Office of Research 

and Development (8101R)

Washington, DC 20460

EPA/600/R-07/028 

January 2007

www.epa.gov

Printed with vegetable-based ink on paper that contains a minimum 

of 50% post-consumer fiber content processed chlorine free.

BioCover_Final.indd 1-2 
EPA/600/R-07/028 

Bioengineering for Pollution Prevention through 

Development of Biobased Energy and Materials 

State of the Science Report

by: 

Dianne Ahmann 

and 

John R. Dorgan 

Colorado School of Mines 

Golden, Colorado 80401 

Contract number 3W-2456NTEX 

April Richards, Project Officer 

National Center for Environmental Research 

Washington, DC 20460 

U.S. Environmental Protection Agency 

Office of Research and Development 

National Center for Environmental Research 

Washington, DC 20460 

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Disclaimer 

The information described in this document does not necessarily reflect the views of the 

Agency, and no official endorsement should be inferred. Mention of trade names or commercial 

products does not constitute endorsement or recommendation by EPA for use. 

Acknowledgements 

This document was reviewed by both internal U.S. Environmental Protection Agency 

(EPA) reviewers and external peer reviewers who were chosen for their diverse perspectives and 

technical expertise in bioengineering and pollution prevention. Richard Engler and Mark Segal 

from EPA’s Office of Prevention, Pesticides, and Toxic Substances and Bob Frederick and 

Diana Bauer from the EPA’s Office of Research and Development provided comments on the 

initial draft. Four external peer reviewers reviewed the final version including: David B. Levin, 

University of Victoria; Anastasios Melis, University of California, Berkeley; Lonnie Ingram, 

University of Florida; and Richard Wool, University of Delaware. 

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iii 

Executive Summary 

Petroleum-based fuels and related materials are central to the economies of developed 

and developing countries around the world. However, these resources are finite and expected to 

enter a period of diminishing availability within the next several decades. 

To move economies based on petroleum and its feedstocks to fuels and materials that are 

renewable, environmentally friendly, and of greater availability, the science and engineering 

communities worldwide are exploring many options. Principal alternative energy resources that 

scientists have been exploring are wind, solar radiation, hydropower, geothermal power, coal 

combined with carbon sequestration, hydrogen, and biomass. In addition, biomass and 

biologically-generated polymers are attractive renewable feedstocks as energy-producing 

materials. It appears likely that no single resource will offer the versatility of petroleum in the 

future. As a result, several complementary technologies are being explored to meet the world’s 

diverse needs for energy and resource materials. 

Biologically based transformations have several potentially favorable attributes. They 

typically operate on renewable resources, at low temperatures, in aqueous environments, and 

produce few byproducts because of the specific nature of enzymatic catalysis. These attributes 

make industrial biotechnology inherently consistent with the principles of Green Chemistry and 

promise industrial commodity production with less environmental impact. 

In this document, the application of industrial biotechnology to the important commodity 

classes of fuels and plastics is reviewed. Where applicable, those areas that have been advanced 

under funding from the joint EPA and National Science Foundation (NSF) program, Technology 

for a Sustainable Environment (TSE), are highlighted. Promising areas for future exploration 

and development are identified as well. 

A. BIOMATERIALS 

The worldwide production of plastics reached 260 billion pounds per year at the end of 

the 20th century, with a value of over $310 billion to the U.S. economy in 2002. Approximately 

one-third of all plastics produced are intended as disposable packaging, and nearly all these 

plastics are derived from petroleum and are highly resistant to natural biodegradation. Because 

plastic is recycled at rates of only a few percent in most countries, plastics are rapidly 

accumulating in unproductive and virtually permanent landfills. Additionally, pollution results 

from the manufacture, use, and disposal of plastic materials. Notwithstanding, plastics have 

significant benefits for society, such as abundant sterile medical supplies; increased agricultural 

production; reduced food spoilage; reduced fuel consumption in lighter-weight vehicles; and, 

low-cost, net-shape manufacturing. Increases in oil prices that consumers are experiencing at the 

gas pump are also impacting the plastics industries where production costs are rising and being 

passed on to the consumer. While energy recovery through combustion, recycling, and 

minimizing plastics use can all aid pollution prevention, the new industrial biotechnology 

paradigm can be a more environmentally benign solution to the societal, economic, and 

environmental impacts of increasing oil consumption. 

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iv 

Economically competitive properties are now well within reach among biomaterials 

including starch cellulosics, proteins, polylactides, soy and plant oil-based plastics, and 

polyhydroxyalkanoates. 

Several research priorities have been identified to improve production of biomaterials. 

The primary need is the development of standards for assessing the energy content and emissions 

profiles for plastic materials. This is essential to ensure the pursuit of truly environmentally 

benign materials. 

Metabolic engineering routes to the synthesis of monomers and polymers should also be 

explored due to the increasing practicality of inducing the necessary genetic changes. Advances 

in metabolic pathway modeling can further enhance these efforts by indicating promising targets 

for genetic engineering. Among these, modifications necessary to enable use of waste biomass 

feedstocks, such as lignocellulosics and waste oils, or to enable biosynthesis within crops that 

can be grown on marginally productive lands, such as switchgrass, would be especially valuable. 

Biopolymers and other bioplastics could be more widely used in place of petroleumbased plastics if their physical properties (such as heat resistance and moisture permeability) 

could be improved. Conventional composites and nanocomposites in which the reinforcing 

agents are also based on renewable resources (biocomposites) are of particular interest in this 

context. In addition, biopolymers based on inexpensive monomers presently available using 

conventional fermentation technologies should be explored and developed where feasible. 

B. BIOFUELS 

Energy is a central issue in economic sustainability. Production and distribution of 

inexpensive energy in a variety of forms (electricity, heating and transportation fuels) is essential 

for maintaining industries and for supporting stable lifestyles for people. Transportation fuels 

dominate use of imported petroleum. 

In the area of biofuels, fostering collaboration of scientists and engineers is critical. The 

absence of interdisciplinary collaboration is repeatedly cited as one of the greatest limits to the 

scale-up and commercialization of bioenergy technologies. Collaborations between those 

attempting to understand and engineer the organisms and those attempting to design optimal 

bioreactors and bioseparation processes should be encouraged and when appropriate, should 

include researchers from industrial laboratories. Such teams are essential to commercialization 

of the advances made in academic and governmental laboratories. 

Within the field of bioethanol production, the most important challenge is the 

development of feedstocks based on waste biomass. Research in this area will end or reduce 

reliance on crops produced with conventional energy-intensive practices that makes bioethanol 

no more sustainable than fossil fuels. Obstacles to waste-based bioethanol include the absence 

of high-performance, low-cost cellulase enzymes and/or cellulolytic organisms; the separation of 

lignin from cellulose; the optimization of simultaneous fermentation of hexoses and pentoses; 

and the purification of the ethanol and recovery of other valuable byproducts. 

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v 

Biodiesel development is also dependent on non-sustainable agriculture. Therefore, 

sustainable production of oilseed crops and the development of technologies to allow use of 

waste oils are important priorities. Following these, bioengineering advances are needed to be 

efficient in transesterification and separations. 

Relative to other biofuels, biohydrogen is still in its infancy. Biohydrogen technology is 

actually five distinct technologies (direct photolysis, indirect photolysis, photofermentation, 

water-gas shift production, and dark fermentation) involving four very different types of 

microorganisms (green algae, cyanobacteria, purple non-sulfur bacteria, and anaerobic 

heterotrophic bacteria, respectively). While each type of technology faces its own particular 

challenges, a few priorities are common to all. 

Of utmost importance to the three photolytic technologies is the reduction of 

photosynthetic antenna pigments. Second, the addition of heterologous pigments to allow 

utilization of photons outside the photosynthetic spectrum is also desirable. Vital to the two nonphotolytic technologies is the development of sustainable organic carbon sources. Current 

efforts underway to use various waste sources as substrates should be strongly encouraged to 

continue. 

Within the realm of biorefinery platform technologies, the development of integrated 

bioreactor-bioseparation unit operations should be supported, especially those that can overcome 

inherent limitations of bioprocessing and other integrated designs that selectively remove the 

components limiting organism growth. Integration offers tremendous benefit for relatively little 

investment. Bioreactor design and operation can be further optimized with the assistance of 

improved theoretical models for reaction kinetics, including structured models. This would 

provide commercial viability for commodity products with narrow profit margins and for 

membrane-based separation technologies. In addition, new benign solvent extraction processes 

are needed for bioseparations to avoid fossil-based and/or toxic solvents−supercritical CO2 is an 

excellent example. The development of new strategies to suppress or control membrane fouling 

in relevant separations would also be immensely useful. 

C. RESEARCH STRATEGIES 

Fostering interdisciplinary research can be accomplished easily by encouraging 

multidisciplinary teams through the research grant award process. Also, certain elements of the 

needed bioengineering platform technologies are already well supported by the USDA and to a 

lesser extent by the DOE. A joint program between the USDA/DOE that supports the 

development of bioengineering for energy production is in place. Additional partnerships with 

these agencies would help to improve the knowledge base of bioengineering for pollution 

prevention. 

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Contents 

Disclaimer...... .............................................................................................................................. ii 

Acknowledgements ....................................................................................................................... ii 

Executive Summary..................................................................................................................... iii 

A. BIOMATERIALS ......................................................................................................iii 

B. BIOFUELS................................................................................................................. iv 

C. RESEARCH STRATEGIES ....................................................................................... v 

List of Acronyms and Abbreviations......................................................................................... xi 

Chapter I Introduction.................................................................................................................1 

A. CURRENT STATUS OF ENERGY AND MATERIALS FEEDSTOCKS............... 1 

1. The Petroleum Resource .................................................................................... 1 

2. Benefits of Petroleum Replacement................................................................... 2 

3. Unique Contributions from Biotechnology........................................................ 3 

B. CURRENT CHALLENGES ....................................................................................... 4 

1. Cellulose Stability .............................................................................................. 4 

2. Engineering Optimal Organisms........................................................................ 5 

3. Bioreactors and Bioseparations.......................................................................... 5 

C. TERMS AND DEFINITIONS .................................................................................... 6 

1. Bioengineering ................................................................................................... 6 

2. Pollution Prevention........................................................................................... 6 

3. Materials............................................................................................................. 6 

4. Biopolymers, Bioplastics.................................................................................... 6 

5. Energy and Fuels................................................................................................ 6 

6. Economics and Commercialization.................................................................... 6 

D. STATEMENT OF PURPOSE..................................................................................... 7 

E. REFERENCES............................................................................................................ 7 

Chapter II Biotechnological Platforms.......................................................................................9 

A. GENETIC ENGINEERING........................................................................................ 9 

1. Introduction ........................................................................................................ 9 

2. State of the Science ............................................................................................ 9 

3. Research Priorities............................................................................................ 20 

4. References........................................................................................................ 20 

B. BIOREACTOR TECHNOLOGIES.......................................................................... 23 

1. Introduction ...................................................................................................... 23 

2. State of the Science .......................................................................................... 24 

3. Research Priorities............................................................................................ 29 

4. Commercialization ........................................................................................... 31 

5. References........................................................................................................ 31 

C. BIOSEPARATIONS AND BIOPROCESSING....................................................... 34 

1. Introduction ...................................................................................................... 34 

2. State of the Science .......................................................................................... 35 

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viii 

3. Research Priorities............................................................................................ 39 

4. Commercialization ........................................................................................... 40 

5. References........................................................................................................ 40 

Chapter III Biomaterials............................................................................................................43 

A. PROBLEM AND SIGNIFICANCE.......................................................................... 43 

1. Overview of Importance................................................................................... 43 

2. Conventional Plastics....................................................................................... 44 

3. Plastics from Renewable Resources................................................................. 44 

4. Renewable Polymer Production ....................................................................... 46 

5. Environmental Benefits of Green Plastics........................................................ 47 

6. References........................................................................................................ 48 

B. POLYLACTIDES ..................................................................................................... 49 

1. Introduction ...................................................................................................... 49 

2. PLA Biosynthesis, Biodegradation, and Environmental Impact: Overview... 50 

3. State of the Science .......................................................................................... 52 

4. Research Priorities............................................................................................ 60 

5. Commercialization ........................................................................................... 61 

6. References........................................................................................................ 61 

C. POLYHYDROXYALKANOATES.......................................................................... 76 

1. Introduction ...................................................................................................... 76 

2. PHA Biosynthesis, Biodegradation, and Environmental Impact: Overview .. 77 

3. State of the Science .......................................................................................... 78 

4. Research Priorities............................................................................................ 83 

5. Commercialization ........................................................................................... 84 

6. References........................................................................................................ 84 

D. STARCHES, PROTEINS, PLANT OILS, AND CELLULOSICS .......................... 88 

1. Introduction ...................................................................................................... 88 

2. State of the Science .......................................................................................... 89 

3. Research Priorities............................................................................................ 96 

4. Commercialization ........................................................................................... 97 

5. References........................................................................................................ 99 

Chapter IV Biofuels..................................................................................................................103 

A. BIOETHANOL ....................................................................................................... 103 

1. Introduction .................................................................................................... 103 

2. State of the Science ........................................................................................ 104 

3. Research Priorities.......................................................................................... 111 

4. Commercialization ......................................................................................... 111 

5. References...................................................................................................... 112 

B. BIODIESEL ............................................................................................................ 117 

1. Introduction .................................................................................................... 117 

2. State of the Science ........................................................................................ 120 

3. Research Priorities.......................................................................................... 125 

4. Commercialization ......................................................................................... 126 

5. References...................................................................................................... 126 

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C. BIOHYDROGEN.................................................................................................... 129

1. Introduction .................................................................................................... 129 

2. State of the Science ........................................................................................ 130 

3. Research Priorities.......................................................................................... 147 

4. Commercialization ......................................................................................... 148 

5. References...................................................................................................... 148 

D. BIODESULFURIZATION OF FOSSIL FUELS.................................................... 155 

1. Introduction .................................................................................................... 155 

2. State of the Science ........................................................................................ 155 

3. Research Priorities.......................................................................................... 161 

4. Commercialization ......................................................................................... 161 

5. References...................................................................................................... 162 

Chapter V Summary of Future Research Priorities..............................................................165 

A. INTRODUCTION................................................................................................... 165 

B. GENETIC ENGINEERING.................................................................................... 165 

C. BIOREACTOR TECHNOLOGIES........................................................................ 165 

D. BIOSEPARATIONS AND BIOPROCESSING..................................................... 166 

E. POLYACTIDES...................................................................................................... 168 

F. POLYHYDROXYAKANOATES .......................................................................... 169 

G. STARCHES, PROTEINS, PLANT OILS, AND CELLULOSICS ........................ 170 

H. BIOETHANOL ....................................................................................................... 171 

I. BIODEISEL ............................................................................................................ 171 

J. BIOHYDROGEN.................................................................................................... 171 

K. BIODESULFURIZATION OF FOSSIL FUELS.................................................... 173 

APPENDIX: Contributions of the NSF/EPA Technology for a Sustainable 

Environment Program 1995–2004................................................................. A-1 

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List of Acronyms and Abbreviations 

13C Carbon 13 

14C Carbon 14 

2D 2-Dimensional 

3D 3-Dimensional 

3G 1,3-Propanediol 

Al Aluminum 

ANNs Artificial Neural Networks 

AOX Alcohol Oxidase 

ATP Adenosine Triphosphate 

ASTM American Society for Testing and Materials 

B20 20 percent Biodiesel combined with 80 percent Petroleum Diesel 

B100 100 percent Pure Biodiesel 

BCI BC International Corporation 

BRI BioEngineering Resources, Inc. 

BSPs Biomass Support Particles 

CA Cellulose Acetate 

CAB Cellulose Acetate Butyrate 

CAFI Consortium for Applied Fundamentals and Innovation 

CAP Cellulose Acetate Propionate 

CBP Consolidated Bioprocessing 

C–C Carbon-carbon Bond 

cDNA complementary DNA 

CDP Cargill with Dow Polymers 

CFD Computational Fluid Dynamics 

CH4 Methane 

CIE Compression-injection Engines 

CO Carbon Monoxide 

CO2 Carbon Dioxide 

CODH Carbon Monoxide Dehydrogenase 

CUBIC Columbia University Bioinformatics Center 

DBT Dibenzothiophene 

DNA Deoxyribonucleic Acid 

DOE Department of Energy 

DSA Diafiltration Saccharification Assay

dsRNA double-stranded Ribonucleic Acid 

Dsz Biodesulfurization 

DXO 1,5-Dioxepan-2-one 

EMBL European Molecular Biology Laboratory 

EPA Environmental Protection Agency 

Fd Ferredioxin 

FFV Flexible-fuel Vehicles 

GCN General Control 

H+ Hydrogen Ion 

H2 Molecular Hydrogen 

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HA Hydroxyalkonate 

IPTG Isopropyl-β-D-thiogalactoside 

ISO International Organization for Standardization 

JGI Joint Genome Institute 

LCA Life-cycle Analysis 

LCIA Life-cycle Impact Assessment 

LDPE Low-density Polyethelyne 

MF Microfiltration 

MFA Metabolic Flux Analysis 

Mg Magnesium 

MG D, L-3-methyl glycolide 

Mg2+ Magnesium (II) Ion 

Mn2+ Manganese (II) Ion

mRNA messenger RNA 

MTBE Methyl Tertiary Butyl Ether 

MW Molecular Weight 

NADPH Reduced form of Nicotinamide Adenine Dinucleotide Phosphate 

NADP Nicotinamide Adenine Dinucleotide Phosphate 

NCBI National Center for Biotechnology Information 

NIH National Institutes of Health 

NMR Nuclear Magnetic Resonance 

NREL National Renewable Energy Laboratory 

NRMRL National Risk Management Research Laboratory 

NSF National Science Foundation 

PCR Polymerase Chain Reaction 

PE Polyethylene 

PEA Polyester Amide 

PET Poly(ethylene terephthalate) 

PHA Polyhydroxyalkanoate 

PHB Polyhydroxybutyrate 

PLAs Polylactic Acids 

Poly (3HB) Poly [(R)-3-hydroxybutyrate] 

PP Polypropylene 

PPM Primary Packaging Material 

ppm parts per million 

PS Polystyrene 

PS I Photosystem I 

PS II Photosystem II 

PVC Polyvinyl Chloride 

[(R)-3HAs] (R)-3-hydroxyalkanoates 

RISC RNA-induced Silencing Complex 

RNA Ribonucleic Acid 

SEM Scanning Electron Microscope 

siRNA short interfering RNA 

Sn Tin 

SO2 Sulfur Dioxide 

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SOx Sulfur Oxides and Dioxides 

SSCF Simultaneous Saccharification and Co-fermentation 

SSF Simultaneous Saccharification and Fermentation 

ssRNA single-stranded RNA 

STRs Stirred-tank Reactors 

SV40 Simian Virus 40 

TCA Tricarboxylic Acid 

TEC Triethyl Citrate 

TMC Trimethylene Carbonate 

TPS Thermoplastic Starch 

TSE Technologies for a Sustainable Environment 

UF Ultrafiltration 

USDA United States Department of Agriculture 

UV Ultraviolet 

VOC Volatile Organic Compounds 

WAXS Wide-angle X-ray Scattering 

Zn Zinc 

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1 

Chapter I 

Introduction 

A. CURRENT STATUS OF ENERGY AND MATERIALS FEEDSTOCKS 

1. The Petroleum Resource 

Resource shortages are a natural consequence of the utility of the resource combined with 

human ingenuity. As a new resource is discovered, people invent uses for it in proportion to its 

adaptability; increased uses typically increase the demand for it often resulting in increased 

production, which thereby increases the opportunity for new uses to be discovered, and so on. In 

the case of petroleum, this cycle has progressed to such an extent that developed world 

economies are dependent on it for heat, food (through agriculture), shelter (through synthesis of 

construction materials), and transportation. 

In the United States, for example, petroleum use has increased steadily from 

approximately 9.8 million barrels per day in 1960 to ~21 million barrels per day in 2005 (1). 

The present rate of demand increase is ~1.5 percent per year resulting in U.S. demand expected 

to increase 37 percent over 2004 levels by 2025 (2). Within this demand, transportation and 

industry, the latter including plastics and materials production, consume the greatest shares at 

~66 percent and ~25 percent, respectively. Comparable increases have been observed in other 

developed countries as well (3, 4).

If petroleum were plentiful, widely distributed, and environmentally benign, this situation 

would be no cause for concern. Unfortunately, though, it is not the case. 

First, the demand of the developed economies for petroleum is now reaching a level that 

is comparable to known reserve limits. For example, an estimated 875 billion barrels of oil have 

been consumed since the dawn of the oil age (5), while 1.7 trillion barrels remain in proven 

reserves (within oil fields discovered but not yet pumped out), and another estimated 900 billion 

remain to be discovered (6). Of the 875 billion consumed, however, greater than 60 percent (550 

billion) have been consumed since 1975. With world demand continuing to rise at ~2 percent 

per year, the world production peak is estimated to occur between 2026 and 2047 (5). Within the 

United States, crude oil production is expected to peak in 2010 (2), and the remainder of the 

petroleum that is easily accessible given both geological and political constraints is expected to 

peak in that approximate time frame as well (6). 

This raises the second issue, that of petroleum accessibility, given both geological and 

political constraints. Geological factors dictate that not all petroleum is equally accessible. 

Fields that may lie in temperate zones within several hundred feet of the surface have 

understandably been the first to be exploited. This leaves oil beneath oceans, in remote Arctic 

regions, tightly associated with sands, and/or laden with impurities as an increasing component 

of that that remains to be exploited. Technological advances have greatly increased the amount 

of petroleum that can be extracted from the earth, once discovered, but often at high operational 

and environmental cost. 

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2 

Political factors also contribute to petroleum accessibility. While the United States was 

once the world’s greatest petroleum producer, it and many other developed countries are now net 

importers: the United States now imports approximately 56 percent of its demand, or ~11.2 

million barrels per day (7, 8). The United States is not only the greatest consumer of world 

petroleum resources–demanding ~25 percent of the global total production (4)–but notable to 

foreign suppliers, the United States is also the greatest importer, with nearly double the net 

imports of second-ranked Japan (9). Of the 1.7 trillion barrels of oil in the world’s proven 

reserves, over half of those are located in the Middle East. Petroleum suppliers are therefore 

becoming increasingly concentrated in regions of the world, particularly the Middle East, that 

have historically been politically unstable and/or unfriendly to western interests (6). 

Finally, petroleum is far from environmentally benign. Petroleum combustion releases 

carbonaceous gases, principally carbon dioxide (CO2), carbon monoxide (CO), and methane 

(CH4), as well as sulfurous gases such as sulfur dioxide (SO2). Decades of climate and 

atmospheric composition data are now confirming the link between increasing concentrations of 

greenhouse gases such as those emitted by combustion of fossil fuels and increasing global 

temperatures (10). In addition, concerns are growing about the volume of discarded wastes that 

the United States and other countries produce. Global consumption of petroleum-based 

thermoplastics, the greatest component, now exceeds 100 million tons per year, of which 

approximately half is discarded within two years of production. Much of the other half, used to 

generate products with longer lifetimes, is just beginning to enter the waste stream, with the 

result that plastic waste generation is expected soon to exceed the growth in consumption (11). 

This, in turn, is expected to create a considerable demand on landfill space (12). An 

accompanying problem is that wealthy countries can export such wastes to poorer countries. 

Although these practices have been addressed through measures such as the Basel Ban, 

diminishing waste volumes is the most straightforward solution to exploitation of vulnerable 

peoples and natural areas (13). As a result, the impetus for transition from fossil fuels to 

renewable energy sources and materials feedstocks is resulting as much from environmental 

considerations as it is from concerns about future conflict over petroleum resources. 

2. Benefits of Petroleum Replacement 

Numerous technologies are under development for the replacement of petroleum as the 

primary energy source and materials feedstock in developed countries. Wind, solar, 

hydroelectric, geothermal, and biomass-derived power will each be called upon to contribute to 

the post-petroleum economy, and conservation measures are also expected to receive greatly 

increased attention. Materials derived from biological molecules are also gaining diversity and 

availability. 

To what extent can petroleum be replaced, and by what alternatives, within the next 

decades? While this potential is debatable, a realistic best-case scenario can be presented given 

recent projections. In April, 2005, a report by the U.S. Department of Energy (DOE) and the 

U.S. Department of Agriculture (USDA) estimated that the United States could quite feasibly 

produce 1 billion dry tons of biomass feedstock (over half of which is waste) per year, enough to 

displace 30 percent or more of the present U.S. petroleum consumption for fuels and materials 

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3 

by 2030 (14). When this is combined with projections of solar and wind energy together 

providing 20 percent of the power demand in the industrialized world by that time, it appears that 

biological resources could become important contributors to the evolution of a post-petroleum 

world. Under these projections, CO2 emissions could peak before 2050 and conventional fossil 

fuel use could be virtually eliminated by 2100 (6). 

3. Unique Contributions from Biotechnology 

The application of biotechnology to the production of commodities–notably fuels, 

chemicals, and structural materials–increases the array of options available to supply sustainable 

resources and preserve the environment. In particular, through the use of biological feedstocks, 

biotechnology has the potential to minimize greatly the overwhelming dependence developed 

countries, particularly the United States, have on petroleum and other non-renewable fossil fuels 

for production of fuels and plastics.

Numerous mechanical, geothermal, and electrical technologies offer valuable 

contributions to issues of energy independence and pollution prevention (15). At the same time, 

several unique advantages are brought by biofuels and bioproducts that will be highlighted in this 

report. 

First, many biotechnologies use the abundant, renewable, and potentially sustainablyproduced resource of plant biomass as the primary feedstock for liquid biofuels, biochemicals, 

and biomaterials. Cellulosic and other biomass is currently available at the commodity scale and 

is increasingly cost-competitive with petroleum, especially when environmental costs are 

included, on both energy and mass bases (16). Indeed, the land resources of the United States 

are capable of producing a sustainable supply of biomass sufficient to displace 30 percent or 

more of the country’s present petroleum consumption, amounting to approximately 1 billion dry 

tons of biomass feedstock per year (14). 

Second, microbiotechnologies have the potential to use simple, organic, and inorganic 

feedstocks in microbe-based bioreactors that generate desired products directly, without plant 

biomass intermediates. For example, photosynthetic microbial biohydrogen production requires 

only sunlight, CO2, salts, and water (17), and bioplastic precursors such as polylactic acid and 

polyhydroxybutyrate can be made directly by microbes as well (18, 19). 

Third, biotechnologies make use of enzymes, proteinaceous catalysts that are often 

exquisitely selective and provide high rates of product generation. Unlike other catalysts, 

enzymes can be manipulated genetically to improve parameters such as substrate affinity, 

specificity, and catalytic rate, as well as tolerance to process conditions, longevity, and even 

production rate of the enzyme itself by the host cell. 

Finally, as a result of the above, many biotechnologies are able to avoid use of toxic 

feedstocks and processing reagents that are necessitated by conventional methods and thereby 

minimize toxic wastes. For example, biosynthesis of the denim dye, indigo, requires only 

glucose as substrate, in contrast to the conventional synthesis that requires benzene or other 

aromatic solvents (20). 

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4 

Bioengineering for pollution prevention is an emerging area of both an intellectual 

endeavor and an industrial practice. The economic driving forces, the importance of feedstock, 

and the scale of production all distinguish this arena of biotechnology from the pharmaceutical 

and nutritional sectors. While fossil fuel-based economies typically evolve from a relatively 

low-value commodity (e.g., kerosene for lighting) to intermediate-value materials (gasoline, 

plastics) and ultimately to valuable specialty chemicals (cosmetics, pharmaceuticals), it appears 

that the biobased economy is progressing from high-value products (pharmaceuticals) to those of 

intermediate value (industrial catalysts, plastics). As biotechnology evolves and matures, the 

production of large-scale, relatively low-value products such as fuels is becoming increasingly 

attractive and economically feasible. 

B. CURRENT CHALLENGES 

1. Cellulose Stability 

The greatest impediment to widespread application of bioengineering for production of 

commodities is currently the general absence of low-cost processing technologies for biomass. 

Challenges associated with the conversion of plant biomass into useful products are dominated 

by the chemical stability of cellulose within biomass (Figure 1), causing it to resist modification 

and therefore require valuable enzymes or other catalysts, as well as special processing 

conditions (21). Advances are therefore greatly needed in both enzymatic and non-enzymatic 

biomass pretreatment technologies, as well as in the development of efficient product-producing 

microbes and fermentation bioreactor technologies, the latter of which would directly benefit 

non-biomass-consuming processes as well. The generation of high-value coproducts has the 

potential greatly to offset expenses of processing any feedstock, showing that exploration of the 

diversity of products that a process or feedstock can yield is also of central importance to the 

realization of true, profitable, economically resilient biorefineries. 

Figure 1. Structure of cellulose as it occurs in a plant cell wall. 

 from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates 

(www.sinauer.com) and WH Freeman (

 

 Reprinted

www.whfreeman.com), with permission. 

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5 

2 Engineering Optimal Organisms . 

niques are now being used to address the 

icrobial and enzym

 lowwell 

ologies must play important roles in the 

evelop

Important considerations in the overall sustainability of both 

iofuels

el 

y 

, 

Genetic and metabolic engineering tech

m atic problems of biocommodity production from hundreds of different 

angles. The central goal, in virtually all cases, is the development of organisms that can use

cost substrates, give high product yields, and/or exhibit robustness in temperature and pH 

extremes characteristic of many industrial environments. The rapidity of development of 

techniques for manipulation and/or analysis of gene sequences and expression patterns, as 

as the exponential rate of accumulation of genetic information about numerous industrial 

microorganisms, are enormous forces propelling the field of bioengineering forward (22). 

3. Bioreactors and Bioseparations 

Similarly, reactor and separations techn

d ment of economically feasible biorefineries, as these processing steps often contribute 

the greatest expense of the final products. At early stages of development, new bioengineering

processes often benefit greatly from combination of separate components. For example, 

integrating unit operations, as into reactor-separators and other novel processes, often provides 

immediate improvements in efficiency. Similarly, integrating individual production steps into a

multi-product biorefinery, and integrating biorefineries into the broader economic and 

environmental systems in which they function, are important avenues to economic feasibility. 

To evaluate energy, material, and cost efficiencies of these systems, Life-cycle Analysis (LCA)

tools are invaluable and are themselves undergoing rapid development (23). An enormous 

barrier exists in the funding and deployment of any pioneer manufacturing plant, and federal 

programs may be necessary to provide loan guarantees and other incentives to encourage 

enterprise in this direction (24). 

Complete sustainability. 

b and biomaterials include the fossil-energy costs of agriculture and processing. For 

example, current practices of conventional agriculture use petroleum-powered machinery and 

fertilizers synthesized with fossil energy. In addition, conventional processing techniques use 

nonsustainable energy sources. Furthermore, the treatment of acidic, alkali, and/or organic 

wastes resulting from processing techniques consumes additional energy. As a result, no biofu

or biomaterial is currently completely sustainable as an energy resource or completely free of 

pollution generation. Bioethanol, in particular, has come under criticism for its use of 

intensively-cultivated food crops, while several bioplastics potentially use as much fossil energ

in their processing as petroplastics use in their feedstocks and processing combined. For this 

reason, development of technologies that use waste or other biofeedstocks with low-embodied 

energy (energy consumed in its production) is a high priority for both biofuels and biomaterials

as is development of efficient bioprocesses, including enzymatic and therefore low-wastegenerating catalysis. Underlying each of these, moreover, is the importance of developing 

sustainable agriculture. 

