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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 BenemannPaul 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

NREL/TP-580-24190

Executive Summary
From 1978 to 1996, the U.S. Department of Energy’s Office of Fuels Development funded a program todevelop 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 theengineering 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 naturaloils. Researchers not only concerned themselves with finding algae that produced a
lot of oil, but also with algae that grow under severe conditions—extremes oftemperature, pH and salinity. At the outset of the program, no collections existed thateither 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 thewest, 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. Afterscreening, 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 algalorganisms. Much of the program’s research focused attention on the elusive “lipid
trigger.” (Lipids are another generic name for TAGs, the primary storage form ofnatural 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 insupport 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 inthe 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 ofnutrient deficiency. Higher levels of oil in the cells are more than offset by lower
rates of cell growth.

National Renewable Energy Laboratory
Breakthroughs in molecular biology and genetic engineering.
Plant biotechnology is a field that is only now coming into its own. Within the field of plantbiotechnology, 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 andgenetics 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 ACCasegene in any photosynthetic organism. With this gene in hand, researchers went on to develop the firstsuccessful 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 theclosing days of the program, researchers initiated the first experiments in metabolic engineering as a meansof increasing oil production. Researchers demonstrated an ability to make algae over-express the ACCasegene, a major milestone for the research, with the hope that increasing the level of ACCase activity in thecells 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 physicalconditions 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 speciesgrown. 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 highproductivities were hampered by low temperature conditions encountered at the site. The desert conditionsof New Mexico provided ample sunlight, but temperatures regularly reached low levels (especially atnight). 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 islittle prospect for any alternatives to the open pond designs, given the low costrequirements associated with fuel production. The factors that most influence costare biological, and not engineering-related. These analyses point to the need forhighly productive organisms capable of near-theoretical levels of conversion ofsunlight to biomass.
Even with aggressive assumptions about biologicalproductivity, we project costs for biodiesel which are two times higher than current
petroleum diesel fuel costs.

A Look Back at the Aquatic Species Program—Executive Summaryii

National Renewable Energy Laboratory
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 lesswater 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 thetechnology 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 Aquatic Species Program—Executive Summaryiii

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 ProgramThis year marks the 20th anniversary of the National Renewable Energy Laboratory(NREL). In 1978, the Carter Administration established what was then called theSolar Energy Research Institute (SERI) in Golden, CO. This was a first-of-its kindfederal laboratory dedicated to the development of solar energy. The formation ofthis lab came in response to the energy crises of the early and mid 1970s. At thesame 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 ofalternative fuels, including ethanol and methanol (alcohol fuel substitutes forgasoline), 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 backto 1978, much of the research from 1978 to 1982 was focused on using algae toproduce hydrogen. The program switched emphasis to other transportation fuels, inparticular 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 forbiodiesel production.
In 1995, DOE made the difficult decision to eliminate funding for algae researchwithin 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 thisreport is to bring closure to the Biofuels Program’s algae research. This report is asummary 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 allorganic life in our environment. These organisms use energy from the sun tocombine 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 wasconcerned 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 partiallysubmerged in bogs and marshes. Microalgae are, as the name suggests, microscopic
photosynthetic organisms. Like macroalgae, these organisms are found in bothmarine and freshwater environments. In the early days of the program, research wasdone on all three types of aquatic species. As emphasis switched to production ofnatural 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 thisoverview).
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. Muchof the work done over the past two decades represents genuine additions to thescientific literature. The limited size of the scientific community involved in thiswork also makes it more difficult, and sometimes slower, compared to the progressseen with more conventional organisms. The study of microalgae represents an areaof 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 ofabundance) are:2•

The diatoms (Bacillariophyceae). These algae dominate thephytoplankton of the oceans, but are also found in fresh andbrackish water. Approximately 100,000 species are known toexist. 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 ofcarbohydrates known as chyrsolaminarin.

•

The green algae (Chlorophyceae).
These are also quite
abundant, especially in freshwater. (Anyone who owns aswimming pool is more than familiar with this class of algae).
They can occur as single cells or as colonies. Green algae are theevolutionary progenitors of modern plants. The main storage
compound for green algae is starch, though oils can be produced
under certain conditions.

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 rolein 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 issimilar 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 infreshwater systems. They are similar to diatoms in pigmentation
and biochemical composition. The golden algae produce naturaloils and carbohydrates as storage compounds.

The bulk of the organisms collected and studied in this program fall in the first twoclasses—the diatoms and the green algae.
Microalgae are the most primitive form of plants. While the mechanism ofphotosynthesis 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 capableof 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 oftaking 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 forrenewable energy technology), the concept of using microalgae as a source of fuel isolder than most people realize. The idea of producing methane gas from algae wasproposed in the early 1950s1. These early researchers visualized a process in whichwastewater 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 andenergy 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 namefor the unit of energy often used by DOE to describe the amounts of energy that agiven technology might be able to displace. Quad is short for “quadrillion Btus”—aunit 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 Summary3

Such algae farms would be based on the use of open, shallow ponds in which somesource of waste CO2 could be efficiently bubbled into the ponds and captured by thealgae (see the figure below).

Water
Nutrients
Motorized
paddle
wheel

Algae

Waste CO2

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 regularfrequency. 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 areconstantly 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 theseponds 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 ofbiomass 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 andother 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 highconcentration 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 approachto 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
SystemAlgae/Oil
Recovery
SystemFuel
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 ofland analogous to traditional farming. Such large farms would be located adjacent topower plants. The bubbling of flue gas from a power plant into these ponds provides asystem 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 ontransportation 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 evaluatedunder a separate program funded by DOE’s Office of Fossil Fuels. The Japanese
have been the most aggressive in pursuing this application. They have sponsoreddemonstrations 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, thethree fuel production options listed previously can be used in a number of
combinations. The most simplistic approach is to produce methane gas, since theboth the biological and thermal processes involved are not very sensitive to whatform the biomass is in. Gasification is a somewhat brute force technology in thesense 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 tomean 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 assources of biodiesel. Biodiesel made from rapeseed oil is now a substantialcommercial enterprise in Europe. Commercialization of biodiesel in the U.S. is stillin 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 theirbody 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 ismostly 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 tobring the viscosity of the oils within the range of current petroleum diesel3. Byreacting these TAGs with simple alcohols (a chemical reaction known as“transesterification” already commonplace in the oleochemicals industry), we cancreate a chemical compound known as an alkyl ester4, but which is known moregenerically as biodiesel (see the figure below). Its properties are very close to thoseof petroleum diesel fuel.
3 molecules of biodiesel

1 TAG

O

CH2

COOH

HC
HC

COOH

HC

COOH

O

CH2

O

CH2

+
3(CH2OH)

3 molecules of alcohol

+
HCOH
HCOH
HCOH

1 molecule of glycerol

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 manyengines. Biodiesel virtually eliminates the notorious black soot emissions associatedwith diesel engines. Total particulate matter emissions are also much lower6,7,8.
Other environmental benefits of biodiesel include the fact that it is highlybiodegradable9 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 avery 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 sametime, algae technology provides a means for recycling waste carbon from fossil fuelcombustion. Algal biodiesel is one of the only avenues available for high-volume re-useof CO2 generated in power plants. It is a technology that marries the potential need forcarbon 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 anddeploying 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 othertechnology 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 ourimports to exceed 60% of our total consumption ten years fromnow.

•

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 anational security issue, there is one more piece to the puzzle that influences ourperspective 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 ourtransportation sector.
Our almost complete reliance on petroleum in transportation comes from the demand forgasoline in passenger vehicles and the demand for diesel fuel in commerce. Bioethanol
made from terrestrial energy crops offers a future alternative to gasoline, biodiesel madefrom algal oils could do the same for diesel fuel.
Climate Change
CO2 is recognized as the most important (at least in quantity) of the atmosphericpollutants that contribute to the “greenhouse effect,” a term coined by the Frenchmathematician 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 lastcentury, 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 theforefront in the scientific community was the publication of Revelle’s data in 1957,
which quantified the geologically unprecedented build-up of atmospheric CO2 thatbegan with the advent of the industrial revolution. Revelle14 characterized thepotential risk of global climate change this way:
“Human beings are carrying out a large scale geophysical experiment ofa 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 andthe 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 changeremains 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 thecurrent build up of atmospheric CO2. Thus, identifying alternatives to fossil fuels must bea 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 madefrom 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 ofconsumption, coal reserves could last for over 200 years.
Regardless of how much faith you put in future fossil energy projections, it is clearthat coal will continue to play an important role in our energy future—especiallygiven the relatively large amounts of coal that we control within our own borders.
DOE’s Energy Information Administration estimates that electricity will become anincreasingly 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, thedemand 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 ofenergy 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 anincreasingly 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 theocean15.
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 andoperating dollars would be spent simply to dispose of carbon. While such Draconianmeasures 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, willcontinue 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 notcompete for the land already being eyed by proponents of other biomass-based fueltechnologies. Biomass power and bioethanol both compete for the same land and forsimilar feedstocks—trees and grasses specifically grown for energy production.
More importantly, many of the algal species studied in this program can grow inbrackish water—that is, water that contains high levels of salt. This means that algae
technology will not put additional demand on freshwater supplies needed fordomestic, 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 applicationof water or land resources. If we were to draw a map showing areas best suited forenergy crop production (based on climate and resource needs), we would see thatalgae 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 theopportunity to utilize land and water resources that are, today, unsuited for any otheruse. Land use needs for microalgae complement, rather than compete, with otherbiomass-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 naturaloils. Researchers not only concerned themselves with finding algae that produced a
lot of oil, but also with algae that grow under severe conditions—extremes oftemperature, pH and salinity. At the outset of the program, no collections existed thateither 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 thewest, 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. Afterscreening, 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 algalorganisms. Much of the program’s research focused attention on the elusive “lipid
trigger.” (Lipids are another generic name for TAGs, the primary storage form ofnatural 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 insupport 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 inthe 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 ofnutrient 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 fieldof 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 themore 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 wasfound 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 ofincreasing 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 oflarge-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 thealgal 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 atnight). 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 theinability 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 aconsistent species of algae was to allow a contaminant native to the area to take overthe 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 islittle prospect for any alternatives to the open pond designs, given the low costrequirements associated with fuel production. The factors that most influence costare biological, and not engineering-related. These analyses point to the need forhighly productive organisms capable of near-theoretical levels of conversion ofsunlight to biomass.
Even with aggressive assumptions about biologicalproductivity, 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 ona year-to-year basis. In order to provide a consistent context and framework forunderstanding 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 maincategories—laboratory studies and outdoor testing/systems analysis. For the sake ofclarity 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 thework 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 morecomprehensive 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 needto 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 tounderstanding 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 behindcollecting 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 thetechnology). 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 aresult, about midway through the collection efforts, the program began studies on thebiochemistry 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
AZ,CA,
Carribean, NM,CO,
NV,NM,
UT, FL, HI, NE, AL
TX,UT
CO, UTLab Studies

CO, AL,MI

Artificial saline
media; TempSalinity Gradient
ScreeningCollection,
Screening,
Characterization

UT, WA, CO, CA,
NV

Nile Red lipid
screening
Biochemistry, Physiology
of Lipid Production
N-deficiency
lipid trigger Si-deficiency
lipid trigger

Link between
Si-deficiency
and ACCase

Outdoor Culture Studies and Systems Analysis
Algae Production in Wastewater
Treatment

Isolation andcharacterizationof ACCase
enzymeGenetic Engineeringof AlgaeTransient
expression offoreign gene inalgae using
protoplasts<100 sq.m. Pond Studies (CA, HI)
1000 sq.m. Pond Study (NM)Systems Analysis and Resource Assessment

By 1987, over
3,000 strains ofalgae had beencollected.
Future efforts

1st successful
genetictransformation of
diatomIn the end, however, the studies conducted both by NREL researchers and programsubcontractors 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 ameans 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 growthrate of the cells dropped. Further studies identified two factors that seemed to be atplay 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 tolipids.

Diatoms store carbon in lipid form or in carbohydrate form. The results of theseexperiments suggested that it might be possible to alter which route the cells used forstorage (see schematic below):

CO2

Photosynthesis

C
Lipid Synthesis

16

Carbohydrate
Synthesis

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 insynthesis 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 theflow 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 endof the program, not only had researchers successfully cloned the ACCase gene, butthey had also succeeded in developing the tools for expressing foreign genes indiatoms.
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, otherapproaches were also developed for foreign gene expression—in green algae as wellas in diatoms. Another interesting sideline in the research involved studies aimed at
identifying key enzymes involved in the synthesis of storage carbohydrates. Insteadof over-expressing these enzymes, researchers hoped to inactivate them. Returning
to our “spigot” analogy, this approach was like shutting off the flow of carbon tocarbohydrates, 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 tothe 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 forenergy 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. Overthe course of several years, the Richmond Field Station demonstrated techniques foralgae 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 farmoperations. 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 ofCalifornia, 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 avariety 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, theprogram 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 forscale-up. The “Outdoor Test Facility” (OTF) was designed and built at the site of anabandoned 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 overin 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 late1980s. This work consisted of a combination of experimental and computermodeling 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 muchthe technology will cost.
As early as 1982, the program began to study the question of resource availability foralgae 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 suitableto support 2 to 7 quads of fuel production annually. The cost of the CO2 wasestimated to range anywhere from $9 to $90 per ton of CO2. This study did notconsider 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 assessland and water availability for algae technology in New Mexico. This study took amore 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 withassumptions of $50 per ton of CO2 as a carbon credit, the cost of biodiesel nevercompetes 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

Funding History for the Aquatic Species Program
3000$1000s per year

2500
2000
1500
1000

1996

1994

1992

1990

1988

1986

1984

1982

1980

0

1978

500

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 and1985, 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 Summary19

relatively low, they were at least steady—providing a desperately needed stability forthe program. The years of higher spending are, for the most part, dominated bycostly demonstration work (the tests carried out in California, Hawaii andculminating in New Mexico), engineering analysis and culture collection activities.
High Return for a Small Investment of DOE Funds

45000

Aquatic Species
Program

40000

Total Biof uels Program$1000s per year

35000
30000
25000
20000
15000
10000

1996

1994

1992

1990

1988

1986

1984

1982

1980

0

1978

5000

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 the20

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 toincrease 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 amolecular 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 somemechanism 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 aswastewater treatment.
Excessive focus on long term energy displacement goals will slow down
development of the technology. A more balanced approach is needed in which morenear 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 awaste 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. 179183.
2Peterson, C. L. (1986) “Vegetable Oil as a Diesel Fuel: Status and Research Priorities,” Transactionsof 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 the1980 Symposium of the South African Institute of Agricultural Engineers.
4Markley, 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.
9Peterson, C.; Reece, D. (1994) “Toxicology, Biodegradability and Environmental Benefits ofBiodiesel,” 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

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 UnitedStates ...........................................................................................................................................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

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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. ................................................................................................202ii

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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.
V.A.
V.B.
V.C.

BIBLIOGRAPHY ........................................................................................................................ 263
SERI/NREL/DOE REPORTS AND PUBLICATIONSADDITIONAL REFERENCESPATENTS

263
293
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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 fortheir 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 theirprimary energy storage compound. In contrast, many microalgae, (microscopic, photosyntheticorganisms 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 thegrowth and chemical composition of macroalgae and emergents will not be discussed in thisreport. 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 focusedon the production of fatty acid methyl esters (FAMEs), which can be used as a substitute forfossil-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 beused in unmodified diesel engines, and has advantages over conventional diesel fuel in that it isrenewable, 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.

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National Renewable Energy Laboratory

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 bysufficient 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 thedesert 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 havehigh 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 shouldhave 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 compositionof selected strains.

This effort involved in-house researchers and subcontractors from academia, industry, and othergovernment 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 thehistory 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, noone algal strain was identified that exhibited the optimal properties of rapid growth and highconstitutive lipid production. Many microalgae can be induced to accumulate lipids underconditions of nutrient deprivation. If this process could be understood, it might be possible tomanipulate 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

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ASP subcontractors to study the biochemistry and physiology of lipid production in oleaginous(oil-producing) microalgae. Work performed by several ASP subcontractors was designed tounderstand the mechanism of lipid accumulation. In particular, these researchers tried todetermine 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 indiatoms also induced lipid accumulation. Unlike N, Si is not a major component of cellularmolecules, 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 andcarbohydrate 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 geneticallyengineer 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 theprelimilary 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 aclear 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 andsome 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 andseawater 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, withthe 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

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3National Renewable Energy Laboratory

1,000 m2 high-rate ponds. This last project represented the culmination of ASP R&D inlarge-scale algal mass culture R&D. These studies are described in Section III.A. Somesupporting 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 technicallyfeasible, 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 greenhousegas (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 insuch cost analyses. Section III.D. provides recommendations for future research that could makethis technology commercially feasible.

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National Renewable Energy Laboratory

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 andscreening. 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 thedevelopment 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 growvia photosynthesis. Many microalgae grow quite rapidly, and are considerably more productive
than land plants and macroalgae (seaweed). Microalgae reproduction occurs primarily byvegetative (asexual) cell division, although sexual reproduction can occur in many species underappropriate 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 importanceto the ASP: diatoms (Class Bacillariophyceae), green algae (Class Chlorophyceae), goldenbrown algae (Class Chrysophyceae), prymnesiophytes (Class Prymnesiophyceae), and theeustigmatophytes (Class Eustigmatophyceae). The blue-green algae, or cyanobacteria (Class
Cyanophyceae), were also represented in some of the collections. A brief description of thesealgal groups follows.
•

Diatoms. Diatoms are among the most common and widely distributed groupsof algae in existence; about 100,000 species are known. This group tends todominate the phytoplankton of the oceans, but is commonly found in fresh- andbrackish-water habitats as well. The cells are golden-brown because of thepresence 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

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5National Renewable Energy Laboratory

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 thisfrom a strain development perspective is that it makes producing improved
strains via classical mutagenesis and selection/screening substantially moredifficult. As a consequence, diatom strain development programs must rely
heavily on genetic engineering approaches.

