBCV-1.” Virology 134:443-449. 

Van Etten, J.L.; Burbank, D.E.; Kuczmarski, D.; Meints, R.H. (1983) “Virus infection of a 

culturable Chlorella-like algae and development of a plaque assay.” Science 219:994-996. 

Van Etten, J.L.; Burbank, D.E.; Meints, R.H (1986) “Replication of PBCV-1 in ultra-violet 

irradiated Chlorella.” Intervirology 26:115-120. 

Van Etten, J.L.; Burbank, D.E.; Schuster, A.M.; Meints, R.H. (1985) “Lytic viruses infecting a 

Chlorella-like alga.” Virology 140:135-143. 

Van Etten, J.L.; Burbank, D.E.; Xia, Y.; Meints, R.H. (1983) “Growth cycle of a virus, PBCV-1, 

that infects Chlorella-like algae.” Virology 126:117-125. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 291 

Van Etten, J.L.; Meints, R.H. (1991) “Viruses and virus-like particles of eukaryotic algae.” 

Microbiological Reviews 55:586-620. 

Van Etten, J.L.; Lane, L.; Meints, R.H. (1991) “Unicellular plants also have large dsDNA 

viruses.” Seminars in Virology 2:71-77. 

Van Etten, J.L.; Meints, R.H.; Burbank, D.E.; Kuczmarski, D.; Cuppels, D.A.; Lane, L.C. (1981) 

“Isolation and characterization of a virus from the intracellular green alga symbiotic with Hydra 

viridis.” Virology 113:704-111. 

Van Etten, J.L.; Meints, R.H.; Kuczmarski, D.; Burbank, D.E.; Lee, K. (1982) “Viruses of 

symbiotic Chlorella-like algae isolated from Paramecium bursaria and Hydra viridis.” Proc. 

Nat’l Acad. Sci. 79:3867. 

Van Etten, J.L.; Schuster, A.M.; Meints, R.H. (1988) “Viruses of eukaryotic Chlorella-like 

algae.” In Viruses of Fungi and Simple Eukaryotes, Mycology Series Vol. 7 (Koltin, Y.; 

Leibowitz, M.J., eds.), Marcel Dekker, Inc., New York, pp. 411-428. 

Van Etten, J.L.; Xia, Y.; Meints, R.H. (1987) “Viruses of a Chlorella-like green alga.” In PlantMicrobe Interactions II (Kosuge, T.; Nester, E.W., eds.), Macmillan Publishing Co., pp. 307-325. 

Van Etten, J.L.; Xia, Y.; Narva, K.E.; Meints, R.H. (1987) “Chlorella algal viruses.” In 

Extrachromasomal Elements in Lower Eukaryotes (Fink, G.; et al., eds.), Plenum Press, New 

York, pp. 337-347. 

Vigon, B.W.; Arthur, M.F.; Taft, L.G.; Clark, R.; Wagner, C.K.; Lipinsky, E.S.; Scantland, D.A.; 

Kresovich, S. (1982a) “Resource assessment for microalgal/emergent aquatic biomass systems 

in the southwestern United States.” Proceedings of the June 1982 SERI Biomass Program 

Principal Investigators’ Review Meeting, Aquatic Species Program Reports, Solar Energy 

Research Institute, Golden, Colorado, SERI/CP-231-1808, pp. 139-149. 

Vigon, B.W.; Arthur, M.F.; Taft, L.G.; Wagner, C.K.; Lipinsky, E.S.; Litchfield, J.H.; 

McCandlish, C.D.; Clark, R. (1982b) “Resource assessment for microalgal/emergent aquatic 

biomass in the arid southwest.” Battelle Columbus Laboratory Report, Solar Energy Research 

Institute, Golden, Colorado. 

Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1989) “The mitochondrial genome of the 

exsymbiotic Chlorella-like green alga N1A.” Aquatic Species Program Annual Report

(Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, 

SERI/SP-231-3579, pp. 91-118. 

Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1990) “The mitochondrial genome of an 

exsymbiotic Chlorella-like green alga.” Plant Mol. Biol. 14:187-195. 
National Renewable Energy Laboratory 

292 A Look Back at the Aquatic Species Program—Technical Review 

Walsh, R.G. (1984) “Microalgal technology research at SERI: The biochemistry of lipid trigger 

mechanisms in microalgae—alteration of lipid metabolism during transition from rapid growth to 

rapid lipid accumulation.” Aquatic Species Program Review: Proceedings of the April 1984 

Principal Investigators’ Meeting, Solar Energy Research Institute, Golden, Colorado, SERI/CP231-2341, pp. 170-183. 

Weissman, J.C. (1984) “Cultivation of microalgae for liquid fuels.” Proceedings of the April 

1984 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy Research 

Institute, Golden, Colorado, SERI/CP-231-2341, pp.132-149. 