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C. TERMS AND DEFINITIONS 

The field of bioengineering for pollution prevention as outlined is immense. A 

tremendous number of talented and energetic people are working to bring the elements outlined 

in the preceding paragraphs into reality. To create a coherent, focused, and thorough document, 

the authors have limited the discussion to the areas of energy and materials, and provide here a 

set of terms that further define, limit, and clarify the topics involved. 

1. Bioengineering 

In its broadest sense, this term is applied to the manipulation, influence, or purposeful 

design of any entity, material, or process involving biological components. For the purpose of 

this document, however, a more restricted definition is adopted, in which the engineering itself 

must involve the manipulation and/or exploitation of biological components themselves to 

accomplish the desired task. 

2. Pollution Prevention 

In the context of this document, “pollution prevention” is restricted to the description of 

processes that deliberately avoid the generation of environmentally deleterious substances. 

Processes that reclaim, recycle, or degrade such substances, once generated, are excluded. 

3. Materials 

This term is broadly used to represent any form of matter. 

4. Biopolymers, Bioplastics 

These terms are used specifically to represent high-molecular-weight structural materials 

that can be shaped or otherwise manufactured into useful articles for human use. 

5. Energy and Fuels 

“Energy” in this document emphasizes biological, biologically-produced, or biologicallymodified materials or processes capable of generating heat or power. Thus, all significant 

biofuels (bioalcohols, biodiesel, biohydrogen) are included, in addition to conventional fuels that 

have been enzymatically treated to reduce the generation of pollutants. 

6. Economics and Commercialization 

In a market economy, the financial aspects of a new technology inevitably have a major 

impact on its adoption. Indeed, the majority of technological limitations and research needs 

described below derive their importance from the need to make pollution prevention 

technologies competitive with their conventional counterparts. Nevertheless, as a review of the 

state of the science, thorough analyses of the process economics of each technology are beyond 

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the scope of this work. Many excellent reviews of this aspect exist however, and are cited in the 

text. 

D. STATEMENT OF PURPOSE 

The purposes of this document are four-fold: first, to explore the realm of current and 

developing technologies in the fields of biomaterials and biofuels that benefit environmental 

integrity through their production and use; second, to identify the most promising and most 

essential areas of endeavor within each field, thus highlighting top priorities for further research 

and development; third, to examine the technological challenges and/or barriers to the progress 

of the given technologies; and fourth, to elucidate the contributions of the NSF/EPA’s 

Technology for a Sustainable Environment Program for each topic area. 

E. REFERENCES 

(1) Energy Information Administration (2005). Annual energy outlook 2005, 

http://www.eia.doe.gov/oiaf/aeo/. 

(2) Reuters (2004). Petroleum demand to grow 37 percent by 2025–EIA, MSNBC, 

Washington, D.C. 

(3) U.S. Department of Energy, Energy Information Administration (2003). Annual energy 

review 2002, Washington, D.C., http://www.bts.gov/publications/

pocket_guide_to_transportation/2004/html/figure_06_table.html. 

(4) Bureau of Transportation Statistics (2004). Overview of U.S. Petroleum Production, 

Imports, Exports, and Consumption, Washington, D.C., http://www.bts.gov/publications/

national_transportation_statistics/2004/html/table_04_01.html. 

(5) Wood, J. H., G. R. Long, and D. F. Morehouse (2004). Long-Term World Oil Supply 

Scenarios, Energy Information Agency, http://www.eia.doe.gov/pub/oil_gas/

petroleum/feature_articles/2004/worldoilsupply/oilsupply04.html. 

(6) Roberts, P. (2004). The End of Oil. Houghton Mifflin, Boston. 

(7) U.S. Department of Energy, Energy Information Administration (2003). Net Imports of 

Crude Oil and Petroleum Products into the United States by Country, 2003, Washington, 

D.C., Table 29 Petroleum Supply Annual 2003, Volume 1, page 68. 

(8) U.S. Department of Energy, Energy Information Administration (2004). U.S. Petroleum 

Imports and Exports, Washington, D.C., http://www.eia.doe.gov/oil_gas/petroleum/

info_glance/importexport.html. 

(9) U.S. Department of Energy, Energy Information Administration (2001). Top Petroleum 

Net Importers, Washington, D.C., http://www.eia.doe.gov/emeu/security/topimp.html. 

(10) U.S. Environmental Protection Agency (2000). Global Warming–Climate, 

http://yosemite.epa.gov/oar/globalwarming.nsf/content/Climate.html. 

(11) Greenpeace (1998). The Plastics Boom and the Looming PVC Waste Crisis, Amsterdam, 

The Netherlands, http://archive.greenpeace.org/comms/pvctoys/reports/

loomingcontents.html. 

bioengineering report_5_x1a.pdf 21 5/25/07 6:44:44 PM
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(12) Manchester Metropolitan University (2004). Atmosphere, Climate, & Environment 

Information Programme: Waste Fact Sheet Series, Manchester, UK, 

http://www.ace.mmu.ac.uk/Resources/Fact_Sheets/Key_Stage_4/Waste/05.php. 

(13) Basel Action Network (1997). The Basel Ban: A Triumph Over Business-As-Usual, 

Basel, Switzerland, http://www.ban.org/about_basel_ban/jims_article.html. 

(14) U.S. Department of Energy and U.S. Department of Agriculture (2005). Biomass as 

Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a 

Billion-Ton Annual Supply, Oak Ridge, TN, DOE/GO-102995-2135. 

(15) Hester, R. E., and R. M. Harrison, Eds. (2003). Sustainability and Environmental Impact 

of Renewable Energy Sources. Springer-Verlag Telos, New York, NY. 

(16) Lynd, L. R., and C. E. Wyman (1999). Testimony Before the Senate Committee on 

Agriculture, Nutrition, and Forestry on Senate Bill S935: Sustainable Fuels and 

Chemicals Act of 1999. Thayer School of Engineering, Dartmouth College, Dartmouth, 

NH. 

(17) Levin, D. B., L. Pitt, and M. Love (2004). Biohydrogen production: prospects and 

limitations to practical application, Intl J Hydrogen Energy 29:173-185. 

(18) Salehizadeh, H., and M. C. van Loosdrecht (2004). Production of polyhydroxyalkanoates 

by mixed culture: recent trends and biotechnological importance, Biotechnol Adv 

22:261-279. 

(19) van Maris, A. J. A., W. N. Konings, J. P. van Dijken, and J. T. Pronk (2004). Microbial 

export of lactic and 3-hydroxypropanoic acid: Implications for industrial fermentation 

processes, Metabo Eng 6:245-255. 

(20) Berry, A., T. C. Dodge, M. Pepsin, and W. Weyler (2002). Application of metabolic 

engineering to improve both the production and use of biotech indigo, J Ind Microbiol 

Biotechnol 28:127-133. 

(21) Klemm, D., B. Philipp, T. Heinze, U. Heinze, and W. Wagenknecht (1998). 

Comprehensive Cellulose Chemistry, Volume 2: Functionalization of Cellulose. WileyVCH. 

(22) Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and 

K. Struhl, Eds. (1988 [updated quarterly]). Current Protocols in Molecular Biology, 

Ringbound Edition. Greene Publishing Associates. 

(23) Hauschild, M. Z. (2005). Assessing Environmental Impacts in a Life-Cycle Perspective, 

Environ Sci Technol 39:81A-88A. 

(24) Ingram, L. (2005). Personal communication. 

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Chapter II 

Biotechnological Platforms 

A. GENETIC ENGINEERING 

1. Introduction 

 The diversity of biological activity and biological products offered by nature is a great 

resource for biotechnology; inevitably, however, the limits of natural products or activities are 

eventually found, and ways are sought to improve them. In these cases, either the environment 

can be manipulated (as in bioreactors, described separately) or the organism itself can be 

manipulated physiologically or genetically. 

 The possibilities offered by genetic engineering have grown dramatically in recent 

decades, and it is currently possible to alter the capabilities of organisms, especially of microbes 

and plants, such that they exhibit activities and generate products vastly different from those of 

their genetic precursors. While a thorough review of current genetic engineering technology is 

beyond the scope of this report, it is nevertheless useful to consider the methods most widely 

utilized in the development of biofuels and bioplastics. These methods include: 

• The cloning of key genes (regions of deoxyribonucleic acid (DNA) that encode 

enzymes) so that they may be moved and changed at will; 

• The detection of gene expression patterns by microarray analysis (facilitating the 

controlled expression or over-expression of desired genes in native or heterologous 

hosts); 

• The corresponding deletion or repression of expression of undesired genes; 

• The use of genomic techniques to predict functions of genetic loci; 

• The mutagenesis of genes to provide variants with altered activities, specificities, 

environmental sensitivities, etc.; and 

• The integration of the above methods to construct new biosynthetic pathways. 

2. State of the Science 

2.1 Cloning and Sequencing 

 The “cloning” of a gene refers to the physical isolation of a sequence of DNA that 

encodes a complete polypeptide in a form that can be replicated and manipulated easily. 

Typically, this involves extraction of total genomic DNA from an organism, use of restriction 

enzymes to cut the DNA into manageable fragments, and ligation of the fragments into DNA 

vectors known as plasmids or cosmids. The collection of fragments is known as a “library,” and 

screening of the library by molecular or genetic methods ultimately leads to identification of the 

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vector bearing the desired gene. Further manipulation, including gene isolation through 

subcloning of the genomic fragment, is then possible (1). 

 Gene cloning is a well-established technology that has nevertheless become far more 

efficient with advances in enzyme and DNA purification technologies. The cloning of 

industrially-important genes is essential to convenient gene expression because, while desirable 

genes may be found in numerous organisms, only a few organisms are sufficiently durable and 

productive for industrial bioprocesses. Because genes are expressed through the action of 

numerous enzymes that are conserved to some extent among organisms, it is often possible to 

express a cloned gene from one organism in another; moreover, it is also often possible to 

control the extent of expression very closely, as described below. 

2.2 Genomics 

The field of genomics–the study of the sequence, structure, and function of an organism’s 

complete set of genetic information (its genome) (2)–is expanding rapidly due to the invention of 

numerous high-efficiency and high-throughput technologies capable of handling large amounts 

of DNA as well as sequence data. A key player in this field, and especially important from the 

perspective of environmental biotechnology, is the Joint Genome Institute (JGI) 

(www.jgi.doe.gov). The JGI was established in 1997 to unite the expertise and resources in 

genome mapping, DNA sequencing, and information sciences among the DOE genome centers 

at the Lawrence Berkeley National Laboratory, the Lawrence Livermore National Laboratory, 

and the Los Alamos National Laboratory. Its mission is to advance high-throughput, genomescale, computational technologies that facilitate understanding of relationships among genome 

structure and function, and it now has the capacity to generate DNA sequences of two billion 

nucleotide bases per month. The JGI contributed complete sequences of Chromosomes 5, 16, 

and 19 to the Human Genome Project but now, in contrast to National Institutes of Health (NIH)-

funded genome sequencers that continue to concentrate on human targets and applications, the 

project has turned its efforts toward the broader biosphere. Importantly, a primary goal of the 

JGI is to make high-quality genome sequence data freely available to the scientific community 

through its Web site (http://www.jgi.doe.gov/sequencing/seqplans.html). The sequences of 

numerous bacteria, fungi, trees, green algae, protists, crop plants, fish, and amphibians, either 

presently available or are planned to be available, will continue to be a tremendous resource for 

the development of bioplastics and biofuels as well as other areas of environmental 

biotechnology. 

Another highly important component of the current genomics landscape is GenBank®, the 

annotated NIH genetic collection that holds all publicly available DNA sequences in a searchable 

database (3). This set includes virtually all sequences published, because many journals require 

submission of sequence information to a database prior to publication and because GenBank 

exchanges data daily with other members of the International Nucleotide Sequence Database 

Collaboration (the DNA DataBank of Japan and the European Molecular Biology Laboratory). 

GenBank is accessible through the National Center for Biotechnology Information (NCBI) 

search engine, which integrates data from the DNA and protein sequence databases with 

taxonomy, genome mapping, protein structure, and domain information, as well as journal 

literature (http://www.ncbi.nlm.nih.gov/Genbank/index.html). 

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The value of complete, annotated, searchable genome sequence data for environmental 

biotechnology cannot be overstated. One example is the discovery of genes analogous to a gene 

of interest that may possess characteristics desirable for industrial processes or that may be 

desirable in combinatorial mutagenesis (see section 2.5.3). Other examples are the location of 

transposon-induced mutations or other mutations that have conferred desirable (or undesirable) 

phenotypes on an organism and the elucidation of the function of a DNA sequence that 

complements a mutant phenotype. Each of these contributes to the understanding of basic 

biochemical processes as well as to the development of industrially-useful bioprocesses. In 

addition, the progress of biotechnology is therefore significantly enhanced by progress in 

genomics (4, 5). 

2.3 Expression Control 

 Gene expression is the process by which a gene’s coded information is converted into 

active ribonucleic acid (RNA) and subsequently polypeptide molecules. Because the timing and 

extent of expression is crucial for cellular energetic and catalytic efficiency, transcription is 

controlled carefully in response to internal and external conditions. Upstream and intron-based 

DNA sequences known as promoters, enhancers, and other regulatory elements, as well as 

regulatory proteins known as transcription factors, are important factors in transcriptional 

control. Transcriptional regulatory elements differ in strength, or the rate at which they direct a 

cell to transcribe a gene into RNA, as well as in inducibility, or the stimuli to which they respond 

via transcription factors (6). 

2.3.1 Expression enhancement: strong and inducible promoters. Close 

control of expression of genes that direct synthesis of a desired product is advantageous and 

often essential to the development of a successful bioprocess, such that the maximum amount of 

substrate is converted to product while maintaining good health of the culture. One of the 

earliest and still widely-utilized promoters engineered for exquisite control of heterologous gene 

expression is the tac promoter, also referred to as Ptac. This sequence was designed from a 

combination of the inducible lac and trp promoters in the bacterium Escherichia coli, forming a 

hybrid that directs transcription up to 11 times more efficiently than the parental sequences. This 

operon is repressible by the lac repressor and is inducible by addition of isopropyl-ß-Dthiogalactoside (IPTG) (7). Numerous other expression systems have since been designed for 

both research and industrial purposes, prominently including those based on viral T4, T7, simian 

virus 40 (SV40), adenovirus, baculovirus, and cytomegalovirus promoters, the bacterial lac,

araB, and xyl promoters, and yeast alcohol oxidase (AOX) and general control (GCN) 4 

promoters (8, 9). 

2.3.2 Expression repression: RNA interference. Alternatively, it is sometimes 

desirable to prevent the expression of a gene altogether, or at least to diminish it significantly. 

While the ideal approach to the problem of gene silencing is the deletion of the gene in question, 

this is often a difficult and time-consuming process. An attractive emerging solution in 

eukaryotic cells is the use of RNA interference, or RNAi, to diminish the expression of particular 

genes with great specificity. This method takes advantage of an antiviral and possibly regulatory 

strategy in which double-stranded RNA (dsRNA), an unusual molecule within a cell, is first 

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bound by a nucleolytic enzyme called Dicer. This enzyme fragments the dsRNA into fragments 

of 21 to 23 nucleotides in length, known as small interfering RNAs (siRNAs), which bind in turn 

to an RNA-induced silencing complex (RISC). RISC then uses one strand of siRNA to bind to 

single-stranded RNA (ssRNA) molecules of complementary sequence, typically messenger 

RNAs (mRNAs), and cleave them. Because such mRNAs become untranslatable once cleaved, 

their expression is effectively blocked. 

Several methods have been developed for delivery of siRNAs to industrial cells: the 

chemical or enzymatic synthesis of siRNAs followed by transfection into the target cells, which 

is rapid and well-suited to preliminary experiments but is expensive and achieves only transient 

interference; the incorporation of siRNA sequences into a DNA plasmid, which also requires 

transfection but achieves stable interference once the vector is integrated into the host genome; 

and incorporation of siRNA sequences into viral vectors, which can achieve stable interference 

and can propagate themselves among target cells but possess potential biohazard risks (10, 11). 

2.4 Microarray Analysis 

Essential to the control of gene expression in complicated pathways, such as those 

involving the transport of mRNA and/or polypeptides among eukaryotic organelles for 

processing, cofactor insertion, and folding, is often the understanding of expression patterns of 

the multiple genes involved in the pathway. A tremendous advance in these efforts has been 

realized recently in the form of gene microarray technology. 

Microarrays are ordered sets of DNA molecules of known sequence applied, often 

robotically, in a grid of tiny spots onto a glass slide coated with an organic compound such as 

aminosilane to enhance DNA binding. Hundreds to thousands of distinct DNA molecules may 

be present in a single microarray, or gene chip, and are most often prepared in one of two ways: 

first, by a photolithographic process in which single-stranded oligonucleotides of unique, desired 

sequence are synthesized directly on the slide, most often employed in industry, or second, by 

physically attaching DNA fragments–such as polymerase chain reaction (PCR)-amplified 

genomic library clones–to the solid substrate, used almost exclusively in academic research. 

While the former allows higher density of features (>280,000 on a 1.28x1.28-cm array) and 

elimination of the need to generate cloned DNA or PCR products, the latter has lower cost and 

greater flexibility (12). 

For analysis of gene expression patterns in eukaryotes, messenger RNA is collected from 

cells of interest under two (or more) conditions of interest. Each sample is then reversetranscribed into complementary DNA (cDNA) using nucleotides labeled with contrasting 

fluorescent dyes. The cyanine dyes Cy3 and Cy5 are popular, although over 70 different dyes 

are available (13). In prokaryotes, a collection of mRNA is impractical due to the absence of 

polyadenylation; therefore, total RNA must be collected and labeled by covalent linkage or by 

using labeled, but random oligonucleotide primers during reverse transcription. 

On printed (non-photolithographic) DNA microarrays, relative transcript abundance is 

then measured by hybridizing cDNA samples to the microarray simultaneously and determining 

the fluorescence ratio, revealing binding of homologous sequences, for each spot on the array. 

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On photolithographic oligonucleotide arrays, in contrast, multiple probes from the same single 

gene of interest, each with a corresponding mismatch probe that serves as internal control, as 

well as known amounts of labeled transcripts for genes that serve as internal standards, are 

hybridized simultaneously to the microarray to enable quantitation of transcript abundance. 

Gene expression experiments can also be performed by hybridizing a single labeled 

mRNA sample to “macroarrays” of DNA elements that are supported on positively charged 

filters. Specialty arrays can be made and analyzed by this method relatively cheaply, and human, 

mouse, and microbial macroarrays are commercially available (SigmaGenosys, The Woodlands, 

TX; Research Genetics, Huntsville, AL; Clontech Laboratories, Palo Alto, CA; Genome 

Systems, St. Louis, MO). The major disadvantages of this format are reduced sensitivity, limited 

numbers of elements, and the need for higher concentrations of labeled cDNA (12). 

Microarray technology is sufficiently promising for medical and pharmaceutical 

applications that it is expected to continue to attract strong commercial interest leading to 

increasing array element density, greater detection sensitivity, and more cost-effective methods. 

Additional details and technical descriptions are available in recent reviews (14, 15). 

2.5 Mutagenesis 

 The DNA molecule consists of two strands of nucleotide bases held together by hydrogen 

bonds between complementary bases: adenosine pairs with thymine, and cytosine pairs with 

guanine. Within the coding region of a gene, each triplet of nucleotides specifies an amino acid, 

and the strand of amino acids in turn folds, sometimes after processing, into a functional protein. 

DNA is replicated during each cell division in a growing organism, and although this process is 

highly accurate, errors can nevertheless occur in which an incorrect nucleotide is inserted into 

the daughter strand. If the incorrect nucleotide is copied faithfully in subsequent replication, a 

mutation is generated that may ultimately affect the function of the resulting protein and give rise 

to a variant organism (16). 

Mutations arise in nature either randomly or as consequences of DNA damage by 

ultraviolet (UV) radiation, chemicals, or other mutagenic agents (16). Researchers seeking 

desirable mutations can accelerate the mutagenic process by four primary methods: stimulating 

replication error-based mutagenesis with chemicals or radiation; generating localized sequence 

alterations through error-prone PCR; inducing combinatorial mutagenesis through directed 

evolution; or using structural information about a protein to change specific amino acids, termed 

“rational design.” Each of the latter three is conducted in vitro and must therefore be followed 

by reintroduction of the mutated sequence into the host organism. If desired mutations show 

phenotypes that are recessive to the wild-type phenotype, then the wild-type gene must first be 

disabled or displaced by integration of the new sequence in its place; often, however, the desired 

mutations have dominant phenotypes (e.g., enabling function under harsh conditions) so that 

such measures are not necessary. Nevertheless, expression of a mutant sequence can be 

vulnerable to the transcriptional environment at its locus of integration. In the case that the 

mutant sequence must be integrated into host DNA to be maintained stably, numerous 

transfections may be required before satisfactory expression is obtained. 

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2.5.1 Chemical and physical mutagenesis. Chemical and physical agents that 

damage DNA induce mutations during subsequent DNA replication. DNA-damaging chemicals 

include alkylating agents such as ethylmethanesulfonate and derivatives of nitrosoguanidine that 

attach alkyl groups to DNA bases, which promotes mispairing during subsequent replication. 

They also include intercalating agents such as ethidium bromide that insert themselves between 

base pairs, which changes the spacing between bases and therefore induces insertion of extra 

nucleotides during replication. Finally, DNA-damaging chemicals also include bulky adductproducing agents such as benzo(a)pyrene that attach themselves to DNA bases, which also 

promotes mispairing during subsequent replication. Physical mutagens include electromagnetic 

radiation such as gamma rays, x-rays, and UV light, as well as particle radiation such as fast and 

thermal neutrons as well as alpha and beta particles (16, 17). 

The gene encoding the protein or RNA molecule of interest need not be cloned or even 

identified for chemical or physical mutagenesis to be used effectively; indeed, only an activity of 

interest is required. Exposure of the experimental organism to the mutagenic conditions is 

followed by screening or, ideally, selection for improvement of the trait of interest, where 

selection refers to a process that favors reproduction of organisms showing the improved trait 

over those without improvement (18). Improved mutants are typically mated, or back-crossed to 

parental strains, to minimize the accumulation of potentially deleterious mutations in non-target 

genes, before successive rounds of mutagenesis are undertaken (19).

Chemical and physical mutagenesis methods are relatively straightforward and 

inexpensive; however, additional methods have been sought because these methods typically 

affect only a few nucleotides at a time and therefore result in limited improvements (20). 

2.5.2 Error-prone PCR. Error-prone PCR is widely used to generate random 

mutants. In this approach, the gene of interest has been identified and cloned, and PCR primers 

are designed for it. During amplification of the gene, however, a number of changes from 

normal PCR are employed: primer annealing temperatures are lowered to diminish fidelity; 

nucleotide ratios may be lowered and/or altered from the normal 1:1:1:1 equivalence; a nonproofreading DNA polymerase is used; high levels of the magnesium (II) ion (Mg2+) and often 

the manganese (II) ion (Mn2+) are used to further diminish replication fidelity; and up to 

80 cycles may be conducted (21–23). Amplified products must then be re-introduced into the 

experimental organism and expressed to allow screening or selection for improved mutants (21, 

22). While this method is rapid, convenient, and generates large numbers of mutants, it 

primarily generates “point” mutations, or mutations in which single, isolated nucleotide changes 

predominate. As a result, it explores only a small sequence space, meaning it is unable, through 

successive rounds of mutagenesis, to converge upon a globally optimal sequence (20). 

Nevertheless, numerous commercial kits are available to facilitate error-prone PCR, and it 

remains a popular method of mutagenesis (www.stratagene.com/products/display; 

www.jenabioscience.com/images/0ea5cbe470/PP-102.pdf). 

2.5.3 Combinatorial mutagenesis. Combinatorial mutagenesis, in all of its many 

forms, attempts to capture the success of the genetic recombination that occurs in sexual 

reproduction: the genes of multiple parents are mixed and matched to yield new combinations 

that are not present even in the parents. 

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A common form of combinatorial mutagenesis, though by no means the only form, is 

known as DNA shuffling or assembly PCR. In this method, as few as one or as many as 20 or 

more parental homologs of the gene of interest are obtained in isolated form, fragmented 

randomly with DNase I, and subjected to replication by PCR in the absence of primers. Prepared 

this way, the fragments prime each other at their overlapping regions, and successive rounds of 

PCR eventually generate full-length products. Ideally, therefore, the resulting chimeric progeny 

incorporate sequence elements that have already been selected for functionality in the parents, 

while suffering much fewer nonsense, frameshift, and other nonproductive mutations that are 

common with random mutagenesis methods. After expression and screening of the chimeric 

progeny, further rounds of combinatorial mutagenesis are often employed until no further 

improvements are obtained–this process is termed directed evolution. 

Directed evolution has achieved extraordinary success in several industrial enzymes, 

resulting in 100- to over 10,000-fold enhancements of activity, altered substrate specificities, and 

stabilities to environmental conditions such as acidity, temperature, and solvent composition (24, 

25). Assembly PCR (26) as well as several other related combinatorial methods known by their 

acronyms as StEP, SHIPREC, ITCHY, SCRATCHY, CLERY, and RACHITT, are described in 

detail in the book, Directed Evolution Library Creation: Methods and Protocols (27). 

2.5.4 Rational design. The mystery surrounding the way in which a onedimensional sequence of amino acids folds into an active three-dimensional (3D) enzyme has 

fascinated researchers for decades. Now, the understanding that has emerged from their work, 

summarized in several excellent books (28–30), in combination with the crystallization and 

structural analysis of a number of industrially important enzymes, is making the rational 

mutagenesis of such enzymes possible. 

Rational design typically begins with the computer-based structural representation of a 

protein of interest, ideally informed by high-resolution, 3D structures of the protein or near 

relatives obtained by x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. 

Structures of enzyme-substrate, enzyme-product, and enzyme-inhibitor complexes are especially 

useful, because they identify the amino acids that comprise the catalytic part of the enzyme, or 

the active site. However, simulation is also possible with knowledge of only the primary 

(sequence) structure, albeit at lower fidelity, based on similarity to homologous proteins and 

current understanding of protein folding kinetics and thermodynamics. Extensive protein 

sequence and structure databases exist and are freely available, most notably including SwissProt and its supplement, TrEMBL, found online at http://us.expasy.org/sprot/. Secondary and 

tertiary structure predictions may then be generated by the following homology modeling 

software: SWISS-MODEL, an automated protein modeling server at the GlaxoWellcome 

Experimental Research Station in Geneva, Switzerland (free online at 

http://swissmodel.expasy.org/); PredictProtein, an online service for sequence analysis and 

structure prediction maintained by the European Molecular Biology Laboratory - Heidelberg 

(EMBL) and the Columbia University Bioinformatics Center (CUBIC) at http://www.emblheidelberg.de/predictprotein/predictprotein.html; and Modeller, maintained by the University of 

California at San Francisco and also free of charge to academic researchers at 

http://salilab.org/modeller/modeller.html. 

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Once the protein of interest has a structural representation, the most promising sites for 

modification can be predicted, often computationally. These are typically close to the active site 

and accommodate or stabilize the proposed reaction mechanism. They are also in the binding 

pocket for the substrate, or make important structural contributions to the enzyme. By sitedirected mutagenesis of the DNA that encodes these amino acids, in which a common approach 

is to allow oligonucleotides bearing the desired mutation(s) to prime PCR amplification reactions 

at the locus of interest, other residues can be substituted in their place(s) (31, 21). Amino acids 

are usually exchanged with one of the other 19 natural residues, but it has recently become 

fashionable to incorporate so-called designer amino acids into proteins as well. The mutated 

gene is then introduced into a suitable organism and expressed, and the new protein product is 

partially or completely purified to reveal whether the amino acid substitution(s) have had the 

desired effect. This process is typically iterative, with multiple rounds of mutation and 

evaluation (32). 

Alternatively, a 3D protein structure can be compared with those of related proteins that 

vary in the parameter of interest, either by models listed above or by a number of other 

homology modeling programs and online servers (see listing of those currently available at 

http://ncisgi.ncifcrf.gov/~ravichas/HomMod/). Comparisons among related proteins with 

variation in thermostability, for example, have revealed that higher stability generally correlates 

with greater proline, arginine, and tyrosine content; lower asparagine, glutamate, cysteine, and 

serine content; increased numbers of salt bridges and stabilizing hydrogen bonds; and a larger 

fraction of residues in alpha helices (33). Unfortunately, however, the effect of a particular 

amino acid substitution on a protein’s thermostability has not proven to be highly predictable 

with current understanding (34), typical of the situation with other parameters of interest as well. 

This limits speed and throughput, which then limits the sequence space amenable to testing. 

Nevertheless, the understanding of factors influencing protein folding is increasing rapidly, 

supported in great part by funding for basic research, and is expected to continually enhance 

efforts directed toward enzyme rational design (32). 

Rational approaches have also entered an era of de novo design in both enzyme active 

sites and catalytic antibodies. In one method, the high-energy state of a reaction is modeled with 

a protein or antibody side chain geometrically oriented for catalysis. Then, a library of rotamers, 

or low-energy side chain conformations, is generated and the novel active sites are tested for 

optimal fit with a carrier or scaffold protein. This approach has been demonstrated successfully 

in the design of a novel active site for ester hydrolysis within the otherwise inert protein, 

thioredoxin (35). This promises interesting future results for activities of industrial interest (36). 

Rational design is clearly a time- and information-intensive process, and our 

understanding of enzymes is far behind that of small molecules. Still, it has succeeded in the 

reconfiguration of substrate specificities in oxidoreductases, hydrolases, transferases, and 

DNases; the alteration of cofactor requirements; the inversion of reaction stereochemistry; and 

the enhancement of enzyme stabilities under various industrial conditions, as well as the 

introduction of novel catalytic activities into existing templates (32). In addition, sufficient 

structural information is accumulating for industrially important enzymes, including lipases and 

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cellulases as well as hydrogenases, that rational design must be considered as one of the 

promising molecular techniques available for bioenergy development. 

2.5.5 Analytical mutagenesis. Mutagenesis is also frequently employed to lend 

insight into the functions of metabolic pathways, specific genes, or specific regions within a 

gene, rather than to create genes with improved function. For these purposes, mutagenic 

methods are employed that frequently eliminate the function of a gene altogether. Transposon 

insertional mutagenesis is quite popular for this purpose, acting by the random insertion of large 

segments of DNA (the transposons) into a genome. Mutants lacking a function of interest are 

then assumed to have received an insertion within a gene essential to that function, and 

transposon sequences can then be used to locate the insertion. Sequences flanking the insertion 

are then investigated, ideally with the help of annotated genomic databases, to reveal the likely 

function of the interrupted gene (21). 