6•

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 incertain 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 notaccumulate lipids, and thus was not considered for use in the ASP. Anothercommon 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 biochemicalcomposition. Approximately 1,000 species are known, which are foundprimarily in freshwater habitats. Lipids and chrysolaminarin are considered tobe 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, consistsof approximately 500 species. They are primarily marine organisms, and canaccount for a substantial proportion of the primary productivity of tropicaloceans. As with the diatoms and chrysophytes, fucoxanthin imparts a browncolor to the cells, and lipids and chrysolaminarin are the major storage products.
This group includes the coccolithophorids, which are distinguished bycalcareous 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.

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National Renewable Energy Laboratory•

Cyanobacteria. This group is prokaryotic, and therefore very different from allother groups of microalgae. They contain no nucleus, no chloroplasts, and havea 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 (thuseliminating the need to provide fixed N to the cells), no member of this classproduces significant quantities of storage lipid; therefore, this group was notdeemed 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 fromthese 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, andtherefore subject to large variations in temperature and salinity; thus, the strains collected in thisregion 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 toaccumulate lipids. There had never been a large-scale effort to collect strains with thiscombination of characteristics; therefore, they were not available from culture collections.
2

The stated objectives of the SERI culture collection and screening effort were to:2•

Assemble and maintain a set of viable mono-specific algal cultures stored underconditions best suited to the maintenance of their original physiological andbiochemical characteristics.

•

Develop storage techniques that will help maintain the genetic variability andphysiological 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 therange of tolerated conditions.

Taken from the Proceedings of the April 1984 Aquatic Species Program Principal Investigators’ Meeting.

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7National Renewable Energy Laboratory

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 hotsprings 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 toinoculate natural collection site water that had been enriched with N (ammonium and nitrate) andphosphate (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 ofcollection. Conductivity ranged from 1.9 mmhos•cm-2 at South Canyon Spring to 85.0mmhos•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 inseveral 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 andcharacterization.” 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, and8

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National Renewable Energy Laboratory

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 technologicaladvance 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 thedevelopment 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 thevarious 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 responsiblefor 40% of the variance; the second factor, divalent ion concentration, for 30%. A plot of thesefactors 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 (averagevalue = 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-, SO42-, 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 ofconductivities 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 wereallowed 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.

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In order to assess whether these media formulations accurately reflected the types of water indesert region surface waters, samples of the water at numerous algal collection sites in thesouthwestern United States were chemically analyzed. The relative compositions of the anionicand cationic constituents were then plotted on separate trilinear plots, which allowed a graphicalrepresentation 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+ thanmost 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 thatthese 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), andvitamins. 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 strainspresent in the tubes were isolated as unialgal cultures by agar plating or by serial dilution inliquid media.
The isolated strains were then tested for their ability to grow in incubators at 25ºC at 150-200µE•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 characterizedwith respect to growth on a temperature-salinity gradient table at a light intensity of 200µE•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.

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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 aparticular combination of temperature and salinity would result in a given growth rate. Manysuch 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, andChlorococcum) 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 Boekeloviasp.) 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 alsoachieved 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 andtemperatures, 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 (300µM) 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 amodification 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 besystematically screened and provided standardized media that could be used in different
laboratories performing ASP-sponsored research. Numerous strains were characterized withrespect to growth at several temperatures and salinities using these new media.

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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 fromshallow, 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. 18:159163.

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Figure II.A.1. Rotary screening apparatus used for microalgal screening.

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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 statisticalanalysis 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).

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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 andEllingson 1987.)

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Figure II.A.4. Growth contour plots. Examples of growth contour plots generated fromdata obtained by the use of a temperature-gradient table. The contour lines representinterpolated 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 theexponential growth of Monoraphidium sp. (S/MONOR-2) in semicontinuous culture. Eachpoint 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

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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 (25and 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 thepredominant 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 thespring. From these 53 sites, 17 promising strains were isolated. An analysis was conductedcomparing 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 theolder 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 wereeven considered to grow well in the collection site water relative to growth in at least one of theartificial 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 thetemperature-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 andCYCLO2 were actually collected from the Florida keys; the remaining strains were collected inColorado and Utah.) All strains exhibited rapid growth over a wide range of conductivities in atleast 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, andmedia 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 the1986 ASP Annual Report (Barclay et al. 1986).

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Table II.A.1. Growth characteristics of various microalgal strains collected in 1985.
StrainMaximum
Growth Rate(doublings•day-1)

AMPHO1AMPHO2CHAET14CYCLO2MONOR2NAVIC1

1.7
2.48
2.87
1.63
2.84
2.77

OptimalTemperature
(ºC)
30
30-35
35
30-35
25-30
30

OptimalConductivity(mmho•cm-1)
10-25
40-70
25-70
10-70
25
10-40

Optimal Medium Type(dependent on temperatureand conductivity used)
Type I, ASW
Type I, Type II
Type II, ASW
Type I, ASW
Type I, II, ASW
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 incontrolling the growth of Boekelovia sp. (BOEKE1) and Monoraphidium (MONOR2). The
growth rate of MONOR2 increased by more than five-fold as the bicarbonate concentration ofType II/25 medium was increased from 2 to 30 mM, and the growth of BOEKE1 byapproximately 60% over this range. These results make sense, since media enriched inbicarbonate 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 thedivalent 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 thatmatching the chosen strain for a particular production site to the type of water available for masscultivation will be important.
Lipid content.
The lipid contents of several strains were determined for cultures in exponential growth phaseand 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 inresponse to Si and N deficiency, increasing from 19% to 39% and 38%, respectively. A moremodest 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.

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These results suggested that high lipid content was indeed achievable in many strains bymanipulating the nutrient levels in the growth media. However, these experiments did notprovide information on actual lipid productivity in the cultures, which is the more importantfactor 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 fordesigning 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 newstrains 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 inthree 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 theinitial 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 useof 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 ofsalinities (e.g., five of the eight strains exhibited a growth rate greater than one doubling•day-1 at

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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 TypeII medium. These results again highlight the need to have a variety of algal strains available forthe 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, aswell as for cells that were grown under nutrient-limited conditions. Nitrogen deficiency led to anincrease 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 organicmass) 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 andprobably 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 focusedon 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 thisregion 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 characterizestrains 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 islonger 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 strainsfrom each other and identifying which were the best for further characterization were moredifficult.

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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 inprevious 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 characterizethese strains with respect to lipid production potential. Future work should look at the fatty acidprofiles of oil found in the cold-water strains. Such cold-water organisms often contain highlevels 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 ofPleurochrysis carterae var dentata nov. (Prymnesiophyceae), a new coccolithophorid from aninland saline pond in New Mexico, USA.” Phycologica 27:78-88.
Johansen, J.; Lemke, P.; Barclay, W.; Nagle, N. (1987) “Collection, screening, andcharacterization 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 ofcentral 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 sothat it could be used as a standard method in numerous laboratories. The researchers hoped thatsuch a screening tool would allow the size of the strain collection to be reduced to a manageable
number (~200) representing the most promising strains.

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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 (9diethylamino-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 microscopicallyassessing the lipid content of algal cells (Lien 1981). The active ingredient in these Nile Bluepreparations 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 cellsuspension. In this species, the fluorescence of the dye in live stained cells was stable for only 27 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 todifferences 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 foundprimarily 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 afixed 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 fluorometerequipped 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 thespecies-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 alarge number of cells using organic solvents, evaporation of the solvent, and determination of the22

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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 anequally 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 thesouthwestern United States, along with data concerning the high rates of evaporation in thisregion, 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 proceduredeveloped 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 withincreasing 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 bemeasured without removing a sample. The tubes also held enough medium to allow samples tobe taken for Nile Red lipid analysis (both for N-sufficient and N-deficient cells), and for ash-freedry 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 dailyfor 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’slaboratory 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.

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23National Renewable Energy Laboratory

Table II.A.2 Fastest growing strains from Arizona State University collection.
Strain

Genus

Class

OSCIL2OSCIL3
CHLOC4

Oscillatoria
Oscillatoria
Chlorococcum/
Eremosphaera
Synechococcus
Oscillatoria
AmphoraNannochloris
Synechococcus
Chlorella
Synechococcus

SYNEC5
ASU0735AMPHO46
NANNO13
POLYC1CHLOR23SYNEC3MediumCyanophyceae
Cyanophyceae
Chlorophyceae

Growth Rate(doublings•day-1)
4.23
3.50
3.47

Cyanophyceae
Cyanophyceae
Bacillariophyceae
Chlorophyceae
Cyanophyceae
Chlorophyceae
Cyanophyceae

3.25
3.06
2.81
2.78
2.73
2.66
2.51

II/55
I/55
I/55
I/55
I/55
I/55
II/55

I/55
I/55
I/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 ofN-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 ofthese strains are diatoms, confirming the propensity of this group to accumulate lipids.

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National Renewable Energy Laboratory

Table II.A.3. Strains from the Arizona State University collection having the highest
Nile Red fluorescence.
Strain

Genus

Class

NITZS54NITZS53NITZS55
ASU3004
NAVIC36AMPHO45FRAGI2AMPHO27NITZS52

Nitzschia
Nitzschia
Nitzschia
AmphoraNitzschia
AmphoraFragilaria
AmphoraNitzschia

Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

Trioleinequivalents(mg•L-1)
Exponential
growth
8
17
37
9
61
39
6
38
24

Trioleinequivalents(mg•L-1)
N-deficient
growth
1003
934
908
593
579
308
304
235
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, theoptimal 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.

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25National Renewable Energy Laboratory

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

AMPHO27
CHLOC4

AmphoraEremosphaera/
Chlorococcum
Synechococcus
AmphoraSynechococcus
AmphoraNitzschia
OocystisNitzschia

Bacillariophyceae
Chlorophyceae

Triolein equivalents(mg•L-1•day-1)
345
117

Cyanophyceae
Bacillariophyceae
Cyanophyceae
Bacillariophyceae
Bacillariophyceae
Chlorophyceae
Bacillariophyceae

86
71
64
63
48
46
45

SYNEC5AMPHO46SYNEC4AMPHO45NITZS55
OOCYS9NITZS52

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 andNile Red fluorescence in exponentially growing cultures and in cultures grown under N-deficientconditions for 4 days. The results of the SERI and Sommerfeld laboratories cannot be compareddirectly, 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 fluorescencenormalized 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 beststrains 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 andscreening 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 todetermine their ability to grow in actual outdoor mass culture ponds. This work is described in
Section III of this report.

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National Renewable Energy Laboratory

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, andcharacterization 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.; andHall, D.O., eds.), Applied Science Publishers, London, pp. 697-702.
Sommerfeld, M.; Ellingson, S.; Tyler, P. (1987) “Screening microalgae isolated from thesouthwest 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 ofseveral 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 theseexperiments, 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 theuse of the Nile Red fluorometric assay.

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27National Renewable Energy Laboratory

The results indicated that the N content and conductivity of the medium were the most importantvariables 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. Theinteraction of N and conductivity was an important determinant of lipid content as well. Siliconlevel 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 cellmass for NAVIC1, and was a major factor for cell mass yield in CHAET9 (along with theinteraction 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, theseexperiments 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 resultscannot 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 andscreening, 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 laterat 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 ofthese analyses, and will provide details about the analytical methods used, as these methods werethe 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, andextracted 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 wasthen 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 withhexane, benzene, chloroform, acetone, and methanol. In this procedure, the lipids are eluted asfollows:
1. hexane: acyclic hydrocarbons

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National Renewable Energy Laboratory

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; and5. 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 (andautoradiography 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 ofmolybdate/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 Dunaliellacells. 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% and10.9% of the lipid mass, respectively. Isoprenoid hydrocarbons (including β-carotene) andaliphatic 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 werepalmitic (20.6%), linolenic (12.5%), linoleic (10.7%) and palmitoleic (7.8%), but no attempt wasmade to ascertain whether any of these fatty acids predominated a particular lipid class. The highhydrocarbon 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 fuelsource, 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 ofTechnology), 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 grownunder 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 thatwere 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

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29National Renewable Energy Laboratory

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 madeidentification 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 andmonounsaturated fatty acids in this alga is considered optimal from a fuel quality standpoint, inthat 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 thelipid 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 underconditions 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 otherlaboratories 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, andNannochloris, the diatom Nitzschia, and the chrysophyte Isochrysis. N deficiency led to anincrease in the lipid content of Ankistrodesmus (from 24.5% to 40.3%), Isochrysis (from 7.1% to26.0%), 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 thequantities of lipids present. However, as stated before, the most important characteristic of alipid 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.

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National Renewable Energy Laboratory

Table II.A.5 indicates the major fatty acids (those at levels exceeding 5% of the total) present inthese 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 bemade 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 thefuel 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 highlevels.
Table II.A.5. Major fatty acids of various microalgae. (Fatty acids in bold are present
at levels of 15% or higher)
StrainAnkistrodesmus
Botryococcus braunii
Dunaliella bardawil

Nitrogen-sufficient cells
16:0, 16:4, 18:1, 18:3
16:0, 18:1, 18:2, 18:3
not determined

Dunaliella salina

14:0/14:1, 16:0, 16:3, 16:4,
18:2, 18:3
14:0/14:1, 16:0, 16:1, 18:1,
18:3, 18:4, 22:6
14:0/14:1, 16:0, 16:1, 16:2,
16:3, 20:5
14:0/14:1, 16:0, 16:1, 16:2,
16:3, 20:6

Isochrysis sp.
Nannochloris sp.
Nitzschia sp.

Nitrogen-deficient cells
16:0, 18:1, 18:3
16:0, 18:1, 18:3, 20:5
12:0, 14:0/14:1, 16:0, 18:1, 18:2,
18:3
16:0, 16:3, 18:1, 18:2, 18:3
14:0/14:1, 18:1, 18:2, 18:3, 18:4,
22:6
not determined
not determined

Publications:
Ben-Amotz, A.; Tornabene, T.G. (1983) “Chemical profile of algae with emphasis on lipids ofmicroalgae.” 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 speciesof 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.

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31National Renewable Energy Laboratory

Tornabene, T.G.; Ben-Amotz, A.; Hubbard, J.S. (1982) “Isolation, analysis and identification oflipids.” SERI ASP Report prepared under contract XK-2-02149-01.
Tornabene, T.G.; Holzer, G.; Lien, S.; Burris, N. (1983) “Lipid composition of the nitrogenstarved 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 andscreening procedures were carried out by the various research groups, as there was no establishedprotocol. 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 strainsdiffered between the laboratories. At the same time, however, this arrangement provided theopportunity 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 thedesert regions of eastern California and western Nevada. Additional microalgal strains fromdesert 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 UnitedStates (Alabama, Mississippi, and Florida). Additional strains from the Florida Keys andEverglades were collected by John Rhyther (Harbor Branch Foundation); Richard York (Hawaii
Institute of Marine Biology) isolated a number of strains from the Hawaiian islands andsurrounding waters. Certain environmental niches were focused on as well; Dr. Keith Cooksey(Montana State University) isolated several strains from thermal springs in Yellowstone National

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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 Compositionof 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 andanalyzed 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 ofan experiment, the cultures were maintained in a batch mode, and then converted to a continuousor 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 theireffects on the productivities and proximate chemical composition of the strains. The results ofthese 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 lightintensities, different culturing methods [batch, semi-continuous, and continuous], different meansof obtaining N-deficient cultures, and inconsistent use of a CuSO4 heat filter, which resulted indifferences in light quality and culture temperature). Nonetheless, the general conclusions of thisstudy 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, theA Look Back at the Aquatic Species Program—Technical Review

33National Renewable Energy Laboratory

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
3
productivity observed for this strain (21 to 22 g dry weight•m-2•d-1) 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 observedwith 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 dryweight•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 cellularcomposition. Growth of P. tricornutum cells under N-deficient conditions resulted in a reductionof 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 duringcontinuous culture at 60% full sunlight under N-sufficient conditions. Doubling the lightintensity 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, thecomposition 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 inshallow 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.

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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 occurredin 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 valueof 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 wasapproximately 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 accumulatehydrocarbons. 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 grownunder 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 wereinvariably 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.