Weissman, J.C.; Benemann, J.R. (1978) “Biomass recycling and species control in continuous 

cultures.” Bioeng. Biotech. 21:627-648. 

Weissman, J.C.; Goebel, R.P. (1985) “Design, fabrication, and operation of innovative 

microalgae culture experiments using open ponds.” Final Report, Subcontract XK-3-03135, 

Solar Energy Research Institute, Golden, Colorado. 

Weissman, J.C.; Goebel, R.P. (1985) “Production of liquid fuels and chemicals by microalgae.” 

Proceedings of the March 1985 Principal Investigators’ Meeting, Aquatic Species Program, 

Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-2700, pp.141-161. 

Weissman, J.C.; Goebel, R.P. (1987) “Design and analysis of pond systems for the purpose of 

producing fuels.” Report to the Solar Energy Research Institute, Golden, Colorado, SERI/STR231-2840. 

Weissman, J.C.; Goebel, R.P. (1987) “Factors affecting the photosynthetic yield of microalgae.” 

FY 1986 Aquatic Species Program Annual Report, Solar Energy Research Institute, SERI/SP231-3071, pp. 139-168. 

Weissman, J.; Goebel, R. (1989) “An outdoor test facility for the large-scale production of 

microalgae.” Energy from Biomass and Wastes XII, Conference Proceedings (Klass, D.L., ed.), 

Institute of Gas Technology, Chicago, pp. 277-287. 

Weissman, J.C.; Goebel, R.P.; Benemann, J.R. (1988) “Photobioreactor design: comparison of 

open ponds and tubular reactors.” Bioeng. Biotech. 31:336-344. 

Weissman, J.C.; Tillett, D.T. (1989) “Design and operation of an outdoor microalgae test 

facility.” Aquatic Species Program, Annnual Report (Bollmeier, W.S.; Sprague, S., eds.), Solar 

Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 41-57. 

Weissman, J.C.; Tillett, T.D.; Goebel, R. (1987) “Design and operation of an outdoor microalgae 

test facility.” FY 1987 Aquatic Species Program, Annual Report (Johnson, D.A.; Sprague, S., 

eds.), Solar Energy Research Institute, Golden, Colorado, SERI/CP-231-3206, pp. 231-252. 
National Renewable Energy Laboratory 

A Look Back at the Aquatic Species Program—Technical Review 293 

Weissman, J.C.; Tillett, D.T. (1992) “Design and operation of an outdoor microalgae test 

facility: large-scale system results.” Aquatic Species Project Report, FY 1989-90 (Brown, L.M.; 

Sprague, S., eds.), National Renewable Energy Laboratory, Golden, Colorado, NREL/MP-232-

4174, pp. 32-56. 

Weissman, J.C.; Tillett, D.T.; Goebel, R.P. (1989) “Design and operation of an outdoor 

microalgae test facility.” Final Report, Solar Energy Research Institute, Subcontract 

XK-7-06113-1. 

York, Jr., R.H. (1987a) “Collection of high energy yielding strains of saline microalgae from the 

Hawaiian Islands.” Final Technical Report, Year 1, Solar Energy Research Institute, Golden, 

Colorado, 15 pp. 

York, Jr., R.H. (1987b) “Collection of high energy yielding strains of saline microalgae from the 

Hawaiian Islands.” FY 1986 Aquatic Species Program Annual Report, Solar Energy Research 

Institute, Golden, Colorado, SERI/SP-231-3071, pp. 90-104. 

York, Jr., R.H. (1987c) “Collection of high energy yielding strains of saline microalgae from the 

Hawaiian Islands.” Abstract, FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; 

Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3206, p. 76. 

Zeiler, K.G.; Kadam, K.L. (1994) “Biological trapping of carbon dioxide.” Draft Milestone 

Completion Report, National Renewable Energy Laboratory, Golden, Colorado. 

V.B. ADDITIONAL REFERENCES 

Asano, Y.; Otsuki, Y.; Ugaki, M. (1991) “Electroporation-mediated and silicon carbide fibermediated DNA delivery in Agrostis alba L. (Redtop).” Plant Sci. 79:247-252. 

Berliner, M.D. (1977) “Protoplast induction in Chlorella vulgaris.” Plant Sci. Lett. 9:201-204. 

Bligh, E.G.; Dyer, D.J. (1959) “A rapid method for total lipid extraction and purification.” Can 

J. Biochem. Physiol. 37:911-917. 

Braun, E.; Aach, H.G (1975) “Enzymatic degradation of the cell wall of Chlorella.” Planta

126:181-185. 