If the identity of a gene is known, but the regions of amino acids that are most essential to 

the activity of the encoded protein are of interest, a technique known as linker-scanning 

mutagenesis may be used to probe the structure-function relationships among the amino acids. 

In one approach to this technique, 10 to 15 base pair oligonucleotides are inserted into the gene 

sequence at random; alternatively, the mutagenesis may be designed such that internal deletions 

occur as well. Insertions or deletions that disrupt the function of the protein are assumed to 

occur in regions essential to the correct folding of the protein, while those that are tolerated are 

assumed to occur on the surface or in less-essential regions. In this way, the functional regions 

of an enzyme can be mapped with high resolution (1, 21, 31, 37). 

Variations on the above-mentioned analytical mutagenesis methods are numerous, highly 

specialized, and evolving rapidly; for current and detailed information, the reader is referred to 

Current Protocols in Molecular Biology, updated quarterly (1). 

2.6 Proteomics 

The cellular proteome is the complete set of proteins found in a particular cell type under 

a specific set of conditions. The complete proteome, in turn, is the complete set of proteins that 

may be synthesized by the set of cellular proteomes, or the approximate protein analog of the 

genome. A cell’s proteome typically possesses a much larger number of elements than does its 

genome, due to alternative processing of gene products and post-translational modifications such 

as glycosylation and phosphorylation. It is also more complex than the genome, in that it 

incorporates functional interactions among distinct proteins, and it is dynamic, in comparison to 

the generally static genome (38). 

Proteomics is the study of proteomes, particularly including proteome structure and 

function, and includes protein separation, identification, quantification, and analysis of protein 

sequences, structures, modifications, and interactions. It therefore encompasses the development 

of technologies used in protein separation and structural analysis, such as two-dimensional (2D) 

electrophoresis and mass spectrometry, as well as the study of protein-protein and protein-DNA 

interactions that influence the synthesis of other proteins (38). Proteomics is a recent addition to 

the landscape of biological research and represents an expansion in biological thought from 

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concentration upon individual proteins and protein assemblages to large systems of interacting 

proteins. Proteomics is directly related to metabolic engineering, discussed below, in that it 

considers systems of enzymes interacting in metabolic pathways, as well as those interacting to 

govern cell division, cell signaling and response to environmental conditions, and transcriptional 

regulation that in turn determines the composition of the proteome itself (39, 40). 

Like genomics, proteomics has emerged from the rapidly expanding set of databases, for 

example, Swiss-Prot, http://us.expasy.org/sprot/; Protein Data Bank, http://www.rcsb.org/pdb/) 

and data-mining tools as an application of bioinformatics that demands powerful computational 

resources and promises insights on greater scales of cellular complexity than have previously 

been possible (40, 41). Since biotechnology relies directly upon the ability to control a cell’s 

proteome, particularly including optimization of the balance between metabolic pathways that 

synthesize desired products and other pathways that maintain cell vigor, advances in proteomics 

will directly benefit all bioengineering endeavors. 

2.7 Pathway Engineering 

Metabolic pathway engineering integrates the approaches and technologies described 

above and has the fundamental goals of modifying biosynthetic pathways, assessing the 

physiological outcomes of the genetic modifications, and using the resulting information to 

improve further the pathways in question (42). The optimization of entire metabolic networks, 

rather than individual enzymes, is often necessary because kinetic control is frequently 

distributed throughout a pathway rather than concentrated in a single reaction. When levels of a 

particular enzyme are altered, the fluxes not only of its direct product(s) and substrates are 

altered, but also of metabolites in related pathways linked to the pathway of interest through 

regulatory and common-substrate relationships. As a result, multiple points of intervention, 

frequently requiring fairly small changes in enzyme activity, may be required to achieve the 

desired metabolic changes (42, 43). 

Pathway engineering is primarily directed toward one of several distinct goals. The first 

of these is the elucidation of pathways of interest, involving both identification of component 

reactions as well as reaction and/or transport bottlenecks (42). Numerous mathematical tools are 

being brought to bear on this goal, in combination with genomic and microarray-generated 

expression data, and have had great success in elucidating the structures of metabolic pathways 

and distribution of kinetic control within them (42). Two recent breakthroughs in this area are 

found in the development of a new kinetics format, termed linear log kinetics, which has proven 

remarkably accurate in describing intracellular kinetic behavior of metabolic networks, and the 

second is the development of a conceptual and experimental framework known as FANCY for 

elucidation of gene function by analysis of the metabolome, or total metabolite composition of a 

cell (43). Additional promising in silico metabolic pathway modeling approaches interpret and 

predict cellular functions within the extremes of allowable possibilities, followed by the use of 

biochemical rationales to select the most reasonable behaviors (43). This quantitative analysis of 

pathways leads to the understanding necessary, in turn, to target specific promising genetic 

modifications (42). 

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Another primary goal of pathway engineering, enabled by the first, is the modification of 

a pathway such that preferred substrates can be used, where preferred substrates are either less 

expensive, more widely available, or more environmentally friendly than the conventional 

substrates. Increased attention is being given to the use of renewable resources for the synthesis 

of specialty and commodity chemicals by so-called green processes. In addition, biomassderived substrates are among the most widely available renewable resources–those generated 

from agriculture and municipal, agricultural, and forest wastes, among others. The majority of 

inexpensive biomass is composed of lignocellulose. However, this contains a significant 

proportion of less-readily fermented five-carbon sugars in combination with the readily utilized 

six-carbon sugars. The great promise of pathway engineering for facilitating utilization of 

renewable agricultural materials is revealed by the development of metabolic pathways for the 

use of biomass-derived mixed sugars for the production of ethanol, described further in Chapter 

IV (43). 

A third goal of pathway engineering is the development of pathways for the synthesis of 

novel chemical structures, particularly antibiotics, carotenoids, and polyhydroxyalkanoates, the 

latter of which are described further in Chapter III. These efforts involve futuristic approaches, 

such as combining genes from different pathways and/or different organisms; inserting genes 

into a pathway or deleting genes from a pathway; combining modules derived from different 

multidomain enzymes to form new enzymes with novel catalytic activities; and engineering 

enzymes (e.g., by directed evolution) with new substrate specificities or catalytic activities in a 

pathway such that new and even non-natural substrates can be introduced, yielding novel 

products (44). 

Pathway engineering has been greatly facilitated by genomic endeavors, which have 

provided access to sequence data not only of structural genes, but also of the genetic control 

elements central to the transcriptional regulation of genes, pathways, and groups of related 

pathways (42). Gene expression databases have been equally valuable. In particular, the DNA 

microarray-based analysis of expression patterns characteristic of different physiological states, 

together with the characterization of transcriptional and post-translational controls within 

metabolic networks, have helped to identify key genetic targets for improvement of the desired 

biocatalysis. Microarray technology is also contributing to the elucidation of pathways by 

validating gene function, determining whether proteins are membrane-bound or cytosolic, and 

characterizing DNA-binding proteins (42). 

 Pathway elucidation is also highly dependent upon the ability to measure accurately the 

fluxes of metabolites within it. Considering the large numbers of metabolites present in a cell at 

any time, and the typically low concentrations of pathway intermediates, such measurements 

represent considerable challenges. To obtain intracellular flux data, Carbon 13 (13C)-labeling of 

specific atoms within substrates and isotopomers (isotopic isomers) of intermediates are 

frequently used in combination with 13C-NMR, 2D-NMR, or mass spectrometry. Alternatively, 

if the bioreaction sequence under investigation does not involve Carbon-Carbon (C—C) bond 

cleavage, uniformly Carbon 14(14C)-labeled tracers can be used to reveal fluxes by tracking 

depletion of the radiolabeled tracer pulsed into the metabolite pool (42, 43). 

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 While most pathway engineering successes are concentrated in pharmaceuticals and other 

medical applications, several examples demonstrate the great potential of pathway engineering in 

pollution prevention as well. Among these, one of the most famous is the development of a 

pathway to synthesize the blue dye indigo from glucose in bacteria, which avoids use of aromatic 

precursors and generation of aromatic wastes necessary in the conventional chemical synthesis. 

In the indigo pathway engineering process, the tryptophan synthesis pathway was first extended 

to indigo with the addition of one enzyme, naphthalene dioxygenase. This addition resulted in 

the attenuation of activity of another important synthase by the conversion of the intermediate 

indoxyl to indigo, which required the increase of the gene dosage of that enzyme, the increase of 

its substrate availability, and the inactivation of a competing enzyme. Finally, to diminish the 

content of indirubin, a product of a non-enzymatic side reaction in the conversion of indoxyl to 

indigo, in the final product, an isatin hydrolase was introduced. This successful example clearly 

illustrates the unexpected effects of altering a native pathway and the great effort needed to 

compensate for them to achieve the desired catalysis (45, 43). 

3. Research Priorities 

 Current research in genetic engineering platform technologies is proceeding at an almost 

incomprehensibly rapid pace under the impetus of medical and basic biological research goals. 

While environmental biotechnology has much to gain from advances in these research goals, the 

support in these technologies and pace of progress are already sufficiently great, but funding for 

environmental goals is limited such that agencies with primarily environmental goals are 

encouraged to direct their support toward research priorities in other areas. 

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(16) Friedberg, E. C., and G. C. Walker (1995). DNA Repair and Mutagenesis. ASM Press, 

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(17) Kodym, A., and R. Afza (2003). Physical and chemical mutagenesis, Methods Mol Biol 

236:189-204. 

(18) Hartwell, L. H., L. Hood, M. L. Goldberg, A. E. Reynolds, L. M. Silver, and R. C. Veres 

(2004). Genetics: From Genes to Genomes, 2nd Edition. McGraw Hill. 

(19) Østergaard, L., and M. F. Yanofsky (2004). Techniques for molecular analysis: 

establishing gene function by mutagenesis in Arabidopsis thaliana, Plant J 39:682. 

(20) Tobin, M. B., C. Gustafsson, and G. W. Huisman (2000). Directed evolution: the 

“rational” basis for “irrational” design, Curr Opin Struc Biol 10:421-427. 

(21) Braman, J., Ed. (2002). In Vitro Mutagenesis Protocols, 2nd Edition. Humana Press, 

Totowa, NJ. 

(22) Cirino, P. C., K. M. Mayer, and D. Umeno (2003). Generating mutant libraries using 

error-prone PCR, Methods Mol Biol 231:3-9. 

(23) Biles, B. D., and B. A. Connolly (2004). Low-fidelity Pyrococcus furiosus DNA 

polymerase mutants useful in error-prone PCR, Nucleic Acids Res 32:e176. 

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of a family of genes from diverse species accelerates directed evolution, Nature 391:288-

291. 

(25) Christians, F. C., L. Scapozza, A. Crameri, G. Folkers, and W. P. C. Stemmer (1999). 

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family 

shuffling, Nat Biotechnol 17:259-264. 

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(26) Joern, J. (2003). DNA Shuffling, in Directed Evolution Library Creation: Methods and 

Protocols (F. H. Arnold and G. Georgiou, Eds). Humana Press, pp 85-90. 

(27) Arnold, F. H., and G. Georgiou, Eds. (2003). Directed Evolution Library Creation: 

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sequence alignments versus rational design and directed evolution, Curr Opin Biotechnol 

12:371-375. 

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D. P. Raleigh (2000). Rational modification of protein stability by the mutation of 

charged surface residues, Biochem 39:872-879. 

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Opin Chem Biol 6:125-129. 

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(39) Liebler, D. C. (2001). Introduction to Proteomics: Tools for the New Biology. Humana 

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(40) Twyman, R. M. (2004). Principles of Proteomics. BIOS Scientific Publishers, New 

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Proteome Analysis. Wiley-VCH, Weinheim. 

(42) Stafford, D. E., and G. Stephanopoulos (2001). Metabolic engineering as an integrating 

platform for strain development, Curr Opin Microbiol 4:336-340. 

(43) Sanford, K., P. Soucaille, G. Whited, and G. Chotani (2002). Genomics to fluxomics and 

physiomics—pathway engineering, Curr Opin Microbiol 5:318-322. 

(44) De Boer, A. L., and C. Schmidt-Dannert (2003). Recent efforts in engineering microbial 

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(45) Berry, A., T. C. Dodge, M. Pepsin, and W. Weyler (2002). Application of metabolic 

engineering to improve both the production and use of biotech indigo, J Ind Microbiol 

Biotechnol 28:127-133. 

B. BIOREACTOR TECHNOLOGIES 

1. Introduction 

All bioreactors have in common the central purpose of maintaining ideal conditions for 

one or more species of microbes, such that the maximum desired activity is promoted. 

Bioreactors are essential elements in all industrial processes that make use of microbial growth 

and metabolism with the expectation that the future success of commercial biotechnology 

depends significantly on the advancement of bioreactor technology (1). Basic bioreactor design 

has changed little, however, in the past 40 years, possibly due to an absence of sufficient market 

forces. Currently, more than 50 percent of all commercial bioreactors are used in the synthesis 

of low-volume, high-value products, such as pharmaceuticals, that may be synthesized profitably 

without stringent process optimization. In contrast, the productions of high-volume, low-value 

products such as biomaterials and fuels carry sufficiently low profit margins that a high degree of 

optimization is essential. Because the latter class of products has the greatest potential for 

positive environmental impact, bioreactor design and optimization represent important priorities 

in the advancement of bioengineering for pollution prevention. 

1.1 Reactor Conditions 

Successful design and operation of bioreactors requires optimization of numerous 

quantities, including such conditions as pH, substrate and product concentrations, cell density, 

oxygen concentration, and temperature. Understanding the roles of each of these in a particular 

bioprocess, as well as predicting and controlling their spatial and temporal variation within 

narrow limits throughout the bioreactor volume, is of central importance to reactor design. 

Consequently, primary goals of current engineering efforts include accurate modeling of 

bioprocesses, real-time sensing of internal reactor conditions, and the design of bioreactors in 

which conditions can be maintained as nearly uniform as possible. 

1.2 Reactor Types 

Numerous varieties of commercial-scale bioreactors are in use, with the majority 

categorized as unstirred vessels, stirred vessels, bubble columns, airlift reactors, membrane 

reactors, fluidized beds, or packed beds (2). While each category presents a unique set of 

advantages and limitations, the considerations relevant to bioprocesses are quite similar 

throughout. 

Large-scale industrial fermentations are typically conducted as fed-batch processes, 

involving intermittent supply of substrate(s), which utilizes substrates that are as inexpensive as 

possible to yield a product density as high as possible. Fed-batch processes offer several 

advantages over traditional batch operations: first, they avoid catabolite repression effects, 

which occurs when excess quantities of a substrate such as glucose can redirect metabolic energy 

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and carbon flow; second, they avoid overflow metabolism, which occurs when substrate in 

excess of the capacity of a particular pathway is converted to undesirable co-metabolites; and 

finally, they minimize product-inhibition effects, which occurs when accumulation of a product 

can inhibit further progress of a desired reaction (3). An example of this is the application of the 

fed-batch process to the culture of baker’s yeast, Saccharomyces cerevisiae. The yeast 

effectively diminishes glucose repression and overflow metabolism occurs leading to ethanol 

formation (4). Similarly, in the industrially important Escherichia coli, fed-batch processing 

diminishes the production of acetate through overflow metabolism and thereby allows greater 

energy flow toward the production of desired recombinant proteins (5). 

1.3 Reactor Models 

The design, optimization, and real-time control of bioreactors depend on the availability 

of good process models. The central role of these models is the establishment of quantitative 

relationships between kinetic processes (those involving enzymes) and transport processes (those 

involving the flow and distribution of substrates, cells, products, and wastes within the 

bioreactor). The former involve enzyme rates and specificity (or selectivity) characteristics, as 

well as kinetic characteristics of microbial growth, substrate uptake, and longevity, which are 

revealed through basic biochemical research. The latter, in turn, involve considerations of fluid 

mechanics and heat and mass transfer that require considerable computational resources. 

Accurate models become especially important when bioprocesses must be scaled up from

bench to larger commercial volumes without losses of product quality, yield, or process stability. 

Heat removal and gas-liquid mass transfer limitations, for example, can present great scale-up 

challenges that are addressed much more readily with the assistance of accurate models. 

2. State of the Science 

2.1 Kinetic Models 

Kinetic expressions describe quantitative relationships among enzyme concentrations, 

substrate concentrations, product concentrations, and rates of product generation. Reliable 

kinetic expressions are highly advantageous to the design of cost-effective bioprocesses, 

especially in the production of comparatively low-value, high-volume products such as biofuels 

and biomaterials. In these cases, in which small losses of substrates to undesired metabolic byproducts can render a process economically impractical, kinetic models are essential to the 

achievement of high substrate conversion efficiency. 

2.1.1 Metabolic flux analysis. In many biosynthetic pathways of interest, simple 

understanding of metabolite transformation kinetics (substrate consumption rates, intermediate 

lifetimes, and product production rates) is sufficient for reactor design, causing understanding of 

individual enzyme kinetics to be unnecessary. In these cases, an approach known as metabolic 

flux analysis, or MFA, is convenient to describe the flow of each metabolite through nodes, or 

enzymatic transformations, throughout an entire biochemical network. The result is a 

comprehensive flux map that shows and quantifies all major anabolic (synthetic) and catabolic 

(degradative) processes of interest, as well as points of sensitivity to perturbation of metabolite 

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or enzyme concentrations, or flux control coefficients, within an organism (6, 7). Once such a 

mathematical framework has been established experimentally, fluxes of particular metabolites 

can be predicted over ranges of conditions and flux maps can be compared among different 

organisms and growth conditions to reveal optimal process conditions. In addition, flux maps 

and comparisons among different flux maps may indicate possible targets for genetic 

modifications within the cultivated organism, reveal the outcome of genetic manipulations that 

have already been performed, or yield more basic insight regarding cellular energy metabolism 

(8). 

 In the initial development of a metabolic flux map, all theoretically possible enzyme 

reactions in the network are considered; postulation of a map appropriate to a particular process 

then involves the designation of some reactions as dormant. Metabolic flux analysis experiments 

are typically performed in stirred-tank reactors (STRs), also known as chemostats, to establish 

steady-state conditions. Concentrations of substrates, intermediates (where possible), and 

products are then monitored over time to reveal their interdependences, and the flux map is 

refined based on the results. Highly reproducible data are prerequisite for the confirmation of a 

postulated metabolic network, however, and until pathways have been rigorously established, the 

uniqueness of the proposed network is typically viewed with caution (9). 

For the analysis of large metabolic networks, the use of isotopically labeled substrates 

(e.g., 13CH3COO-

) is often required to trace the flows of compounds present at low 

concentrations. The great amount of data generated by such experiments can lead to sizable 

computational challenges, because fluxes through individual pathways must then be calculated 

by solving large sets of non-linear equations. Fortunately, new quasi-linear algebra methods 

have been developed to calculate fluxes from large data sets and, importantly, to estimate 

sensitivities to measurement errors (10). A number of systematic descriptions of metabolic 

pathways for E. coli have been developed using these methods (11-13). 

2.1.2 Enzymatic kinetic models. Detailed kinetic models, involving descriptions of 

enzyme characteristics, fall into three broad categories. The most detailed and comprehensive 

are referred to as mechanistic or structured models. In such models, rate expressions are 

proposed that attempt to describe the mechanistic role of each enzyme in the metabolic pathway. 

A fundamental structured model also includes information about characteristic microbial cell 

dimensions and effects of rate-limiting mass transfer across cell walls. 

In a structured model, the limiting substrate participates in the first reaction pathway; the 

product of this reaction participates as a reactant in the next pathway, and so on to capture the 

full cascade of transformations as well as regulatory feedback reactions. Certain structured 

models have been successful in predicting important effects, such as the differential uptake of 

two separate substrates. This confirms the value of such models when they can be properly 

validated (14). Currently, however, the use of structured models is limited by the lack of 

rigorous verification, which in turn results from the considerable experimental challenges 

involved in determining numerous enzymatic parameter values. Accordingly, utilization of such 

models for design purposes is restricted to the range of parameters that can be validated by 

experimental data (15). 

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 The second, probably most widely-used set of kinetics models comprises those that 

assume the presence of a single growth-limiting substrate. Among these so-called black-box 

models, the most prominent is the Monod equation, in which the biomass specific growth rate, μ, 

is related to the substrate concentration, S, in a nonlinear form (Equation 1). The parameters KS

and μmax, describing the half-saturation constant and maximum specific growth rate, respectively, 

are determined experimentally (16). 

[ ]

[ ] SK

S

S + = μμ max (Equation 1) 

Extensions of the Monod equation to capture effects of metabolite repression and other 

inhibitory and/or limiting effects are available. Black-box models can be extremely useful in 

their estimations of overall fermentation behavior in early stages of process design. At the same 

time, they lack the level of detail often necessary for precise optimization of bioreactor function 

(15). 

Another class of black-box models comprises those based on artificial neural networks 

(ANNs). ANNs structure is characterized by the total number of nodes, responsiveness of each 

node to an average input, and the response function for each node (17). Model parameters for 

ANNs are termed weights, and the process of determining weights from experimental data is 

called the training procedure. While utility of these models is again restricted to the region over 

which experimental data are available, they offer the valuable ability to accommodate increased 

metabolic complexity compared to limiting-substrate models (18). 

 Finally, so-called gray-box modeling is also applied to bioreactor design, referring to 

strategies that attempt to combine both fundamental knowledge and empirical data to obtain 

models of moderate complexity with qualitative behavior in reasonable agreement with 

experimental data. This class of models is best represented, both conceptually and industrially, 

in the form of fuzzy-rule systems. Fuzzy-rule theory was developed by Zadeh (19, 20) and has 

become increasingly important in practical process modeling and control. Relationships between 

fuzzy variables are, in turn, formulated using fuzzy logic operators to reflect the common 

practices used by operators in everyday bioreactor operation. Numerous kinetic expressions for 

bioreactors have been formulated using this approach (21, 22), and fuzzy modeling and control 

are now regarded as promising methods for automating industrial bioprocesses in which 

experienced operators play significant roles in their successful operation (22). 

2.2 Transport Phenomena and Models 

Transport equations predict gradients of dissolved substances, temperature, etc. within 

fluids. They are based on principles of conservation of mass, momentum, and energy. When 

applied to bioreactor operation, they can be used both to discover and explain phenomena of 

interest, as well as to guide bioreactor design and scale-up. Because of the central importance of 

reactor uniformity, transport modeling is frequently used to address issues of mixing, mass 

transfer between gases and liquids, heat removal and/or maintenance of optimal microbial 

growth temperatures, and biofilm formation on reactor surfaces. The distribution of fluid 

velocities, or velocity profile, within a bioreactor is especially important in the calculation of gas 

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transport and heat transfer patterns as well as shear stresses that influence locations of biofilm 

formation. 

The complete simulation of mass and energy transport throughout all parts of a reactor, 

showing consequences of adjusting design variables, would be ideal for reactor design. While 

the typical two-phase gas-liquid media composition, locally turbulent flows, and limitations of 

kinetic models greatly complicate the calculations involved in traditional transport models, new 

approaches are being developed that hold great promise. In recent years, computational fluid 

dynamics (CFD) in particular has enabled the capture of salient features in bioreactors. For 

example, the simulation of a bubble column fermenter is shown in Figure 2. Instantaneous 

values are shown on the left, while time averages are shown on the right; velocity profiles are 

plotted with arrows while oxygen distributions are shown in color. For these calculations, the 

two-phase gas-liquid system has been treated as a homogeneous medium with a variable density 

that depends on the gas retention in the column, resulting in visibly turbulent flow at short 

timescales. In this example, quantification of the spatial variation of the oxygen combined with 

a kinetic model based on oxygen provided the rate of product generation. 

Figure 2. Distribution of fluid velocity (arrows) and oxygen distribution (color). 

 Instantaneous results are shown on the left, time averages on the right. 

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 Other reactor types present even more challenging modeling problems. For example, in 

STRs, involving impellers that rotate rapidly through a two-phase flow, boundary conditions on 

the transport equations require the use of moving boundary computational grids (23, 24). 

Despite the challenges, describing and predicting details of mass and energy flow throughout 

bioreactors mathematically–including the realistic representation of fluids that exhibit nonNewtonian behavior due to the suspension of cells and particulates–remain important goals due 

to their great potential to facilitate reactor design (25). 

2.3 On-line Sensing 

Real-time sensing of bioreactor conditions, involving spatially resolved measurements of 

fluid velocities and reaction components, is essential for both the experimental validation of 

bioreactor models and for monitoring of ongoing performance. Even the most perfectlydesigned and thoroughly-modeled bioreactor is expected to experience unforeseen conditions 

occasionally, particularly given the presence of mutable microorganisms, with the result that 

real-time or on-line sensing of bioreactor conditions during operation is essential. On-line 

sensing is also important, of course, in validating models during development. Especially 

important is the potential of on-line sensing to allow precise, automated, feedback control 

devices to maintain reactor homeostasis. 

Conventional reactor sensors are often not ideal for bioprocess measurements, however, 

due to their vulnerabilities to interference by biofilm growth, inabilities to resolve overlapping 

signals generated in complex culture media, and inabilities to be sterilized to avoid 

contaminating culture media, for example (26, 27), and ongoing research into sensor design is 

important to the future of bioprocessing. 

At the same time, sufficient progress has been made in several key areas that a number of 

important variables may be monitored adequately. Dissolved oxygen and pH, for example, are 

frequently monitored on-line using electrochemical sensors contained within steam-sterilizable 

glass electrodes (28). Dissolved oxygen may also be measured by a recently-commercialized 

method based on fluorescence quenching (29); available sensors are described at 

www.oceanoptics.com and www.fluorometrix.com. On-line measurements of cell mass are also 

desirable and can now be made indirectly by electrical or optical means: capacitance and 

permittivity measurements, for example, provide electrical quantitation (30), while light 

absorbance, light scattering, or a combination of the two provide optical indications of cell mass 

(31). 

 Carbon dioxide, a waste product of cell respiration, is an important indicator of bioreactor 

status and can be measured on-line by means of sterilizable electrode sensors, optical sensors, 

and sensors based on gas permeation through selective polymer membranes (32, 33), serving as 

the basis for process control loops (33). 

 Several spectroscopic techniques are also available to quantify the presence of numerous 

organic and inorganic species simultaneously, due to recent advances in optics and computing. 

An example is shown in Figure 3, in which a noninvasive sensor has measured the whole-cell 

biotransformation of L-serine and indole to tryptophan (34). This approach is also applicable to 

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Figure 3. Two-dimensional fluorescence spectra of a fermentation broth (34). 

the processing of sugar beet molasses at an industrial scale (35). Glucose, fructose, glutamine, 

ammonia, CO2, and phosphate are among the many compounds that can be measured by either 

near or mid-infrared spectroscopy. Recent developments of improved, low-cost optical sensors 

are also promising (27, 36, 37). Further development for miniaturization, improved robustness, 

and sensitivity are expected (38, 39). 

 A separate approach is based on the attachment of the gene for green fluorescent protein 

onto a protein of interest present during manufacture (40). This is particularly attractive if the 

tagged molecule is the product of interest, such that product concentration can be measured 

directly. A number of other color probes are also currently available, opening a promising 

avenue for real-time monitoring of the expression of multiple genes simultaneously (41). 

 Improved real-time sensing of bioreactor conditions is essential to model validation and 

process control during operations, and future bioreactors are expected to be massively 

instrumented to provide detailed real-time information of vital interest. Advances in sensing 

technologies are urgently needed, especially in the design of bioprocesses for commodity 

products where efficiency is paramount, and this area should be considered a top priority within 

bioengineering for pollution prevention. 

3. Research Priorities 

Because bioengineering for pollution prevention involves relatively low-value products, 

requiring optimal bioprocessing for commercial feasibility, improvements in bioreactor 

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technology should be a high priority in general in this field. In addition, several areas are worthy 

of specific mention: 

3.1 Sensing 

Real-time sensing of gases and aqueous metabolites is of central importance because it 

allows or has the potential to facilitate model validation, development of descriptive kinetic 

expressions, and real-time process control based on sensor feedback alone and in combination 

with model predictions. Biosensors suitable for monitoring bioconversions in bioreactors have 

been previously identified as a bottleneck in the development of high-volume, low-cost 

processes (42), indicating that the development of promising emerging technologies should be 

encouraged in every instance possible. 

3.2 Modeling 

In process design and optimization, the utility of a mathematical model lies in its ability 

to predict the operating characteristics in regions for which experimental data do not exist. 

Present kinetic models generally do not allow such procedures in great detail. Detailed kinetic 

models are also useful for capturing the dynamic responses of bioreactors to external stimuli, a 

set of important concerns in process control. Accordingly, a promising area of investigation is 

the development of more-detailed structural models that capture the salient features of complete 

metabolic pathways through the integration of biochemistry, molecular biology, and 

computational techniques. In particular, new approaches to structural kinetic modeling that 

include transcriptional and post-translational regulatory effects are needed. 

In addition, new developments are needed in computational methods to capture effects of 

turbulence in bioreactors, effects of shearing and mechanical stresses on cellular growth and 

death, and the non-Newtonian nature of cellular media. These must involve multi-scale 

modeling to capture details at small spatial scales and must also be able to transfer relevant 

information to lower-resolution models that describe greater volume and time scales. CFD 

models are now able to establish hydrodynamic profiles among different zones of a reactor and 

could serve as the basis for such models, describing concentration and temperature gradients 

both instantaneously and over time. Such models could be extended to bridge length, volume, 

and time scales, linking detailed calculations at smaller scales or in critical areas with lowerresolution models that track averaged quantities. Given recent and continuing increases in 

inexpensive computing power, such models could contribute greatly to reactor optimization. In 

addition, they have the potential to guide the scale-up of industrial processes by revealing 

important mass and heat transfer limitations as reactor configurations are changed. 

3.3 Applications of Genetic Engineering 

Certain challenges inherent in bioreactor operation can be greatly alleviated by the 

skillful application of genetic engineering technologies. For example, substrate and productbased inhibitions are common phenomena which occur when enzyme activities diminish in the 

presence of locally high concentrations of certain metabolites. Increasing reactor-mixing can 

often alleviate these problems, but diminishing the inhibitory mechanisms genetically can offer a 

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31 

more convenient solution. An example of this approach is presented by Agger and colleagues, 

who disrupted the gene responsible for glucose repression so that the glucose conversion rate did 

not decrease with increasing glucose concentrations that were, in turn, needed to work at high 

biomass concentrations (43). The ability of genetic engineering to solve bioreactor-based 

problems offers a set of great opportunities for improvements in reactor productivity. 

4. Commercialization 

Commercial bioreactor usage is growing rapidly, as shown in Figure 4, largely due to increases 

in pharmaceutical and food-based biotransformations, which account for over half and 

approximately one-quarter of the total biotransformations, respectively (44). An important issue 

for the commercialization of bioreactors for pollution prevention goals is simply capitalization: 

entry into established markets for fuels and materials will require large economies of scale, 

meaning that production facilities must be constructed on a large scale to be profitable. At the 

same time, investors are typically reluctant to take risks with newer technologies, which results 

in bioreactors producing biofuels and biomaterials that may face significant initial barriers to 

market entry. The establishment of pilot plants for both biofuel and bioplastics production, 

described in subsequent sections, indicates that these barriers are nevertheless far from 

insurmountable. 