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35National Renewable Energy Laboratory

The results also pointed to the importance of identifying strains that are not photoinhibited atlight intensities that would occur in outdoor ponds. Finally, this work highlighted the fact thatsome microalgae accumulate carbohydrates during nutrient-deficient growth; such strains areclearly 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 andnitrogen 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 collectmicroalgae 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 theseprocedures. Of the 100 strains isolated, 42 were grown under standardized conditions in various

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National Renewable Energy Laboratory

artificial media that were designed to mimic the water from which the strains were originallyobtained. The pH of these various media formulations was typically high because of thepresence of high levels of carbonate and bicarbonate, and the total dissolved solids ranged fromapproximately 1.5 g•L-1 to over 260 g•L-1. The growth of the cultures was visually scored, andnine 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 densityof the cultures increased. The strains tested in this manner included Nitzschia, Ankistrodesmus,
Nannochloris, Oocystis (two strains), Chlorella (three strains), and Selenastrum. The estimated4
productivities ranged from 8.8 g dry weight•m-2•d-1 for for Nitzschia S-16 (NITZS1 ) grown in-2 -1
the presence of urea to 45.8 g dry weight•m •d for Oocystis pusilla 32-1, which was also grownwith 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 aparticular 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 thatof 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 amatrix chart of the various temperature/salinity combinations. This approach was later used byother subcontractors and SERI in-house researchers to determine the optimal growth
characteristics of numerous promising algal strains. Results from this analysis were reported foreight 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 strainstested, 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 thatwere placed at varying distances from a tungsten lamp source. Neutral density filters were
4

NREL Microalgae Culture Collection strain designations are provided when relevant.

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37National Renewable Energy Laboratory

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, theproductivity fell off to 14.7 g dry weight•m-2•d-1 (because of light limitation), and at 70% fullsunlight, the productivity was reduced to 19.0 g dry weight•m-2•d-1 (most likely because ofphotoinhibition). 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 and23.2 g dry weight•m-2•d-1, respectively. These productivity values are believed to be moreaccurate 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, andwere bubbled vigorously with 1% CO2 in air. The results of these experiments indicated thatgrowth rate, when expressed as g dry weight•L-1•d-1, was highest in the 5-cm thick culture andlowest in the 15-cm thick culture. However, when productivities were expressed as g dryweight•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 were0.72, 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 dryweight•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 importantconsideration, although less water (and consequently less water handling) is required when
dealing with more dense cultures. The productivities reported for these experiments may beoverestimates 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
5grow in “SERI standard media “ were investigated. This selection procedure resulted in theisolation 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 originallyisolated. Twelve of the strains that grew best under these conditions were then tested under thesame temperature and light conditions in an early version of standard SERI media (Type I andType 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.

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National Renewable Energy Laboratory

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 ofcells 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 setof experiments. Conversely, Chlamydomonas HL-9 grew much more quickly in the second setof 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 ofthese 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 dryweight•m-2•d-1 under these conditions, and Nannochloris MO-2A had a productivity of 31.9 g dryweight•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 promisingstrains 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: anannotated bibliography.” Report for Subcontract XK-2-0270-01. Solar Energy ResearchInstitute, 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.

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39National Renewable Energy Laboratory

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 ResearchInstitute, 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 theSoutheastern 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 thesoutheastern 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 fromrivers, 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, thecultures 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 thesepreliminary 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.

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National Renewable Energy Laboratory

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 usedby Dr. William Thomas (discussed earlier) and SERI researchers for screening purposes. Growthof 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 100µE•m-2•s-1, and a salinity of 15 ppt (parts per thousand). Growth was nearly as good at a lightintensity of 200 µE•m-2•s-1 and a temperature of 35°C, and substantial growth occurred at asalinity 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 tosupport 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 ofwhich 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 atlight 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 160µE•m-2•s-1 illumination than at 80 µE•m-2•s-1; and even higher growth rates might well have beenobtained 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 to37.1% 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%).

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41National Renewable Energy Laboratory

NAVIC8 grew most rapidly (3.8 doublings•d-1) at 35°C and 45 mmho•cm-1. Nitrate andammonium 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 inthis 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), andCylindrotheca 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, thenature 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 thesoutheastern 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 thesoutheastern 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.

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II.A.2.e.

Collection of High Energy Strains of Saline Microalgae from SouthwesternStatesSubcontractor:

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 growunder 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 toscreen 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 andNew Mexico that were deemed suitable for microalgal mass culture, based in part on theavailability 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 thePecos 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), andspecific 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 TypeI or Type II medium having a conductivity similar to that of the collection site water. In somecases, an enrichment step was performed before streaking out the cells, wherein the samples wereplaced 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 inthis 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 growthat 25°C at 200 µE•m-2•s-1 in tubes containing SERI Type I/40 and Type II/40 media. Strains thatgrew the most rapidly were subjected to further characterization, including analysis of the effects

A Look Back at the Aquatic Species Program—Technical Review

43National Renewable Energy Laboratoryof temperature, salinity, light intensity, and N source on growth rates. Cultures were grown inseveral 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 themean 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 thehigher 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 groupwere the strains mentioned earlier along with Synechococcus ASU0075 (CHROC2) andDunaliella ASU0038. Some strains were clearly euryhaline (i.e., able to tolerate a wide range ofsalinities), 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 grewmuch 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 growthrate 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 wasdetermined 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 strainstested, Franceia ASU0146 (FRANC1) had the highest lipid content under normal growth
conditions (26.5% of the AFDW). The strains were not evaluated under nutrient-deficientconditions, 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 the44

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National Renewable Energy Laboratory

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 theuse of a fluorometer. This latter work was discussed in Section II.A.1. along with the results ofsimilar efforts carried out by SERI researchers and other subcontractors.
This subcontract was somewhat unusual in that many of the strains were collected from theactual 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 thesouthwest 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 resourcesin 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 ofeach 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 thefull 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 tothrive under outdoor mass culture conditions. The glass tubes were incubated at 25°-26°C at 40µE•m-2•s-1 under a 16h:8h light:dark regime; these conditions were less stressful than the outdoor

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45National Renewable Energy Laboratory

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 andmaintained 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 werereported 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 ResearchInstitute, 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) andthat 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 mLeach) 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 naturallighting 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 tostimulate 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 thesurface 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 microscopicallyexamined, and cultures that were dominated by diatoms, cyanophytes, and flagellates were46

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National Renewable Energy Laboratory

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, andseveral representatives of other genera. Stichococcus, Nannochloris, and Chlorella are allchlorophytes. Because cyanophytes typically do not accumulate lipids, they were eliminatedfrom further study in this subcontract. The researchers anticipated that isolating strains in thismanner would enrich for lipid-accumulating microalgae.
The isolated strains were tested for the ability to grow in freshwater; all the Stichococcus andChlorella strains grew well in freshwater, suggesting perhaps a brackish water origin for thesestrains. 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, thelipid 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 averageof 33%. The lipid contents of the 21 Nannochloris strains ranged from 6% to 63%, with anaverage 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 averageof 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 nitrateas 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 beenobtained 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 atemperature 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 ina 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, thisA Look Back at the Aquatic Species Program—Technical Review

47National Renewable Energy Laboratory

property might facilitate harvesting. Outdoor testing of the most promising strains would help toevaluate this group of microalgae.
Publications:
Lewin, R.A. (1985) “Production of hydrocarbons by micro-algae; isolation and characterizationof 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
FloridaSubcontractor:

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 waterfrom the Keys (35.6 mmho•cm-1) was somewhat higher than that of the Everglades (25.7mmho•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 fullsunlight) 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 in48

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National Renewable Energy Laboratory

the Harbor Branch algal collection) were screened in this manner. In general, the strains thatwere newly isolated under the selection scheme outlined above grew more rapidly than theculture collection strains. Members of the Prymnesiophyceae, particularly coccolithophorids andochromonads, tended to grow well in most media types, but the dinoflagellates isolated via theseprocedures 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 inthis paragraph except for HYMEN3).
Several of the most promising strains were examined in more detail; they were grown in a matrixof five different salinities (8-60 mmho•cm-1) at five different temperatures (15°-35°C) by the useof 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 strainstested (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 agrowth 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 bystaining 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 andcharacterization of saline microalgae from South Florida.” FY 1986 Aquatic Species Program
Annual Report, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3071, pp. 122136.

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49National Renewable Energy Laboratory

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 highlipid content, and to use this method to select microalgae with potential for liquid fuelproduction. 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 massculture 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 oftemperature 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.

50

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National Renewable Energy Laboratory

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 toprovide a central repository for strains that were believed to have potential as biomass fuelproduction 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 indifferent 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 ofimportance):•

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 thestrains fairly extensively before a decision could be made as to whether they should be includedin the collection. This detailed characterization became increasingly difficult as the number ofstrains available increased. As a consequence, many strains were maintained that were notofficially 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.

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51

National Renewable Energy Laboratory

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 wereraised 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; thismoratorium 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 predominanceof chlorophytes (green algae) and diatoms. These two groups tended to dominate under the hightemperature/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 threeculture collection catalogs published during the course of the ASP.
The first culture collection catalog (1984-1985) listed 11 strains, which included fivechlorophytes, four diatoms, one chrysophyte, and one eustigmatophyte. Some of these strains
were obtained from other culture collections, including the University of Texas algal culturecollection. 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.
SpeciesAnkistrodesmus falcatus
Botryococcus braunii
Chaetoceros gracilis
Chlorella sp.
Isochrysis galbana
Nannochlropsis salina
Nitzschia sp.
Oocystis pusilla
Phaeodactylum
Phaeodactylum
Platymonas sp.
(later Tetraselmis suecia)

52

Original SERI
StrainDesignation
S/ANKIS-1

Final SERI
StrainDesignation
ANKIS1

Strain Alias

Collector

Pyramid Lake 91-

W. Thomas

S/BOTRY-1
S/CHAET-1
S/CHLOR-1
S/ISOCH-1
S/NANNO-1
S/NITZS-1
S/OOCYS-1
S/PHAEO-1
S/PHAEO-2
S/PLATY-1

BOTRY1CHAET1CHLOR1ISOCH1
NANNP1NITZS1OOCYS1PHAEO1PHAEO2TETRA1UTEX #572
CHGRA
S01
Tahitian T-ISO
GSBSTICHO
Mono Lake
Walker Lake
TFX-1
BB
---

--R. York
S. LienJ.-L.
J. Rhyther
D.
W. Thomas--W. Thomas
E. Laws

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National Renewable Energy Laboratory

The next edition of the SERI Microalgae Culture Collection Catalog (1985-1986) included thefollowing additional strains:

Table II.A.7. Microalgal strains added to the SERI Culture Collection Catalog, 19851986.
Species

Final SERI
StrainDesignation
AMPHO1

Strain Alias

Collector

Amphora coffeiformis

Original SERI
StrainDesignation
S/AMPHO-1---

W. Barclay

Boekelovia hooglandii
Chaetoceros sp.
Chlorella ellipsoidea
Chlorella sp.
Cyclotella cryptica
Monoraphidium sp.
Monoraphidium sp.
Nannochloropsis sp.
Nitzschia dissipata

S/BOEKE-1
S/CHAET-2
S/CHLOR-2
S/CHLOR-3
S/CYCLO-1
S/MONOR-1
S/MONOR-2
S/NANNO-2
S/NITZS-2

BOEKE1CHAET14CHLOR2CHLOR3CYCLO1MONOR1MONOR2
NANNP2NITZS2--SS-14
BL-6
SC-2
DI-35
Mom’s Ranch--Nanno-Q
DI-160

W. Barclay
W. Thomas
W. Thomas
W. Thomas
M. Tadros
W. Barclay
W. Barclay
R. Lewin
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, 19861987.
Species

Strain Alias

Collector

Amphiprora hyalina
Amphora sp.

Final SERI
StrainDesignation
ENTOM3
AMPHO27

BB-333
MLS-1, ASU 0032

M. Tadros
M. Sommerfeld

Amphora sp.
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri

AMPHO28
CHAET6CHAET9CHAET10GR-2, ASU 3001
NM-6
UT-147
S/CHAET-4, UT-27

M. Sommerfeld
W. Barclay
S. Rushforth
S. Rushforth

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53National Renewable Energy Laboratory

Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Cyclotella cryptica
Cyclotella cryptica
Ellipsoidon sp.
Franceia sp.
Nannochloris sp.
Nannochloris sp.
Navicula saprophila
Navicula acceptata
Navicula sp.
Navicula acceptata
Navicula saprophila
Nitzschia dissipata
Nitschia communis
Pleurochrysis carterae
Tetraselmis sp.
Thalassiosira weissflogii
Thalassiosira weissflogii
Unidentified Prasinophyte

54

CHAET15CHAET38CHAET58
CHAET61
CHAET63
CYCLO2CYCLO4ELLIP1
FRANC1NANNO2NANNO12NAVIC1NAVIC6NAVIC7
NAVIC8NAVIC24NITZS13NITZS28
PLEUR1TETRA4
THALA2
THALA6
GREEN349-1A
SH9-1------F-1
UT-65
70-01
LCC-1, ASU 0146
HB44
120-01
F-2
SB-264
BB 260SB-304--SB-307----HB47
CO-F15
SH14-89---

W. Thomas
R. York
J. Johansen
S. Rushforth
S. Rushforth
P. Roessler
W. Barclay
R. Lewin
M. Sommerfeld
R. Carlson
R. Lewin
P. Roessler
M. Tadros
M. Tadros
M. Tadros
J. Johansen
M. Tadros
J. Johansen
W. Barclay
R. Carlson
W. Barclay
R. York
J. Johansen

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National Renewable Energy Laboratory

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
StrainACHNA1
ACHNA2

Species
Achnanthes orientalis
Achnanthes orientalis

Class
Bacillariophyceae
Bacillariophyceae

AMPHO1AMPHO2AMPHO3
AMPHO5AMPHO6AMPHO7AMPHO8AMPHO10AMPHO11AMPHO12AMPHO13AMPHO14AMPHO18
AMPHO21
AMPHO22AMPHO23AMPHO24
AMPHO25
AMPHO26
AMPHO28
AMPHO29AMPHO30

Amphora coffeiformis
Amphora coffeiformis
Amphora coffeiformis
Amphora coffeiformis
Amphora coffeiformis
Amphora delicatissima capitata
Amphora coffeiformis punctata
Amphora delicatissima
Amphora coffeiformis
Amphora coffeiformis punctata
Amphora coffeiformis punctata
Amphora coffeiformis
Amphora coffeiformis
Amphora coffeiformis linea
Amphora coffeiformis
Amphora delicatissima
Amphora coffeiformis
Amphora sp.
Amphora coffeiformis tenuis
Amphora coffeiformis
Amphora coffeiformis
Amphora coffeiformis

Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

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55

National Renewable Energy Laboratory

StrainAMPHO31AMPHO32AMPHO33AMPHO34AMPHO35AMPHO36AMPHO37AMPHO38AMPHO40AMPHO46
ANKIS1
BOROD2
BOTRY1CARB
CHAET1CHAET3CHAET5CHAET6CHAET7CHAET9CHAET10CHAET11CHAET14CHAET15CHAET17CHAET18
CHAET19CHAET20CHAET21CHAET22CHAET23CHAET24
CHAET30CHAET39

56

Species
Amphora coffeiformis
Amphora coffeiformis
Amphora delicatissima
Amphora coffeiformis
Amphora coffeiformis taylori
Amphora coffeiformis taylori
Amphora coffeiformis linea
Amphora delicatissima
Amphora delicatissima
Amphora sp.
Ankistrodesmus falcatus
Borodinella sp.
Botryococcus braunii--Chaetoceros muelleri
Chaetoceros gracilis
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros sp.
Chaetoceros muelleri

Class
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae--Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

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StrainCHAET40CHAET41
CHAET43CHAET44CHAET45CHAET46
CHAET47
CHAET48
CHAET50
CHAET51
CHAET54
CHAET55
CHAET57
CHAET58
CHAET59
CHAET60
CHAET62
CHAET64
CHAET66
CHAET67
CHAET68
CHAET69
CHAET73
CHAET75
CHAET76
CHAET78
CHLOC1
CHLOC2CHLOC3
CHLOC6
CHLOC7
CHLOC8CHLOC10
CHLOC11Species
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri
Chaetoceros muelleri subsalsum
Chaetoceros muelleri subsalsum
Chaetoceros muelleri-trans
Chaetoceros muelleri muelleri
Chaetoceros muelleri subsalsum
Chaetoceros muelleri subsalsum
Chaetoceros muelleri subsalsum
Chaetoceros muelleri subsalsum
Chaetoceros muelleri
Chaetoceros sp.
Chaetoceros sp.
Chaetoceros sp.
Chaetoceros sp.
Chaetoceros sp.
Chaetoceros sp.
Chaetoceros sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.
Chlorococcum sp.