Daboussi, M.J.; Djeballi, A.; Gerlinger, C.; Blaiseau, P.L.; Bouvier, I.; Cassan, M.; Lebrun, 

M.H.; Parisot, D.; Brygoo, Y. (1989) “Transformation of seven species of filamentous fungi 

using the nitrate reductase gene of Aspergillus nidulans.” Curr. Genet. 15:453 

Global, E.; Aach, H.G. (1985) “Studies of Chlorella protoplasts. II. Isolation and fusion of 

ptotplasts from Chlorella saccharophila (Krüger) nadson strain 211-1a.” Plant Sci. 39:213-218. 
National Renewable Energy Laboratory 

294 A Look Back at the Aquatic Species Program—Technical Review 

Greenbaum, E.; Lee, J.W.; Tevault, C.V.; Blankinship, S.L.; Metz, L.J. (1995) “Carbon dioxide 

fixation and photoevolution of hydrogen and oxygen in a mutant of Chlamydomonas lacking 

photosystem I.” Nature, August 3. 

Greenspan, P.; Mayer, E.P.; Fowler, S.D. (1985) “Nile Red: A selective fluorescent stain for 

intracellular lipid droplets.” J. Cell Biol. 100:965-973. 

Greenspan, P.; Fowler, S.D. (1985) “Spectrofluorometric studies of the lipid probe, Nile Red.” J. 

Lipid Res. 26:781-. 

Hoffman, C.S.; Winston, F. (1987) “A ten-minute DNA preparation from yeast efficiently 

releases autonomous plasmids for transformation of Escherichia coli.” Gene 57:267-272. 

Kaeppler, H.F.; Gu, W.; Somers, D.A.; Rines, H.W.; Cockburn, A.F. (1990) “Silicon carbide 

fiber-mediated DNA delivery into plant cells.” Plant Cell Rep. 9:415-418. 

Kindle, K. (1990) “High frequency nuclear transformation of Chlamydomonas reinhardtii.” 

Proc. Nat. Acad. Sci. USA 87:1228-1232. 

Kindle, K.L.; Schnell, R.A.; Fernández, E.; Lefebvre, P.A. (1989) “Stable nuclear transformation 

of Chlamydomonas using the Chlamydomonas gene for nitrate reductase.” J. Cell Biol

109:2589-2601. 

Mitra, A.; Higgens, D.W. (1994) “The Chlorella virus methyltransferase gene promoter is a 

strong promoter in plants.” Plant Mol Biol. 26:85-93. 

Murray, H.G.; Thompson, W.F. (1980), “Rapid isolation of high molecular weight plant DNA.” 

Nuc. Acids Res. 8:4321-4325. 

Nei, M. (1972) Amer. Natur. 106:283. 

Siver, P. (1983) “A new thermal gradient device for culturing algae.” British J. Phycol. 18:159-

163. 

V.C. PATENTS 

Dunahay, T.; Roessler, P.G.; Jarvis, E.E. “Method to Transform Algae, Materials Therefor and 

Products Produced Thereby.” U.S. Patent 5,661,017, issued 8/26/97. 

Jarvis, E.E.; Roessler, P.G. “Isolated-Gene Encoding an Enzyme with UDP-Glucose 

Pyrophosphorylase Activities from Cyclotella cryptica.” Allowed 10/24/96. 

Roessler, P.G; Ohlrogge, J.B., “Gene Encoding Acetyl-Coenzyme A Carboxylase.” U.S. Patent 

5,559,220, issued 9/24/96. 
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1. AGENCY USE ONLY (Leave blank) 2. REPORT DATE

July 1998 

3. REPORT TYPE AND DATES COVERED 

Technical Report 

4. TITLE AND SUBTITLE

A Look Back at the U.S. Department of Energy’s Aquatic Species Program: Biodiesel from Algae 

6. AUTHOR(S) 

J. Sheehan, T.G. Dunahay, J.R. Benemann, P.G. Roessler, J.C. Weissman 

5. FUNDING NUMBERS

BF886001 

7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 

National Renewable Energy Laboratory 

1617 Cole Blvd. 

Golden, CO 80401-3393 

U.S. Department of Energy 

U.S. Department of Agriculture 

Ecobalance, Inc. 

Colorado School of Mines 

Colorado Institute for Fuels and Engine Research 

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National Renewable Energy Laboratory 

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13. ABSTRACT (Maximum 200 words) The Aquatic Species Program was a relatively small research effort intended to look at the use of 

aquatic plants as sources of energy. Its history dates back to 1978, but much of the research from 1978 to 1982 focused on using 

algae to produce hydrogen. The program switched emphasis to other transportation fuels, particularly biodiesel, beginning in the early 

1980's. This report summarizes the research activities carried out from 1980 to 1996, with an emphasis on algae for biodiesel 

production. 

15. NUMBER OF PAGES

14. SUBJECT TERMS 

biodiesel, algae, aquatic species, raceway ponds 16. PRICE CODE

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18. SECURITY CLASSIFICATION 

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