Figure 4. The growth of commercially practiced biotransformations (44). 

5. References 

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(3) Hochfeld, W. L. (1994). Growth & Synthesis: Fermenters, Bioreactors, & Biomolecular 

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(4) Rose, A. H. (1979). History and scientific basis of large-scale production of biomass, in 

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(6) Liao, J. C., and E. N. Lightfoot (1988). Characteristic reaction paths of biochemical 

reaction systems with time scale separation, Biotechnol Bioeng 31:847-854. 

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(8) Stephanopoulos, G., A. Aristidou, and J. Nielsen (1998) Metabolic Engineering: 

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(9) Nielsen, J., and J. Villadsen (1994). Bioreaction Engineering. Plenum Press, New York. 

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Biodirectional reaction steps in metabolic networks: III. Explicit solution and analysis 

of isotopomer labelling systems, Biotechnol Bioeng 66:69-85. 

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coli glucose catabolism under various oxygenation rates, Appl Environ Microbiol 

59:2465-2473. 

(12) Varma, A., and B. O. Palsson (1993). Metabolic capabilities of E. coli II. Optimal 

growth patterns, J Theor Biol 165:503-522. 

(13) Varma, A., and B. O. Palsson (1994). Stochiometric flux balance models quantitatively 

predict growth and metabolic by-product secretion in wild-type E. coli W 3110, Appl 

Environ Microbiol 60:3724-3731. 

(14) Sonnleitner, B., and O. Kappeli (1986). Growth of S. cerevisiae is controlled by its 

limited respiratory capacity: formulation and verification of a hypothesis, Biotechnol 

Bioeng 28:927-937. 

(15) Ishii, N., M. Robert, Y. Nakayama, A. Kanai, and M. Tomita (2004). Toward large-scale 

modeling of the microbial cell for computer simulation, J Biotechnol 113:281-294. 

(16) Bitton, G. (1999). Wastewater Microbiology. Wiley-Liss. 

(17) Bhat, N., and T. J. McAvoy (1990). Use of neural nets for dynamic modelling and 

control of chemical process systems, Comput Chem Eng 14:573-582. 

(18) Gadkar, K. G., S. Mehra, and J. Gomes (2005). On-line adaptation of neural networks 

for bioprocess control, Comput Chem Eng 29:1047-1057. 

(19) Zadeh, L. A. (1965). Fuzzy Sets, Inform Contr 8:338-353. 

(20) Zadeh, L. A. (1973). Outline of a new approach to the analysis of complex systems and 

decision processes, IEEE Trans Sys Man Cybern 3:28-44. 

(21) Lee, B., J. Yen, L. Yang, and J. C. Liao (1999). Incorporating qualitative knowledge in 

enzyme kinetic models using fuzzy logic, Biotechnol Bioeng 62:722-729. 

(22) Horiuchia, J.-I. (2002). Fuzzy modeling and control of biological processes: review, 

J Biosci Bioeng 94:574-578. 

(23) Reuss, M., and R. Bajpai (1991). Stirred tank models - Measuring, modelling, and 

control, in Biotechnology Vol 4. (H. J. Rehm, Ed.). VCH, Weinheim, pp 299-348. 

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(24) Reuss, M., S. Schmalzriedt, and M. Jenne (2000). Application of Computational Fluid 

Dynamics (CFD) to modeling stirred tank bioreactors, in Bioreaction Engineering (K. 

Schugerl and K. H. Bellgart, Eds.). Springer, Berlin, pp 207-246. 

(25) Leib, T. M., C. J. Pereira, and J. Villadsen (2001). Bioreactors: a chemical engineering 

perspective, Chem Eng Sci 56:5485-5497. 

(26) Gastrock, G., K. Lemke, and J. Metze (2001). Sampling and monitoring in bioprocessing 

using microtechniques, Rev Molec Biotechnol 82:123-135. 

(27) Bellon-Maurel, V., O. Orliac, and P. Christen (2003). Sensors and measurements in solid 

state fermentation: a review, Process Biochem 38:881-896. 

(28) Johnson, M. J., J. Borkowski, and C. Engblom (2000). Steam sterilizable probes for 

dissolved oxygen measurement, Biotechnol Bioeng 67:645-656. 

(29) Bambot, S. B., R. Holavanahali, J. R. Lakowicz, G. Carter, and G. Rao (1994). Phase 

fluorometric sterilizable optical oxygen sensor, Biotechnol Bioeng 43:1139-1145. 

(30) Zeiser, A., C. B. Elias, R. Voyer, B. Jardin, and A. A. Kamen (2000). On-line 

monitoring of physiochemical parameters of insect cell cultures during the growth and 

infection process, Biotechnol Prog 16:803-808. 

(31) Janelt, G., N. Gerbsch, and R. Buchholz (2000). A novel fiber optic probe for on-line 

monitoring of biomass concentrations, Bioprocess Eng 22:275-279. 

(32) Chang, Q., L. Randers-Eichhorn, J. R. Lakowicz, and G. Rao (1998). Steam-sterilizable, 

florescence lifetime-based sensing film for dissolved carbon-dioxide, Biotechnol Prog 

14:326-331. 

(33) Pattison, R. N., J. Swamy, B. Medenhall, C. Hwang, and B. T. Frohlich (2000). 

Measurement and control of dissolved carbon dioxide in mammalian cell culture process 

using an in situ fiber optic chemical sensor, Biotechnol Prog 16:769-774. 

(34) Skibsted, E., C. Lindemann, C. Roca, and L. Olsson (2001). On-line bioprocess 

monitoring with a multi-wavelength fluorescence sensor using multivariate calibration, J 

Biotechnol 88:47-57. 

(35) McGovern, A. C., D. Broadhurst, J. Taylor, N. Kaderbhai, M. K. Winson, D. A. Small, J. 

J. Rowland, D. B. Kell, and R. Goodacre (2002). Monitoring of complex industrial 

bioprocesses for metabolite concentrations using modern spectroscopies and machine 

learning: application to gibberellic acid production, Biotechnol Bioeng 78:527-538. 

(36) Bashir, R. (2004). BioMEMS: state-of-the-art in detection, opportunities and prospects, 

Adv Drug Deliv Sys 56:1565-1586. 

(37) Pons, M.-N., S. Le Bonte, and O. Potier (2004). Spectral analysis and fingerprinting for 

biomedia characterisation, J Biotechnol 113:211-230. 

(38) Doak, D. L., and J. A. Phillips (1999). In situ monitoring of an E. coli fermentation using 

a diamond composition ATR probe and mid-infrared spectroscopy, Biotechnol Prog 

15:529-539. 

(39) Vaidyanathan, S., A. Arnold, L. Matheson, P. Mohan, G. Macaloney, B. McNeil, and L. 

M. Harvey (2000). Critical evaluation of models developed for monitoring an industrial 

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submerged bioprocess for antibiotic production using near-infrared spectroscopy, 

Biotechnol Prog 16:1098-1105. 

(40) Chen, H. J., M. P. DeLisa, H. L. Cha, W. A. Weigand, G. Rao, and W. E. Bentley (2000). 

Framework for on-line optimization of recombinant protein expression in high-celldensity E. coli cultures using GFP-fusion monitoring, Biotechnol Bioeng 69:275-285. 

(41) Li, J., S. Wang, W. J. Van Dusen, L. D. Schultz, H. A. George, W. K. Herber, H. J. 

Chae, W. E. Bentley, and G. Rao (2000). Green fluorescent protein in real-time studies 

of the GAL1 promoter, Biotechnol Bioeng 70:187-196. 

(42) Lubbert, A., and S. B. Jorgenson (2001). Bioreactor performance: a more scientific 

approach for practice, J Biotechnol 85:187-212. 

(43) Agger, T., A. B. Spohr, and J. Nielsen (2001). Alpha-amylase production in high cell 

density submerged cultivations of Aspergillus oryzae, Appl Microbiol Biotechnol 55:81-

84. 

(44) Straathof, A. J. J., S. Panke, and A. Schmid (2002). The production of fine chemicals by 

biotransformations, Curr Opin Biotechnol 13:548-556. 

C. BIOSEPARATIONS AND BIOPROCESSING 

1. Introduction 

 The bioproducts considered in this report, primarily including materials either made by 

living organisms or derived from biomass, typically require extraction from either a whole 

organism, such as a plant; from aqueous bioreactor media (also referred to as fermentation broth 

or culture media; or from downstream processing solutions. A prevalent challenge presented by 

bioreactor-based processes, in particular, is the generation of products within relatively dilute 

aqueous solutions. Bioreactor media usually must remain dilute, however, to prevent inhibition 

of enzyme activity by accumulated products and to prevent cell mortality due to accumulated 

wastes. Nevertheless, the design of bioreactors to allow higher solute concentrations while 

maintaining cell health and activity is worthy of considerable effort. 

Traditional separation techniques are well-developed, widely practiced, and 

comprehensively described in standard references (1). The techniques are categorized according 

to their fundamental mechanisms as physical (adsorption, crystallization, extraction, etc.), 

mechanical (filtration, centrifugation, etc.), thermal (distillation), or chemical (chemisorption, 

chromatography). Because separations often dominate the economics of biochemical processing, 

development of energy-efficient separation methods is of especially great importance to the 

commercial success of the low-value, high-volume bioproducts most relevant to pollution 

prevention. 

Downstream processing of bioreactor contents involves special challenges due to the 

presence of biomass and non-product biomolecules such as proteins and sugars, which promote 

fouling (coverage with biofilms or clogging with bioparticles), and also due to the necessarily 

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dilute nature of fermentation broths, which causes the use of energy-intensive distillation to be 

prohibitively expensive (1). 

A generalized block diagram of downstream processing of bioreactor media is shown in 

Figure 5 (2). During primary recovery, preliminary separation of solid (biomass) and aqueous 

(product) phases, as well as product extraction, occur. The fermentation broth is also reduced 

significantly in volume during this stage. Products are further concentrated during intermediate 

recovery, which also involves, in some cases, the redissolution of materials before further 

concentration is possible. In the final purification stages, a series of polishing steps are used to 

raise product purity to the final specifications. 

2. State of the Science 

Among separation techniques, mechanical filtrations are especially important in 

bioprocessing. These include micro-, ultra-, and nanofiltration; deep bed filtration; static and 

dynamic cross-flow filtration; electrofiltration; and centrifugation, involving both filtration and 

sedimentation. In these filtrations, fouling remains a major technical obstacle. In addition, the 

chemical and thermal separation techniques of ion exchange, chromatography, electrophoresis, 

crystallization, and extraction are also commonly used in bioprocessing. Research is currently 

underway in improving many bioseparation techniques, offering hope that major improvements 

in the efficiency of bioengineered processes can be achieved by incorporating new bioseparation 

technologies into process design, and several recent, excellent compilations of modern 

techniques are now available (3-5).

2.1 Physical Separations 

Emerging physical bioseparation techniques include aqueous two-phase extraction, 

reverse micellar extraction, cloud point extraction, and magnetic and electrophoretic separation 

(6, 7). 

2.1.1 Two-phase partitioning bioreactors. Among emerging techniques, the two-phase 

partitioning bioreactor appears to have great potential in enhancing the productivity of many 

bioprocesses. The approach integrates fermentation with a primary product recovery step by 

incorporating both organic and aqueous phases simultaneously, such that microbial growth 

occurs in the aqueous media and substrates and products partition into the organic phase based 

on their affinities for it. This approach allows controlled substrate delivery to the fermentation 

broth and effectively lowers product concentrations in the fermentation broth as well, promoting 

microbial health and activity. Although it is already practiced commercially, its effectiveness 

could still be improved by the discovery of low-cost solvents that are non-toxic for microbial 

growth (8, 9).

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Figure 5. Overview of bioseparations (2). 

Final Purification 

•Chromotography 

(affinity, reversed 

phase, ion exchange, 

size exclusion, etc.) 

•Crystallization 

•Fractional 

precipitation 

•Diafiltration 

Intermediate 

Recovery 

Stages 

Bioreactor 

Intracellular products Extracellular Products 

Cell harvesting 

•Centrifugation 

•Microfiltration 

•Ultrafiltration 

Cell disruption 

•Homogenization 

•Bead milling 

•Osmotic shock 

Cell debris removal 

•Centrifugation 

•Microfiltration 

•Vacuum filtration 

•Press filtration 

Biomass removal 

•Vacuum filtration 

•Centrifugation 

•Microfiltration 

•Ultrafiltration 

•Press filtration 

•Candle filtration 

Product extraction by •Flotation 

•Aqueous two phases 

•Organic solvents 

•Expanded-bed absorption 

•Batch absorption 

•Supercritical fluids 

•Reverse micelles 

•Extractive distillation 

Renaturation 

•Dissolution 

•Refolding 

Concentration 

•Ultrafiltration 

•Evaporation 

•Reverse osmosis 

•Precipitation 

•Extraction 

•Adsorption 

•Distillation 

Dehydration or 

solvent removal 

•Spray drying 

•Freeze drying 

•Tray drying 

•Fluid bed drying 

•Drum drying 

High purity required Low purity required 

Primary 

Recovery 

Stages 

Final 

Purification 

Stages 

IBs 

For solid final form

2.1.2 Non-solvent-based processing. A primary purpose of nonpolar organic 

solvents in bioprocessing is the dissolution of cell membranes to release intracellular products. 

However, organic solvents are undesirable for several reasons: they are frequently derived from 

non-renewable resources such as petroleum; they are frequently toxic and/or carcinogenic, and 

consequently expensive to treat in disposal; and they frequently form non-aqueous phases that 

hinder biodegradation. As a result, efforts to develop efficient, non-solvent-based approaches to 

cell disruption and product purification deserve high priority. 

The purification of polyhydroxyalkanoate (PHA) polymers provides an excellent 

example of the benefits possible with such approaches. Following fermentation, PHAcontaining cells are separated from culture media by centrifugation, filtration, and/or 

flocculation, and cells are then disrupted to recover the polymer. Subsequent recovery, 

unfortunately, typically involves extraction of the polymer from biomass with large amounts of 

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toxic and inflammable organic solvents (e.g., chloroform, methylene chloride, propylene 

carbonate, or dichloroethane). Another established method, again involving environmentally 

undesirable reagents, uses sodium hypochlorite for the digestion of non-PHA cellular materials, 

carrying the additional disadvantage that it partially degrades the PHA (10). 

To improve the environmental friendliness of PHA processing, a non-solvent-based 

process has been developed by Zeneca Agrochemicals (now part of Syngenta; 

www.syngenta.com) to assist in the commercial production of poly[(R)-3-hydroxybutyrate] 

[poly(3HB)] and poly(3HB-co-3V) by Alcaligenes eutrophus. In this process, cells are brought 

to 80°C and treated with a mixture of hydrolytic enzymes, including lysozyme, phospholipase, 

and lecithinase that hydrolyze cellular components without degrading the polymer. The polymer 

can then be recovered as a white powder after washing and drying, demonstrating the technical 

feasibility of performing novel separations utilizing advanced bioengineering techniques. Cost 

of necessary reagents, such as the enzymes involved in this case, remains an important 

consideration, however, and must be addressed to enable such techniques to compete with 

existing routes to synthesis of plastics based on petrochemical sources (11). Additional nonsolvent based methods of cell lysis, including modern developments in enzymatic, chemical, and 

mechanical cell disruption, are considered in detail in the recent book, Bioseparations Science 

and Engineering (5). 

2.2 Mechanical Separations 

A great variety of membrane-based techniques are not only under development, but are 

already central to commercial integrated bioprocesses, enabling further avoidance of 

environmentally deleterious reagents in bioprocessing. These approaches separate products from 

culture media based on hydrophobicity, volatility, and/or affinity for membrane components, and 

offer the potential for great selectivity as well as low-energy and waste-disposal requirements in 

biorefining (12). 

2.2.1 Pervaporation. Pervaporation is the separation of various liquid mixtures by 

partial vaporization through a non-porous membrane. The membrane acts as a selective barrier 

between the two phases: the liquid feed and the vapor permeate phases. It allows the desired 

component(s) of the liquid feed to dissolve within it and then to transfer through it by 

vaporization, resulting in a separation based primarily on differences in polarity rather than 

volatility. Pervaporation has shown great promise in separating alcohols such as ethanol and 

butanol from bioreactor media, as in the production of ethanol from rice straw by Pichia stipitis

(13), as well as other azeotropic and close-boiling mixtures, including isomers (14). A challenge 

associated with the use of pervaporation is the accumulation of less volatile components (higher 

alcohols) in fermentation media that can cause microbial growth inhibition (9). Nevertheless, 

industrial uses for this method are widespread, and membrane technologies as well as integration 

of pervaporation with other unit operations are still showing promising improvement (14).

2.2.2 Size-based separations. Membranes have traditionally been used for sizebased separations that require high throughput but relatively low resolution. Examples include 

microfiltration (MF) for clarification and sterilization as well as ultrafiltration (UF) for product 

concentration and buffer exchange. 

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Microfiltration is competitive with depth filtration, centrifugation, and expanded-bed 

chromatography for the initial harvest of products from bacterial, yeast, and mammalian cell 

cultures (15, 16, 5). Use of 0.2 μm-rated MF membranes can yield a particle-free harvest 

solution requiring no additional clarification prior to purification, while larger pore-size 

membranes can be used to improve product yield and throughput when the filtrate is 

subsequently treated with a normal flow filter for final clarification. MF systems are generally 

operated at constant flux instead of constant transmembrane pressure to improve product yield 

and throughput, although this practice tends to exacerbate fouling problems (17). An emerging 

solution to this problem is high-frequency, back-pulsing air scrubbing to clean the surface of the 

membrane continuously (18, 5). 

Ultrafiltration, in turn, has become the method of choice for protein concentration, 

replacing size-exclusion chromatography in this application (19). Plasmid DNA (20) and viruslike particles (21) can also be purified using UF. UF membranes are typically composed of 

polysulfone, polyethersulfone, or regenerated cellulose, and experience fouling problems 

analogous to those that plague MF membranes. Newly-developed composite cellulose 

membranes are less susceptible to fouling than are many synthetic polymers, and are more easily 

cleaned, but still possess excellent mechanical strength, thus outperforming other membrane 

materials. Nevertheless, cellulose has had a much longer development history than have 

synthetic polymers in membrane applications, and promising developments in the latter are still 

expected (22). 

2.2.3 Membrane chromatography. Recent studies are generating renewed interest 

in membrane chromatography (23). Diffusion limitations are less pronounced in membranes 

than in conventional bead packings, thus providing, in principle, a binding capacity independent 

of flow rate. Although the high, internal surface area provided by small pores in membranes is 

often offset by the reduction in convective flow, and it is challenging to achieve binding 

capacities competitive with bead packings, research into optimization of membrane 

chromatography continues (24, 25). Especially encouraging are manifold designs with high 

membrane density and low retention volumes that can accomplish up to 100-fold concentrations 

in a single stage (5).

Flow distribution within membrane modules is an important consideration for optimal 

performance, especially in manifold designs, and careful design of entrance and exit regions is 

essential to provide even, well-distributed flow. In addition, sensing and maintenance of 

constant concentration of the retained species at the membrane surface is becoming a new robust 

control strategy, enhancing product yield and providing greater operational robustness with 

respect to variations in feed quality (26). Finally, additional efforts are directed toward 

improving binding capacity, membrane selectivity, flow distribution, and flow rate, through 

adjustments in pore size, membrane chemistry, and membrane morphology (27, 28). 

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39 

2.3 Microbe Engineering 

Sometimes bioprocessing procedures can be simplified greatly by engineering an 

organism itself to produce a more convenient product, as in the development of DuPont’s biobased 1,3-propanediol (3G) process (29). In nature, two separate microorganisms are required to 

convert glucose to glycerol and then to convert glycerol to 3G. To avoid the difficulty of 

purifying the glycerol, a genetically-engineered microorganism was developed that converted 

glucose directly to 3G. With increasing advances in metabolic pathway engineering (see section 

II.A.2.7), such avenues are expected to become increasingly available. 

3. Research Priorities 

Separation technologies to facilitate commercial success of biomass conversions include 

those suitable for low-molecular-weight organic acids, organic esters, diacids, and alcohols; 

gases such as H2; and biobased oils such as biodiesel and biolubricants. Among these, advances 

in membrane technologies and in processes utilizing environmentally-benign solvents promise 

especially great benefits. 

3.1 Membrane Techniques 

The development of new and/or improved membrane materials that provide increased 

selectivity and specificity for the desired substances, as well as increased flux with stability and 

robustness, is of central importance to the membrane-based techniques discussed below (5). 

3.1.1 Pervaporation. The use of pervaporation to remove either water or bioproducts 

from bioreactor media appears promising. Continued support for new membrane materials, new 

module and process designs, and improved theoretical understanding and modeling of the 

pervaporation process should therefore be pursued. The work of Vane and colleagues at the EPA 

National Risk Management Research Laboratory (NRMRL) is a noteworthy example of efforts 

in the development of pervaporation modeling and performance prediction software (30). 

3.1.2 Micro- and ultrafiltration. Microfiltration and ultrafiltration promise to 

become major unit operations in the emerging biorefinery arena. The development of new 

materials for UF and MF, including porous metals and ceramics as well as polymers, is therefore 

an important priority. Similarly, nanofiltration and reverse osmosis are becoming increasingly 

important, with recent developments in nanotechnology promising to yield new materials with 

significantly improved fluxes and selectivities (5). 

3.1.3 Membrane chromatography. An improved understanding of the interactions 

between culture media components and synthetic polymers suitable for membranes would 

greatly facilitate the design of synthetic substrates for use in membrane chromatography. 

Among those, ligand-binding and sterically-interacting species should be investigated closely to 

improve the selectivity of membrane chromatography while maintaining acceptably high 

throughput. 

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3.1.4 Antifouling techniques. Fouling is a persistent problem among membrane 

technologies, with the result that methods to diminish fouling of membranes and ion exchange 

materials, as well as to remove impurities such as salts or acids that cause complications in 

downstream processes, are high priorities in the advancement of bioseparations. 

3.2 Environmentally Benign Solvents 

New renewable, biodegradable solvents are needed to support environmentally-friendly 

extraction processes. Supercritical CO2, a highly compressed phase of CO2 possessing properties 

of both liquid and gas phases, is one benign solvent that has already achieved great popularity 

and that has the potential to contribute performance, cost-effectiveness, and sustainability to 

separations of both biofuels and biomaterials (31). 

3.3 Integrated Modules 

 Combined- or hybrid-unit operations in which a bioreactor is integrated with a 

bioseparation module, as in two-phase reactor systems, are particularly attractive as means to 

overcome limitations inherent to bioprocessing. These are particularly desirable for their 

potential to remove products as they are synthesized, alleviating the nearly universal problem of 

product inhibition in culture media (5). 

4. Commercialization 

One of the primary limitations to commercialization of biobased products is the high cost 

of isolating the desired products. Accordingly, development of advanced separation 

technologies should be an integral part of a comprehensive program for reducing costs and 

encouraging commercialization. Affinity-based separations, environmentally-benign extractions, 

and membrane separations hold the promise of operating at low temperatures and thereby 

reducing the demand for expensive energy presented by thermal separation processes such as 

distillation. Currently, fouling, other flux limitations, and sub-optimal selectivities limit the 

larger scale deployment of the more sustainable separation techniques, with the implication that 

research should focus on these areas. 

5. References 

(1) Perry, R. H., and D. W. Green, Eds. (1997). Perry’s Chemical Engineers’ Handbook. 

McGraw Hill, New York. 

(2) Petrides, D. P., C. L. Cooney, and L. B. Evans (1989). An introduction to biochemical 

process design, in Chemical Engineering Problems in Biotechnology (M. L. Shuler, Ed.). 

American Institute of Chemical Engineers, New York, pp 351-391. 

(3) Keller, K., T. Friedmann, and A. Boxman (2001). The bioseparation needs for 

tomorrow, Trends Biotechnol 19:438-441. 

(4) Ladisch, M. R. (2001). Bioseparations Engineering: Principles, Practice, and 

Economics. Wiley-Interscience. 

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41 

(5) Todd, P., S. R. Rudge, D. P. Petrides, and R. G. Harrison, Eds. (2003). Bioseparations 

Science and Engineering. Oxford University Press. 

(6) Karumanchi, R. S. M. S., M. Dueser, and P. W. Todd (2000). Multistage magnetic and 

electrophoretic extraction of cells, particles and macromolecules, Adv Biochem Eng 

Biotechnol 68:143-190. 

(7) Karumanchi, R. S. M. S., S. N. Doddamane, C. Sampangi, and P. W. Todd (2002). 

Field-assisted extraction of cells, particles and macromolecules, Trends Biotechnol 

20:72-78. 

(8) Gu, T. (2000). Liquid-liquid partitioning methods for bioseparations, in Handbook of 

Bioseparations (S. Ahuja, Ed.). Academic Press, San Diego, pp 329-364. 

(9) Malinowski, J. (2001). Two-phase partitioning bioreactors in fermentation technology, 

Biotechnol Adv 19:525-538. 

(10) Williamson, D. R., and J. F. Wilkinson (1958). The isolation and estimation of the polybeta-hydroxybutyrate inclusions of Bacillus species, J Gen Microbiol 19:198-209. 

(11) Byrom, D. (1987). Polymer synthesis by micro-organisms: technology and economics, 

Trends Biotechnol 5:246-250. 

(12) Bailly, M., H. Balmanna, P. Aimar, F. Lutin, and M. Cheryan (2001). Production 

processes of fermented organic acids targeted around membrane operations: design of 

the concentration step by conventional electrodialysis, J Membr Sci 191:129. 

(13) Nakamura, Y., T. Sawada, and E. Inoue (2001). Mathematical model for ethanol 

production from mixed sugars by Pichia stipitis, J Chem Technol Biotechnol 76:586-592. 

(14) Smitha, B., D. Suhanya, S. Sridhar, and M. Ramakrishna (2004). Separation of organicorganic mixtures by pervaporation: a review, J Membr Sci 241:1-21. 

(15) van Reis, R., L. C. Leonard, H. C. Chung, and S. E. Builder (1991). Industrial scale 

harvest of proteins from mammalian cell culture by tangential flow filtration, Biotechnol 

Bioeng 38:413-422. 

(16) Dorgan, J. R. (1992). Polymer Membranes for Bioseparations, in Polymer Applications 

for Biotechnology (D. S. Soane, Ed.). Prentice Hall, Englewood Cliffs, pp 314. 

(17) Belfort, G., R. H. Davis, and A. L. Zydney (1994). The behavior of suspensions and 

macromolecular solutions in crossflow microfiltration, J Membr Sci 96:1-58. 

(18) Levesley, J. A., and M. Hoare (1999). The effect of high frequency backflushing on the 

microfiltration of yeast homogenate suspensions for the recovery of soluable proteins, J

Membr Sci 158:29-39. 

(19) Kurnik, R. T., A. W. Vu, G. S. Blank, A. R. Burton, D. Smith, A. M. Athalye, and R. van 

Reis (1995). Buffer exchange using size exclusion chromatography, countercurrent 

dialysis, and tangential flow filtration: models, development, and industrial application, 

Biotechnol Bioeng 45:149-157. 

(20) Kahn, D. W., M. D. Butler, G. M. Cohen, J. W. Kahn, and M. E. Winkler (2000). 

Purification of plasmid DNA by tangential flow filtration, Biotechnol Bioeng 69:101-106. 

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42 

(21) Cruz, P. E., C. C. Peixoto, K. Devos, J. L. Moreira, E. Saman, and M. J. T. Carrondo 

(2000). Characterization and downstream processing of HIV-1 core and virus-Iikeparticles produced in serum free medium, Enzyme Microb Technol 26:2661-2670. 

(22) van Reis, R., and A. L. Zydney (1999). Protein ultrafiltration, in Encyclopedia of 

Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation (M. C. 

Flickinger and S. W. Drew, Eds.). John Wiley & Sons, New York, pp 2197-2214. 

(23) Zeng, X., and E. Ruckenstein (2000). Membrane chromatography: preparation and 

applications to protein separation, Biotechnol Prog 15:1003-1019. 

(24) Schweitzer, P. A. (1997). Handbook of Separation Techniques for Chemical Engineers. 

McGraw-Hill. 

(25) Noble, R. D., and P. A. Terry (2004). Principles of Chemical Separations with 

Environmental Applications. Cambridge University Press. 

(26) van Reis, R., E. M. Goodrich, C. L. Yson, L. N. Frautschy, R. Whiteley, and Z. Al 

(1997). Constant Cwall ultrafiltration process control, J Membr Sci 130:123-140. 

(27) Sarfert, F. T., and M. R. Etzel (1997). Mass transfer limitations in protein separations 

using ion-exchange membranes, J Chromatogr A 764:3-20. 

(28) Klein, E. (2000). Affinity membranes: a 10 year review, J Membr Sci 179:1-27. 

(29) Laffend, L. A. (1997). Bioconversion of a Fermentable Carbon Source to 1,3-

propanediol by a Single Microorganism, E.I. DuPont de Nemours and Company, Patent 

5686276. 

(30) Pend, M., S. X. Liu, and L. M. Vane (2003). Profiling concentration gradient in a 

tubular membrane pervaporation module: a modeling approach, Intl Comm Heat Mass 

Transfer 30:755-764. 

(31) Kemmere, M. F., and T. Meyer, Eds. (2005). Supercritical Carbon Dioxide in Polymer 

Reaction Engineering. John Wiley & Sons. 

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Chapter III 

Biomaterials 

A. PROBLEM AND SIGNIFICANCE 

1. Overview of Importance 

The worldwide production of plastics reached 260 billion pounds per year at the end of 

the 20th century, with a value of over $310 billion to U.S. economy in 2002 (1). Large quantities 

of petroleum are used to produce present-generation plastics, but oil is of finite supply, and as 

world economies continue to develop oil it will become more and more expensive (2). 

Additionally, pollution results from the manufacture, use, and disposal of plastic materials. As 

the world’s finite supplies of petroleum are used up, and as the growing industrial economies of 

China and other regions continue to rapidly boost demand, oil prices are skyrocketing, along 

with the prices for all products that rely on oil supplies. 

Moreover, the increased demand is driving us to drill in sensitive areas and to use lowergrade crude oils that are less economical and that contain contaminants that threaten the 

environment. However, plastics offer profound societal benefits, including increased agricultural 

production, reduced food spoilage, reduced fuel consumption in lighter-weight vehicles, better 

health care, and low-cost, net-shape manufacturing. We need plastics, but the financial hardship 

consumers are feeling at the gas pump from increasing prices is also sharply impacting the 

plastics industries where production costs are rising and being passed on to the consumer. What 

will happen to our environment, to human and animal health, and to the plastics industries–the 

fourth largest manufacturing sector of the U.S. economy employing more than 1.2 million 

citizens (1)–if sustainable technologies are not developed and deployed? While energy recovery 

through combustion, recycling, and minimizing use of plastics all aid pollution prevention, a new 

paradigm is emerging that holds great promise—namely the production of plastic materials using 

renewable resources as feedstocks. This approach is becoming increasingly viable due to 

advances in industrial biotechnology. 