Class
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae

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57National Renewable Energy Laboratory

StrainCHLOR1CHLOR2CHLOR3
CHLOR5CHLOR6CHLOR8CHLOR9CHLOR10CHLOR11CHLOR12CHLOR13CHLOR14CHLOR15
CHLOR16
CHLOR17
CHLOR18
CHLOR20
CHLOR24
CHLOR27
CHOOC3
CHOOC4
CHOOC5
CHOOC7
CHOOC8CHOOC10
CHOOC13
CHOOC14
CHOOC16
CHROO1CHRYS1CHRYS2CRICO1CRYPT1CYCLO1

58

Species
Chlorella sp.
Chlorella ellipsoidea
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella salina
Chlorella salina
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
Chlorella sp.
(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)(Chlorococcales)
Chroomonas sp.
Chrysosphaera sp.
Sarcinoid chrysophyte
Cricosphaera sp.
Cryptomonas sp.
Cyclotella cryptica

Class
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Chlamydophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

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National Renewable Energy Laboratory

StrainCYCLO6CYCLO8CYCLO9CYCLO10CYCLO11
DIATO1DUNAL1DUNAL2ELLIP1
ENTOM1EUSTI1EUSTI2EUSTI3EUSTI5EUSTI6EUSTI7EUSTI8EUSTI9EUSTI10EUSTI11EUSTI12EUSTI13EUSTI14EUSTI15EUSTI16EUSTI17EUSTI18EUSTI19EUSTI20EUSTI21EUSTI22EUSTI23
FLAGE1
FLAGE2

Species
Cyclotella cryptica
Cyclotella meneghiniana
Cyclotella meneghiniana
Cyclotella sp.
Cyclotella cryptica
Navicula sp. nov
Dunaliella sp.
Dunaliella sp.
Ellipsoidon sp.
Amphiprora hyalina(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)(Eustigmatophyte)
Flagellate
Unknown flagellate

Class
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Eustigmatophyceae
Bacillariophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae
Eustigmatophyceae-----

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59National Renewable Energy Laboratory

StrainFLAGE3
FLAGE6FRANC1GLECA2
GLOEO1
GREEN1
GREEN2
GREEN4
GREEN6
GREEN7
GREEN8GREEN9GREEN10
GREEN11
HYMEN2
ISOCH1MONOR1MONOR2MONOR3
MONOR4NANNO2NANNO3NANNO5NANNO7NANNO8NANNO9NANNO10NANNO12NANNO13NANNP1
NANNP2NAVIC1NAVIC2NAVIC3

60

Species
Pleurochrysis sp.
Green Flagellate
Franceia sp.
Gleocapsa sp.
Gloeothamnion sp.
Green unicell
Green unicell
Green coccoid
Green coccoid
Green coccoid
Unknown green coccoid
Unknown green coccoid
Unknown green coccoid
Unknown green coccoid
Hymenomonas sp.
Isochrysis aff. galbana
Monoraphidium sp.
Monoraphidium sp.
Monoraphidium sp.
Monoraphidium minutum
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloris sp.
Nannochloropsis salina
Nannochloropsis sp.
Navicula saprophila
Navicula saprophila
Navicula saprophila

Class----Chlorophyceae
Cyanophyceae--------------------Prymnesiophyceae
Prymnesiophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Eustigmatophyceae
Eustigmatophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

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StrainNAVIC5NAVIC7
NAVIC9NAVIC10NAVIC12NAVIC13NAVIC14NAVIC15NAVIC16
NAVIC17NAVIC20NAVIC21
NAVIC22NAVIC23NAVIC24
NAVIC26
NAVIC28
NAVIC31
NAVIC32
NAVIC33
NAVIC35NEPHC1NEPHR1NITZS1NITZS3NITZS4NITZS5NITZS6NITZS7NITZS9NITZS10NITZS12NITZS13NITZS14

Species
Navicula saprophila
Navicula saprophila
Navicula pseudotenelloides
Navicula biskanterae
Navicula acceptata
Navicula saprophila
Navicula pseudotenelloides
Navicula pseudotenelloides
Navicula pseudotenelloides
Navicula pseudotenelloides
Navicula pseudotenelloides
Navicula pseudotenelloides
Navicula saprophila
Navicula saprophila
Navicula saprophila
Navicula saprophila
Navicula saprophila
Navicula acceptata
Navicula acceptata
Navicula pseudotenelloides
Navicula acceptata
Nephrochloris sp.
Nephroselmis sp.
Nitzschia pusilla monoensis
Nitzschia pusilla elliptica
Nitzschia alexandrina
Nitzschia quadrangula
Nitzschia pusilla monoensis
Nitzschia quadrangula
Nitzschia inconspicua
Nitzschia microcephala
Nitzschia pusilla
Nitzschia dissipata
Nitzschia communis

Class
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae----Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae

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61National Renewable Energy Laboratory

StrainNITZS17NITZS19NITZS20NITZS23NITZS24NITZS30NITZS35NITZS37NITZS38NITZS39NITZS40NITZS43NITZS44NITZS45NITZS49
OCHRO2
OOCYS1OOCYS3
OOCYS5
OOCYS9
OOCYS11
OOCYS14OSCIL1
OSCIL3
OSCIL8OSCIL9OSCIL10OSCIL11OSCIL12OSCIL13OSCIL14OSCIL15PAVLO2PHAEO1

62

Species
Nitzschia hantzschiana
Nitzschia inconspicua
Nitzschia inconspicua
Nitzschia intermedia
Nitzschia hantzschiana
Nitzschia hantzschiana
Nitzschia inconspicua
Nitzschia pusilla
Nitzschia pusilla
Nitzschia pusilla
Nitzschia pusilla
Nitzschia pusilla
Nitzschia frustulum
Nitzschia inconspicua
Nitzschia sp.
Ochromonas sp.
Oocystis pusilla
Oocystis parva
Oocystis sp.
Oocystis sp.
Oocystis sp.
Oocystis sp.
Oscillatoria limnetica
Oscillatoria subbrevis
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Oscillatoria sp.
Pavlova sp.
Phaeodactylum tricornutum

Class
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Chrysophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Chlorophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Chrysophyceae
Bacillariophyceae

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StrainPHAEO2PLATY1
PLEUR1
PLEUR4
PLEUR5
PLEUR6PRYMN2PSEUD1PSEUD4PYRAM2
STICH1
STICH2SYNEC1SYNEC3SYNEC5TETRA1TETRA2TETRA3TETRA4TETRA5TETRA6TETRA7TETRA8TETRA9TETRA11
THALA6
THALA7THALA14
THALA15
THALA16
UNKNO1UNKNO5UNKNO6UNKNO8Species
Phaeodactylum tricornutum
Platymonas sp.
Pleurochrysis dentata
Pleurochrysis dentata
Pleurochrysis sp.
Pleurochrysis sp.
(Prymnesiophyte)
Pseudoanabaena sp.
--Pyramimonas sp.
Stichococcus sp.
Stichococcus sp.
Synechococcus sp.
Synechococcus sp.
Synechococcus sp.
Tetraselmis suecica
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Tetraselmis sp.
Thalassiosira weissflogii
Thalassiosira weissflogii
Thalassiosira weissflogii
Thalassiosira weissflogii
Thalassiosira weissflogii--Unknown olive-green unicell
Unknown coccolithophorid
Unknown coccolithophorid

Class
Bacillariophyceae
Chlorophyceae
Prymnesiophyceae
Prymnesiophyceae
Prymnesiophyceae
Prymnesiophyceae
Prymnesiophyceae
Cyanophyceae--Prasinophyceae
Chlorophyceae
Chlorophyceae
Cyanophyceae
Cyanophyceae
Cyanophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Prasinophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae
Bacillariophyceae--Prymnesiophyceae
Prymnesiophyceae

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63National Renewable Energy Laboratory

StrainUNKNO10UNKNO24
UNKNO36
UNKNO52
UNKNO58
VW291

Species
Nitzschia sp.
-----------

Class
Bacillariophyceae----Cyanophyceae-----

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 andRecommendations

The collection, screening, and characterization of microalgal strains represent a major endeavorof 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 culturecollections. This was the first major collection of microalgae that emphasized strains suitable forcultivation 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 tofunding cutbacks. However, the approximately 300 strains remaining in the collection will betransferred to the University of Hawaii, and should be available to interested researchers.

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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 thesetypes 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 basedon 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 informationis critical, and has a profound impact on the type of screening that should be conducted.

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65National Renewable Energy Laboratory

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 ofisolates, 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 takeup 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, thistechnique seems readily adaptable for use as a powerful screening tool.
For future screening endeavors, we recommend that an effort be made to naturally select strainsat the locations that would likely be commercial microalgal production sites. In this manner, thealgae 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 ofindigenous 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 thismethod is that the dominant strains may not be good lipid producers. For this reason, geneticallymanipulating the dominant strains by classical or recombinant means may be necessary, such thatthey 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 thepreceding 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 thatpowerful methods for generating genetic sequence information are becoming routine.

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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 toincrease the yield of oil from microalgae for cost-effective liquid fuel production. Initially, alarge 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 theUnited States (a region targeted as a feasible location for large-scale microalgal culture), marine
environments, and established culture collections. These organisms were then screened andnumerous 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 deprivationor other conditions that inhibited cell division. Unfortunately, these conditions are the opposite ofthose 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 andphysiology of lipid accumulation, with emphasis on understanding the “lipid trigger”, amechanism 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, thedevelopment of methods for screening for high lipid strains, and attempts to understand thebiochemical lipid trigger. The research performed by SERI/NREL subcontractors on thephysiology, biochemistry, and molecular biology of lipid production in oleaginous microalgae

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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 andTypes of Lipids

Subcontractor:
Principal Investigator:

Selma University
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 ofnutrient 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 effectof 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 theoptical 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 apercentage 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 theauthors 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, andHantzchia 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 tonutrient-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-deficientconditions 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 toincreases in both the neutral lipid and polar lipid fractions. In several cases, the ratio of neutral

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lipids to polar lipids increased significantly in the nutrient-stressed cells (i.e., in Hantzschia andC. 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 undernutrient-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 anincrease 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 thevalidity 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-sufficientand 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 todetermine 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 forneutral lipid production. Many species produce constitutively high levels of lipid, and the levelof 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.

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Publications:
Sriharan, S.; Bagga, D. (1987a) “Effects of induction strategies on Chaetoceros (SS-14), growthwith 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 thequantity 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, andGuillard.” Appl. Biochem. Biotech. 28/29:317-326.
Sriharan, S.; Bagga, D.; Sriharan, T.P. (1989) “Environmental control of lipids and fatty acidproduction 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 andfatty 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 YorkPrincipal 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

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has implications for algal collection strategies, for strain selection for high lipid producers, andfor 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 physiologicalvariability between isolates of a single species. In these studies, various isolates of a speciesgrown 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 comparedelectrophoretic banding patterns of specific proteins to obtain quantitative estimates of thegenetic 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 byenvironmentally induced changes in gene expression. Each strain was examined using lightmicroscopy (LM) and scanning electron microscopy (SEM) to look for morphologicaldifferences and to confirm species identity. Crude protein extracts from each strain wereseparated 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, andtetrazolium 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 oneach 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 tothe migration of the known enzyme from Skeletonema. An example of this type of experiment isshown 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 thatallele.
For the studies of Amphora, Dr. Gallagher obtained 47 isolates, 32 of which were isolated from asalt 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 inculture, 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

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variation was observed in the morphology of the frustule between strains, for example, in thepresence 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 unialgalclones 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 siteon the same day. These differences were not correlated with the extent of morphologicalvariation, 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 waslimited due to the high percentage of “null” alleles (no staining of some enzymes) in someisolates. It was unclear whether this was caused by undetermined genetic differences between theisolates (and between Amphora and Nannochloropsis), or due to difficulties in extraction of theproteins 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 suggestthat 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 datasuggested significantly less genetic variation between isolates of Skeletonema, even between
strains isolated from different locations, than was seen between Amphora strains isolated fromthe same environment. This difference was attributed to the fact that Amphora is an attached,benthic organism that produces amoeboid gametes, whereas Skeletonema is planktonic, andproduces swimming sperm. These “lifestyle” differences would result is lower potential for geneflow between Amphora populations, although the presence of heterozygotes indicatesinterbreeding 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 canoften express genes from very different species, so these differences between strains probably
will not affect the expression of genes transferred between these similar organisms.

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While working under the SERI subcontract, Dr. Gallagher also participated in a study thatprovided 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, fluorescencequenching, 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 thefirst 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 thatdrive 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 thanchlorophyll, 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 ofviolaxanthin in Nannochloropsis spp. (Eustigmatophyceae).” J. Phycol. 23:79-85.
Additional References:Nei, M. (1972) Amer. Natur. 106:283.

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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).

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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 accumulationin 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; and2. 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 thistechnique, 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 bestudied. This is often problematic for microalgae, presumably due to the chemical and physicalproperties 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 ofstandard methods of fixation using glutaraldehyde and osmium tetroxide, dehydration with anorganic solvent, embedding of the cells in an acrylic resin, and poststaining of the sections withuranyl acetate and lead citrate. Initially, Dr. Solomon reported that the best fixation ofAnkistrodesmis and Isochrysis was achieved by exposing the cells briefly to glutaraldehyde andosmium 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 andresins.
Nitrogen deprivation was used to trigger the production of lipids in the cells. The cells weregrown 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.

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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 thechloroplast 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 structuresconsisting 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 ofthe fixation process, was unclear.
It is difficult to conclude much more about ultrastructural changes that might have been inducedin these cells following N deprivation. The sample size was very small. One hundred-nm thicksections 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 highlikelihood that the chemical fixation methods used in the study can create artifacts that are notrelated 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 thepresence of a discrete lipid trigger mechanism within each cell, as lipid did not appear toaccumulate 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 Analysesof Lipid Accumulation in Microalgae: A Subcontract Report.” Solar Energy Research Institute,Golden, Colorado, SERI/STR-231-3089.

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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. Scalebars = 0.5 µm. The numbers in the lower left corner of each figure are from the original
publication (Solomon et al.1986b).

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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 amethod 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 ininternal 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 awavelength 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 thismethod 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 foundto be appropriate for flow cytometric analysis. The cells of this strain are small and spherical, theoptimal 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 fluorescencealone. 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 wasachieved 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 fromundissolved 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

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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 theresults to account for differences in cell size and age and allow detection of individual cells withunusually 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 thedaughter 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 interestingobservation 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 theentire 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 tolipid 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 ofexperiments 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 orfrom 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 forhigh-lipid cells using flow cytometry was related to the cell cycle. The exponential culturescontained 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 sizeand 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 (NileRed fluorescence only) or using the ratio of chlorophyll to Nile Red fluorescence to control forcell 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

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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 eachsorting 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 (orbetween) 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 achievedby using stationary or stressed cells as the parent population. In addition, cells should be selectedbased 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 notproduced 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, thatthe stressed cells from very old cultures had all entered a similar metabolic state so that size andlipid 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 oflipid 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 Analysesof 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.

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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 (describedabove 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 inbatch cultures of Chlorella over time. As the N became depleted, lipids accumulated in thecultures, 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 enterstationary phase and cell division ceases; the timing of this event would be different forindividual 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 accumulationin 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 levelof 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, thecultures generally showed a significant increase in lipid levels that was accompanied by adecrease 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'slaboratory 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 otherphotosynthetic systems studied, the data indicated that cells synthesize triglycerides in the lightand utilize these lipids as energy stores in the dark and during cell division. If division were

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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 lightmicroscopy 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 proportionof the cells was in the form of autosporangial complexes, i.e., their nucleii had divided, but theautospores 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 theautospore 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 affectlipid 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 affectthe biochemistry of lipid biosynthesis. Analysis of the lipid classes present in the cells at the endof the 10-day growth period showed accumulation of triglycerides in cells at high pH, with adecrease in glycolipids and polar lipids. The nonpolar storage lipids predominantly contain 16and 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 ahigher proportion of polyunsaturated fatty acids. However, analysis of the fatty acid compositionof the storage lipids showed that at higher pH, more of these precursor lipids were seen in thepolar lipids and glycolipids. This suggests a switch in the lipid synthesis patterns that results inless 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, andprovided 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 oflipid 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 thephotosynthetic 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 andcaused 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

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National Renewable Energy Laboratorythat are coupled to cell growth and division. It will also be important to consider not only factorsthat 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 cyclein 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 foranalysis 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 thebiochemistry 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. Theseexperiments were directed at understanding how these processes are affected by N limitation in
the algal cells.

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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 culturescontaining 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 asteady 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, thelipid content of the culture per mL (or percentage of the AFDW composed of lipid) did notchange. 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 cangrow heterotrophically using ethanol as the sole carbon source. The growth of cells in thepresence 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 andcarbohydrate 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 acarbon 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 of84

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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 ofcarbohydrate 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 ofthe 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 areapparently 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 monitorchanges in the levels of chloroplast and mitochondrial proteins under N-deficient conditions(Coleman et al., 1988a). Under photosynthetic growth conditions (high light), exposure of thecells to N-deficient conditions resulted in a decrease in the levels of 37 proteins identified ascomponents 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 unstablechloroplast. 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 theactivation of the oxygen-sensitive pyruvate dehydrogenase in the mitochondria. This led toincreased levels of acetyl CoA in the mitochondria, which activates the mitochondrial fatty acidsynthesis 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 thelipid biosynthetic pathway suggested that lipid synthesis could be increased by increasing thelevels 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 thebiochemical factors that limit production of the lipid precursors could lead to biochemical or

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85National Renewable Energy Laboratory

genetic engineering strategies to increase the activity of these enzymes that could produce anorganism 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 thelate 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 theimportanance of understanding the biochemical mechanisms by which algae accumulate lipids.
For Euglena and the Nannochloropsis strains described here, N deprivation does not seem tofunction 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, withno net accumulation in the culture. This result confirms the conclusions of Cooksey andcoworkers. 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 thisarticle was also submitted as a SERI Report, 47pp.)
Coleman, L.W.; Rosen, B.H.; Schwartzbach, S.D. (1988b) “Environmental control ofcarbohydrate 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.

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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 andcharacterizing 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 incollaboration with Dr. James Van Etten, in the Department of Plant Pathology at the Universityof 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 thatlive in a symbiotic relationship within cells of the protozoan Hydra viridis. They found that thealgal 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 andreleased 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

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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 anddevelopment. 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 viralprogeny. 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 (seebelow). Several of the most interesting and possibly relevant findings are summarized here.