 It can be argued that using bioengineering techniques to make plastic materials is more 

economically favorable than using these same techniques to make fuels. The reason for this 

economic advantage is simply that plastics have a greater value than fuels. Consider gasoline at 

a retail price of $3 per gallon; the price before taxes would be approximately $2.25–$2.65 per 

gallon. This means that for approximately $2.50, about 7 pounds of gasoline can be purchased; 

that is, the price per pound is about $0.35. For comparable crude oil prices, commodity plastic 

resins would cost anywhere from $0.50 per pound to upwards of $3.00 per pound. As discussed 

further below, biobased plastics are already economically competitive with conventional 

petroleum based plastics because of the higher price they can command when they have specific 

materials properties. It can be argued that industrial bioengineering for commodity production 

will first be utilized on the most economic targets and that plastics are more economically 

attractive than fuels. 

 Pursuing economically viable plastics using bioengineering techniques can also affect

fuel prices to a substantial degree. This economic impact is due to several factors. Presently, 

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about 65 percent of petroleum goes to transportation fuels, (3) however, if the 5–10 percent of 

petroleum going into plastic materials can be substituted, the marginal cost of petroleum could 

decrease significantly. This is due to the fact that market surpluses and shortages are, in fact, 

relatively small perturbations on a large base number. Perhaps more significantly, producing 

bioplastic materials provides an opportunity for economic integration. That is, by having 

integrated biorefineries in which high-margin plastics are coproduced with biofuels, economic 

production of the lower-margin fuel products can be produced. 

 There are a number of materials that can be made that start with renewable resources and 

use bioengineering techniques. These include thermoplastics, thermosets, foams, pressure 

sensitive adhesives, various biocomposites, and coatings. This document focuses on the 

emerging material classes where bioengineering techniques play the largest role; that is, where 

engineered organisms play a prominent role. Only a brief synopsis of materials available from 

bioresources using more traditional chemical routes is given; several excellent monographs are 

available that discuss biobased plastics and composites (4-6). 

2. Conventional Plastics 

Considerable environmental pollution occurs as a result of the production, use, and 

disposal of conventional plastics. The absolute mass of plastics produced in a given year is small 

compared to that of fuels; however, due to their persistence they present unique challenges when 

released into the environment. Current polymer materials are nearly all derived from 

petrochemical sources, contributing significantly to greenhouse gas emissions during both 

production and incineration (7, 8). Because producing plastics from renewable resources 

provides a real economic opportunity, perhaps the best possible substitution strategy would be to 

make non-degradable, persistent plastics from renewable resources; this strategy could provide at 

least a relative sequestration of CO2 from the atmosphere, thereby mitigating effects of global 

climate change (9). 

3. Plastics from Renewable Resources 

Through application of bioengineering in conjunction with traditional chemical 

processing techniques, bioplastics production is proving viable in a number of commercially 

produced materials. Most notable are the production of PLA by Cargill-Dow under the trade 

name of NatureworksTM (http://www.packexpo.com/ve/82489/main.html); the production of 

SoronaTM polyesters, which contain a 1,3-propanediol monomer derived from renewable 

feedstocks, by DuPont (http://www.dupont.com/sorona/backgroundsoronapolymer .html); and 

the production of PHA by Metabolix, Inc. (www.metabolix.com). 

Other environmentally benign plastic materials include those available directly from 

plants, such as starches, starch-protein composites, triglycerides, and cellulosics 

(www.greenplastics.com). It is important to understand that many such plant-based plastics are 

presently competitive with petroleum-based materials on a cost-performance basis. A clear 

example of this competitiveness is the case of Nylon-11 (polyamide-11), which is made from 

castor oil extracted from castor beans. This tough and resilient material is marketed as Rilsan by 

the Arkema Company (www.arkema-inc.com) and is widely used in powder-coating 

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applications. These demanding applications include protective coverings for submerged 

pipelines and other industrial piping. In these uses, it is the superior properties of the plastic, 

rather than its renewable nature, which has developed the market. While biodegradable plastics 

are also available from petroleum-derived chemicals, this is not accomplished using 

bioengineering techniques and so these materials fall outside the scope of this document. 

Polymer Nomenclature 

Because the challenges associated with bioplastics development depend on the 

detailed chemistry and physics peculiar to polymers, a set of useful terms is presented here for 

readers less familiar with this specialized topic. 

Polymer. A high-molecular-weight organic compound with a repeating unit (a 

monomer) that constitutes its structure. While typically represented by a long linear structure, 

different chain architectures are possible by including branching points and other monomers. 

If two or more monomers are present in the molecule it is referred to as a copolymer. Block 

copolymers consist of long strings of the same monomer connected to other long stretches of a 

different monomer whereas random copolymers have a random sequential arrangement of the 

different monomers. Branched polymers consist of linear sections that diverge at a point so 

that three or more strands emerge from the common branch point. In hyperbranched

polymers the divergence from one branch point leads to another and each of those branches 

leads to another branch point and so on to create a highly branched or arborescent (tree like 

branching) polymer. 

 Glass Transition Temperature (Tg). The temperature at which the reversible change in 

an amorphous polymer or in the amorphous regions of a semi-crystalline polymer change to 

(or from) a hard and brittle glassy material to a soft, viscous, rubbery material. Hardness, 

thermal expansibility, and specific heat all change abruptly at Tg, however, it is not a true 

thermodynamic transition as it shows a dependence on cooling rate. 

 Melting Temperature (Tm). The temperature where all crystalline structure is lost to 

yield a liquid. Scientifically, it is incorrect to talk about the melting temperature of a fully 

amorphous polymer, however, in practice a melt-flow temperature of 50 °C above the Tg is 

sometimes used. 

Polydispersity Index (PDI). The ratio of weight averaged to number averaged 

molecular weight. This index measures the breadth of the molecular weight distribution in a 

polymer sample. If all polymer chains in a sample had exactly the same length, the PDI 

would be 1.0; typical polymer samples have values from 1.5 to 30.0. 

Tacticity. Refers to the geometric arrangement of substituent groups (e.g., a methyl 

group) along a polymer chain backbone. Syndiotactic indicates the substituents alternate 

regularly on opposite sides, isotactic means the substituents are all on the same side, and 

atactic means the order is random. 

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4. Renewable Polymer Production 

Conceptually, three tiers of polymer production from renewable resources exist, as shown 

in Figure 6 (10). Polylactic acids (PLAs) and Sorona polymers are produced in a three-stage 

process. The feedstock is first harvested in the form of crops, crop residuals, or other biomass, 

and refined to yield plant sugars (primarily glucose and xylose). A secondary stage of 

production consists of forming the subunit monomers via fermentation and separation. Finally, 

the third stage consists of traditional chemical processing to form the polymers that will 

ultimately be molded into plastic parts. 

Figure 6. Systems for the production of materials by bioengineering (10). 

Systems for Bioplastics Production

1. 3-Stage Production ( Polylactides )

CO2 + H2

O

Plants

Photosynthesis

Sugars

Bacteria

Fermentation

Lactic acid

Catalyst

Chemosynthesis

Polylactide

2. 2-Stage Production ( Biopolyesters )

CO2 + H2O

Plants

Photosynthesis

Sugars, Plant oils

Bacteria

Fermentation

Biopolyesters

3. 1-Stage Production ( Biopolyesters )

CO2 + H2

O

Plants, Algae

Photosynthesis

Biopolyesters

Systems for Bioplastics Production

1. 3-Stage Production ( Polylactides )

CO2 + H2

O

Plants

Photosynthesis

Sugars

Bacteria

Fermentation

Lactic acid

Catalyst

Chemosynthesis

Polylactide

2. 2-Stage Production ( Biopolyesters )

CO2 + H2O

Plants

Photosynthesis

Sugars, Plant oils

Bacteria

Fermentation

Biopolyesters

3. 1-Stage Production ( Biopolyesters )

CO2 + H2

O

Plants, Bacteria, Algae

Photosynthesis

Biopolyesters

Systems for Bioplastics Production

1. 3-Stage Production ( Polylactides )

CO2 + H2

O

Plants

Photosynthesis

Sugars

Bacteria

Fermentation

Lactic acid

Catalyst

Chemosynthesis

Polylactide

2. 2-Stage Production ( Biopolyesters )

CO2 + H2O

Plants

Photosynthesis

Sugars, Plant oils

Bacteria

Fermentation

Biopolyesters

3. 1-Stage Production ( Biopolyesters )

CO2 + H2

O

Plants, Algae

Photosynthesis

Biopolyesters

Systems for Bioplastics Production

1. 3-Stage Production ( Polylactides )

CO2 + H2

O

Plants

Photosynthesis

Sugars

Bacteria

Fermentation

Lactic acid

Catalyst

Chemosynthesis

Polylactide

2. 2-Stage Production ( Biopolyesters )

CO2 + H2O

Plants

Photosynthesis

Sugars, Plant oils

Bacteria

Fermentation

Biopolyesters

3. 1-Stage Production ( Biopolyesters )

CO2 + H2

O

Plants, Bacteria, Algae

Photosynthesis

Biopolyesters

In a two-stage production system, plant sugars are used as substrates to support growth of 

bacteria that synthesize polymeric materials directly to store their own excess energy, much as an 

animal synthesizes glycogen. Such materials are being developed by a number of groups and 

corporations around the globe, including Proctor and Gamble (under the trade name Nodax, 

www.nodax.com) and Metabolix, Inc. (www.metabolix.com)—both of which now claim the 

ability to provide commercial scale samples. It can also be argued that the many interesting and 

useful materials being made from soybean and other plant oils (6) fall into the two-stage 

production scheme, but with the second stage consisting of chemical rather than biochemical 

transformation. 

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In single-stage production, the material of interest is grown directly within the plant. 

This technology is the most futuristic of the three but holds the greatest promise for additional 

environmental benefits, particularly the direct capture of atmospheric CO2 within the plastic; it is 

now being pursued energetically by Metabolix, Inc. for production of PHA within both the 

genetic model organism, Arabidopsis, and within the hardier switchgrass. While technical issues 

regarding both genetic engineering of the plants and separation of product polymers from plant 

tissues persist, ongoing research efforts have provided steady progress in these areas (11, 12). 

5. Environmental Benefits of Green Plastics 

Since 1993, the International Organization for Standardization (ISO) has been developing 

LCA programs to analyze material and energy requirements of various products (13). Life-cycle 

Impact Assessment (LCIA), in turn, is the part of LCA in which the inventory of a product’s 

material flows is translated into environmental impacts and resource consumption (14). 

Although the LCIA of plastic products is still in the early stages of development, it will soon be 

possible to compare the environmental impacts of various green plastics with one another and 

with the conventional polyolefins that make up more than 90 percent of current plastics 

production in a quantitative, reliable way. 

Controversy does exist regarding the extent to which the production of bioplastics serves 

the principles of environmental sustainability. Gerngross and Slater, for example, have argued 

that the conversion of biomass to biopolymers is energy-intensive (involving fertilizer 

production, farming, corn wet-milling, fermentation, and polymer purification, for example) and 

results in significantly greater net CO2 emissions than the synthesis of petroleum-based plastics 

(9). While conceding that renewable energy sources (e.g., solar, wind, geothermal, etc.) could be 

used to improve the environmental profiles of these processes, they argue that greater 

environmental benefit could be obtained by using that energy to displace fossil fuels in other 

applications. Gerngross and Slater also question the benefits of biodegradability, with the 

rationale that non-degrading polymers produced from renewable resources could be used to 

sequester atmospheric CO2. 

 The gaseous emission resulting from biodegradation of any biopolymers also represents 

an important consideration, although few agree that the alternative, accumulation of plastic 

debris, is desirable. In landfills, oxygen is limited, and organic matter is often degraded 

anaerobically to yield a mixture of CO2 and CH4 rather than pure CO2. Because CH4 is a 23-fold 

more potent greenhouse gas than CO2 (15), anaerobic degradation of bioplastics is potentially 

quite deleterious to the environment. In a composting environment, however, in which regular 

mixing ensures aerobic degradation, only CO2 is released, causing a process supported by 

sustainable agriculture and composting to be effectively CO2-neutral (16). In addition, another 

promising disposal option for bioplastics may be waste incineration with recovery of the energy 

generated, which then can be used to displace fossil fuel-derived energy (17). 

 Gruber has noted that a life-cycle assessment performed by Gerngross considered only 

the production of PHAs by microbial fermentation, and that other materials such as PLA hold the 

promise of lower energy requirements for production. This perspective has, in fact, been 

supported by other more recent life-cycle analyses (18). A review of 20 such LCA studies of 

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biodegradable polymers indicates that starch, the major component of approximately 75 percent 

of green plastics production, offers important environmental benefits compared to conventional 

polymers (19). Concerning commercially produced biodegradable polymers, the environmental 

benefits of PLA, currently accounting for 10–15 percent of production, and of biodegradable 

polymers made from nonrenewable resources, accounting for approximately 10 percent of 

production, seem to be smaller than starch-based thermoplastics, but still greater than 

conventional polymers. For microbial polyesters, which currently make up a very small part of 

total green plastics production, the environmental advantage seems to be small (or perhaps 

nonexistent), but the fermentation technologies for producing them are among the most recently 

developed, and both the production method and the scale of production can influence evaluations 

of the overall environmental balance (20). Additionally, the prospect of producing these plastics 

in transgenic plants completely alters the environmental consequences of production, thereby 

opening up improved life-cycle possibilities (12, 21). 

 Given the inherent problems associated with persistent plastics in the environment–

increasing pressure on landfill space, concerns over climate change, and the economic reality 

that biobased plastics are already competing in the market without subsidies–applying the tools 

of industrial biotechnology to the production of environmentally benign plastics is a particularly 

vibrant area of scientific and commercial activity. 

6. References 

(1) Society of the Plastic Industries (SPI) (2003). Size and Impact of the U.S. Plastics 

Industry. Executive Summary, http://www.plasticsindustry.org/industry/impact.htm. 

(2) Dreffeyes, K. S. (2001). Hubbert’s Peak: The Impending World Oil Shortage. 

Princeton University Press, Princeton, NJ. 

(3) Board of Transportation Statistics (2004). Overview of U.S. Petroleum Production, 

Imports, Exports, and Consumption, Washington, D.C., http://www.bts.gov/publications/ 

national_transportation_statistics/2004/html/table_04_01.html. 

(4) Kaplan, D. L. (1998). Biopolymers from Renewable Resources. Springer-Verlag, 

Berlin. 

(5) Stevens, E. S. (2002). Green plastics: An Introduction to the New Science of 

Biodegradable Plastics. Princeton University Press, Princeton, NJ. 

(6) Wool, R. P., and X.S. Sun (2005). Bio-Based Polymers and Composites. Elsevier 

Academic Press, Burlington, MA. 

(7) Harper, C. A. (2000). Modern Plastics Handbook. McGraw-Hill Professional, New 

York. 

(8) Strong, A. B. (1999). Plastics: Materials and Processing, 2nd Edition. Prentice Hall, 

New York. 

(9) Gerngross, T. U., and S.C. Slater (2003). Biopolymers and the environment, Science 

299:822-825. 

(10) Doi, Y. (2004). In Life Cycle Assessment of Microbial Polyesters, BioEnvironmental 

Polymer Society Meeting, Denver. 

bioengineering report_5_x1a.pdf 62 5/25/07 6:45:07 PM
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(11) Skraly, F. (2002). Bioplastics, in Encyclopedia of Environmental Microbiology. John 

Wiley & Sons, New York. 

(12) Snell, K. D., and O.P. Peoples (2002). Polyhydroxyalkanoate polymers and their 

production in transgenic plants, Metab Eng 4:29-40. 

(13) International Organization for Standardization Environmental Management (1997). Life 

Cycle Assessment—Principles and Framework. Geneva, Switzerland, ISO 14040. 

(14) Hauschild, M. (2005). Assessing environmental impacts in a life-cycle perspective,

Environ Sci Technol. 39:81A-88A. 

(15) U.S. Department of Energy (2003). U.S. Climate Change Technology Program: 

Research and Current Activities, Washington, D.C., http://www.climatetechnology.gov/

library/2003/currentactivities/car24nov03.pdf DOE/PI-0001. 

(16) Schlesinger, W. H. (1997). Biogeochemistry: An Analysis of Global Change. 

Academic Press, San Diego, CA. 

(17) Holmgren, K., and D. Henning (2004). Comparison between material and energy 

recovery of municipal waste from an energy perspective: a study of two Swedish 

municipalities, Res Cons Recyc 43:51-73. 

(18) Vink, E. T. H., K. R. Ra ́ bago, D. A. Glassner, and P.R. Gruber (2003). Applications of 

life cycle assessment to NatureWorks polylactide (PLA) production, Polymer Degrad and 

Stab 80:403–419. 

(19) Patel, M. (2002). Do Biodegradable Plastics Fulfill the Expectations Concerning 

Environmental Benefits? 7th World Conference on Biodegradable Polymers & Plastics, 

Tirrenia, Italy. 

(20) Stevens, E. S. (2005). Green plastics: Environment-friendly technology for a 

sustainable future, http://www.greenplastics.com/. 

(21) Martin, D. P., O. P. Peoples, and S. F. Williams (2000). Methods for Isolating 

Polyhydroxyalkanoates from Plants. Patent US 6,709,848. 

B. POLYLACTIDES 

1. Introduction 

The family of bioplastics known as PLA encompasses the set of polymers of lactide, a 

cyclic dimer produced by the dehydration of lactic acid, which represents a highly promising and 

versatile category of biomaterials. The development of PLA into a commodity polymer has 

spanned over six decades of research and design from inception to the present commercial 

utility. The recorded history of PLA development began in 1932 when Carothers and colleagues 

documented the earliest attempted polymerization and depolymerization of oligomeric lactides in 

the Journal of the American Chemical Society (1). In 1954, the DuPont Corporation synthesized 

high molecular-weight PLA with improved lactide purification techniques, as well as antimony 

trioxide and antimony trihalide as polymerization catalysts (2). Later, methods were developed 

to produce high molecular weight PLA with properties sufficient for competition with traditional 

oil-derived polymers. Higher molecular-weight PLA was found to have interesting properties, 

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50 

but its production was prohibitively expensive. In the 1960s, several researchers investigated 

relationships between the chemical structures of lactide monomers and the configurations and 

crystalline structures of resulting PLAs (3-7). Because of its inherent biodegradability, PLA 

was one of the earliest polymers used in biomedical applications; Kulkarni et al. demonstrated 

the human body’s ability to absorb PLA-based sutures in 1966(8). Work in the 1970s and 1980s 

focused primarily on discovery of new catalyst systems for polymerization, improving 

characterization with new analytical techniques, and investigation of new medical applications 

(9). 

Recently, the need for more biofriendly polymers has led to the study of additional 

catalyst systems, new types of copolymers, and polymerization mechanisms. In the early 1990s, 

methods were developed for the continuous production of both lactide and PLA. A major step in 

the commercialization of PLA occurred when Cargill Corporation developed its method to 

produce PLA in a continuous process (10-13). A joint venture of Cargill with Dow Polymers 

(CDP) was then formed in 1997 to develop further the potential of PLA as a commodity 

polymer. A production facility opened in 2002 and was built in Blair, Nebraska with the 

capability of producing approximately 300 million pounds of PLA per year. Large-scale 

production of PLA has dramatically decreased the cost of PLA resins and is now enabling it to 

compete with established petroleum-based materials (Wall Street Journal, “One Word of Advice: 

Now it’s Corn,” October 12, 2004). Dow Chemical recently exited the joint venture in 2004 and 

the Blair facility is operated by a wholly owned subsidiary of Cargill called Natureworks 

(www.natureworksllc.com). This corporation is now producing and selling large quantities of 

plastics being used in products ranging from clothing to food packaging. 

2. PLA Biosynthesis, Biodegradation, and Environmental Impact: Overview 

PLA is presently commercially produced utilizing corn as the feedstock according to the 

process diagramed in Figure 7. Corn first undergoes the traditional milling process to produce 

unrefined glucose (dextrose), after which microorganisms ferment the glucose to lactic acid. 

Importantly, this fermentation process is anaerobic: a wide variety of literature suggests that 

anaerobic fermentations hold great advantage over aerobic fermentations when CO2 balance is of 

concern (discussed below). After fermentation, the resulting lactic acid is formed into cyclic 

lactide through dimerization using reactive distillation. The lactide ring is then polymerized to 

produce PLA. 

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51 

Figure 7. Commercial route to biobased PLA plastics. 

The degradation of polymeric materials in the environment is a critical area of concern 

due to the large quantity of plastics generated. Each year, over 70 million tons of polymers are 

produced which end up in landfills (14, 15). Breakdown of PLA in the environment can occur 

biotically or abiotically (16). In the absence of sufficient microbial activity or oxygen, 

hydrolysis becomes the predominant pathway for degradation, while in aerated composting 

environments, biotic processes can degrade PLA rapidly (within weeks) and completely (17, 18). 

Regardless of the process, degradation is also subject to the form of the plastic and purity of the 

PLA used (19). As an alternative to biodegradation, waste PLA can be recycled into lactic acid, 

which can then be reformed into lactide and repolymerized (16, 17). 

Figure 8. Life cycle analysis results for energy content of various thermoplastic 

polymers (20). 

 PLA1 represents present technology; PLA Bio/WP is the projection for the production of 

PLA from agricultural waste using wind power. 

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To assess the environmental impact of PLA synthesis and use, a comprehensive LCA 

enabling apples-to-apples comparisons with petrochemical-based thermoplastics was recently 

undertaken (Figure 8) (20). Among the most notable benefits of PLA shown were reductions in 

both fossil-fuel use and global warming potential, even assuming use of fossil-based energy 

sources for agriculture and processing. Compared to most traditional hydrocarbon-based 

polymers, PLA uses 30–50 percent less fossil-fuel energy and results in lower CO2 emissions by 

50–70 percent. Whereas conventional thermoplastic polymers require oil as their source of 

monomers and additional fossil fuels for processing, solar energy provides approximately onethird of the gross energy requirement in PLA production; in addition, processing of oil into 

conventional plastics releases even greater amounts of CO2 than does PLA production (20). On 

October 11, 2005 Natureworks announced that they would purchase renewable energy 

certificates to offset the fossil energy being used in the production of PLA. By doing so, they 

claim to be producing the first-ever greenhouse gas-neutral commercial plastic (see 

www.natureworks.com). 

3. State of the Science 

3.1 Physical Properties 

The bulk properties of PLA are greatly affected by the molecular weight of the polymer, 

the chain architecture (branched vs. linear), and the degree of crystallinity in the polymer (21, 

22). The amount of crystalline character within a type of PLA, in turn, is determined by the 

relative proportions of L- and D-lactide in the polymer backbone. A diagram of representative 

backbones is provided in Figure 9. 

Figure 9. Stereochemistry of L, D, and D,L-PLA backbones. 

n

O

H CH3

H3C H O

O

H

O

H CH3

H3C H O

O

O O O

H

H CH3

H3C H O

O

O

O O

H3C H

H CH3

O

O

H

O O

H3C H

H CH3

O

O

O O

H

H3C H

H CH3

O

O

O O

H3C H

H CH3

O

O

O

Poly(L-lactide)

Poly(D-lactide)

n

O

H CH3

H CH3 O

O

H

O

H3C H

O

O O

H H3C H

H CH3O

O

Poly(D,L-lactide)

bioengineering report_5_x1a.pdf 66 5/25/07 6:45:09 PM
53 

PLA samples containing 87.5 percent L-lactide are completely amorphous, while samples 

with 92 percent L-lactide possess some crystallinity (14). Polymers from 100 percent L -lactide 

can be nearly half-crystalline (16). The Tm range of crystalline PLA is 145-186°C (21), although 

a blend of 100 percent D-lactide and 100 percent L-lactide polymers in a stereo-complex form a 

closely packed crystalline structure that increases the Tm to 230°C (23). The appearance of the 

PLA is also affected by the crystalline content. Amorphous PLA and low-crystalline PLA are 

clear materials with high gloss, while highly crystalline PLA is an opaque white material. 

The molecular weight, structure, and crystallinity of PLA play important roles in its 

mechanical properties as well, including tensile strength, tensile modulus, and percent elongation 

to break. These are comparable to those of poly(ethylene terephthalate) (PET), PP, and 

polystyrene (PS), fortunately, presenting the possibility that PLA variations may replace 

thermoplastics in many applications. Investigation of such applications is an active area of 

research in Japan, North America, and Europe (17, 21, 22). 

3.2 Applications 

A wide range of products can be produced from PLA and PLA derivatives, including 

structural support and drug delivery in medical applications, tough fibers in woven and nonwoven products, and in molded or extruded consumer products. 

In initial medical applications, lactide and glycolide copolymers were used to make 

absorbable sutures capable of replacing denatured collagen or catgut (14). Materials based on 

PLA and PLA copolymers have also been designed to replace metal and other non-absorbable 

polymers as therapeutic aids in surgery, including pins (24), plates (25), screws (25, 26), suture 

anchors (27), and intravascular stents (9, 28). The advantages of PLA and PLA glycolide 

copolymers for these applications comes from the ability of the body both to degrade the 

polymers and to metabolize the degradation products over time, leaving no residual foreign 

material in the body (8). The ability to tune PLA degradation times is also relevant to the field of 

drug delivery, as drugs encapsulated in polymers can be released based on the known 

degradation time of PLA (29) or PLA copolymers (30, 31), even allowing the specific targeting 

of organs (32). Other medical applications currently being pursued include dressings for burn 

victims (33), substrates for skin grafts (24), and dental applications (9). Medical usage in 

devices, sutures and drug delivery systems, while technologically important, nevertheless 

represents only a small opportunity for displacing less environmentally benign plastics. 

Fortunately, PLA also has properties suitable for commercial fiber products. Fibers can 

be produced from either pure liquid polymer, in a melt-spun process (34, 35), or from polymer 

dissolved in an organic solvent, in a solution-spun process (36, 37). Advantageously, fibers 

produced from PLA have lower processing temperatures than PET fibers and therefore require 

lower energy input during processing. In applications, non-woven fibers are able to wick 

moisture without absorbing it and can therefore be used in products such as diapers. Materials 

produced from woven fibers have additional desirable properties, including favorable hand and 

touch, drape, wrinkle resistance, rapid wicking, and low-moisture absorbance. Woven PLA 

fiber-based materials are highly resilient to wear, have excellent UV resistance, and low 

inflammability. Applications for the PLA fibers or blends of PLA fibers with other natural 

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54 

fibers, such as silk, cotton, or wool, therefore include clothing, carpets, upholstery, and draperies 

(38). 

The greatest opportunity for PLA to displace less benign materials is in the area of 

packaging for both food and consumer products. This opportunity arises because the properties 

of PLA allow improved function as well as diminished environmental impact. PLA can be used 

in traditional polymer processing operations such as injection molding, blow molding, extrusion, 

and extrusion coating. As a result, lids, trays, and clamshells used in food handling can be 

thermoformed from extruded sheet PLA, even yielding products with higher flex-crack 

resistance in living hinges (thin sections of plastic that bridge two parts) than those made of 

polystyrene. In addition, thin sheets of many PLA variants possess high gloss, excellent heat 

sealability, and clarity, allowing extruded thin sheets of PLA to replace cellophane and PET in 

transparent packaging (39). Bags for yard and/or food wastes form another set of applications in 

which the physical properties as well as the biodegradable nature of PLA can be used to 

advantage; bags are tough and puncture resistant but are completely degraded within 4 to 6 

weeks in a composting environment (39). 

Extrusion coating of PLA for paper products is another set of applications with several 

benefits. For example, paper coated with PLA does not require pretreatment for ink adherence, 

whereas polyethylene (PE) paper coatings often do. PLA coatings also possess higher gloss, 

greater clarity, lower coefficients of friction, and greater stiffness than PE, allowing thinner 

paper to be used (18). 

Some limitations do still exist, however, to the use of PLA in polystyrene-like 

applications. These include primarily the low-melt strength (extensibility without breaking of 

the molten state) and the relatively low temperature at which heat distortion begins to occur. 

The drawbacks of PLA are overshadowed by its advantages in products with short lifecycles 

(39). 

3.3 Synthesis 

The two general synthetic routes for PLA include the condensation polymerization of 

lactic acid and the ring-opening polymerization of lactide. Condensation polymerization 

methods have produced high molecular weight PLA through chain extension and, more recently, 

through dehydration using azeotropic high-boiling (boiling point > 150°C) aprotic solvents and 

vacuum techniques. Polymerization utilizing a ring-opening mechanism is the preferred method 

of synthesis, however, and is the basis of the commercial Natureworks process. 

Condensation polymerization has recently become a reliable method for production of 

high molecular-weight PLA. Attempts as recently as the mid-1990s to produce high molecularweight PLA through simple dehydration without catalysis were unsuccessful, primarily due to 

side reactions (40). To reduce the interfering side reactions, two methods are currently available. 

The first, developed by Ajioka et al. in 1995 (40), involves the dehydration of lactic acid using 

high-boiling aprotic solvents, such as diphenyl ether, that form azeotropes with water under 

vacuum conditions, as well as a catalyst. The second method involves using a dual catalyst 

system (41). Other methods have been attempted with the goal of producing high molecularbioengineering report_5_x1a.pdf 68 5/25/07 6:45:10 PM
55 

weight PLA directly from lactic acid monomers, but to date have yielded only moderate 

molecular weight materials (42, 43). 

Chain extension of oligomeric PLA is another approach, within the category of 

condensation polymerization, used to produce higher molecular weight PLA. Two basic 

methodologies have been developed. The first uses an additive to promote esterification of two 

PLA chains into one continuous chain (44, 45) while the other uses a linking agent to couple two 

or more chains together. Chain-linking agents such as diisocyanates (46, 47), thiirane (47), and 

diacidchlorides (44) are more economical than esterification promoting agents due to fewer 

purification steps and the important ability to run the reactions in the bulk. Problems associated 

with linking agents, however, include the persistence of unreacted chain-linking agents, residual 

metals, and the non-biodegradability of the linking agent, all of which diminish the 

environmental advantages of the resultant PLA. 

Although the straight dehydration of lactic acid does not produce high molecular-weight 

polymers, the process is important in the production of lactide. The amount of lactide produced 

is influenced by temperature, pressure, and the types of catalysts added to the system (48). 

Multiple purification methods are used industrially, including Cargill’s multiple reflux controlled 

columns (11, 12), multi-step recrystallization reactors (49–56), and direct vapor-phase reaction 

of lactic acid (57, 58). Formation of lactide occurs through a back-biting reaction involving two 

lactic acid monomers to form the six-member lactide ring. Three types of lactide can be formed: 

L-lactide or D-lactide with melting point = 97°C, or Meso-lactide with melting point = 52°C, 

depending on the composition of the starting material and extent of racemization (Figure 10). A 

fourth type, D,L-lactide, is formed when a racemic stereocomplex of D- and L-lactide are 

crystallized together; this form has a much higher melting point of 126–127°C (16). 

Figure 10. Types of lactide: Meso-lactide, L-lactide, and D-lactide. 

O

O

O

O

H

CH3

H

H3C

O

O

O

O

CH3

H

H

H3C

O

O

O

O

H

CH3

H3C

H

Meso-lactide L-lactide D-lactide

poly(Meso-lactide) poly(L-lactide) poly(D-lactide)

O O

H3C H

H CH3

O

O

O O

H CH3

H CH3

O

O

O O

H CH3

H3C H O

O n n n

Lactide, once formed, can be polymerized by three general mechanisms: anionic (33, 59-61), 

cationic (33, 62, 63), and coordination insertion (see Figure 11). Of these, coordinationinsertation is the most prevalent and industrially most important. Polymerization reactions may 

bioengineering report_5_x1a.pdf 69 5/25/07 6:45:11 PM
56 

be performed in bulk or in solution. The type of lactide, reaction temperature, and catalyst 

system determine the stereochemistry at each carbon-carbon bond along the backbone of PLA, in 

turn determining the properties of the resulting material. Impurities present in the lactide such as 

water, lactic acid, and lactyl-lactic acid decrease the molecular weight of the end polymer (64, 

65). 

Figure 11. Coordination insertion polymerization of lactide with 

Sn(Oct)2 (73).

O

O O

O

Sn

Tin(II) bis(2-ethyl-hexanoate)

 Sn(Oct)2

O

O

O

O

Oct2 Sn R O

H

O

O O

O

Sn

Oct2

R

O H O O O

O

H O

R Oct2 Sn n

R = H or Benzyl

The coordination insertion mechanism is the most common method used to produce high 

molecular weight PLA with high optical purity and crystallinity. Many catalytic or initiator 

systems have been developed for the coordination insertion polymerization of lactide. Oxides, 

halides, and alkoxides of metals possessing free p-, d-, or f- orbitals such as magnesium (Mg) 

(66-68), zinc (Zn) (66, 69-73), aluminum (Al) (74-83), tin (Sn) (64, 65, 73, 80, 82, 84-94), and 

yittrium (Y) (95, 96) effectively catalyze the ring-opening polymerization of lactide. In general, 

the most useful catalysts possess highly covalent metal-ligand bonds (33). 

The general catalytic mechanism involves coordination of the lactide carbonyl group to 

the catalyst metal, followed by cleavage of the ring acyl-oxygen bond and attachment of the 

growing, catalyst-attached polymer (Figure 11). The terminally bound catalyst then promotes 

the addition of successive monomers. Because each catalyst or initiator molecule facilitates the 

extension of a single polymer chain, molecular weights of PLA are controlled by varying the 

proportion of catalyst to lactide. 

Among the coordination insertion catalysts, tin 2-ethylhexanoate (tin octoate, or 

Sn[Oct]2) is the most widely used and studied due to its ability to produce highly crystalline PLA 

in relatively short periods of time with high conversion and low racemization up to 180°C. It has 

also been approved by the U.S. Food and Drug Administration for food contact (16), making it 

ideal for many packaging applications. 

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Both kinetics and mechanisms of tin octoate polymerizations have been well 

characterized at both low (64) and high (≥180°C) temperatures (65). Water, lactic acid, and 

lactyl lactate can also form other species with tin octoate, including tin oxide, tin hydroxides, tin 

lactate, all of which have been shown to catalyze the ring opening of lactic acid (65, 97, 98). 

Lactide polymerized with tin octoate is best described by a second-order insertion mechanism 

(64) that is first order in monomer concentration (73). 

Recently, single-site catalysts prepared by the addition of bulky side groups to metals 

have produced PLA with stereochemically-controlled structures (99). Highly syndiotactic PLA 

has been formed from meso-lactide using bulky racemic aluminum catalysts (100). Using the 

same catalyst, D,L-lactide was polymerized to form “stereoblocks” of PLA, possessing blocks of 

isotactic D- and L-PLA (101). The stereoblock copolymer had a Tg slightly higher than isotactic 

L- or D-PLA, but lower than the stereocomplex formed between D- and L-PLA. Similar results 

were observed for the polymerization of D,L-lactide (102) using the same catalyst. Single-site 

catalyst work thus demonstrates a method to produce PLA with good control of the polymer 

backbone stereochemistry. This is a promising development, now opening the possibility of 

forming stereocomplexes between copolymer blocks that can withstand higher temperatures 

before heat distortion occurs at the interfaces between blocks. 

3.4 Architectural Variations 

Using techniques developed for polymerization of linear PLA, it has been possible to 

synthesize random copolymers, block copolymers, and branched polymers to modify the 

properties of the materials produced. 

Random copolymers are produced by polymerization of two or more species together 

using a catalyst functional with both. Monomers that fit the criteria for ring-opening 

copolymerizations with lactide are glycolide (103-105), glycolide derivatives (106-109), lactones 

(30, 110-112), cyclic amide ethers (113), cyclic amide esters (114, 115), cyclic ether esters (31, 

116, 117), cyclic phosphates (118), cyclic anhydrides (119) or cyclic carbonates (120-122). 

Additional copolymers have been made through transesterification reactions of PLA with other 

polyesters, including PET (123). By adjusting the proportions of each monomer, properties such 

as Tg, ease of biodegradation, and backbone flexibility can be altered (31, 117). 

Block copolymers can also be produced. In one method, a ring-opening polymerization 

mechanism is employed using either coordination insertion catalysis or anionic catalysis (124). 

Monomer addition sequence is important in this method, those that produce primary hydroxyl 

groups, such as lactones (113, 125-129), trimethylene carbonate (TMC) (122, 128), and 1,5-

dioxepan-2-one (DXO) (130) must generally be polymerized first. Exceptions to the order of 

addition involve both glycolide (131) and D,L-3-methyl glycolide (MG) (109), which can be 

polymerized sequentially before or after PLA to form block copolymers because they possess the 

same type of propagating chain end as lactide. Ring-opening polymerization from polymeric 

initiators, a second method to produce block copolymers, employs polymeric initiators that 

possess hydroxyl groups on their chain ends. These groups initiate polymerization with the same 

catalysts used in polymerization of linear PLA. Effective polymeric initiators include 

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poly(ethylene glycol) (124, 132-140), poly(ethylene glycol-co-propylene glycol) (141), 

poly(propylene glycol) (105), poly(tetrahydrofuran) (133, 142), poly(dimethyl siloxane) (133, 

143-145), hydroxyl functionalized polyethylene (146), and hydroxy functionalized 

poly(isoprene) (147, 148). Dendritic molecules with a single hydroxyl group have also been 

used to produce linear/dendritic di-block copolymers (149). 

Several methods for synthesis of highly branched polymers through the ring opening of 

lactones have likewise been developed. Star formations are produced either by divergence, 

where polymer chains grow from a central core, or through convergence in which a termination 

reaction couples together individual chains (150-152), glycerol (133, 152-155). Comb-shaped 

copolymers consisting of linear backbones with pendent linear chains may also be produced 

(156-162). Lactides have also been polymerized into hyperbranched structures, in which many 

branch points lead to other branch points (155, 163-166). Star-shaped copolymers are generally 

produced in a similar manner to star-shaped homopolymers, with hydroxyl groups on a 

multifunctional initiator used to initiate polymerization (167). 

3.5 Fundamental Chain Properties 

The characterization of PLA depends on the accurate knowledge of its fundamental 

properties. These include such parameters as Mark-Houwink constants that relate intrinsic 

viscosity to molecular weights, theta-condition front factors (K) used to calculate single chain 

properties, and characteristic ratios (C) that give an indication of the bonding structure of 

polymers. Unfortunately, each of these is reported inconsistently in the literature (5, 6, 150, 168-

174). 

A recent study (175) has addressed these inconsistencies experimentally. PLA 

homopolymers and copolymers spanning wide ranges of molecular weight and stereoisomer 

proportion were prepared by ring-opening polymerizations of L- and D-lactides using tin 

octanoate as the catalyst. Samples were then characterized by means of dilute-solution 

viscometry in three different solvents; size-exclusion chromatography; static multiangle light 

scattering; variable-angle spectroscopic ellipsometry; and melt rheology. Data provided by these 

experiments include values of characteristic ratios as well as Schulz-Blaschke and MarkHouwink constants, all of which show consistently that polylactides are typical linear flexible 

polymers, in excellent agreement with recently published theoretical simulation results (176). 

3.6 Processing Properties 

Plastics are typically fabricated into useful articles in the molten state, thereby causing 

the melt flow, or rheological, properties of a polymer to be of great importance. As discussed 

above, PLAs have physical properties useful in fibers, packaging, and other applications 

traditionally dominated by petroleum-based resins. Although the general literature on 

polylactides is extensive, only a few articles (177-181) have considered rheological properties. 

Measurements of dynamic, steady, and transient shear viscosities have been presented and the 

extensional data on PLA showed a strong strain hardening behavior (181). These studies did not, 

however, capture a systematic description of PLA rheology across a broad range of stereooptical 

composition, as materials studied to date have usually possessed high (>90 percent) 

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L-stereochemical center content. However, a recent study (182) has provided a comprehensive 

evaluation of the linear viscoelastic properties of PLA across a wide range of molecular weights 

and stereochemical compositions. The rheology of blends of linear and branched-PLA 

architectures has also been comprehensively investigated (180, 183). The results suggest that 

excellent control over rheological behavior of PLA is possible through blending chain 

architectures without compromising mechanical properties. 

Mechanical properties of solution-spun (36, 37, 184, 185) and melt-spun (34, 36, 186-

189) PLA fibers have been thoroughly investigated. It has been found that these properties are 

roughly equivalent to other polyesters meaning that PLA can replace textiles based on nonrenewable resources. In addition, scanning electron microscopy (SEM) (34, 37, 184, 186) and 

wide-angle x-ray scattering (WAXS) (36, 190) have been useful in examining surface structure 

with respect to roughness and fracture surfaces. Understanding surface properties is important 

for dying and other textile finishing operations. Cicero et al. have provided a complete 

characterization of the hierarchical fiber morphology from linear PLAs (35, 191), determining 

thermal, mechanical, and morphological properties of the fibers and showing that properties can 

be widely manipulated through a combination of processing temperature and draw ratio (the 

amount of stretching the fiber undergoes). The same researchers have studied the effects of 

branching on fiber properties and morphology (192) and investigated the improvement of fiber 

properties specifically when thermally stabilized PLA is used (193). These research studies 

provide several routes for optimizing the performance of PLA when used as a textile fiber in 

place of conventional, fossil resource based polyesters such at PET. 

3.7 Permeation Properties 

Because of the desirability of using PLA in packaging applications, understanding the 

permeation properties of PLA with respect to various gases and vapors, especially those of 

interest to the food industry, is extremely important. PLA is, unfortunately, a relatively poor 

barrier to water vapor and CO2, with a water-vapor transmission rate significantly higher than 

those of PET, polypropylene (PP), or polyvinyl chloride (PVC), (194) resulting in some limitated 

highly environmentally beneficial applications of PLA to packaging. For example, PLA’s high 

permeability to water prevents it from being used to bottle water over long durations, despite the 

fact that increasing sales of bottled water have created considerable pressure on landfill space, 

particularly in California. 

In response, Natureworks has developed several biaxially-oriented PLA films to improve 

PLA barrier properties, including two with trade names of PLA 4030-D and PLA 4040-D. 

Permeation of nitrogen, oxygen, CO2, and CH4 in very thin (5 μm) amorphous films of various 

grades of PLA (L:D ratios from 95:5 to 98:2) cast from solution have also been examined (195). 

Notably, changes in polymer chain branching and L:D ratios had no effect on the permeation 

properties of PLA with respect to small gases. 

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4. Research Priorities 

4.1 Development of LCIA Tools 

One of the greatest challenges facing the production of truly environmentally-benign 

plastic materials, PLA and otherwise, is the evaluation of net environmental impacts, beginning 

with feedstock production (agriculture or collection of biomass wastes), including processing 

steps (production of lactide and subsequent polymerization) and ending with the emissions 

resulting from biodegradation. Specific processes chosen at each stage, particularly concerning 

conventional vs. sustainable methods, are likely to have dramatic impacts on the net 

environmental profiles of individual materials, yet the tools with which to evaluate these 

differences are not yet fully refined (196). These metrics are needed urgently, both to guide 

research and development of bioplastics and to advocate use of the truly environmentally 

beneficial materials. Consequently, a top priority in the development of environmentally benign 

plastics is the continuation of efforts to develop tools and standards within the context of LCIA 

that will make comparisons transparent and meaningful. 

4.2 Improvement of Physical Properties 

Presently, PLA and other biopolyesters suffer from two important deficiencies that limit 

their use. The first of these is their relatively low heat distortion temperatures, and the second is 

their relatively high permeabilities toward a number of substances, particularly water. As 

current, best-available LCIA analyses have indicated that PLA is indeed environmentally benign, 

continued research into biological, chemical, and physical transformations of PLA-based 

materials to improve these properties is warranted. In particular, nanocomposite technologies 

(Chapter III.D.) hold promise of improving both temperature distortion and permeation 

characteristics, as they have in conventional plastics, and should be investigated. 

Microcomposite technologies are related, already well-established approaches to achieve similar 

improvements in conventional plastics. In addition, plant microparticles derived from waste 

agricultural residues and simple grasses can be used directly as microparticles, providing both 

economic and environmental advantages (197, 198). Alternatively, blending and trans-reacting 

PLA-based plastics with starch- or triglyceride-based materials (Chapter III.D.) may improve 

performance while maintaining biodegradability, with the result that these techniques also 

deserve further investigation (199-205). Recently, copolymerization of cellulose acetate with 

PLA has demonstrated that the heat distortion temperature can be increased (206). In this 

interesting case, both constituents of the plastic material come from renewable resources. This 

suggests that copolymerization of PLA, especially with other polymers based on renewable 

resources, can provide a viable route towards improved performance. 

4.3 Exploration and Development of New Polyesters 

A recent comprehensive study by the DOE (207) has identified 12 promising low 

molecular-weight materials that can be produced by fermentation in commercial quantities from 

plant sugars (succinic, fumaric, malic, 2,5-furandicarboxylic, 3-hydroxypropionic, aspartic, 

glutaric, glutamic, itonic, and levulinic acids, and the alcohols 3-hydroxybutyrolactone, glycerol, 

sorbitol, and xylitol). Combination of these acids and alcohols can produce polyesters by direct 

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61 

condensation (44, 208, 209). In particular, reactive intermediates that can be produced by 

anaerobic fermentations are desirable, because anaerobic processes typically lose much less of 

the feedstock carbon to CO2 than do aerobic processes (210). The success of the DuPont 

SoronaTM material, a polyester of such low molecular-weight precursors (1,4-

benzenedicarboxylic acid-dimethyl ester with 1,3-propanediol) shows that development of 

sustainable processes to take advantage of readily available, renewable substances to produce 

additional biodegradable plastics deserves high priority for its great potential to yield both 

homopolymeric and copolymeric materials with new ranges and combinations of desirable 

properties. 

5. Commercialization 

 Polymers based on polylactic acid are the leading success stories in bioplastics, moving 

from the laboratory into the market within the last decade. PLA-based polymer synthesis is 

protected by numerous patents to several different entities (10, 47, 211-214) and Natureworks 

appears to have taken the lead in the United States with its opening in 2002 of the first 

commercial manufacturing facility for PLA in Blair, Nebraska. Natureworks continues to be the 

most internationally visible PLA producers, but Japan and Germany are showing interest in 

developing PLA commercially as well (http://www.friendlypackaging.org.uk/materialslist.htm). 

Beyond issues of heat distortion and permeability described above, the greatest current 

obstacle to greater market penetration of PLA is cost. Increasingly, higher oil prices are enabling 

PLA to compete directly with petroleum-based plastics despite the fact that PLA is a new entity 

in the plastics marketplace, but cost reductions would nevertheless allow greater utilization. 

Accordingly, renewable materials such as starches, cellulose, and wood flours and fibers that 

could be used in PLA blends to decrease costs without significantly degrading performance are 

of great commercial interest. In this respect, bioplastics have a general advantage over biofuels–

they are already clearly cost competitive with fossil-resource based materials. 

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C. POLYHYDROXYALKANOATES 

1. Introduction 

A variety of bacteria synthesize PHAs as intracellular carbon and energy storage 

materials, much as higher organisms synthesize starch, glycogen, or fat for energy storage. 

PHAs were first observed as dark-staining bodies within microbes observed under microscopes, 

and in 1925, poly(3HB), was first isolated from Bacillus megaterium by Lemoigne (1, 2). 

Because they are bacterial storage polymers, PHAs are of necessity biodegradable, and further 

study revealing their plasticity spurred commercial interest. In 1974, Wallen and Rohwedder 

predicted that novel hydroxyalkanoate (HA) subunits could become important to further 

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development of the microbial polyester, due to the ability of varying structural units within 

poly(3HB) to modify polymer properties (3). 

Research investigating the identities and characteristics of various units within microbial 

PHAs began in earnest during the 1980s, when a number of bacteria were found to synthesize 

optically active homopolymers and copolymers of (R)-3-hydroxyalkanoates [(R)-3HAs] ranging 

from 4 to 14 carbon atoms (4-9). Saturated, unsaturated, halogenated, branched, and aromatic 

side chains in (R)-3HA monomeric units have now been found within microbial PHAs; at 

present, at least 140 different monomeric units have been found as constituents of microbial 

PHAs (10-12). In addition, certain bacteria also produce copolymers containing 

hydroxyalkanoate repeat units with side chains such as 3-hydroxypropionate and 

4-hydroxybutyrate (13, 14). Subsequently, many different approaches have been taken to 

produce PHA materials with desirable physical properties (15-17). 

2. PHA Biosynthesis, Biodegradation, and Environmental Impact: Overview 

The biosynthetic routes to PHA monomer synthesis are interconnected with several 

central metabolic pathways: the tricarboxylic acid (TCA) cycle, fatty acid degradation 

(β-oxidation), and lipid biosynthesis, through common intermediates and cofactors such as 

acetyl-CoA, other fatty acyl CoAs, NADH, and NADPH. 

PHA is synthesized under the direction of a family of enzymes that shows some diversity 

among microorganisms but nevertheless possesses several conserved members. β-ketothiolases 

harvest acetyl CoA from the TCA cycle, condensing it into acetoacetyl CoA; transacylases 

similarly harvest intermediates from lipid biosynthesis, and enoyl CoA hydratases divert enoyl 

CoA intermediates from β-oxidation. Where necessary, as with acetoacetyl CoA, reductases 

then reduce keto groups to hydroxy groups. Each of these enzymes thus provides monomers for 

PHA synthesis by the final enzyme in the pathway, PHA synthase. The β-ketothiolases, 

transacylases, hydratases, reductases, and PHA synthases are encoded by well-understood genes: 

PhaA, PhaG, PhaJ, PhaB, and PhaC, respectively. The genetic understanding of this system, as 

well as its connectedness to other well-understood metabolic pathways, has made PHA 

biosynthesis an ideal target for metabolic engineering. 

Once the PHA is synthesized, the microbes are harvested, disrupted, and fractionated to 

isolate the polyester. To produce PHA materials with desirable physical properties, a variety of 

approaches including copolymerization, blending with other polymers, cross-linking, and the 

introduction of functional groups have been studied extensively. PHAs can be produced in a 

wide variety of molecular configurations; homopolymers, copolymers, and functionalized 

polymer chains may all be created by utilizing various microorganisms and fermentation 

conditions. A representative sample is shown in Figure 12 (18). 

PHAs are benign materials from an environmental perspective for several reasons. First, 

substrates necessary for bacterial growth of PHA-producing bacteria are available from 

renewable resources, and second, PHAs are highly biodegradable, not only by their bacterial 

producers, but also by numerous other aquatic and terrestrial microorganisms. Chowdhury first 

isolated poly(3HB)-degrading bacteria in 1963 (19), and additional studies on the isolation and 

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characterization of PHA-degrading microorganisms began to appear in the 1990s (18). Since 

that time, concern about the environmental impacts of plastic wastes has led to expanded 

investigations into PHA-degrading microorganisms. At this point, a number of extracellular 

PHA-degrading enzymes from various microorganisms have been purified and characterized, 

and PHA-derived products appear to decompose readily in both composting and activated sludge 

systems (20, 21). 

Figure 12. Chemical structures of PHAs and degradation rates in aqueous 

solutions at 37 °C containing depolymerases (18). 

3. State of the Science 

3.1 Synthesis 

Poly (3HB) is the most common biological polyester and is produced by numerous 

microorganisms in nature (22). From this basic starting point, however, three primary 

approaches have been investigated to produce novel polymers with a wide range of properties. 

3.3.1 Feedstock manipulation. Because the physical and thermal properties of 

PHA polymers and copolymers can be regulated by varying their molecular structure and 

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copolymer compositions, the simplest metabolic engineering strategy is to provide specific 

carbon sources to the microbes that bias the monomer production in favor of desired compounds. 

Polymer composition can be further controlled by varying the feed ratio of various substrates. 

For example, a random copolymer of (R)-3HB and (R)-3-hydroxyvalerate, 

poly(3HB-co-3HV), has been produced in Ralstonia eutropha by feeding pentanoic acid and 

butyric acid as the carbon sources (5). The poly(3HB) homopolymer was produced from butyric 

acid, while a poly(3HB-co-3HV) copolymer was produced from pentanoic acid. By varying the 

ratio of pentanoic acid to butyric acid in the feed, variable composition copolymers were 

produced. Similarly, using 3-hydroxypropionic acid as the substrate, R. eutropha produced a 

random copolyester of (R)-3HB and 3-hydroxypropionate, poly(3HB-co-3HP) (23). The same 

copolymer can be produced by Alcaligenes latus (24). Using olive oil as a substrate, Aeromonas 

caviae produced a random copolymer of (R)-3HB and (R)-3-hydroxyhexanoate, poly(3HB-co3HHx) (25). Feeding 4-hydroxybutyric acid, 1,4-butanediol, or butyrolactone as the carbon 

source produces a random copolyester of (R)-3HB and 4-hydroxybutyrate, poly(3HB-co-4HB) 

when R. eutropha, (5) A. latus, (26) or Comamonas acidovorans (27) are utilized. Recently, a 

number of unusual sulfur-containing polymers have been generated by feeding alkylthionates to 

R. eutropha, and external substrate manipulation has even been used to generate block 

copolymers by intermittent addition of one substrate. In the latter example, R. eutropha was fed 

pulses of valerate–a precursor substrate of 3-hydroxyvalerate–to medium containing an excess of 

fructose–a precursor of PHB–to generate the block copolymer (12). 

PHAs with functional groups in the side chains can also be produced when functionalized 

organic substrates are employed, allowing the engineering of specific physical properties and the 

provision of reactive sites for applications such as adhesives and coatings. Representative side 

chains that have been incorporated into 3HA include unsaturated (8, 28), halogenated (10), 

branched (29), and aromatic (30) moieties. 

 The addition of inhibitors, reducing the function of metabolic pathways that compete 

with PHA synthesis, is another feeding strategy that has been employed to improve PHA yield 

and/or to facilitate incorporation of longer monomers. In one example, acrylate was used to 

inhibit β-oxidation in R. eutropha, such that the microbe accumulated a copolymer of both shortand medium-chain length monomers rather than exclusively short chain-length monomers (12). 

3.1.2 Genetic engineering. Extensive investigations into PHA synthesis has led to 

the identification, cloning, and sequencing of approximately 40 responsible genes from various 

gram-positive and gram-negative bacteria, providing a great deal of material to support efforts in 

genetic engineering (31). 

Among the most commonly-used heterologous hosts, including Ralstonia eutropha, 

Pseudomonas putida, Pseudomonas oleovorans, and Escherichia coli, the latter deserves special 

mention for its advantages as a host. It has an extremely thoroughly understood physiology; it is 

not a native PHA producer, with the result that productivity is not limited by natural regulation; 

it harbors no native machinery for PHA degradation; and its cells are easily disrupted, facilitating 

PHA recovery (12).

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One straightforward approach to increasing PHA synthesis is simply increasing gene 

dosage or providing additional copies of the PHA synthetic enzymes. While this has shown 

success in efforts with Aeromonas punctata and R. eutropha, not all attempts have been 

successful. This shows that the effectiveness of gene dosage depends on the limiting factor for 

polymer synthesis in each individual case. 

A more involved but increasingly popular approach is the expression of heterologous 

genes for polymer precursor production in a desirable host, with the goals of facilitating 

synthesis of polymers that would not naturally accumulate and/or that might have desirable 

structures and properties, as well as facilitating use of simple, inexpensive carbon sources for 

production of the desired polymers. In one encouraging example, the incorporation of three 

enzymes into a recombinant Salmonella allowed the synthesis of propionate, an expensive but 

previously necessary substrate for the synthesis of PHB-co-PHV, from succinyl-CoA—an 

intermediate in the TCA cycle. The microbe was then able to synthesize PHB-co-PHV from 

glycerol, a significantly less expensive carbon source. A number of similar efforts have also 

been successful (12). 

In the realm of pathway engineering, competing pathways can also be eliminated and 

regulatory systems can be altered to facilitate PHA synthesis. Related to the example above 

involving propionate, propionate-degrading enzymes have been deleted, and propionate 

synthesis machinery has been placed under the control of an IPTG-inducible promoter, allowing 

the composition of PHB-co-PHV polymers to be adjusted at will (12). 

Finally, PHA biosynthetic enzymes are amenable to protein engineering, and PHA 

synthase in particular is the subject of an on-going effort to develop a complete understanding of 

its structural and mechanical characteristics. In the case of the Pseudomonas 61-3 PHA 

synthase, error-prone PCR mutagenesis revealed two primary sites that affected PHB 

accumulation, allowing subsequent site-directed mutagenesis to test all possible amino acid 

combinations at those sites. The optimal combination of amino acids at those sites yielded a 

synthase that promoted the accumulation of up to 400-fold more PHB in the microbe (12). 

Given the number and diversity of PHA synthase genes now available, family shuffling 

approaches are being explored as well, and transacylase and hydratase genes are expected to be 

targets in the near future (12). 

Mathematical modeling, informed by microarray analysis, proteomics, and 

metabolomics, is also suggesting new targets for additional metabolic engineering efforts. Flux 

analysis, for example, recently elucidated the role of the Entner-Doudoroff pathway in PHB 

production in E. coli, while other mathematical models have identified optimal substrate 

switching strategies for the production of desired block copolymers (12). 

3.1.3 Transgenic plants. The detailed genetic understanding of PHA biosynthesis 

pathways offers hope for the cost-effective production of PHAs in transgenic plants (32, 33). 

Initial success in Arabidopsis thaliana suffered from a depletion of suitable substrate for growth 

(34), but genetic manipulation led to more effective production in plant plasmids (35). The 

construction of transgenic A. thaliana using the PHA syntheses gene of P. aeruginosa indicates 

that plant fatty acids can generate a range of (R-)3-hydroxyalkanoate monomers that can be used 

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to synthesize medium molecular weight PHAs (36). From a commercial perspective, the 

copolymers P(3HB-co-3HV) and P(3HB-co-4HB) are attractive, and recent reports show 

promise for the production of both monomers and polymers plants (16, 14). 

Development of PHA-producing transgenic switchgrass is also underway, with the goal 

of incorporating the synthesis into a more productive, easily-grown plant (37). In 2001, 

Metabolix Inc. was awarded a $15 million “Industries of the Future” cost-shared grant from 

DOE to help fund the development of a biomass biorefinery based on switchgrass. The goal of 

this program is to produce PHAs in plants and, after polymer extraction, use the residual plant 

biomass for fuel generation, thereby generating both materials and fuels from a sustainable 

resource (http://www.metabolix.com/biotechnology percent20foundation/plants.html). Efforts 

are also underway to produce PHAs/PHBs in photosynthetic bacteria. 

3.2 Physical Properties 

The molecular weight (MW) of poly(3HB) produced from wild-type (unmodified) 

bacteria usually ranges from 104

 to 106 grams per mole, with a polydispersity of about 2 (38). 

Within the bacteria, the polymers remain amorphous and are found as water-insoluble inclusions 

(39, 40). This fact is surprising, as the polyester only has (R)-configuration stereochemical sites 

in the backbone and thus exhibits a perfectly isotactic structure. Crystallinity is typically 55-80 

percent in poly(3HB) isolated from bacteria (7). 

3.2.1 Homopolymers. The physical properties of amorphous, crystalline, and ultrahigh MW poly(3HB) are tabulated below, showing comparisons with isotactic PP (Table 1) 

(41, 9, 42). The primary difficulty with ordinary poly(3HB) is that it is a relatively brittle plastic, 

as shown by the low extension to break value in comparison to polypropylene (43). Films have 

been prepared from ultra-high molecular-weight poly(3HB) that show improved mechanical 

properties when stretched, however, raising both the elongation to break and tensile strength 

values (44, 45). Mechanical properties have been improved further upon annealing, as well as by 

the incorporation of structural variations made possible by genetic engineering, with the result 

that new PHAs are promising candidates for further commercial exploitation. 

Table 1. Physical properties of PHA homopolymers. 

Plastic 

Density 

g/cm

Tg 

°C 

Tm 

°C 

Crystal 

structure 

Conformation Morphology 

Young’s 

modulus 

GPa 

Tensile 

strength 

MPa 

Extension

to break

percent 

poly(3HB), 

amorphous 1.18 

poly(3HB), 

crystalline 1.26 

4 180 

orthorhombic 

left-handed 

21 helix 

lamellar 

3.5 43 5 

ultra-high Mw

poly(3HB) lamellar 1.1 62 58 

PP 400 

3.2.2 Copolymers. Random copolymers of (R)-3HB and (R)-3HV have been 

investigated extensively with regard to various physical properties. In the case of random 

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copolymers of (R)-3HB with (R)-3HHX (3-hydroxyhexanoate), the repeat unit contains a propyl 

side chain that strongly affects properties. Most notably, in solution-cast films, 

poly(3HB-co-3HHx) becomes soft and flexible with increased proportion of (R)-3HHx, as 

shown by the diminished Tm, Tg, crystallinity, and tensile strength, and by the increased 

elongation to break factor (Table 2). An additional example is provided by poly(3HB-co-4HB), 

in which crystallinity, elongation to break, and other physical properties can vary widely by 

adjusting proportions of the component monomers (Table 2). These data show that PHA 

copolymer properties, like those of other plastics, can be regulated by their composition; in 

addition, the range of materials observed, from hard crystalline plastics to elastic rubbers, show 

that these plastics hold great promise for a wide variety of applications. 

Table 2. Physical properties of PHA copolymers. 