88•

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 wallat this site, and the viral DNA is released into the cell. Living algal cells are notrequired for virus attachment and wall degradation (viruses can attach to anddegrade isolated wall fragments), but living cells are necessary for release of theviral 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. Viralgene expression entails transcription of early and late genes, and may include

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National Renewable Energy Laboratory

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 andcytosine residues. The data suggested that the virus produces a uniquerestriction enzyme that is specific for non-methylated sequences for degradationof 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 fromdegradation 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 geneticallymanipulating 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 theseprocedures, 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 wereidentified. 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 inthat 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 avirus 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 werediscontinued.
In a separate, but related, series of experiments, Dr. Meints received 250 water samples collectedby Dr. Ralph Lewin of the Scripps Institute in La Jolla, California. These were also screened forthe 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 tohave potential for biodiesel production. These included Chlorella 501 and A. falcatus (A record

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89National Renewable Energy Laboratoryof 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 thealgal 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 usedin studies of somatic cell fusion (genetic improvement by fusion of two individuals with usefultraits 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 andvirus. 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 throughdiscrete 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 theviral-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 oflysin to produce algal protoplasts, but the article was never published in a technical journal.

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Characterization of the chloroplast and mitochondrial genomes of microalgal strains.
The goal of this research was to characterize the organellar genomes of Chlorella and othermicroalgae. As organellar DNA is thought to be highly conserved evolutionarily, the idea was touse 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 thesubcontract.
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 withheterologous chloroplast DNA markers. The chloroplast genome of Chlorella N1a was found tobe 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 withchloroplast 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 areflanked 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 thatchloroplast 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 themitochondrial genome isolated was first presumed to be a plasmid. Unlike some species inwhich 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,A Look Back at the Aquatic Species Program—Technical Review

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heterologous probes were used to identify the mitochondrial DNA and to localize specificmitochondrial genes on the restriction map. The gene organization in the Chlorella N1amitochondrial 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 andgreen 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 taskother related research efforts in his laboratory toward the development of a genetictransformation 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 sequencesthat 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 theviral DNA was prepared in a lambda vector, which allowed for the sequencing of the viralgenome 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 thealgal 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/algalinteractions, the results obtained were not directly applicable to the development of atransformation 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).

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Publications:
Anberg, S.M.; Meints, R.H. (1991) “Nucleotide sequence of the genes for ribulose-1,5bisphosphate 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 invitro 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 sizeclasses.” 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 zincfinger motif.” Virology 219:307-.
Lee, A. M.; Ivey, R.G.; Meints, R.H. (1995) “Characterization of a repetitive DNA element in abrown 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 algawith 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.

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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 167:143149.
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 geneencoding 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-likealga 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 aculturable 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.

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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-likealgae.” 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 theexsymbiotic Chlorella-like green alga N1a.” Aquatic Species Program Annual Report,
September 1989, Solar Energy Research Institute, Golden, Colorado, SERI/SP-231-3579, pp. 91118.
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 andphotosynthetic 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

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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 boththe 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 fromreduced 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 Lienand 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 (seeSection 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 thecultures as long as N was present. When N was depleted, cell division stopped, althoughbiomass accumulation continued for several days. The major portion of the new biomass wascomposed of lipids and storage oils. N depletion resulted in a rapid decrease in the level ofchlorophyll 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 inthe treated cells (discussed in more detail below).

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II.B.2.c.

Studies on Photosynthetic Efficiency in Oleaginous Algae

SERI researchers Lien and Roessler (1986) tried a somewhat different approach to understandthe processes affecting lipid accumulation (Lien and Roessler 1986). A recently publishedtechnical evaluation (Hill et al. 1984) identified two major requirements for economic feasibilityof biodiesel production:
1. Photosynthetic efficiency (which can simply be thought of as the percentage ofincident 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 ofnitrogen 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, andyield 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 amaximum 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 andthe 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 theearly stages after the N was depleted, the cultures showed a decrease in energy efficiency withrespect 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 therelationship 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 theperiod 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

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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 underintermittent light conditions is similar to that attained under constant light of the same averageintensity. 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 underflashing 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 lightdecreased. Although these data were preliminary, they supported the proposal thatphotosynthetic 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-houseresearch 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 ismajor 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 anaccumulation of lipid within the cells. However, Si (unlike N) is not a component of othercellular macromolecules (enzymes, membranes) or cell structures such as the photosynthetic

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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 thebiochemistry 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 thecells were still growing exponentially. Cell growth, chlorophyll a content, AFDW, lipid, andphotosynthetic 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 littlechange 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 percentageof 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 ofneutral lipids increased from 17%-20% to 57% in Si-deprived cultures.

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Based on these studies, C. cryptica was identified as the best candidate for further studies on thebiochemistry 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 majorcarbohydrate 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 labeledwith 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 thenonlipid 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 isapparently 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 4hours 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; and14:0, 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 predominantfatty 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

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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 usuallymuch slower, as the cells could utilize internal N stores). This result again emphasizes the needto 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.

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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 effectsof 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 thechloroplasts 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 andfree 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 malonylCoA. Malonyl-CoA is a substrate for fatty acid synthase and this reaction is considered to be thefirst committed step in fatty acid synthesis.
These pathways had not previously been well-characterized in diatoms. To better understand thelipid 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, followedby the transfer of the carboxyl group from biotin to acetyl-CoA. In bacteria, the enzyme iscomposed of four non-identical subunits. However, in eukaryotes, biotin binding, biotin
carboxylation, and carboxyl-transfer all occur on a single large multifunctional protein; thefunctional ACCase occurs as a multimer of this polypeptide. ACCase had previously beenshown to play a key regulatory role in the rates of fatty acid synthesis in both animal and plantsystems. 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 themicroalgal enzyme with those isolated from plants, animals, yeast, and bacteria.
The enzyme was purified from C. cryptica by a combination of (NH4)2SO4 precipitation, gelfiltration 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

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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 aslightly alkaline pH for optimum activity (pH 8.2), although the enzyme was most stable whenstored 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 werealso had little or no effect on C. cryptica ACCase. Thus, the ACCase from this diatom wasfound 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).

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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 isno 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).

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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 thatshowed 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 ACCasegene 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 sabbaticalfrom 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 thiscollaboration 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); thepeptides generated were separated by SDS-PAGE, purified, and several of these peptides wereanalyzed 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 toscreen 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 fromthe 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 themolecular 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 resultin 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 theprotein) 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. Thissuggests that the central region of the protein probably functions primarily as a linker or spacerthat 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

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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 beeninterest from at least one major plant biotechnology company in using this gene to manipulateoils 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, inwhich 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. ACCasegene 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 thelevel 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 anincrease 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 wasa major premise of the genetic engineering experiments discussed in Section II.B.3.

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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 arepresent in all three sequences and that exhibit statistically significant homology (p<10-6) areindicated by thickened areas.
(Source: Roessler and Ohlrogge 1993).

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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 fundedby 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 understandthe biochemistry of lipid synthesis in the eustigmatophyte Nannochloropsis sp., particularly withrespect 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 thequantity and quality of lipids in algae; so there are likely to be significant differences in thebiochemical pathways. In addition, like many algae, Nannochloropsis contains a high proportionof 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 andpharmaceutical applications for humans. Understanding the details of the biochemistry of lipid
accumulation in microalgae could help researchers develop strategies for genetic manipulation oflipid synthesis pathways to affect not only the quantity but also the quality (chain length, degreeof 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 adifferent distribution of labeled carbon in the lipids and fatty acids. The work demonstrated theprobable existence of two pools of malonyl-CoA used as substrates for fatty acid synthesis, andresulted 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 thisresearch did not come from DOE. In addition, it would require a lengthy discussion of the detailsof 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 tolimit the flow of newly assimilated carbon into other cellular pathways. Many diatoms, including

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C. cryptica, can produce a significant amount of a storage carbohydrate called chrysolaminarin, aβ-(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 diphosphatesugars; for example, starch is formed in plants from ADPglucose, and UDPglucose is used toform 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 extractsof C. cryptica, and identified significant amounts of UGPase activity. The enzyme activity wascharacterized to optimize in vitro assay conditions. The enzyme was activated in the presence ofMn2+ and Mg2+ but was not affected by 3-phosphoglycerate or inorganic phosphate; thesechemicals 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 specificactivity 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 todecreased 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 genetictransformation 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.

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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 thepathways 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 genefrom 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 andsequenced; the derived amino acid sequence showed 37% identity with the corresponding
sequence from potato UGPase, confirming that a C. cryptica UGPase gene fragment had beencloned. 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 toknown 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 molecularweight 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 thepresence of both enzyme activities in C. cryptica on a single multifunctional protein. A patentsubmitted 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.

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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 methylationin 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 offuels 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 andMolecular 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.

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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 thediatom Cyclotella cryptica.” Aquatic Species Program Annual Report, Solar Energy ResearchInstitute, (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 thediatom Cyclotella cryptica.” Plant. Physiol. 92:73-78.
Roessler, P.G.; Bleibaum, J.L.; Thompson, G.A.; Ohlrogge, J.B. (1994) “Characteristics of thegene 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. 268:1925419259.
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, theNetherlands, pp. 105-107.

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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 nitrogenstarved 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 torapid 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 toenvironmental fluxes, and high production of TAG storage lipids. Although numerous promisingorganisms 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 adecrease in cell division and total productivity.
During the late 1980s and 1990s, the direction of the laboratory research efforts at NREL shiftedto 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 withconstitutively 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

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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 highlipid algae by genetically manipulating selected oleaginous strains. Microalgae generally
reproduce asexually by simple fission. Many strains can also produce sexually, but theconditions 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 (PaulRoessler and Eric Jarvis).
II.B.3.b.

Mutagenesis and Selection

Work by SERI/NREL subcontractors in the early 1980s supported the idea that there issignificant 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 ofnatural genetic variants with desired traits, such as high lipid levels or increased tolerance to highsalinity 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 somepotential 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 subpopulationsof 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 withheritable traits, often the result of a mutation within a single gene. As a prelude to the initiationof 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 toform colonies on agar plates, sensitivity to herbicides and other growth inhibitors, and thesensitivity 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 threeclasses: the chlorophyceae (M. minutum MONOR1 and MONOR2), the eustigmatophyceae(Nannochloropsis (NANNP1 and NANNP2), and the bacilliarophyceae (C. cryptica T13L, C.

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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 alarge 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 growheterotrophically would be important for the isolation of photosynthetic mutants.
Predictably, antibiotics that inhibit bacterial cell wall synthesis such as ampicillin andcarbenicillin 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 andstreptomycin. 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 toother 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 tocompounds 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 ofGalloway 1990). Many of the growth inhibitors used in this study affect specific proteins in thetarget organism, and many of these proteins have been well characterized in a number ofsystems. 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 fortransformation 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 withfluorodeoxyuridine. 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 themutagen, 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 withtraits thought to be due to recessive nuclear gene mutations (i.e., glyphosate resistance orphotosynthesis mutants). On the other hand, in Navicula, the only diatom in which mutants weregenerated, the types of mutations produced were indicative of dominant mutations, i.e.,

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atrazine/diuron resistance (resulting from a chloroplast gene mutation) or resistance totunicamycin, an inhibitor of n-glycosylation. These results indicate that Monoraphidium andNannochloropsis are probably haploid; the diatoms are diploid. Design of strategies forgeneration 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 haploidstrains, 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 algalmutants 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 thegrowth conditions for the target strains. The conditions to be used for selection (inhibitor
specificities and concentrations) should be determined for each strain. However, the generationof 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 mutantsin 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 eachsample 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 lookfor changes in a quantitative trait. This result suggests that, with the right method to screen formutants 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 becomeroutine 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

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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 thehighest 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 withthe 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 tofunction 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 membraneusing 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 theunderstanding of the processes involved in DNA uptake and expression and to increase theprobability 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 algaltransformation,•

the development of methods to introduce DNA into algal cells through the cellwall, 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 thetransformation system to manipulate lipid content in the algae by overexpressing or
downregulating key genes. In addition, the transformation system was used to introduce areporter gene under the control of various regulatory sequences, to better understand theregulation of gene expression in microalgae.

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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 thefirst 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 avery 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 ofphenol: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 andThompson 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 ofeach nucleoside (often represented as %GC) is variable between species. Restriction enzymes
cut DNA at specific nucleotide sequences, generally recognizing 4-6 bp motifs. Therefore, thefrequency of cutting by a particular enzyme will be affected by the total nucleotide compositionof 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 witha 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 5hydroxymethyldeoxycytidine (hm5dC) and 5-hydroxymethyldeoxyuridine (hm5dU), although thebiological significance is unclear. Another common modification is the presence of methylated
nucleosides, in particular 5-methyldeoxycytidine (m5dC) and 6-methyldeoxyadenosine (m6dA).
The degree of methylation has been associated with levels of gene expression. In addition, somemicroorganisms use DNA methylation as a defense mechanism, in that methylated DNA
sequences are often not recognized by endonucleases from invading pathogens. Although thepresence 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.

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DNA was isolated from microalgae strains, including 10 species from 5 classes. The nucleosidecomposition 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 thediatoms showed a GC content typical for most eukaryotes (42%-48% GC), the GC content of thegreen algae (excepting Stichococcus) was significantly higher. In particular, Monoraphidium
DNA contains 71% GC. The table also shows the presence of m5dC in the algal DNA. All
species tested contained some level of this modified base, although once again Monoraphidium
stands out with approximately 11% m5dC. The only other unusual feature was the presence of
12% hm5dU 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 theseorganisms. 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 ofhomologous selectable markers (discussed in more detail later). Also, GC content should beconsidered when designing synthetic DNA probes based on protein sequences, i.e., for isolationof 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)
M5dC

5GC

Chlamydomonas reinhardtii

0.16

61.6

Chlorella ellipsoidea

1.48

51.6

Monoraphidium minutum

11.2

70.9

Cyclotella cryptica

1.95

43.2

Navicula saprophila

0.20

46.2

Nitzschia pusilla

0.78

45.4

Algal species
Chlorophyceae

Bacillariophyceae

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Phaeodactylum tricornutum

0.14

48.0

0.30

44.8

3.32

57.5

1.54

43.7

Charophyceae
Stichococcus sp.
Prasinophyceae
Tetraselmis suecica
Dinophyceae
Crypthecodinium cohnii

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 beremoved 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 ofprotoplast 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 theseprocesses, 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.

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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 wasmonitored 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 theluciferase 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 inprotoplasts treated with the luciferase plasmid, but not in protoplasts that had not been exposed toplasmid 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 theluciferase 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, theintroduction 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 achievetransformation in another green alga (Chlamydomonas). The dogma in the field was thatheterologous 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

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project manager at the time, was that this was “an endless pit of fruitless endeavor”, and thedecision 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 isolationof very rare individual transformants within a population of microalgal cells. This will be
discussed in the following section.

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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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Development of Homologous Selectable Markers for Monoraphidium and Cyclotella:
Transient expression assays can be useful for the rapid assessment of DNA uptake andexpression 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 toknow 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 fromdegradation 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 toenvironmental 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 literaturesuggested that this unusual GC content would inhibit the expression of foreign genes, such asbacterial antibiotic resistance genes that had been used successfully as transformation markers inplant 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. Althoughsome 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 atransformation 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, from1989 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 undermodified growth conditions. This will allow for positive selection of transformed cells. Then thecorresponding wild-type gene is isolated and inserted into a plasmid vector. The wild-type geneis 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 ofa 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 goodpotential for use as selectable markers should not only code for a protein essential for growth124

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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) andseveral 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 ofnitrogen 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 willtake 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 isadded 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 atleast 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 alambda phage vector. Although the library appeared to be representative of the algal genome inthat it contained approximately 300,000 separate clones of about 20,000 bp each, repeatedscreening 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, andhad 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 synthesisof pyrimidines. Organisms with defects in the OPDase gene will only grow if pyrimidines suchas 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

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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 besusceptible 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 weremutagenized 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 andspectrophotometric 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 sequenceconservation between genes from different organisms. Dr. Jarvis made a number of attempts toisolate 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 PCRtechnique 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 aselectable marker (discussed in the following section); however, there was still interest in
producing a selectable marker system that would work for high lipid (although geneticallyrecalcitrant) 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 aselectable marker. ALS is an enzyme involved in the synthesis of branched-chain amino acidsuch 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 anddemonstrated 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 someorganisms, in particular, those with unusual DNA compositions, and for haploid organisms forwhich 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

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solved if time had permitted the generation of a cDNA library, or a new genomic DNA libraryusing 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 throughthe 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 plasmidDNA, glass beads, and polyethylene glycol (Kindle 1990). This method was reported to work forwalled cells, but at a very low frequency. DNA could also be introduced into walled cells ofChlamydomonas 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 etal. 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 thewild-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 plasmidDNA, 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 NRgene) appeared in 1-2 weeks.
Attempts to transform walled cells of Chlamydomonas using SiC were made in parallel withglass bead-mediated transformation to compare the two procedures. The results of a typicalexperiment are shown in Figure II.B.7. The number of transformants obtained using SiC varied

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between experiments, but generally were in the range of 10-100 per 107 cells, comparable totransformation 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 generatetransformants 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 ofinterest 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, thesuccessful development of a transformation system for Cyclotella using biolistics (discussed
later) precluded further work on SiC-mediated algal transformation. A few attempts were madeto 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 ofthe 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 arevery small and may not be a good target for biolistics, but might be readily pierced by SiC fibers.