Plastic 

non-HB mole 

fraction 

 percent 

Crystallinity 

 percent 

Tm

°C 

Tg

°C 

Tensile 

strength 

MPa 

Elongation to 

break 

percent 

poly(3HB-co-3HV)a various 50-70 

poly(3HB-co-3HHx)b 0 60 177 4 43 6 

17 20 850 

25 18 52 -4 

poly(3HB-co-4HB)c 0 60 43 5 

16 26 444 

49 14 

64 17 

100 104 a

(46) 

b

(47) 

c

(27)

3.2.3 Blends. An established method for changing plastic properties is through 

blending with other materials. Several detailed investigations of blends containing poly(3HB) 

with other biodegradable polymers have appeared. Such blends are physical mixtures of 

different polymers; however, sometimes the two polymers react with one another, 

compatibilizing the blend. Blends can be either homogeneous, forming a single thermodynamic 

phase, or heterogeneous, comprising two or more phases. Blend properties are dependent, in 

turn, on such phase behavior. 

Miscible blends containing poly(3HB) have been formed with poly(ethylene oxide) 

(48, 11, 49), poly(vinyl alcohol), (50) PLA (51, 52), poly(ε-caprolactone-co-lactide) (53), 

poly(butylene succinate-co-butylene adipate) and poly(butylene succinate-co-ε-caprolactone) 

(54). Immiscible blends are formed in mixtures of poly(3HB) with poly(β-propiolactone) 

(55, 13), poly(ethylene adipate) (55), poly(butylene adipate) (56), and poly(ε-caprolactone) (55). 

For miscible blends of poly(3HB) with atactic poly(3-hydroxybutyrate), increasing the 

weight content of the latter from 0–76 percent increases the elongation to break from 

5–500 percent with an accompanying decrease in the Young’s modulus and tensile strength (15). 

For the immiscible system of poly(ε-caprolactone) with poly(3HB), in contrast, the decrease in 

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Young’s modulus and tensile strength is not accompanied by an increase in elongation to break 

due to macroscopic phase separation (56). These studies show the continuing importance of 

physical property modification by blending as a valuable route towards improving the properties 

and therefore increasing the utilization of bioplastics. 

4. Research Priorities 

 PHA development is proceeding in a number of promising directions on both metabolic 

engineering and chemical engineering fronts. Fortunately, most of these have the potential for 

success both individually and in combination with others, such that no particular obstacle is 

currently forming a bottleneck to further progress. The range of physical and thermal properties 

achievable with PHAs is still expanding rapidly as new configurations of copolymers and blends 

are explored. An on-going challenge will be the ability of the metabolic engineers to keep pace 

with the discoveries of the materials scientists, enabling microbes to synthesize the desired 

polymers both conveniently and inexpensively. These efforts can be categorized as follows. 

4.1 Investigation of Novel Polymers and Properties 

Clearly, a number of modifications of PHA composition have the potential to improve the 

plasticity, moldability, heat tolerance, and durability of the resulting plastics to approach those of 

conventional thermoplastics. Because of the promising availability and flexibility of routes to 

PHA synthesis, and because of increasing oil prices that will enable PHA polymers to become 

increasingly cost-competitive, it is a valuable effort to explore the properties of new PHA-based 

homopolymers, copolymers, and blends even before microbial pathways to their syntheses are in 

place. 

4.2 Metabolic and Genetic Engineering 

The increasing availability of mathematical modeling tools, genomic and proteomic data 

and techniques, and microarray and antisense RNA technology will allow increasingly accurate 

prediction of useful targets for metabolic engineering. At the same time, genetic manipulation 

within both microorganisms and plants is becoming increasingly possible and rapid. Several 

enzymes central to PHA synthesis are just beginning to be explored through combinatorial and 

rational design mutagenesis approaches, and efforts to understand their catalytic mechanisms, 

substrate specificities, modes of competition with other enzymes, and regulation are likely to 

contribute greatly to the microbial or plant-based syntheses of novel polymers. 

4.3 Reactor and Processing Technology 

As described previously, gains in commercial feasibility are often found in improving 

bioreactor yield and in diminishing processing costs. Without reiterating topics addressed 

previously, it is nevertheless important to include these in the research priorities for PHA efforts, 

with special note that the transfer of PHA synthesis to plants may circumvent many limitations 

of both reactor efficiency and processing costs. 

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5. Commercialization 

PHA-based materials were originally produced primarily under the trade name of 

BiopolTM by ICI, Zeneca, and Monsanto. Metabolix recently acquired the BiopolTM patents from 

Monsanto, however, and is now producing several PHA polymers in bacteria and plants. 

Independently, Proctor and Gamble developed PHA copolymers of short and medium chainlength monomers under the trade name NodaxTM and licensed the technology to the Kaneka 

Corporation, a Japanese manufacturer of plastics and resins that is focusing on the production of 

P(3HB-co-3HHX). 

Nevertheless, Metabolix, Inc. currently possesses a virtual monopoly on the commercial 

research and development of PHA polymers, holding approximately 90 issued U.S. patents and 

approximately 40 additional pending U.S. patents, as well as their foreign equivalents, protecting 

methods of PHA isolation, purification, and processing; use of several preferred metabolic 

pathways for PHA copolymer production; and several novel PHA compositions and specific 

applications. Most basic to Metabolix’s position are the patents that give Metabolix exclusive 

rights to the genes within the PHA biosynthesis pathway, as well as the use of the genes in any 

combination for the preparation of PHAs (e.g., [57, 58]). As the company states itself on its Web 

site, “Metabolix owns the genes that encode the basic PHA pathway.” 

(http://www.metabolix.com/publications/patents.html). 

This comprehensive ownership of intellectual property has given Metabolix the freedom 

and protection to invest in research to improve PHA biosynthesis and processing technologies, 

with which it has greatly advanced these fields, as evident from the preceding discussion. At the 

same time, the present situation effectively diminishes or even deprives potential competitors of 

the ability to use either microbes or plants to produce PHAs commercially. 

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polyhydroxyalkanoates, Process Biochem 40:607-619. 

(44) Kusaka, S., T. Iwata, and Y. Doi (1998). Microbial synthesis and physical properties of 

ultrahigh molecular weight poly[(R)-3-hydroxybutyrate], J Macromol Sci Pure Appl 

Chem A35:319-335. 

(45) Kusaka, S., T. Iwata, and Y. Doi (1999). Properties and biodegradability of ultrahigh 

molecular weight poly[(R)-3-hydroxybutyrate] produced by recombinant Escheria coli, 

Intl J Biol Macromol 25:87-94. 

(46) Orts, W. J., R. H. Marchessault, and T. L. Bluhm (1991). Thermodynamics of the melting 

point depression in poly(b-hydroxybutyrate-co-b-hydroxyvalerate) copolymers, 

Macromolecules 24:6435-6438. 

(47) Doi, Y., S. Kitamura, and H. Abe (1995). Microbial synthesis and characterization of 

poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), Macromolecules 28:4822-4828. 

(48) Avella, M., and E. Martuscelli (1988). Poly-D-(3-hydroxybutyrate)/poly(ethylene oxide) 

blends: phase diagram, thermal and crystallization behavior, Polymer 29:1731-1737. 

(49) Kumagai, Y., and Y. Doi (1992). Enzymatic degradation of poly(3-hydroxybutyrate)-

based blends: poly(3-hydroxybutyrate)/poly(ethylene oxide) blend, Polym Deg Stabil 

35:253-256. 

(50) Azuma, Y., N. Yoshie, M. Sakurai, Y. Inoue, and R. Chujo (1992). Thermal behaviour 

and miscibility of poly(3-hydroxybutyrate)/poly(vinyl alcohol) blends, Polymer 33:4763-

4767. 

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(51) Blumm, E., and A. J. Owen (1995). Miscibility, crystallization and melting of poly(3-

hydroxybutyrate)/poly(L-lactide) blends, Polymer 36:4077-4081. 

(52) Koyama, N., and Y. Doi (1997). Miscibility of binary blends of poly[(R)-3-

hydroxybutyric acid] and poly[(S)-lactic acid], Polymer 38:1589-1593. 

(53) Koyama, N., and Y. Doi (1996). Miscibility, thermal properties, and enzymatic 

degradability of binary blends of poly[(R)-3-hydroxybutyric acid] with poly[ecaprolactone-co-lactide], Macromolecules 29:5843-5851. 

(54) He, Y., T. Masuda, A. Cao, N. Yoshie, and Y. Doi (1999). Thermal, crystallization and 

biodegradation behavior of poly(3-hydroxybutyrate) blends with poly(butylene succinateco-butylene adipate) and poly(butylene succinate-co-e-caprolactone), Polym J 31:184-

192. 

(55) Kumagai, Y., and Y. Doi (1992). Enzymatic degradation of binary blends of microbial 

poly(3-hydroxybutyrate) with enzymatically active polymers, Polym Deg Stabil 37:253-

256. 

(56) Kumagai, Y., and Y. Doi (1992). Enzymatic degradation and morphologies of binary 

blends of microbial poly(3-hydroxybutyrate) with poly(e-caprolactone), poly(1,4-butylene 

adipate) and poly(vinyl acetate), Polym Deg Stabil 36:241-248. 

(57) Peoples, O. P., and A. J. Sinskey (1996). Polyhydroxybutyrate polymerase, U.S. Patent 

5,534,432. 

(58) Skraly, F. A., and O. P. Peoples (2001). Polyhydroxyalkanoate biopolymer compositions, 

U.S. Patent 6,323,010. 

D. STARCHES, PROTEINS, PLANT OILS, AND CELLULOSICS 

1. Introduction 

Polysacharides, plant proteins, plant oils and lignin are four of the most widely available 

naturally occurring biomaterials, and they have therefore been the earliest materials utilized in 

bioplastics production. In fact, in their raw forms these plant-based materials have been 

exploited by humankind for millennia. Presently, starch is still the major component of 

approximately 75 percent of green plastics production if all biodegradable plastics are 

considered. Soy proteins have potential as adhesives and tremendous progress has been made in 

the past decade in using chemical methods to convert soy and other plant oils into useful 

materials. Cellulose, in turn, is emerging as a valuable component of bioplastics as a reinforcing 

member of composites, lending strength, durability, and heat tolerance to various biobased 

polymers. A brief discussion of these bioresources is presented here for completeness, however, 

for many of the materials discussed in this section, chemical manipulation is the predominant 

technology used to produce useful plastic materials. Accordingly, the emphasis is placed on the 

more promising areas of investigation involving industrial bioengineering techniques. For many 

of the materials within this broad class, the predominant bioengineering occurs in the genetic 

manipulation of the plant to produce a more desirable starting feedstock. DuPont’s high oleic 

soybean is a good example of this type of manipulation; the soybean has been genetically 

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engineered to produce a more desirable distribution of soy oils–in this case, one particularly rich 

in oleic oil. 

2. State of the Science 

2.1 Thermoplastic Starch (TPS) 

Starch, an energy storage material found abundantly in cereals and tubers, was one of the 

first natural polymers studied for the production of biodegradable materials and is widely used in 

the food, paper, and textile industries. It is composed of amylose and the branched polymer 

amylopectin, both are polysaccharides of alpha-D-glucopyranosyl units linked by (1-4) and (1-6) 

linkages (1). 

Starch was first used in biodegradable materials in the 1970s as a filler in synthetic 

polymers such as polyethylene or, in its gelatinized form, as a component of blends with watersoluble or water-dispersible polymers. These were best described as bio-disintegratable rather 

than biodegradable; data showed that only the surface starch was decomposed, leaving behind 

recalcitrant polyethylene fragments. Because products made from these resins did not meet the 

criteria of biodegradability for defined disposal systems like composting, further applications 

were soon sought (2). 

More recently, starch has been used as the primary component in thermoplastic 

compositions. Although starch is not itself a thermoplastic material, at moderately high 

temperatures (90–180°C), under pressure and shear stress, starch granules melt and flow to give 

an amorphous material called TPS, which can be processed just like a thermoplastic synthetic 

polymer. Conventional plastic processing techniques such as injection molding and extrusion 

can then be applied successfully. The ability to mold TPS, and the advantages of starch in terms 

of low cost and high availability from renewable resources, have made it a highly attractive 

resource for the development of biodegradable polymers. Unfortunately, however, the use of 

regular TPS has been limited by its brittleness, by degradation under conditions of normal use, 

and by hydrophilicity (1, 2). The latter is especially problematic because water is a plasticizer of 

starch, with the result that the performance of regular TPS is unstable at sufficiently great 

relative humidity (3). 

To address these problems, a number of modifications of TPS have been attempted to 

improve its material properties. Surface modifications form one class of approaches; in these, 

the superficial hydroxyl groups of the material are derivatized, especially to reduce 

hydrophilicity, without changing the bulk composition and characteristics of the TPS. This type 

of approach has also been used with cellulose fibers, considered below, to improve their 

compatibilities with polymer matrices when they are used as natural reinforcements in 

composites (1). 

 The use of biodegradable plasticizers has also been investigated, with the goals of 

increasing durability as well as diminishing brittleness. Polyols such as glycerol, which is often 

used with biodegradable polymers, effectively reduce degradation of thermoplastic starch with 

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increasing glycerol content corresponding to diminishing starch degradation under conditions of 

normal use (4). 

The starch structure can also be modified directly, particularly to increase durability and 

plasticity. Among the many possibilities of modification, esterification is one of the most 

important ones. Direct esterification is the simplest route, but the unavoidable degradation of 

starch chains diminishes mechanical strength of the end product. Alternatively, starch can be 

covalently linked to other materials during a polymerization process; starch-poly(vinyl acetate) 

materials, for example, have been prepared via in situ polymerization of vinyl acetate in the 

presence of starch with a ferrous ammonium sulfate-hydrogen peroxide redox initiator system. 

Other methods include melt blending of starch with synthetic polymers, such as poly(ethyleneco-vinyl acetate) and polyethylene with anhydride functionality (3). 

Current work in this area is directed toward the modification of TPS by reactive blending 

with polymers containing functional groups, which are intended to bond with starch hydroxyl 

groups during the blending procedure by means of a polymer analog (trans-esterification) 

reaction. Poly(vinyl acetate) and poly(vinyl acetate-co-butyl acrylate) are of special interest 

because of their potentials to diminish moisture sensitivity and the glass transition temperature of 

resulting blends. Preliminary experiments have shown successful reactive blending, increases in 

thermal stability, and decreases in swelling due to water, although work remains to be done to 

improve the processability of the blends (3). 

 In addition, mathematical and computational efforts involving models such as the latticefluid hydrogen-bonding model are assisting experimental work by facilitating the prediction of 

mechanical and volumetric properties of starch-based polymers and water (3). 

 Transgenic plants again offer the hope of improving polymer properties. Transgenic 

plants have been studied to understand the biosynthesis of starch (5, 6). Manipulation of these 

biosynthetic pathways provides a means for affecting the distribution of amylose and 

amylopectin, potentially providing exquisite control over material properties within a single crop 

without the need to blend different varietals. 

 Several other polysaccharides are valuable bioresources from a materials point of view 

(7). Naturally this includes cellulose, but also includes pectins. Pectins are used in the food 

industries as coatings and additives and can be extracted from apple pomace and citrus peels; 

pectin is partially methylated poly-α-1,4-D-galacturonic acid. Konjac (a copolymer of mannose 

and glucose with a ratio of about 1.6:1) is derived from plant tubers and can be formed into 

films. Alignates are also film formers that are derived from the cell wall of brown seaweed; 

structurally alignate is poly(1,4 uronic acid). Guar gum and gum Arabic are two widely 

recognized materials from the family of plant gums that consist of hydrateable polysaccharides; 

these gums find application as binders, adhesives, flocculants, emulsifiers, and even lubricants in 

the food, papermaking, and petroleum industries. 

Polysacharides are also available from animal sources; the primary example is chitin. 

Chitin is the second only to cellulose in its natural abundance in biomass being found in the 

exoskeletons of crustacean and insects. Because chitin and the related chitosan are available 

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from shellfish waste, they are inexpensive in their raw forms and have been widely studied (8). 

Applications include absorbants used in wastewater treatment, films used as membranes, beads 

for metal chelation, and coatings for improved seed germination. To date, most manipulation of 

the raw materials to final products has involved chemical methods with relatively little emphasis 

on bioengineering techniques. Given the abundance of chitin, better more benign processing 

techniques are warranted. 

From the perspective of bioengineering, bacterial polysaccharides are the most interesting 

as they lend themselves to the host of metabolic engineering techniques. For example, xanthan 

gum is a high molecular weight branched polysaccharide extracted from Xanthomonas 

campestris that is used as a viscosity modifier in drilling fluids. By introducing mutants in 

various parts of the biosynthetic pathway through genetic engineering, a series of polymers with 

different rheological properties can be produced by fermentation (9, 10). 

2.2 Plant Proteins 

Several potentially useful plant proteins are available as byproducts of biorefining. For 

example, corn zein (the alcohol soluble fraction of corn gluten) is becoming increasingly 

available as a result of expansion in the production of bioethanols. Protein based adhesives 

appear promising because many proteins in nature are used for adhesion, for example, marine 

mussels use proteins to adhere to surfaces. 

Widespread cultivation of soybeans in the United States has encouraged a great deal of 

research into the development of biopolymers derived from soy protein. Soy proteins are 

complex macromolecules with many sites available for interaction with plasticizers and other 

copolymeric constituents, enabling soy protein to be converted to soy protein plastic through 

extrusion with a plasticizer or cross-linking agent. Although the mechanical properties of soy 

protein plastic can be controlled and optimized by adjusting the molding temperature and 

pressure and initial moisture content, applications are limited because of its low strength and 

great tendency to absorb moisture. The most effective method is to blend soy protein plastic 

with biodegradable polymers to form soy protein-based biodegradable plastics. Currently, the 

biodegradable plastics being used to blend soy plastic include polyester amide, polycaprolactone, 

Biomax®, and poly(tetramethylene adipate-co-terephthalate) (11, 12). While these approaches 

have been effective, other promising options are found in the use of soy proteins in structural 

composites, addressed below. 

Proteins can be synthesized using recombinant DNA technologies. Because of this there 

is great interest within the polymer science community in exploiting proteins to make specialized 

supramolecular structures. The primary protein sequence of amino acids (residues) dictates its 

molecular conformation and resulting supramolecular structure. If this structure formation can 

be controlled novel and interesting materials will result. It is unclear however, if such materials 

will have widespread utility as commodity plastics. 

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2.3 Plant Oil-based Polymers 

Soy bean oil and other plant oils have already proven useful as plastic materials when 

converted using chemical techniques. Their great utility lies in the fact that the much of the oil 

present in plants is unsaturated (i.e., the oils contain reactive carbon double bond) and contain 

ester linkages. These chemical functionalities allow a range of polymerization chemistries to be 

practiced leading to a wide variety of plastic resins. From a bioengineering point of view, 

genetic engineering techniques may be used to manipulate and control the distribution of the type 

of oil present in the plant. DuPont has developed a soybean that contains in excess of 80 percent 

oleic within the fatty acid distribution. In 2002, 75 percent of U.S.-soybean acres were planted 

with biotech soybeans–up from 68 percent in 2001, according to statistics released by the USDA 

(13). The widespread acceptance of transgenic crops likely means that plant-based oils of 

defined character will become increasingly available in the future. Once again, the conversion of 

these specialty plant oils to plastics is expected to be more economical than conversion to 

biodiesel. 

 Natural oils are comprised of triglycerides and are abundant in many areas of the world. 

Triglycerides consist of three fatty acids (i.e., carbon numbers from 22 down to 14 and double 

bonds down from 3 to 0) covalently linked to a central glycerol. A variety of chemistries is 

available for turning such oils into useful polymeric products and these are discussed at length in 

the recent compilation by Wool from which Figure 13 is taken (14). The various functionalities 

available include amines (structure 2), monoglycerides (3A, 3B), polyols (4), maleates (5), 

acrylates (6), epoxy (7), hydroxyl (8), and maleate half esters (9, 10, 11). Such monomers are 

easily incorporated into well established vinyl ester resin formulations and can serve to decrease 

volatile organic compound (VOC) emissions in styrene containing formulations. Other 

polymeric materials that can contain significant plant-oil content include polyurethanes. 

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Figure 13. Pathways for the chemical conversion of plant 

triglycerides (1) into different classes of chemically 

reactive species useful for making plastics 

(from Wool [14]). 

2.4 Cellulose, Lignin, and Reinforced Composites 

 As is evident from the preceding sections, many plastics can be improved substantially 

for materials applications by the introduction of reinforcing constituents to form composites. 

Indeed, certain biosubstances such as proteins can only form usable materials as components of 

composites. In the interest of minimizing environmental impact, natural cellulose-based fiber 

reinforcements have become especially attractive. 

2.4.1 Cellulose. Cellulose, the most abundant biological substance on Earth, is a 

polymer of glucose with both amorphous and crystalline regions in its native state (see Chapter 

IV. Bioethanol for additional structural details). Although cellulose is most widely applied as a 

reinforcing agent in composites, it can also be used directly or as an esterified derivative in place 

of petroleum as a feedstock to make cellulosic plastics, particularly as flexible films. The most 

widely used cellulose esters are cellulose acetate (CA), cellulose acetate propionate (CAP), and 

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cellulose acetate butyrate (CAB), (15) although cellulose fatty esters are under investigation as 

well (16). These later esters can be produced from wastes such as recycled paper and bagasse, 

and have found applications in film substrates for photography, toothbrush handles, selective 

filtration, adhesive tapes, cellophane, various semi-permeable and sealable films, and automotive 

coatings (17) (http://www.innoviafilms.com/product/summary_datasheets.htm). 

2.4.2 Lignin. The pulp and paper industries produce in the vicinity of 20 million tons 

per year of lignin in the United States alone (18). Along with cellulose and hemicellulose, lignin 

constitutes a major component of the cell wall in both hardwoods and softwoods; lignin content 

ranges from 15–40 percent by weight (19). Lignins are complex heteropolymers of p-hydroxy 

cinnamyl alcohols. Lignin is derived by treatment of pulp with sodium hydroxide and sodium 

sulfide (kraft pulping liquor) which cleaves linkages in the protolignin structure (20). Glasser 

and Lora have recently published a survey of lignin useages (21). Lignin can substitute for 

petroleum based materials in a number of ways and an excellent overview of these uses is 

available (14). Lignin can serve as a filler in thermoplastics, thermosets, and rubbers; it has also 

been converted into carbon fibers, a potentially high value-added application. While most efforts 

in lignin modification have relied on chemical methods, enzymatic grafting has been reported 

(22). In the context of the integrated biorefinery, if forest resources (hardwoods and softwoods) 

are to be exploited in the future, additional uses for lignin will be needed. Presently it is 

typically burned as a fuel but higher value added uses are clearly desirable but technically 

challenging. 

 For example, downregulation of lignin content is of considerable interest. Transgenic 

aspen trees in which expression of a lignin biosynthetic pathway has been downregulated by 

antisense inhibition have been developed (23). However, while these trees produced up to 

45 percent less lignin, this was accompanied by a 15 percent increase in cellulose. That is, the 

lignin to cellulose mass ratio remained essentially unchanged. 

2.4.3 Natural fiber-reinforced composites. Natural fibers such as kenaf, flax, 

jute, hemp, sisal, and henequen are attractive options for reinforcing starch- and protein-based 

composites because they are renewable and sustainable, as well as low cost, with acceptable 

mechanical properties, ease of separation, and biodegradability. Additionally, these fibers have 

excellent thermal and sonic insulation properties. Natural fibers from grass, hemp, and ramie 

have been reported as reinforcements for soy-based matrices, compared with glass fibers, and 

improved in terms of physical properties via surface treatments (11, 12, 24, 25). 

Although both soy plastics and natural fibers possess hydroxyl and carboxyl groups that can 

interact during processing, these interactions seem to be limited and typically do not lead to 

significant improvements in performance. However, enhancing these interactions through the 

use of compatibilizers, molecules such as polyester amide (PEA) that interact with both the fiber 

and the polymer matrix, is of current interest in the synthesis of natural fiber-reinforced soy 

biocomposites. Among reinforcing fibers, pineapple leaf fiber is an attractive option due to its 

high tensile strength (400–1600 MPa) and modulus (59 GPa), that result in turn from its high 

cellulose content (70–82 percent) and high degree of crystallinity. In addition, its cultivability in 

the southern United States and Central and South America cause it to be widely available, and it 

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has shown previous success in strengthening low-density polyethelyne (LDPE), rubber, 

polyester, and polyhydroxybutyrate (PHB) (11, 12). 

Hemp fibers and related grass fibers are additional promising natural reinforcement 

agents. Hemp is an important ligno-cellulosic natural fiber that contains (by weight) 70.2–70.4 

percent cellulose, 3.7–5.7 percent lignin, 17.9–22.4 percent hemicellulose, 0.9 percent pectin, 

0.3 percent wax, and 10.8 percent moisture. Natural waxy substances on the fiber surfaces 

diminish fiber-matrix bonding, leading to the necessity of investigation of various surface 

treatments to improve fiber-matrix adhesion of the resulting biocomposites. In addition, hemp, 

as a natural fiber, starts to decompose at 300°C, with the consequence that its use is limited to 

plastics that can be processed at lower temperatures. Fortunately, that is not an obstacle for soyand starch-based plastics (17). 

2.4.4 Nanocomposites. Particles are often incorporated into plastics to improve 

stiffness and toughness, to enhance barrier properties or fire resistance, or simply to reduce cost. 

Unfortunately, however, brittleness and opacity are sometimes imparted to the resulting 

composites. Nanocomposites are a new class of plastics that incorporate nanoparticles, or 

particles having at least one dimension in the nanometer range, in attempts to reduce these 

undesirable side effects. 

2.4.4.1 Classes. Three types of nanocomposites are distinguished based on the number 

of dimensions of the dispersed particles that are in the nanometer range. If all three dimensions 

are in the nanometer range, the nanoparticles are isodimensional; examples include spherical 

silica (26) and other (27) nanoparticles. Nanotubes or nanowhiskers are particles in which only 

two dimensions are in the nanometer scale; examples include carbon nanotubes (28) and 

cellulose whiskers (29–31). If only one dimension is on the nanometer scale, the filler is present 

in the form of sheets of a few nanometers thick. These are polymer-layered crystal 

nanocomposites and are almost exclusively obtained by the intercalation of the polymer inside 

galleries of layered host crystals. A wide variety of both synthetic and natural crystalline fillers 

are able to intercalate as polymers. Those based on clays and other layered silicates are most 

widely investigated because the starting materials are widely available and inexpensive. In 

addition, the intercalation chemistry of clays has been well-studied (32, 33). When such sheetshaped nanofillers are successfully dispersed in a polymer, the resulting nanocomposites exhibit 

markedly improved mechanical, thermal, optical, and physio-chemical properties when 

compared with the pure polymer or conventional (microscale) composites. For example, the first 

profound demonstration provided by Kojima and coworkers (34–37) for Nylon6-clay 

nanocomposites showed that improvements can include increased moduli, strength, and heat 

resistance, as well as decreased gas permeability and inflammability. 

2.4.4.2 Cellulose nanocomposites. In recent years, natural cellulose fibers have 

gained attention as reinforcing phases for polymer nanocomposites (30, 31, 38–46). Several 

approaches to the production of such microfibers are known, including chemical treatments and 

steam explosion of cellulose starting materials. Their low density, gentleness toward processing 

equipment, and relatively reactive surfaces hold great promise for excellent property 

improvement. In addition, they are abundant and inexpensive, and most notably, they retain their 

biodegradability (47). 

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In cellulose nanocomposites, the polymer modulus can be increased by more than threefold at 6 percent loading levels due to the long aspect ratio of the cellulosic fillers. These fibers 

reach the percolation threshold at relatively low loading, causing the modulus to increase rapidly. 

Cellulose nanocrystals can exhibit high aspect ratios, whereby the length divided by thickness 

can approach 500 for some agricultural fibers such as sugar beet and the giant reed Arundo 

donax. Proper treatment can also lead to the desired platelet-like morphology similar to the very 

successful clay nanofillers (48). 

In efforts to synthesize completely biodegradable nanocomposites, so-called green 

nanocomposites have been successfully fabricated from cellulose acetate powder, biodegradable 

triethyl citrate (TEC) plasticizer, and organically modified clay. Varieties with 20 weight 

percent plasticizer and 5 weight percent organoclay showed better intercalation and an exfoliated 

structure than the counterpart having 30–40 weight percent plasticizers, while the tensile 

strength, modulus, and thermal stability diminished with increasing plasticizer content from 

20-40 weight percent. Of special note, the nano-reinforcement at the lower volume fractions 

(phi ≤ 0.02) reduced the water vapor permeability by 50 percent (49). 

2.4.4.3 Polysaccharide nanocomposites. Renewable nanocomposites have also been 

made using microbially-derived polysaccharide polymers, such as polysaccharide fillers as the 

reinforcing phase for a poly(3HO) (45, 50). The mechanical properties of poly(3HO) are 

significantly improved by the addition of certain polysaccharides (up to 50 percent starch by 

weight or up to 6 percent cellulose by weight). The aspect ratio of the reinforcing phase is a 

critically important variable. Improved nanocomposites are possible by transreacting the 

hydroxyl groups of polysaccharides with functional groups in PHA, again demonstrating the 

importance of chain functionalization (45, 50). 

3. Research Priorities 

3.1 Basic Biosciences 

From a long-term perspective, continued support for basic biosciences that allow the 

manipulation of plants at the genetic level is absolutely essential. Only through continued 

development of genetic and physiological engineering techniques will the possibility of inducing 

organisms to produce polymers directly, and thereby reducing the cost of bioplastics by 

minimizing processing steps, be realized. In addition, the preference for anaerobic processes 

among microbial fermentations should be recognized due to the minimization of carbon loss 

from feedstocks. In the context of the plant-based plastics described in this section, the required 

tools include the genetic engineering of the cellulose-lignin and oil distribution in plants. Such 

basic genetic manipulation of plants is supported by the USDA. Specific strategies for pollution 

prevention are necessarily more narrow and should focus on developing cost-effective methods 

for producing plastics from plant-based matter. 

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3.2 Biodegradable Plastics 

Within the realm of starch-based plastics, commercial interest and success is currently 

carrying the development of compostable disposable packaging, including garbage bags, food 

wraps, diapers, as well as disposable food service items such as plates, cups, and utensils. 

Commercial research and development is even leading to improvements in water resistance and 

durability of starch-based materials, with the result that these are not considered to be high 

priorities for federal research funding. In contrast, the possibility of vastly improved strength, 

lightness, durability, and heat and water resistance offered by natural fiber-reinforced 

composites, particularly nanocomposites, cause this area to be highly attractive for additional 

effort. Low-cost polysaccharide-based plastic materials have the greatest potential to displace 

significant amounts of petroleum-based plastics. 

Similarly, more benign greener chemical processes should be investigated for 

transforming the available renewable resources. Such activities should include enzymatic 

transformations when they are less energy intensive than existing chemical routes. 

Genetic engineering of plant proteins for specific functionality is a lower priority simply 

because the potential application, adhesives, is small relative to the packaging and structural uses 

for plastics. If plant proteins can be engineered for larger volume applications, for example, into 

thermoplastic film or sheet materials, the promise of coproducing both fuels and materials in an 

integrated biorefinery would be supported. Specialized materials produced through recombinant 

DNA technologies are expected to be of high commercial value but of relatively small volume. 

4. Commercialization 

Commercialization of many of the bioplastics discussed in this section is well 

established. Chemically modified cellulosic esters are used in a wide range of products. 