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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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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 thedevelopment of the transformation system were:•the cloning of the acetyl-CoA carboxylase gene from C. cryptica, and thus theavailability 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 particlegun) 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 todeliver foreign DNA into the cells through the algal cell wall, or inefficient expression of theforeign gene, is not clear.
As discussed in the previous section of this report, a significant amount of work went intodeveloping 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 genetictransformation in higher plants. These antibiotics function by binding to 30S ribosomes andinhibiting protein synthesis. Resistance to kanamycin or G418 can be induced in cells byexpressing 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 theosmoticum 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 aconfluent lawn of colonies under the same conditions.
Early attempts to use the nptII gene as a selectable marker used a plasmid construct that had beenused 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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pCaMVNeo contains the nptII gene driven by the cauliflower mosaic virus 35S ribosomal genepromoter (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 todrive expression of nptII in T13L. A plasmid (pACCNPT10) was constructed that contained achimeric 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, wasconstructed 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 thisprocedure, 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 toaccelerate the particles. Because of prohibitive costs and restrictive licensing agreements, ahomemade version of the particle gun was designed and built at NREL. No transformants weregenerated using this device, but as these experiments were performed before the acc1-nptII
chimeric plasmids where constructed, whether the device actually functioned as planned isunclear. Ultimately, a commerical microprojectile accelerator was purchased. This device was
optimized for very simple operation and used helium pressure to propel the DNA-coatedparticles. 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 thecenter 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 werethen propelled into the cells using the microprojectile accelerator. The exact parameters used aredescribed 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 washedfrom 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

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National Renewable Energy Laboratoryfrom the plates and tested for continued resistance to G418. The presence of the foreign gene wastested 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-specificantibody (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 independentsites, often in form of tandem repeats. The nptII DNA remained stably integrated into the hostgenome 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 majorachievement 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 weregenerated. 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 patentdescribing this technology was applied for and issued in August 1997. Diatoms represent a verylarge 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 ofthese organisms. The availability of a genetic transformation system for diatoms could have amajor 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 genetictransformation protocol to manipulate levels of storage lipids in C. cryptica.

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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 theacetyl-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).

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Figure II.B.9. Simplified schematic showing the protocol for transformation of diatoms by
microprojectile bombardment (“gene gun”).

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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) weredigested 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).

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Figure II.B.11. Western blot, showing the presence of the 30-kDa NPTII protein intransformed cells of C. cryptica T13L and N. saprophila.
In this example, C. cryptica and N. saprophila were transformed with pACCNPT10 andpACCNPT5.1, 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).

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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 saturationand 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 cellsby 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 (seeSection 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 abacterial 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, theintrons 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 wasintroduced 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 theseshortened peptides; therefore, they were presumed to be the result of truncated transcription ortranslation. 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 madeto 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 ofincreasing 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 aplasmid containing a selectable marker gene such as nptII. Although the exact mechanism for

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this phenomenon is not clear, it is believed that during a given transformation procedure, aparticular subpopulation of the cells becomes “transformation competent”. These cells may thentake 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 indicatedthat 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, andthere 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 iscompensated 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. saprophilatransformants 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 notdetected in any of the N. saprophila strains tested. Whether this result was due to inefficienttranslation 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 characterizationof 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 tolipids.
Two techniques that are becoming widely used for gene inactivation are ribozymes and antisenseRNA. 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

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National Renewable Energy Laboratoryin 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 thatparticular protein in the cell. Ribozymes are also RNA molecules produced by synthetic genesthat can bind to, and cleave, very specific RNA sequences. Ribozymes can be designed todegrade 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 shownto 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 theacc1 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 toG418. 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 tocontinue 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 genetictransformation was not possible in this group of algae before NREL’s transformation system wasdeveloped. The availability of this transformation system now allows the study of the roles ofregulatory DNA sequences in gene expression. As a first step toward a better understanding ofgene transcription in diatoms, NREL researchers Paul Roessler and Steve Milstrey used thefirefly 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 otherorganisms. 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 ACCasepromoter and the UDP-glucose pyrophosphorylase/phosphoglucomutase promoters from C.
cryptica. Also tested were the simian virus 40 (SV40) promoter, which drives high levels ofgene 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 mannercontained the luc gene, as determined by PCR analysis. Based on past results, it is expected thatthe plasmid DNA was integrated into the genome of the cells. Luciferase activities in randomly

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chosen transformants (eight from each plasmid type) were determined by the use of aluminometer.
As expected, the promoter regions of both C. cryptica genes drove luciferase expression in thetransformed 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 wasmuch 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 ofgene transcription efficiency.
Previous results at NREL indicated that Si deficiency may affect the expression of the acc1 genein 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 thesame extent (approximately twofold) 6 hours after transfer into Si-free medium. This suggeststhat the Si-responsive elements are either within the shortest (396-bp) promoter region tested orin 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 siliconcarbide 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.

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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 thegreen alga Chlorella ellipsoidea.” Curr. Genet. 19:317-321.
Jarvis, E.E.; Dunahay, T.G.; Brown, L.M. (1992) “DNA nucleoside composition and methylationin 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. 268:1925419259.
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

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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 transformationof 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 andProducts 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 acommercial organism. Although much remains to be done, significant progress was made in theunderstanding 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 ishappening 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 allcell 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

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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 isthat, 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 fuelproduction 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 theratio 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 moreclear cut. An enzyme that appears to play a key regulatory role in the synthesis of lipids inplants, and likely in algae, ACCase, was purified from the diatom C. cryptica. The gene thatcodes 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 genetictransformation 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 andreproducible, and should work for a variety of diatom strains, as long as the cells can formcolonies 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 geneticallyengineered algal strains for commercial applications, the ability to transform these algae shouldhave positive ramifications for the algal biotechnology community.

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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.

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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 firstsuggested 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 asource of nutrients (Golueke et al. 1957; Golueke and Oswald 1959).
Oswald and Golueke (1960) presented a conceptual analysis of this process, in which large (40ha) 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 productionof 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 alaboratory study of microalgae fermentations to methane gas (Uziel et al.1975). Using both freshand dried biomass of six algal species, roughly 60% of algal biomass energy content converted tomethane 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 fuelproduction process. This project, started in 1976, was carried out at the Richmond Field Stationof 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 bioflocculationin 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 thisproject was the removal (harvesting) of the algal biomass from the effluents. Not only did thealgal 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-costand less energy-intensive microalgae harvesting technologies and wastewater treatment processes

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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 frommicroalgae 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 ofa 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 developmentof larger, stand-alone systems. Although this argument was controversial at the time, theUniveristy 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 ofbiophotolysis.” Enzyme and Microbial Technology 2:103-111.
Benemann, J.R.; Weissman, J.C.; Koopman, B.L.; Oswald, W.J. (1977) “Energy production withmicroalgae.” Nature 268:19-23.

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Golueke, C.G.; Oswald, W.J. (1959) “Biological Conversion of light energy to the chemicalenergy 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 forcontrol 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 forbiomass conversion would require a universally applicable harvesting technology, such as
centrifugation or chemical flocculation, to enable the recovery of any algal type. However, theseprocesses 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 grownthat would be easier and cheaper to harvest using microstrainers. Microstrainers, which arerotating screens (typically 25 to 50 µm openings) with a backwash, are already widely used forremoving filamentous algae, mainly filamentous cyanobacteria (blue-green algae) from potablewater 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), andunmixed. 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 ofshallow, mixed, raceway-type ponds, the “high rate pond” of Oswald (1963), for microalgae

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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 thisresearch.
A fundamental theory of species control was developed based on selective recycle of harvestablebiomass (Figure III.A.1.). The concept is that harvesting filamentous algae and recycling part ofthe biomass back to the ponds (similar to the process of activated sludge), would favor theslower-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 experimentallydemonstrated in the laboratory in competition experiments with mixed cultures of Chlorella (aunicellular green alga) and Spirulina (a filamentous blue-green alga). Without biomass recycle
Chlorella always out-competed Spirulina; with biomass recycle Spirulina could be made todominate 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, thefirst 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 enoughOscillatoria biomass was collected and returned to the Richmond Field Station to inoculate oneof 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 inoculationsof 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

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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 harvestablecultures 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 forconversion 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.

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Figure III.A.1. Selection process through biomass recycle.
A theoretical model of selective cell harvesting and recycling resulting in dominance of theslower-growing over the faster-growing species. (Source: Benemann et al. 1978.)150

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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 heightindicates filaments shorter than 150 µm; vertical stripes indicate filaments between 150 and 375µm; 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: Benemannet al. 1977).

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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, andthus 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 rectangularponds 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 ofsuspended 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 withmicrostrainers. 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 andclimatic 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 toinduce 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 tozooplankton 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 highalgal productivity and effective harvesting process.
After growing and harvesting an algal culture on sewage, enough nutrients remain to grow asecond 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

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limitation, CO2 must be supplied to these cultures. Such a process, which would achieveso-called tertiary wastewater treatment (nutrient removal) is shown schematically in Figure
III.A.3., and was demonstrated during this project. Considerable problems were encounteredwith the secondary ponds (shown as a small box labeled “green algae” in Figure III.A.3.), due toculture 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 otheroperational problems, the results were “reasonably consistent with the smaller 12-m2 ponds, bothin 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.

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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 forcomplete 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 theinitial pond. (Source: Benemann et al. 1978.)154

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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 5day culture period appears to be optimal. (Source: Benemann et al. 1978.)

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III.A.4.

Large-Scale Freshwater Microalgal Biomass Production for Fuel andFertilizer

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 foundbetween numbers of algal grazers and the large colonial algal types easy to harvest withmicrostrainers. Apparently the grazers preferentially consumed the smaller algae. Overall, theharvestability results with the microstrainers continued to be poor, so this line of research wasabandoned during the initial period of this project.
Simultaneously with these studies, another project was being carried out under EPA sponsorshipto 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 whensewage inflow was stopped (Koopman et al. 1978, 1980). Although generally long times wererequired 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 bywhich 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 ofbioflocculation involve extracellular polymers excreted by the algae, but the details remain to beinvestigated.
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 toremove 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 settlingin an “Imhoff Cone,” was high, about 90%.

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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 andchlorophyll. 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 themethod 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 highrate ponds. Table III.A.2. summarizes productivity and settleability for more than 1 year for thetwo 0.1-ha ponds. The 24-h laboratory settling tests correlated well with settling in the large (32m3) settling ponds. Overall, a settling efficiency of greater than 85% was achieved, if the betterof 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 ofcontinuous 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 culturecollection 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 ofN-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 removalfrom wastewaters is of interest.
This project also investigated the anaerobic digestion of algal biomass using both a large (1,400L) 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 tomethane. 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 particularmixing, were studied. The shaft power input required for mixing followed closely the predictedcube 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

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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, withthe 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 Benemannet 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 andenergy conservation in microalgae sewage ponds.” Biotech. Bioeng. Symp., No. 11, pp. 429-449.

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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 underotherwise similar conditions except that one pond was screened to remove zooplankton and theother 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.)

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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 theculture is removed from the mixed ponds.

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III.A.5.

Other Microalgae Projects During the ERDA/DOE Period

During the 1975-1979 period, several other projects on microalgae fuels production were fundedby 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 lightsaturation 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-houseproject that was very similar to the University of California-Berkeley project, including the useof microstrainers for harvesting filamentous microalgae, biomass recycle, and even abiophotolysis 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 atthe 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 thetheoretical 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 theanalysis 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 ofbiophotolysis.” Enzyme and Microbial Technology 2:103-111.
Goldman, J.R. (1979a) “Fuels from solar energy photosynthetic systems—state of the art andpotential 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.

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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 laboratoryof 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 hightemperatures: 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 massculture projects. However, for the most part, the laboratory and outdoor projects were notintegrated into a strongly unified program. This reflects in large part the difficulty of suchintegration. 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 oralgal 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 theisolates 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.

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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 otherclaimed attributes (Figure III.B.1.). Very high productivities were claimed. But a review of thework (Raymond 1979), in which P. tricornutum was grown in a 0.5-m2 system, revealed that thisprojection 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 acomparative 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 bydescriptions 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.

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Figure III.B.1. The algal raceway production system.
(Source: Raymond 1981.)

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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 systemcompleted 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 andlipid 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, comparedwith the original reports by Raymond for the ARPS system. Researchers tried to increase celldensity 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 wasagain 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 theRaymond prediction. However, this extrapolation was based on the invalid assumption that suchlaboratory 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, “asit 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 sdark), 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 fivetimes 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 notstated.
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 theculture, 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 lighton the system will allow the extra production to be achieved,” so the reliability of this prediction
is questionable.

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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 massculture production” (Laws, 1982; see also Laws et al. 1983). The basic idea was that a “foilarray” in the pond culture would generate a vortex that would create organized mixing in theponds, 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 flashinglight 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 wasrecorded for the 50-m2-flume reactor, a very low value for Hawaii, even in winter. Afterinstallation 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 notallow 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 infestationof 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 ofcases.

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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.)

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Figure III.B.3. Five-day running productivity averages for the Hawaii system. (Source:
Laws 1982.)

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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 ofvariables, 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), and8. 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 orabsence 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” wasculture 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 andexperience, which assumes that, everything else being equal, depth should not affectproductivity. Of course, depth can affect pH, temperature, pO2, and other variables, which if notheld 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.

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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 thecultures as a function of several variables, this time studied independently (Laws 1984b,c; seealso 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 theexperiments 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 sameas observed with P. tricornutum with temperature control.
Another question raised was the reproducibility of the data obtained with the flumes. During thisyear 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 atvarious 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 theeffects of N and P limitation on algal lipid content. Lipids increased from less than 20% toalmost 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 achievedby diluting the culture every 3 days. Further investigation of this phenomenon suggested that thiswas 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, andcontroversial, result, and a major focus for the following years of this project.

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Figure III.B.4. Productivity as a function of dilution. (Source: Laws 1984.)

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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 andType 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 ofearlier 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 thepO2 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 toincrease 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 tobe 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 pondwith and without the foils for 2-week periods. The presence of foils increased productivity byalmost 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, andthus resulted in lower productivities.

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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 andcontroversy. 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 tosupraoptimal density conditions” was speculated to account for this phenomenon. The classicaltechnique 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, theresults 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, andSynechococcus 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, and9.6% 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), theyobserved 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 initialconcentration 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 thecells to divide 2.5 times the first day, and once the second day. Not impossible, certainly, butsomewhat 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, eventhough 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 (2day batch cycle) productivity of about 30 g/m2/d was measured. This is, indeed, a high sustained
productivity.

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III.B.2.h.

Hawaii ARPS Project, Conclusions

This project evolved from one that focused on a demonstration of the ARPS concept using asingle 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 highproductivities achieved by two methods: organized mixing in ponds (e.g., the foils), and optimalbatch dilution (2- or 3-day intervals, depending on species). However, the basis for theseproductivity enhancements was speculative, and it proved difficult to demonstrate thereproducibility of these effects. The effects of foils could be better ascribed to degassing ofoxygen from the ponds with foils (e.g., higher mixing power inputs) and the results from the 3day dilution experiments to some uncontrolled factors, in addition to possible methodologicalproblems (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 highcosts, 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.” Proceedingsof 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 ResearchInstitute, 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.

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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 massculture.” 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 highproductivity.” FY 1986 Aquatic Species Program Annual Report, Solar Energy ResearchInstitute, Golden, Colorado, SERI/SP-231-3071, pp. 209-225.
Laws, E.A. (1987) “Productivity optimization of saline microalgae grown in outdoor massculture.” 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 ashallow outdoor flume.” Biotech. Bioeng. 28:191-197.
Laws, E.A.; Taguchi, S.; Hirata, J.; Pang, L. (1988) “Optimization of microalgal production in ashallow 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 ResearchInstitute, 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.

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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 (asEcoenergetics, Inc., later renamed EnBio, Inc.) to Fairfield, California, some 30 miles north ofBerkeley, 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 thescale-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 detailon 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 reinstatedin August 1982, and actual pond operations were initiated.

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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.)

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Table III.B.1. Microalgae pond carbonation requirements. (Source: Benemann et al.
1981.)

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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 aSelenastrum 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 poorthat 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 theculture, 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 weresatisfactory 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 foreach pond size and design to obtain even mixing.
As expected, productivities were rather low in the initial experiments carried out during Octoberand 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 ofspecies 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 newcompany, 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 forthis 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

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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 badsituation and cultivate the invading organisms. This was also the experience and approach of thisproject.
After inoculation of Scenedesmus obliquus strain 1450 from the SERI Culture Collection, astrain 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 Marchthrough 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 (20g/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 withproductivity 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-monthgrowing 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.

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6. There is a significant decrease in productivity in the afternoons, compared to themornings, in the algal ponds.
7. Oxygen levels can increase as much as 40 mg/L, over 450% of saturation, andhigh 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 evenupon 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 wasincreased 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 andone-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 initiallycontroversial 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 wastaken 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 forby 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 wasthat mixing power inputs make any mixing speed much above about 30 cm/s impractical, as theenergy 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.

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Another major conclusion was that competitive strains would be required to maintainmonocultures. 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 yearof this project, described in Section III.B.3.d.

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Figure III.B.6. Long-term productivity of S. quadricauda in freshwater (100-m2 pond).
(Source: Weissman and Goebel 1985.)

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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 productivityof the control. Max/min temperatures 20/30 oC (15/25 oC for the 60 cm/s data set). (Source:
Weissman and Goebel 1986.)

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III.B.3.d.

Completion of the California HRP Project, 1985-1986

Numerous microalgal strains were obtained from the SERI Culture Collection and tested insmall-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 notnecessarily correlated with dominance or persistence. However, these factors may be related tooxygen 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 theoxygen 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 (andantifoam) to reduce DO levels, from typically 400% to 500% of saturation without air sparing, to150% 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 thesame productivity with or without sparging despite the 20%-30% opaque foam cover, suggestingsome 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 wasnoted.
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 theconfounding factors of CO2 supply, temperature, and light intensity. In general the diatoms wereinsensitive to high DO; most, but not all, of the green algal strains exhibited marked inhibition byhigh oxygen levels (Figure III.B.8.). None of the oxygen-sensitive algae could be grownoutdoors, 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, withgrowth rates also affected. Of course, the actual density of the culture is a major factor

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determining productivity, and dense cultures avoid most, if not all, the deleterious effects of highlight 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 wherechlorophyll 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 tothe 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 moredetailed 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 amuch 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 toremove 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 havesignificant 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 alsopresented.
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.

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Publications:
Benemann, J.R.; Goebel, R.P.; Weissman, J.C. (1981) “Production of lipid hydrocarbon fuels andchemicals 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 andchemicals 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 asource of liquid fuels.” Proceedings of the June 1982 SERI Biomass Program Principal
Investigators' Review Meeting, Aquatic Species Program Reports, Solar Energy ResearchInstitute, 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 ResearchInstitute, 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 ofopen ponds and tubular reactors.” Bioeng. Biotech. 31:336-344.

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Table III.B.2. Outdoor results summary for California pond operations.
Data from 3.5-m2ponds. (Source: Weissman and Goebel 1986.)

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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.)

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III.B.4.

The Israeli Microalgae Biodiesel Production ProjectIn 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 ofunicellular 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 compositionand 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 0.5Liter, pH-controlled chemostats for effects of temperature, light intensities, nutrients (Fe andnitrate), 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 inalgae 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 onlipid 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 withlaboratory 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 highrate ponds. Among other factors, the effects of culture density on productivity and lipid content

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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 andlipid 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 ofalgal 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 ofchemical 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, themethod 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, theIsraeli project provided another dimension to the ASP effort, generally supporting theconclusions 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-2312700, pp. 228-229.

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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-richhalotolerant microalgae.” Proceedings of the March 1985 Principal Investigators’ Meeting,
Aquatic Species Program, Solar Energy Research Institute, Golden, Colorado, SERI/CP-2312700, pp. 230-243.
Ben-Amotz, A. (1987) “Development of outdoor raceway capable of yielding oil-richhalotolerant 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 forproduction 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 ResearchInstitute, 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 fromsaline 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.

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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 thesouthwestern 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 thiscompetition, with a proposed facility consisting of two 1,000-m2 ponds, one plastic lined andanother unlined, as well as supporting R&D using six small, 3-m2 ponds, continuing andextending 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 andsupplies of saline groundwaters. The primary limitation of this site was temperature, which, inretrospect, 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 andconstructing 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 (15and 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 ofthe layout of the completed facility.

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Figure III.B.9. Schematic of the microalgae OTF in Roswell, New Mexico.
(Source: Weissman and Goebel 1988.)

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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 theconclusion that differences between the different treatments should be judged significant only ifthese 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 ofMONOR2 fell to about 10 g/m2/d, and was very low (3.5 g/m2/d) in December in unheatedponds. Remarkably, despite these ponds freezing over repeatedly, the culture survived andexhibited some productivity. During August and September, productivities for CYCLO1 andAmphora 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 ofTechnology, combining climatic, design, operation, physico-chemical and biological processcharacteristics, 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 madeit 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 termsof 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 fromthe 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 highmixing 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 thesmaller ponds.

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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 withthree 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 withnutrient 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 thisprocess, though probably not at this site because of low seasonal productivities.
6. Very high, 50 g/m2/d, single day, productivities were observed on someoccasions.
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, continuationof 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 three196

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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 affectoverall 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 ispreferred, 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 assoon 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 asa 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
YES
NO
NO
YES
NO

Dates
Productivity
CO2 use(std.m3/d)(gm afdw/m2/d)
15.2
10/1/88-9/30/89
9.8
13.4
10/1/88-9/30/89
8.3
14.6
10/1/89-9/30/90
10.5
22.0
6/1/90-10/30/90
1919.2
5/1/90-9/30/90
18
Notes:
std m3/d: standard cubic meters per day.

Carbon Use
Efficiency
59
50
82
81
88

Water Loss
5.7 mm/d
6.2 mm/d

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.

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III.B.5.d.

Conclusions

The performance of the large-scale system improved considerably in all aspects during the 2years of operations. The parallel use of the smaller-scale ponds helped guide this research, inparticular 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 initialsuboptimal 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 amajor 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 morefavorable 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 processdevelopment 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 inperformance and increased understanding of the overall process in specific critical areas ofculture 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 evenbasic 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 ofthe data would suggest considerable success with species control, with several species cultivatedsuccessfully for relatively long periods. However, considerably more research will be requiredon 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 ofspecies 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.

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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 Roswellproject, John R. Benemann and Dr. David Tillett carried out a research project in support of theseoutdoor pond projects (Benemann and Tillett 1987, 1990). This project included studies of theeffect 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 developmentof 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 ofvariables, specifically light intensity, temperature, pH/pCO2, and pO2. The first two are
essentially uncontrolled, although somewhat predictable, variables. The second two factors areconsequences 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 beaveraged over the time scales of interest (>1 h). With this assumption it would be possible topredict, and reproduce on a small-scale, the key environmental parameters of outdoor ponds towhich microalgae likely would respond, determining productivity and culture stability.

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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 easilyinterpretable 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.

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Table III.B.4. Scale down parameters for microalgae culture.
Feasiblity of scaling pond environmental parameters to laboratory reactors.
(Source: Benemann and Tillett 1987.)

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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, andbiological 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 forsmaller 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 andmonthly 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, andlength-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 parametera 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 forphotosynthesis (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 ambientconditions. 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 pondfor 2 weeks.
Simulations were also run for a larger, 1,000-m2 earthen pond as built at Roswell (an arid andcool climate), which were then compared with a site in West Palm Beach, Florida (a humid,

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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 100cm 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 formost 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 (bothgave about 15.3 g/m2/d), this was probably fortuitous, as the Ik actually used in the model waswell 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 over85%.
A final important parameter is dissolved O2, which was predicted to accumulate within 1 hour toa 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 andsmall-scale outdoor research in making these more representative of the outdoor pond
environments. Laboratory applications are discussed in the following section.

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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 andunheated 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.

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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 ecologicaland 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 thatshould 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 ofenvironments. Indeed, even Hutchins did not realize the greatness of microbial biodiversity innature, 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 toperiodic 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 toappear 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 wereobserved 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 ofdifferent light spectral use of the two types of algae (greens and diatoms). Many otherexperiments were carried out, with fluctuating pH, dilution rates, and light intensities and evengas 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 notclearly 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 lightsaturation 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.

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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 muchas 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 andChisholm (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 cellmass. 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 inthis 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 andthen 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 andcellular 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 therate 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 possibilityof producing high lipids content by nutrient limitation while achieving a substantial increase in
overall lipid productivity. Experiments with continuously diluted cultures, however, did notexhibit such responses, indicating the necessity to carefully control and modulate conditions tomaximize lipid production.

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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.)

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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 ofcompetitiveness in mixed cultures. That is, the most productive culture will also be the mostcompetitive. Another assumption is that productivity and competitiveness will be affected byfluctuations in environmental variables and cannot be predicted from single parameter variations
or steady-state operations. Testing these hypotheses was a central objective of theseexperiments, 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, a14-h constant light period, 0.6-0.7 d-1 dilution, and 6-h gassing with pure O2 rather than air). Asin earlier studies, Chlorella and Monoraphidium exhibited higher productivities (250-300
mg/L/d) than the other strains tested (Porphyridium, Ankistrodesmus, and Chaetaceros, withsome 150-200 mg/L/d productivity). Under constant conditions, similar ranking and differenceswere 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 theexperimental 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, fortemperature 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 theoutcome of species competition experiments. Still, much more experimental work is required onthese problems.
Publications:
Benemann, J.R.; Tillett, D. (1987a) “Effects of fluctuating environments on the selection of highyielding microalgae.” FY 1986 Aquatic Species Program Annual Report, Solar Energy ResearchInstitute, Golden, Colorado, SERI/SP-231-3071, pp. 285-300.

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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 ofhigh yielding microalgae.” Final Report, Subcontract XK-4-04136-06, Solar Energy ResearchInstitute, Golden, Colorado.
Benemann, J.R.; Tillett, D.M. (1987d), “Effects of fluctuating environments on the selection ofhigh 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 ofhigh yielding microalgae.” Final Report, Subcontract XK 4-04136-06, Solar Energy ResearchInstitute. 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 effectsof nitrate, silicate and light-dark cycles.” J. Phycol. 17:374.
Sphoer, H.A.; Milner, H.W. (1956) “Production of protein, lipids and carbohydrates by culturesof algae.” U.S. Patent 2,732,661.
Spoehr, H.A.; Milner, H.W. (1949) “The chemical compostition of Chlorella; effect ofenvironmental conditions.” Plant Physiol. 24:120.

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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.)

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III.C.

Resource Analyses

III.C.1.

Introduction

One major concern of the ASP, as for any renewable energy option being developed by DOE, isthe 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 isrequired both by the mission of the DOE as well as the inherent need to justify budgetarydecisions. However, potential contributions to fuel supplies should be only one, albeit animportant, 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 ona few technologies with a low probability of succeeding in practice.
Microalgal biodiesel is one of many different biomass energy options, extending from co-firingof wood in power plants to energy farms and a myriad of fuel conversion options (Hughes andBenemann 1997). One important attribute of microalgae systems is that they need not competewith other biomass alternatives, but must be able to use water and land resources generally notconsidered for crop production. Also, microalgae fuel production systems could meet otherobjectives, from waste treatment to salinity management. Resource potentials are important, andhave 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 potentialof 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 geographicalarea. In fact, a significant activity of the ASP was to document this resource potential. It was nota trivial task to assess these resource potentials. This required the development of suitableassessment techniques. This was a major accomplishment of the ASP, as reviewed in thissection. However, estimating an actual resource potential is not a simple matter, as thejuxtapositions of the requirements for microalgae production—water, land, CO2, and climate—are very difficult to quantitate, and would greatly reduce the resource potential estimated fromsingle 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).

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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 thatshould and could be used to identify available water, land and other resources, to estimate theirrelative 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 waterrights issues are addressed, which are certainly important, but are difficult to extrapolate overlarge 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 andtransportation to arrive at permissible lift and distance criteria for water resources. Water liftingof 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 ashigh 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 forwater supplies. This report did not arrive at a prediction for the resource base, but was animportant 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 aquaticbiomass in the arid southwest.” Battelle Columbus Laboratory Report, Solar Energy ResearchInstitute, 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 todeliver CO2 for EOR. One study even considered the production of CO2 from a power plant fluegas 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 forthe 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.

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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 theSouthwestern 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, andsupplies 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 andpower.
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 andwater availability, were prepared, including a final overall suitability assessment (Figure
III.C.1.). The authors proposed continuing such an assessment, pointing out the many limitationsof 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-freedays, 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.

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Figure III.C.1. Overall suitability map for microalgae culture in the Southwestern UnitedStates.
Zones of relative suitability for microalgae biomass production based on compositing of climate,land and water suitability maps. (Source: Maxwell et al. 1985.)

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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 andauthoritative source of information on this subject, from merchant CO2 supplies and costs topotential 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 expensivefrom cement, refineries, or power plants.
After a detailed review of the options, the authors estimated that the potential CO2 resource basewas 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-specificopportunity, 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.

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Table III.C.1. Summary of Availability and Cost of CO2 Sources(Source: Feinberg and Karpuk, 1990.)

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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, andclimatic 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 ofvarying 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 wellyields, size of reserves, and chemical composition. A detailed analysis of six groundwater basinsof 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 wereidentified as available. This report is an excellent example of the challenges of developing sucha 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.

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Table III.C.2. Suitable water resources in New Mexico.
(Source: Landsford et al. 1990.)

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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 thefocus 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 andsaline 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, oreven 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% ofclimatically 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 makesome 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/costprojections for large-scale microalgae biofuels production. These analyses generally supported
the view that microalgae biomass production could be performed at sufficiently low cost as toplausibly 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 mostR&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, andsupplied 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

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219National Renewable Energy Laboratory(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 acovered 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 thewoody 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 typicalmunicipal 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 growthpotential of the wastewater, containing 65 mg/L of useable N (as organic N and ammonia). Thisrequired 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 (thisanalysis 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 forsuch large algal ponds and of the transfer requirements for CO2 to the algal culture. A 160-haalgal pond system was required to treat this wastewater flow year-round. This was about threetimes larger than a conventional oxidation pond system. Costs were projected to be competitive
with conventional wastewater treatment systems.

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Energy outputs were twice the energy inputs, based on digester gas production and requirementsfor 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 locallyavailable municipal wastes could supply only a small fraction of fertilizer needs for the verylarge (>10,000 ha) energy plantations being projected. It does, however, point to the potential ofthis 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 toInter-Technology Solar Corp., Sanitary Eng. Res. Lab., Univ. of Calif.-Berkeley.
InterTechnology/Solar Corp. (1978) “The photosynthesis energy factory: analysis, synthesis anddemonstration.” 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 fora “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 forsuch 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.

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Based on these assumptions, designs of the various system components were carried out, andsupporting 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 theanalysis. 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 costcenters 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-scalealgal (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.

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Table III.D.1. Design assumptions for a microalgae production system.
(Source: Benemann et al. 1978.)

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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 waspointed 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 highprobability that land-based aquatic biomass growth systems can be designed which aretechnically 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 ofsome 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 andfertilizer resources for microalgae ponds would be prohibitive. However, Oswald and Benemann(1977) countered arguments, pointing out, for example, that such a simplistic analysis failed toconsider water and nutrient recycling. In this connection, Goldman (1979a,b) also reviewed thefundamental 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.

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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 andsystems 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 somemicroalgae 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 somewhatmoot, and the final report (Benemann et al. 1982a) covered only the HRP system in detail, withthe 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 withsome significant differences and much greater details for the engineering designs and costestimates. 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 toconcentrate the biomass to a paste-like consistency. A solvent extraction process as used forsoybean 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 befeasible for such high moisture biomass.) As before, the residual biomass was to beanaerobically digested in covered ponds to produce methane gas, with the nutrients (and C) fromthe 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 carriedout the engineering designs and calculations for CO2 supply systems for the present report. A

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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 ofthis nutrient. Various scenarios were analyzed for CO2 sources, including purchase andcombustion of coal (e.g., co-siting a power plant). Another major variable analyzed was thecapital and operating costs factors, such as labor costs and overhead, utilities and fuel costs, landcosts, factors for buildings and power supplies, contingencies and contracting, architect andengineering 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 theengineering 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 “basecase” 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 theflue 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 CO2case, 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% perannum). 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 forthe 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 tosuch 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

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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 inlarge-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 byRaymond (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 forthis 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 forfuel 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 bewell 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 asource 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.

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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 thedigesters and use of either pure CO2 delivered to the site for $22/mt, or generation of CO2 on sitefrom 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.)

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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 andcosts involved in microalgae biomass production systems (Neenan et al. 1986). The report
reviewed the various system components and requirements for algal biomass production, andsummarized 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 fuelproduct 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 authorsraised 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. Somemodifications were made in the engineering analyses, such as replacing channel dividers byplastic 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 designand 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 tomethane 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 thereference 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 evenless for ethanol). Table III.D.3. provides a summary of this analysis, for a 36,000 mt/y (33,000t/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 theconclusion that such a process “is currently not commercially viable.” In fact, the main and

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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 costswith 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 incombination could do so. In particular, by increasing productivity to as high as 8% of total solar
conversion efficiency and 50% lipids (50 g/m2d), 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 fuelproduct 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 wererather high, and might be reduced in the future. But, as a central conclusion, productivity wasagain 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 chemicalcompositions.” 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 ResearchInstitute, Golden, Colorado, SERI/SP-231-2550, 158 pp.

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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 fuelprocessing 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 feedsockb. Process and cooling waters
c. By-product prices: $0.07/m3 for CO2 (captive use only), $7.40/MMBtu formethane, 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 $1.75/gal forbiodiesel (methyl ester) or PVO
e. The value ($/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 ofglycerol for export (plus 11 million m3 CO2 and 2,400 tons N, recyled
internally) at prices listed in c. and d. above.

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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:

232a.

Includes microalgae biomass feedstock production costs of $211/mtb.

Process and cooling watersc.

By-product prices: $0.07/m3 for CO2 (captive use only), $7.40/MMBtu formethane, fuel gas, or LPG, and $13.30/MMBtu diesel fuel (exports)d.

Main product, million gallons/yr.

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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 thedevelopment 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 forscale-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 aclosed photobioreactor process for cultivating Haematococcus pluvialis, an alga high inastaxanthin.
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 wereavailable 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 morethan 10,000 ha of algal ponds, as such ponds could help manage the salinity of this inland sea, amajor 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 thealgal 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 mostdetailed, 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%, withinparameters that would allow high microalgae productivity. Another innovation was the use ofsmall amounts of high molecular weight polymers to improve the flocculation, settlingcharacteristics and harvesting efficiency of the basic bioflocculation process. The polymers canbe 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

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things, higher costs were assumed for rough and fine (laser) grading, which depends on the typeof 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 thesehigher 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 morethan twice the cost derived from the “conservative” case in the earlier study (Benemann etal.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.

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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

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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)

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III.D.8.

Systems and Economic Analysis of Microalgae Ponds forConversion 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 equivalentamount 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 systemcomponents. For example, the costs of laser grading and earthworks were independently costestimated 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. Amongthe 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 thecentrifuge earlier specified for final concentration. However, overall this analysis was derivativeof 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 lightharvesting (“antenna”) pigments would increase the photosynthetic efficiencies at high lightintensities. Microalgae with reduced antenna pigments would, however, not be very competitivein 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.

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Both direct flue-gas utilization near the power plant and remote use of CO2 captured from fluegas and piped to the algal ponds were considered. With projected oil prices of $25/bbl andproductivities 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 withmicroalgae 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 overinoculum 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 forconversion 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 fuelpower plants.” Report, U.S. Dept. of Energy, DOE/ER-30194, vol. 1 and 2.

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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.)

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Table III.D.7. Greenhouse gas balances and mitigation costs.
400-ha system, @$0.065/kWh.
(Source: Benemann and Oswald 1996.)

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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 fluegases. The general arguments for use of microalgae in CO2 mitigation, from high productivity torelatively 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 ofthe 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 ofexcessive 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., whichsummarizes current and long-term projections for such a process. Although there are somesignificant 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.

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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.

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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.)

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Table III.D.8. Summary of costs for microalgae CO2 mitigation.
Economic performance of processes with different maturities.1(Source: Kadam 1995.)

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III.D.10.

Conclusions.

The cost analyses for large-scale microalgae production for fuels reviewed earlier evolved fromthe 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 thefundamental 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, andare 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 productionof Dunaliella, and in several countries in wastewater treatment. Thus, although someuncertainties remain (such as allowable channel width and wind fetch effects), in general thebasic engineering designs and assumptions for the microalgae cultivation ponds appear wellestablished.
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 ofthese 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 (Benemannand 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 designand system construction approaches were based on agricultural engineering practices, rather thanthose 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 theculture 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

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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) thanopen 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 overenvironmental conditions (pH, temperature) and biological contaminants, and higher cellconcentrations, reducing liquid handling and harvesting costs. Thus, it would be theoreticallypossible to grow algal strains not able to dominate in open ponds, at higher productivities andreduced 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 geneticallyengineered 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 productionin 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 notthe engineering designs, or even the cost estimates, but the biological assumptions on which suchdesigns 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 theinitial 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 ofphotosynthetic 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 (seeSection IV.A.2.).

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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 inthe 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 isthe 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 settlingprocess, followed by further mechanical concentration and processing.
The major conclusion of these analyses is that microalgae production for fuels is currently notlimited 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.

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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 andcontractors to identify or develop microalgal strains that demonstrated properties conducive tocost-effective biomass and lipid production. The characteristics deemed desirable in these strains
include high productivity, high lipid content, competitiveness in outdoor culture, and tolerance tofluctuations 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/NRELMicroalgae Culture Collection was established as a valuable genetic resource and was the firstmicroalgal 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 theBacilliarophyceae (diatoms). Organisms were identified in both classes that showed highproductivity, 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 accumulationin diatoms. In green algae, lipid accumulation is induced by N starvation. N is a component ofmany 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 useof strains with lightly silicified walls. Some green algal strains, for example M. minutum, areadvantageous for mass culture applications in that they can survive temperature fluctuations thatare 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,

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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 asite, to maximize productivity at different times of the year.
Significant progress was made during the last 15 years in the understanding of lipid accumulationin 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 cellularenergy 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. Manyof the green algae were found to contain DNA with unusually high GC ratios, and often withunusual 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. Workat 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 theseorganisms. The successful development of the transformation system led to an increased
understanding of the factors involved in introducing and expressing foreign genes in theseorganisms, 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 thefollowing sections.

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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 andaspects, from species control to lipid (oil) yield and harvesting, is therefore recommended as acentral 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 evenoutdoor 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 oflarge-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 otherbiological 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 andother 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 selectstrains 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 alongsidewith genetic engineering studies to demonstrate productivity enhancements using microalgal
strains with already well-developed genetic systems. Such improvements would be in theefficiency of photosynthesis, described next, and lipid productivities, extending the ASP researchreviewed in Section II.
IV.A.2.b.

Maximum Efficiency of Photosynthesis

Many environmental factors affect the performance of the complex photosynthetic machinery inmicroalgae, reducing its efficiency to well below the maximum at which photosynthesis canperform. That maximum is dictated by the underlying mechanisms, biophysical constraints, andphysiological 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.

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However, somewhat surprisingly, there is still argument about the maximum limit forphotosynthetic 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-photosystemmechanism (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). Suchefficiencies 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 fullsunlight, 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 iswasted. 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, thephotosynthetic apparatus cannot keep up with the high photon flux and most of the photons arewasted, 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 andlight 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.

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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 chlorophyllantenna 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 perunit 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. Anotherapproach, 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 tophotoinhibition, has been recognized for many years (e.g., Kok 1973): reduce the number ofchlorophyll molecules cooperating in photosynthesis (the so-called “antenna” chlorophylls) froma 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 (seeBenemann and Oswald 1996 for a recent discussion).

However, it has only recently become possible to consider achieving this objective, through thedetailed 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 workby Melis et al. (1998) and Neidhardt et al. (1998) demonstrated, at the physiological level, thefeasibility 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 the252

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desired high photosynthetic efficiencies by algal mass cultures. Such tools can also be used todirect 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 strainsrequired for large-scale, low-cost microalgae cultivation. The ASP spent considerable effort inthis area, with the isolation, screening, maintenance, laboratory studies, outdoor cultivation, andgenetic 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 masscultures 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 highsalinity, respectively. Scenedesmus was mass cultured at the pilot scale in Germany and
Chekoslovakia, and other countries, with the cultures obtained from isolates that invaded anddominated the ponds. Commercial Chlorella production, using selected strains, has sufferedfrom culture instabilities, requiring frequent inoculation and short production runs, greatly
increasing the costs of the process. Thus, commercial-scale production of microalgae does notprovide 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 notneed 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 anddemonstrate their survival and efficacy.
Within this context, the demonstration of the ability to mass culture at least some algal strains ona 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

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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 criticalparameters—temperature, light intensity, pH fluctuations—can be modeled rather easily at amodest 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 usuallyconsequences of, not fundamental reasons for, loss of culture competitiveness. Further, sometechniques 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 bedemonstrated 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 masscultures. 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 highbiomass 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 toincrease photosynthetic efficiencies or to optimize lipid quantity or quality, achieved withlaboratory 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.

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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 massculture 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 performanceprojections 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 late1970s. Competing within current market realities is not plausible for most renewable energy
technologies. Indeed, electric industry deregulation is removing price supports for suchtechnologies 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.

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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 fuelcosts 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 anactivated 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., theCity of Sunnyvale, California) the algal biomass is not harvested, and thus it is discharged to thenearest body of water (river, lake, etc.), used for irrigation, groundwater recharge, or it settles tothe 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, theireconomics would not be dictated, except marginally, by their waste treatment functions, and theirimpacts on U.S. greenhouse gas emissions and fuel resources would be modest, at most a fractionof 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 thefollowing 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 producedin 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 economicallydependent 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 driedgrain, is the drying costs. Overall, higher-value feed coproducts cannot, and should not, be amajor 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

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National Renewable Energy Laboratory(from the high 20s to the mid teens in dollars per kg) during the past 2 years. If this trend were tocontinue, to below $10/kg, this algal biomass could become a significant ingredient inaquaculture 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 tobe 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 ofsuch 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 theJapanese 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 andIndustry (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 andacademic 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 theJapanese 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 andprivate 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 theability 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 atleast 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

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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 programand 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 asalgae-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 electricutilities, 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 culturecollection, and then with strains that spontaneously appeared and dominated the cultures at thissite (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, thecomplication 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 specifictechnical 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 andimpurities thereon.” Biochem Biotech. 34/35:681-692.

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Negoro, M.; Hamasaki, K.A.; Ikuta, Y.; Makita, T.; Hirayama K.; Suzuki, S. (1993) “CO2fixation 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 UnitedStates 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 anapproximately equivalent amount of current transportation fuels. Herzog (1995) argued that sucha potential, in fact, anything less than 10%, is not sufficient to justify a R&D effort, and thatscarce resources should be devoted only to potentially high-impact technologies, such as thedisposal of CO2 in the oceans or geological formations. However, Benemann (1995) counteredthat such a resource-only argument is too limited, as it ignores the issue of economics andtechnological risks. For example, the technical feasibility of ocean disposal is far fromestablished, and the costs of such a process are not currently constrained by credible engineeringand 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 formicroalgae 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.

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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 briefsummary 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 andengineering/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 evenprojected before the ASP, but still well below the theoretical potential, and therequirements 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 thesecond conclusion, the emphasis of any R&D effort must be on more fundamental andearly-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 suchcases the economic and resource constraints are relaxed, allowing for such processes to beconsidered 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 thedemonstration 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 stillrequired in algal mass culture. These, however, cannot be demonstrated in the laboratory. They
include competitiveness, predation resistance, and harvestability. The issue thus arises of theneed to carry out such algal mass culture research in parallel with the laboratory studies.

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The outdoor projects of the ASP demonstrated the ability to mass culture microalgae underrelatively 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 thisresearch to mass culture systems. This is not to exclude some supporting outdoor studies, forexample 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, noton outdoor culture technology development.
However, a strictly laboratory-based R&D program, may rapidly loose touch with the realities ofthe eventual applications. Thus, some outdoor mass culture R&D is recommended, specificallyfor 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 athigh 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, haverevealed 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), CO2transfer (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 ofinterest to determine the practical limits of such systems. Can single raceway pond scales belarger than 5 ha? What are the wind effects in such large systems? But overall, the engineeringand 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 thepresent context. Although such devices could have a role in the buildup and production of

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inoculum (starter cultures), they are not likely to be an essential or crucial component oflarge-scale, low-cost microalgae culture processes for energy production.
Any future R&D program for microalgae CO2 capture and biofuels production must start with thedevelopment 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.

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V.

BIBLIOGRAPHY

V.A.

SERI/NREL/DOE REPORTS AND PUBLICATIONS

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Benemann, J.R.; Baker, D.; Koopman, B.L.; Oswald, W.J. (1977) “A systems analysis of
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Colorado, SERI/SP-231-3206, p. 269.
Karpuk, M. (1987b) “CO2 sources for fuels synthesis.” Abstract, FY 1986 Aquatic Species
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Kok, B. (1973) “Photosynthesis.” Proceedings of the Workshop on Bio Solar Hydrogen
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Kresovich, S.; Wagner, C.K.; Scantland, D.A.; Groet, S.S.; Lawhon, W.T. (1982) “Theutilization of emergent aquatic plants for biomass energy systems development.” Report, Solar
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Lansford, R.; Hernandez, J.; Enis, P.J. (1987) “Evaluation of available saline water resources in
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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,
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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 ResearchInstitute, Golden, Colorado, SERI/CP-231-1808, pp. 31-42.
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the April 1984 Principal Investigators’ Meeting, Solar Energy Research Institute, Golden,
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Laws, E.A. (1984c) “Research, development, and demonstration of algal production raceway(ARP) systems for the production of hydrocarbon resources.” Subcontractor Report, Solar
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Laws, E.A. (1987b) “Continuous microalgae production in shallow raceways at highproductivity.” FY 1986 Aquatic Species Program Annual Report, Solar Energy ResearchInstitute, 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 ashallow outdoor flume.” Biotech. Bioeng. 28:191-197.
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1983 Principal Investigators’ Meeting, Aquatic Species Program, Solar Energy ResearchInstitute, 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
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the July, 1981, Subcontractors’ Review Meeting, Aquatic Species Program, Solar Energy
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SERI/CP-624-1228, pp. 59-65.
Lien, S. (1982) “Studies on the production and accumulation of oil and lipids by microalgae.”
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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.
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FY 1987 Aquatic Species Program Annual Report (Johnson, D.A.; Sprague, S., eds.), Solar
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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.
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Methods and Applications (Levin, M.A.; Seidler, R.J.; Rogul, M., eds.), McGraw-Hill, Inc., New
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Proceedings of the March 1983 Principal Investigators’ Meeting, Solar Energy ResearchInstitute, Golden, Colorado, SERI/CP-231-1946, pp. 69-77.
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Owens, T.G.; Gallagher, J.C.; Alberte, R.S. (1987) “Photosynthetic light-harvesting function ofviolaxanthin in Nannochloropsis sp. (Eustigmatophyceae).” J. Phycol. 23:79-85.
Pratt, D.C. (1981) “Wetland biomass production.” Proceedings of the July 1981, Subcontractors’
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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.
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Raymond, L.P. (1984) “Microalgal technology research at SERI.” Proceedings of the April 1984
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microalgae.” Proceedings of the April 1984 Principal Investigators’ Meeting, Aquatic Species
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and carbohydrate biosynthesis in Cyclotella cryptica.” FY 1987 Aquatic Species Program Annual
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enzymes in the diatom Cyclotella cryptica in response to silicon deficiency.” Arch. Biochem.
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Roessler, P.G. (1988b) “Characteristics of abrupt size reduction in Synedra ulna
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35pp.
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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.
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Schuster, A.M.; Burbank, D.E.; Meister, B.; Skrdla, M.; Meints, R.H.; Hattman, S.; Swinton, D.;
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exsymbiotic Chlorella-like alga.” Plant Mol. Biol. 14:859-862.
Schwegler, B.R., Jr. (1983) “Water hyacinth production primary and advanced treatment ofwastewater.” 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,
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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
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Solomon, J.A (1985) “Ultrastructure evaluation of lipid accumulation in microalgae.” Aquatic
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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.

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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 thequantity 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, andGuillard.” Appl. Biochem. Biotech. 28/29:317-326.
Sriharan, S.; Bagga, D.; Sriharan, T.P. (1989) “Environmental control of lipids and fatty acidproduction 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 andfatty 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 geneencoding the adenine methyltransferase M. CviRI from Chlorella virus XZ-6E.” Nuc. Acids Res.
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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 thesoutheastern 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 thesoutheastern 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.

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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 ofthe 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
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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.
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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: anannotated bibliography.” Report for Subcontract XK-2-0270-01, to the Solar Energy ResearchInstitute, 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

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Review: Proceedings of the April 1984 Principal Investigators’ Meeting, Solar Energy ResearchInstitute, 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
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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 andnitrogen 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,
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Timurian, R.; Ward, R.L.; Jeffries, T.W. (1977) “Growth of filamentous blue-green algae at hightemperatures: a source of biomass for renewable fuels.” IZ Int. Seaweed Symp. Aug 20-27.
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Tornabene, T.G. (1984) “Chemical profile of microalgae with emphasis on lipids.” Aquatic
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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 oflipids.” 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,
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Van Etten, J.L.; Meints, R.H. (1991) “Viruses and virus-like particles of eukaryotic algae.”
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Principal Investigators’ Review Meeting, Aquatic Species Program Reports, Solar Energy
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Vigon, B.W.; Arthur, M.F.; Taft, L.G.; Wagner, C.K.; Lipinsky, E.S.; Litchfield, J.H.;
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Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1989) “The mitochondrial genome of theexsymbiotic Chlorella-like green alga N1A.” Aquatic Species Program Annual Report(Bollmeier, W.S.; Sprague, S., eds.), Solar Energy Research Institute, Golden, Colorado,
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Waddle, J.A.; Schuster, A.M.; Lee, K.; Meints, R.H. (1990) “The mitochondrial genome of an
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Walsh, R.G. (1984) “Microalgal technology research at SERI: The biochemistry of lipid trigger
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Weissman, J.C. (1984) “Cultivation of microalgae for liquid fuels.” Proceedings of the April
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Weissman, J.C.; Goebel, R.P. (1987) “Factors affecting the photosynthetic yield of microalgae.”
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Weissman, J.C.; Tillett, T.D.; Goebel, R. (1987) “Design and operation of an outdoor microalgae
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Weissman, J.C.; Tillett, D.T. (1992) “Design and operation of an outdoor microalgae test
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Weissman, J.C.; Tillett, D.T.; Goebel, R.P. (1989) “Design and operation of an outdoor
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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
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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.;
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V.C.

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294

A Look Back at the Aquatic Species Program—Technical Review

REPORT DOCUMENTATION PAGE

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1. AGENCY USE ONLY (Leave blank)

4. TITLE AND SUBTITLE

2. REPORT DATEJuly 1998

3. REPORT TYPE AND DATES COVEREDTechnical Report
5. FUNDING NUMBERS

A Look Back at the U.S. Department of Energy’s Aquatic Species Program: Biodiesel from Algae

BF886001

6. AUTHOR(S)J. Sheehan, T.G. Dunahay, J.R. Benemann, P.G. Roessler, J.C. Weissman
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)8. PERFORMING ORGANIZATIONREPORT NUMBER

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
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES)10. SPONSORING/MONITORINGAGENCY REPORT NUMBER

National Renewable Energy Laboratory
1617 Cole Blvd.
Golden, CO 80401-3393

NREL/TP-580-24190

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National Technical Information Service
U.S. Department of Commerce
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DISTRIBUTION CODE

UC-1503

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.

13. ABSTRACT (Maximum 200 words)

15. NUMBER OF PAGES

14. SUBJECT TERMS

biodiesel, algae, aquatic species, raceway ponds
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Unclassified

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Unclassified

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