Importantly, starch- and cellulose-based products are meeting increasingly high standards of 

biodegradability and compostability, as prescribed by the American Society for Testing and 

Materials (ASTM), DIN CERTCO in Germany, and EK certification in Norway. Representative 

of these, ASTM D6400-99 “Specifications for Compostable Plastics” or ASTM D6868 

“Specification for Biodegradable Plastic Coatings on Paper and other Compostable Substrates” 

certification designates “a plastic or substrate that undergoes degradation by biological processes 

during composting to yield CO2, water, inorganic compounds, and biomass at a rate consistent 

with other known compostable materials and leaves no visible, distinguishable or toxic residue” 

(www.astm.org). Commercial starch- and cellulose- based materials are highly visible 

worldwide, and their producers and distributors, including BASF, Novamont, PolarGruppen, 

Bio-Bag Canada, Innovia Films, and Biosphere Products Inc., to name a few, are prominent 

members of the Biodegradable Products Institute (www.BPIWorld.org). 

4.1 Mater-BiTM 

Mater-BiTM is one of the most prominent starch-based plastics, manufactured by 

Novamont Corporation in Terni, Italy. This product is composed primarily of cornstarch, 

complexed with proprietary natural and/or synthetic polymers to impart water-resistance and 

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durability, and is produced in pellets of three grades that can be processed by distributors using 

common transformation techniques. For recommended uses, including packaging, disposable 

plastics, toys, and biofillers, the company claims that Mater-BiTM plastics have “characteristics 

[that] are identical to those of traditional plastics, but are perfectly biodegradable and 

compostable” (www.materbi.com). After use, products made of Mater-BiTM biodegrade on 

average, in the time of one composting cycle. Although Mater-BiTM was developed only in the 

late 1980s, and Novamont was founded only in 1990, Mater-BiTM products have already become 

internationally visible. The product called Green Pen, made of Mater-BiTM by the company 

Lecce Pen (Turin, Italy), was chosen to be the official pen of the United Nations Earth Summit in 

Rio De Janeiro in 1992, and in September of the same year, the production of the first MaterBiTM bags for separate waste collection began in Germany. Research collaborations with 

Goodyear Tire began in 1995, and in 2001 production of the GT3 tire began, using Biotred 

technology that uses biofillers made of Mater-BiTM. Production of Mater-BiTM diapers, by the 

Swedish company Naty, began in 1999, and in 2000, the Olympics in Sydney, Australia used 

catering products and compost bags made of Mater-BiTM (www.materbi.com). 

 Mater-BiTM is processed and distributed by numerous companies worldwide, including, 

in addition to those named above, NAT-UR in California, which also distributes NatureWorks 

PLA-based materials (www.nat-ur.com), as well as PolarGruppen and Bio-Bag Canada, which 

manufactures biodegradable, compostable bags and packaging from Mater-BiTM 

(www.polargruppen.com; www.biobag.ca) that are largely ASTM, DIN CERTCO, and EK 

certified. 

4.2 Ecoflex®

In 1998, the German company BASF introduced a family of biodegradable aliphaticaromatic copolyesters, including formulations consisting of thermoplastic starch, under the brand 

name Ecoflex®. Ecoflex® is recommended for trash bags and disposable packaging, as well as 

for starch composites, since it degrades completely in compost within just a few weeks. 

Homopolymeric Ecoflex®, lacking the starch component, has been developed specifically for 

flexible film applications, such as those commonly filled by polyethylene, and is now established 

as one of the first completely biodegradable flexible films. Like polyethylene, Ecoflex® is waterproof, tear resistant, flexible, fusible, and imprintable, and appears to have high processing 

flexibility (http://www2.basf.de/basf2/html/plastics/englisch/pages/biokstoff/ecoflex.htm). In 

2005, BASF commercialized blends of Ecoflex® with PLA under the tradename Ecovio®. 

4.3 PPM 

PPM was developed by Biosphere Industries from cellulose-reinforced starch to meet 

rigid packaging needs and has two primary grades, PPM100 and PPM200, which are currently 

used primarily in food-service items and general packaging. PPM100 biodegrades 98 percent in 

28 days (100 percent in less than 40 days) and can resist warm-water extended periods. For 

greater heat and moisture resistance, the higher-grade PPM200 is used; this material requires 

more than 40 days for complete compostability but safely withstands hot liquids for extended 

periods. In January 2005, PPM100 received Biodegradable Products Institute certification, 

demonstrating that the material meets the ASTM D6868 specifications and will biodegrade 

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swiftly and safely during municipal, commercial, or household composting 

(www.biospherecorp.com). 

4.4 Cellulose Films 

Innovia Films, Inc. was formed in 2004 and is one of the producers of cellulose-based 

packaging films. The variety of products offered includes nitrocellulose-coated films, laminating 

films, films for adhesive tape, photographic masking, and heat-sealable films for microwave and 

oven applications (http://www.innoviafilms.com/product/summary_datasheets.htm). 

4.5 Soy-based Plastics 

The Soy Works Corporation in Illinois holds a number of patents for soy-protein-based 

products and has developed a soy-protein-based plastic known as SoyPlusTM. Resins are made of 

soy protein and other unidentified ingredients, yielding a moldable resin that is being developed 

for a variety of applications, including garden supplies, food-service items, industrial packaging, 

mulch, toys, golf tees, and building materials (http://soyworkscorporation.com/). 

 Another soybean product, based from oil rather than protein, was developed by the 

University of Delaware and is a primary ingredient in molded fiberglass-reinforced farm 

equipment parts being tested by the John Deere Company (http://www.unitedsoybean.org/

feedstocks/fsv2i6b.html). This work has continued and resulted in the founding of Cara Plastics 

(http://www.caraplastics.com). Cara Plastics is pursuing market opportunities in structural 

composites, primarily with polyesters and vinyl esters where Cara's resins would be used as the 

matrix of a fiber-reinforced material. Markets are estimated to be 2 billion pounds worldwide 

compared to 500 million pounds for biodegradable plastics. Cara hopes to ultimately capture at 

least 10 percent of these markets, or 250 million pounds annually with a target of 75 million 

pounds annually within 5 years. 

5. References 

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composites from soy based plastic and pineapple leaf fiber: fabrication and properties 

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Wood and Cellulosic Chemistry (David Hon, N. S., Ed.). Marcel Dekker, New York, 

NY. 

(16) Satgé, C., R., B. Granet, P. Verneuil, Branland, and P. Krausz (2004). Synthesis and 

properties of biodegradable plastic films obtained by microwave-assisted cellulose 

acylation in homogeneous phase, Comptes Rendus Chimie 7:135-142. 

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engineering on the performance of natural fiber reinforced cellulose acetate 

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(SERI, now NREL) - SERI R-234-3610. 

(19) Fujita, M., and H. Harada (2001). Ultrastructure and Formation of Wood Cell Wall, in

Wood and Cellulosic Chemistry (David Hon, N. S., Ed.). Marcel Dekker, New York, 

NY. 

(20) Sakakibara, A., and Y. Sano (2001). Chemistry of Lignin, in Wood and Cellulosic 

Chemistry (David Hon, N. S., Ed.). Marcel Dekker, New York, NY. 

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(21) Lora, J. H., and W. G. Glasser (2002). Recent industrial application of lignin: A 

sustainable alternative to nonrenewable materials, J Polym Environ 10:39-48. 

(22) Mai, C., O. Milstein, and A. Hüttermann (2000). Chemoenzymatical grafting of 

acrylamide onto lignin, Journal of Biotechnology 79:173-183. 

(23) Hu, W.-J., S. A. Harding, J. Lung, J. L. Popko, J. Ralph, D. D. Stokke, C.-J. Tsai, and 

V. L. Chiang (1999). Repression of lignin biosynthesis promotes cellulose accumulation 

and growth in transgenic trees, Nature Biotechnology 17:808-812. 

(24) Joshi, S. V., L. T. Drzal, A. K. Mohanty, and S. Arora (2004). Are natural fiber 

composites environmentally superior to glass fiber reinforced composites? Composites 

Part A: Applied Science and Manufacturing 35:371-376. 

(25) Mishra, S., A. K. Mohanty, L. T. Drzal, M. Misra, and G. Hinrichsen (2004). A review 

on pineapple leaf fibers, sisal fibers and their biocomposites. Macromolecular Materials 

and Engineering 289:955-974. 

(26) von Werne, T., and T. E. Patten (1999). Preparation of structurally well defined 

polymer-nanoparticle hybrids with controlled/living radical polymerization, J Am Chem

Soc 121:7409-7410. 

(27) Reynaud, E., C. Gauthier, and J. Perez (1999). Nanophases in polymers, Rev Metall/Cah 

Inf Tech 96:169-176. 

(28) Calvert, P. (1997). Potential applications of nanotubes, in Carbon Nanotubes (T.W. 

Ebbesen Ed.). CRC Press, Boca Raton, FL, 277-292. 

(29) Chazeau, L., J. Y. Cavaille, G. Canova, R. Dendievel, and B. Boutherin (1999). 

Viscoelastic properties of plasticized PVC reinforced with cellulose whiskers, J Appl 

Polym Sci 71:1797-1808. 

(30) Favier, V., G. Canova, S. Shrivastava, and J. Cavaille (1997). Mechanical percolation in 

cellulose whisker nanocomposites, Polymer Engineering and Science 37(10):1732-1739. 

(31) Favier, V., H. Chanzy, and J. Y. Cavaille (1995). Polymer nanocomposites reinforced by 

cellulose whiskers, Macromolecules 28(18):6365-6367. 

(32) Grim, R. E. (1953). Clay Mineralogy. Mc-Graw Hill, New York, p 384. 

(33) Theng, B. K. G. (1974). The Chemistry of Clay-Organic Reactions. Wiley, New York. 

(34) Kojima, Y., A. Usuki, M. Kawasumi, A. Okada, Y. Fukushima, T. Karauchi, and 

O. Kamigaito (1993). Mechanical properties of nylon-6-clay hybrid, J Mater Res

6:1185-1189. 

(35) Kojima, Y., A. Usuki, M. Kawasumi, A. Okada, T. Kurauchi, and O. Kamigaito (1993). 

Synthesis of nylon 6-clay hybrid by Montmorillonite Intercalated with e-Caprolactam, J. 

Poly Sci A: Poly Chem 31:983-986. 

(36) Kojima, Y., A. Usuki, M. Kawasumi, A. Okada, T. Kurauchi, and O. Kamigaito (1993). 

One-pot synthesis of nylon 6-clay hybrid, J Poly Sci A: Poly Chem 31:1755-1758. 

(37) Kojima, Y., A. Usuki, M. Kawasumi, A. Okada, T. Kurauchi, O. Kamigaito, and K. Kaji 

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(38) Angeles, M. N., and A. Dufresne (2000). Plasticized starch/tunicin whiskers 

nanocomposite materials, Macromolecules 33:8344. 

(39) Angeles, M. N., and A. Dufresne (2001). Plasticized starch/tunicin whiskers 

nanocomposite materials. 2. Mechanical Properties, Macromolecules 34. 

(40) Cavaille, J. Y., H. Chanzy, V. Favier, and B. Ernst (2000). Cellulose microfibrilreinforced polymers and their applications, U. S. Patent 6,103,790. 

(41) Cavaille, J. Y., H. Chanzy, E. Fleury, and J. F. Sassi (2000). Surface-modified cellulose 

micro fibrils, method for making the same, and use thereof as filler in composite 

materials, U.S. Patent 6,117,545. 

(42) Cavaille, J. Y., and A. Dufresne (1998). Nanocomposite materials of thermoplastic 

polymers reinforced by polysaccharide fillers, in Biopolymers: Utilizing Natures 

Advanced Materials (Greene, R. V., S. H. Imam Eds.). ACS Publishing, New York. 

(43) Dufresne, A. (1998). High performance nanocomposite materials from thermoplastic 

matrix and polysaccharide fillers, Recent Research Developments in Macromolecules 

Research 3(2):455-474. 

(44) Dufresne, A., J.-Y. Cavaille, and W. Helbert (1996). New nanocomposite materials: 

microcrystalline starch reinforced thermoplastic, Macromolecules 29:7624-7626. 

(45) Dufresne, A., M. B. Kellerhals, and B. Witholt (1999). Transcrystallization in mclPHAs/Cellulose whisker composites, Macromolecules 32:7396-7401. 

(46) Dufresne, A., and M. R. Vignon (1998). Improvement of starch film performances using 

cellulose microfibrils, Macromolecules 31:2693-2696. 

(47) Frisoni, G., M. Baiardo, and M. Scandola (2001). Natural cellulose fibers: 

Heterogeneous acetylation kinetics and biodegradation behavior, Biomacromolecules

2:476-482. 

(48) Grunert, M., and W. Winter (2000). Progress in the development of cellulose reinforced 

nanocomposites, Polymeric Materials, Science and Engineering 82:232-238. 

(49) Park, H. M., M. Misra, L. T. Drzal, and A. K. Mohanty (2004). "Green" nanocomposites 

from cellulose acetate bioplastic and clay: Effect of eco-friendly triethyl citrate 

plasticizer, Biomacromolecules 5:2281-2288. 

(50) Dubief, D., E. Samain, and A. Dufresene (1999). Polysaccharide microcrystals 

reinforced amorphous poly(b-hydroxyoctanoate) nanocomposite materials, 

Macromolecules 32:5765-5771. 

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Chapter IV 

Biofuels 

The worldwide depletion of fossil fuels and widespread concern over increasing 

atmospheric CO2 have sparked interest not only in biomaterials but also in sustainable, nonfossil-based fuels. Political instability in petroleum-producing regions has further increased the 

desirability of domestic fuel sources, particularly for transportation (1). Solar and wind power 

are well-suited to sustainable generation of electricity, including electricity for charging vehicle 

batteries, but most modern vehicles are designed for liquid fuels that are best simulated by two 

biofuels: bioethanol and biodiesel (2). In addition, biohydrogen is an emerging biofuel that 

carries energy from sunlight or organic matter, rather than petroleum, in clean-burning hydrogen 

(H2), Finally, biodesulfurization of petroleum products may offer a way to mitigate some effects 

of petroleum use during a transition and is discussed as well. 

A. BIOETHANOL 

1. Introduction 

Proponents of ethanol as a use for fuel highlight the apparent net-zero contribution of fuel 

ethanol combustion to the global carbon cycle, in that feedstocks for ethanol production derive 

their carbon from atmospheric CO2, and that ethanol combustion simply returns the fixed carbon 

to its atmospheric source (3). Others, however, make the valid counterpoints that the conversion 

even of agricultural wastes to ethanol is, itself, an energy-intensive process that frequently makes 

use of fossil energy sources, and that growth of crops dedicated to energy production must also 

be conducted in a sustainable manner for fuel ethanol use to carry a net environmental benefit 

(4). 

Petroleum currently supplies 97 percent of the energy consumed for transportation (5), 

and transportation accounted for two-thirds of U.S. petroleum use in 2002. This trend is 

expected to continue until 2025 (6). This need not continue, however, as all automobile 

manufacturers produce flexible-fuel vehicles (FFVs) that can use 10 percent or 85 percent 

ethanol blends with gasoline, and ethanol can also replace diesel fuel in heavy vehicles (5). The 

United States also now has 199 fueling stations for ethanol, as well as extensive online services 

for planning travel between stations (7). Although ethanol is limited in availability in some 

states, the transportation market for ethanol could expand to as much as 38–53 billion liters per 

year, if all available agricultural residues were converted to ethanol (8). Ethanol is also being 

used as a replacement for methyl tertiary butyl ether (MTBE), the fuel oxygenate that is being 

phased out due to its widespread contamination of groundwater (9). 

The majority of ethanol, approximately 62 percent of the world total, is currently 

produced in Brazil, primarily from cane sugar (~12.5 billion liters in 2002), and in the United 

States, primarily from corn (~5 billion liters per year) (5, 10). However, these feedstocks are 

expensive and are useful as foods, causing a great deal of research to be focused on the 

development of biomass such as corn cobs and stalks, sugar cane waste, wheat and rice straw, 

other agronomic residues, forestry and paper mill discards, paper municipal waste, and dedicated 

energy crops into ethanol (11). While the use of such non-food substrates helps the economics 

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of ethanol production substantially, the high cost of production, especially relative to gasoline, 

remains the primary obstacle to bioethanol commercialization (5). 

Lignocellulosic (non-food) raw materials, such as agricultural, wood chip, and paper 

wastes, can yield approximately 100 billion gallons of fuel-grade ethanol per year in the United 

States alone (12). The projected cost of bioethanol has dropped from about $1.22 per liter to 

about $0.31 per liter based on consistent improvements in pretreatment, enzyme application, and 

fermentation (13). If additional specific improvement targets are met, this cost could drop to as 

low as $0.20–$0.12 per liter by 2015 (14). For transportation fuel, therefore, ethanol has real 

potential to replace gasoline, even in the absence of governmental support. 

2. State of the Science 

2.1 Ethanol Biosynthesis: Overview

2.1.1 Feedstocks. Biological production of ethanol first requires that atmospheric 

CO2 be fixed into organic carbon (biomass) through photosynthesis. While agricultural crops 

and residues currently form the vast majority of feedstock for ethanol production, other biomass 

sources such as wood chips, sawdust, industrial organic wastes, and municipal organic wastes are 

important for the commercial development of fuel bioethanol (11). Agricultural plant matter 

contains approximately 10–15 percent lignin, a polymer of phenolic subunits that is highly 

resistant (although far from impervious) to enzymatic attack. Lignin typically surrounds and 

protects the more enzymatically-vulnerable components of cellulose and hemicellulose, which 

comprise approx. 20–50 percent and 20–35 percent of the remaining plant material, respectively 

(9). 

The next challenge in bioethanol synthesis is therefore the release of fermentable sugars 

from the biomass, or conversion of the feedstock into fermentable substrates. This phase 

involves both the separation of lignin from cellulosic and hemicellulosic polymers and the 

hydrolysis of the polymers into monomeric sugars, primarily glucose and xylose. This phase is 

termed pretreatment, and a variety of biotic and abiotic approaches are currently under 

investigation; abiotic approaches have been recently reviewed (15). 

2.1.2 Mechanical and chemical disruption. Lignin is typically dissociated from 

the carbohydrates by mechanical and/or thermochemical means, including hot water, steam 

explosion, and/or acid treatments in either batch or flow-through reactors. Many variations have 

been explored. While it was once difficult to compare the performance and economics of the 

various approaches due to differences in feedstocks tested, a group of pretreatment researchers 

has formed in North America to facilitate such comparisons. This group, the Biomass Refining 

Consortium for Applied Fundamentals and Innovation (CAFI), has the goal of advancing the 

efficacy and knowledge base of pretreatment technologies (3), reviewed in (16, 17). 

Mechanical lignin disruption effectively hydrolyzes a significant fraction of the 

hemicellulose, but is less effective in hydrolyzing cellulose. This difference is caused by the 

different structures of the two polymers: hemicellulose is a highly branched, typically 

amorphous polymer that is therefore relatively easy to hydrolyze into its component sugars 

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(pentoses D-xylose and L-arabinose; hexoses D-galactose, D-glucose, D-mannose; and uronic 

acid; all highly substituted with acetic acid). Hemicelluloses from hardwoods are typically high 

in xylose, while those in softwoods contain more hexoses (3). 

Cellulose, in contrast to hemicellulose, is a semicrystalline polymer of pure glucose 

linked by beta-glucoside bonds. The beta linkages form linear strands that establish extensive 

H-bonds between them, leading to a highly stable structure that is quite resistant to degradation. 

Many chemical approaches have been explored to hydrolyze cellulose, though none are 

completely satisfactory; currently, dilute acid hydrolysis procedures are being proposed for 

several near-term commercialization efforts until more effective technologies are available (5). 

2.1.3 Enzymatic cellulose hydrolysis. Enzymatic hydrolysis by cellulases is the 

ultimate goal in biomass processing for fermentation: this method has the advantages of reduced 

sugar loss through side reactions and it is less corrosive of process equipment (18). In addition, 

the hydrolyzed product requires no neutralization prior to fermentation (19). 

Cellulases consist of multicomponent enzyme complexes acting synergistically: 

complete cellulose hydrolysis requires the activity of an endoglucanase, which cleaves interior 

regions of cellulose polymers; an exoglucanase, which cleaves cellobiose units from the ends of 

cellulose polymers; and a beta-glucosidase, which cleaves cellobiose into its glucose subunits 

(20). Because of the complexity and insolubility of the substrate, cellulase catalysis is not only 

relatively slow, but it is also understood much less completely than other enzymes, despite over 

four decades of cellulase research (8). One of the most important organisms in the development 

of cellulase enzymes is Trichoderma reesei, the “ancestor of many of the most potent enzymeproducing fungi in commercial use today” (3). By 1979, genetic enhancement had produced 

mutants with up to 20 times greater cellulose productivity than the original organisms found in 

World War II; today, surprisingly, the most lucrative cellulase market is in the manufacture of 

stone-washed jeans (3). 

2.1.4 Fermentation. Fermentation of glucose to ethanol is performed by numerous 

bacteria, yeasts, and other fungi, and several yeasts have also been identified that can convert 

xylose to ethanol. Pentose fermentation to ethanol does not commonly co-occur with hexose 

fermentation to ethanol, however, spurring efforts to combine these two fermentation pathways 

into single organisms. Genetic engineering has since provided both bacteria and yeasts capable 

of fermenting both 5-carbon and 6-carbon sugars (21, 22). 

Although hydrolysis of biomass cellulose by cellulases was once performed as a distinct 

step between pretreatment and fermentation, fermentation can begin as soon as glucose subunits 

are released from cellulose. The realization of this led to the development of the Simultaneous 

Saccharification and Fermentation (or Co-Fermentation) Process (SSF or SSCF), which now 

provides the great advantage of simultaneous cellulose hydrolysis and glucose fermentation (13). 

This process enhances cellulase activity by relieving the product inhibition of beta-glucosidase 

by glucose, since the products are consumed as soon as they are produced. SSF has been 

patented by the Gulf Oil Company and the University of Arkansas (3). With the availability of 

organisms that can ferment both pentoses and hexoses, all biomass sugars may now be 

simultaneously fermented in SSF/SSCF processes (23, 24). 

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Product inhibition of exocellulases is not eliminated, however, unless the glucose dimer 

cellobiose is also consumed or hydrolyzed. Since conventional S. cerevisiae strains do not 

metabolize cellobiose, and since cellulase preparations with sufficient beta-glucosidase activity 

to hydrolyze all the cellobiose are expensive to produce, much research has been directed toward 

the use of native cellobiose-utilizing yeast strains in SSF, either independently or in co-culture 

with S. cerevisiae (18). 

2.1.5 Fermentation following gasification. A radically different approach to 

preparing biomass substrates for fermentation is found in biomass gasification. In this process, 

biomass is converted to synthesis gas, consisting primarily of CO, CO2, and H2, in addition to 

CH4 and N2 (25). After gasification, anaerobic bacteria such as Clostridium ljungdahlii can

ferment the CO, CO2, and H2 into ethanol by an acetogenic process (26-29). One advantage of 

the process is that, unlike acid and enzymatic hydrolysis methods, gasification can convert 

essentially all of the biomass, including lignin, to syngas that can be potentially fermented by 

bacteria (30). Higher rates of fermentation are also achieved because the process is limited by 

the transfer of gas into the liquid phase instead of the rate of substrate uptake by the bacteria. 

2.2 Feedstock Options 

Corn and sugar cane are not long-term options for ethanol generation because of their 

value as foods. Exploration of various non-food forms of biomass, principally wastes, is 

therefore an active area of research. 

Worldwide, rice straw has the greatest quantitative potential for bioethanol production, 

estimated at 205 gigaliters per year; this potential is concentrated in Asia, which as a region 

could produce up to 291 gigaliters per year of ethanol from rice straw in combination with wheat 

straw and corn stover. Europe has the next-largest supply of agricultural wastes, primarily in the 

form of wheat straw (69.2 gigaliters per year potential ethanol production); followed by North 

America, in which corn stover forms the majority of agricultural wastes and could supply an 

estimated 38.4 gigaliters per year of ethanol (11). Bagasse, or waste derived from sugar cane, is 

widely available in tropical areas and is being explored by BC International Corporation (BCI), 

while municipal solid waste has attracted the attention of Masada Resources Group, LLC; these 

two companies are currently planning construction of unique biomass-to-ethanol plants (31). 

Corn stover or fiber, a by-product of the corn wet-milling industry consisting of corn 

hulls and residual starch, is the subject of great interest as a possible substrate for ethanol 

production in the United States. Conversion of the starch along with the lignocellulosic 

components in the corn fiber could increase ethanol yields from a corn wet mill by 13 percent. 

In a recent study utilizing the bioethanol process development unit at the U.S. National 

Renewable Energy Laboratory (NREL), corn fiber was used to support continuous, integrated 

operation of the plant. The fiber was pretreated by high-temperature, dilute sulfuric-acid 

hydrolysis, and the cellulose was converted to ethanol using simultaneous saccharification and 

fermentation using a commercially-available cellulase and conventional Saccharomyces 

cerevisiae yeast that did not utilize 5-carbon sugars. Despite difficulties with bacterial 

contamination, which are expected to diminish with the use of recombinant, xylose- and 

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arabinose-utilizing fermentative organisms, the attempt was successful and indicates that corn 

fiber could become a valuable feedstock in the United States (31). The use of agricultural wastes 

is not without potential drawbacks, however. Most crop residues are currently plowed into the 

soil to sustain soil quality by increasing the soil organic carbon pool, enhancing activity of soil 

fauna, and minimizing soil erosion, and soil scientists caution that diversion of waste biomass for 

fuel must be undertaken cautiously (32). 

Softwood forest thinnings are also being explored as potential feedstocks. Lumber 

manufacturing, timber harvesting, and thinning of forests to prevent wildfires generate a large 

quantity of softwood residues that require environmentally sound and cost-effective methods of 

disposal. Research in this area in the United States is currently focusing on dilute sulfuric acid 

hydrolysis and SO2-steam explosion pretreatments, followed by fermentation by a 

Saccharomyces cerevisiae mutant yeast adapted to the inhibitory extractives and lignin 

degradation products present in the softwood hydrolysates (33). Testing of recombinant xylosefermenting yeasts is also planned, and investigation is underway by Kemestrie, Inc. (Sherbrooke, 

QC, Canada) to identify high-value coproducts that may be derived from softwoods, focusing on 

antioxidants and other extractives (3). 

2.3 Cellulase Engineering 

The second important area in which improvement is needed for the commercialization of 

fuel ethanol is the conversion of lignocellulosic feedstock into the sugars to be fermented. Most 

current work in this area concentrates on improvement of cellulase expression, activity, and 

production efficiency, with the goal of reducing the cost and increasing the extent of cellulose 

hydrolysis. 

Cellulase cost is a critical limiting factor in lignocellulose feedstock preparation. Current 

estimates of cellulase cost range from 30–50 cents per gallon of ethanol; a goal of 5 cents per 

gallon of ethanol is envisioned (3). Thus, a 10-fold improvement in specific activity, production 

efficiency, or some combination thereof, is required. 

Cellulase improvement in any of the following five critical areas could substantially 

improve the feasibility of bioethanol commercialization: thermostability, acid tolerance (to 

withstand pretreatment acidification), cellulose binding affinity, specific activity, and reduced 

nonspecific binding to lignin (14). While these features are theoretically approachable by 

genetic engineering techniques, use of these techniques is presently limited by the incomplete 

understanding of cellulase catalysis. A primary reason for this is that cellulose-cellulase systems 

involve soluble enzymes working on insoluble substrates, which represents a substantial increase 

in complexity from homogeneous enzyme-substrate systems. In addition, the catalytic system 

involves the synergistic activities of three different enzymes (3). Still, a number of promising 

avenues are currently being explored. 

2.3.1 Cellulase component engineering. The most fundamental improvements 

that are needed are within the cellulase components themselves, these are the endoglucanases, 

exoglucanases, and cellobiohydrolases (or beta-glucosidases). Using Trichoderma, Clostridium,

Cellulomonas, and Thermobifida, among others, efforts are underway to improve activity, 

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expression, and specificity of these components through site-directed mutagenesis, use of 

heterologous promoters to direct transcription, and modeling to reveal structure-function 

relationships (34–39). 

One of the most active cellulase components known is the endoglucanase E1 from 

Acidothermus cellulolyticus. Two leading industrial enzyme producers, Novozymes 

(www.novozymes.com) and Genencor International (www.genencor.com), are currently 

contributing to the cellulase improvement effort with support from DOE. In 1998, J. Sakon and 

colleagues at the University of Arkansas showed that performance of a ternary system was 

improved 13 percent by site-directed modification of one active site amino acid in Acidothermus

E1; currently they are pursuing E1 mutations that modify the biomass interactive surface. 

2.3.2 Chimeric cellulase systems. Cellulase components from diverse organisms, 

primarily bacteria and fungi, are being combined in ways that yield overall improved activity. 

Baker and colleagues have successfully combined bacterial and fungal cellulases in vitro (40), 

showing that these mixtures can be competitive with a native ternary system from T. reesei (41). 

Work with expansins, proteins that enable extension of plant cell walls during plant cell growth, 

has also shown enhancement of hydrolysis of microcrystalline cellulose in a mixed Trichoderma

cellulase preparation (42). The initial approaches to developing artificial cellulase systems, still 

instructive after nine years, are reviewed in (43). 

2.3.3 Heterologous expression. The next logical step in chimeric cellulase 

systems is the cloning of cellulases from one organism into another; this avenue is being 

explored as well, as shown by the expression of the T. reesei cellobiohydrolase I in Pichia 

pastoris (44). Especially important for commercial production, cellulases are being expressed in 

plants such as tobacco and potatoes, potentially providing more abundant sources of the enzymes 

(45). 

Another innovative approach to the heterologous expression of cellulases is the 

expression of heat-activated cellulases within biomass crops themselves, with the idea that the 

plants grow normally until harvested and exposed to elevated temperatures, at which point heatactivated cellulases hydrolyze the cellulose without need for externally added enzymes (46). 

2.3.4 Cellulase performance assays. Convenient, accurate, efficient assays are 

central to the development of any new technology. The diafiltration saccharification assay 

(DSA) developed at the NREL produces precise, detailed progress curves for enzymatic 

saccharification of cellulosic materials under conditions that mimic those of SSF. From this 

method, it is possible to describe the performance of a given cellulase preparation over a wide 

range of loading and reaction times with comparatively little data (47, 48, 3). 

2.3.5 Proteomic analysis, microarray analysis, and modeling. Proteomics is 

an emerging set of techniques that has proven extremely useful in understanding the interactions 

of multienzyme systems. Hydrolysis of complex organic substrates is an ideal candidate for 

proteomic analysis, as it involves a number of enzymes: β-1,4-endoglucanases, β-1,4-

cellobiohydrolases, xylanases, β-glucosidases, α-L-arabinofuranosidase, acetyl xylan esterase, βmannanase, and α-glucuronidase in T. reesei, for example. At the NREL, the expression of these 

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enzymes is being investigated under various conditions by proteomic methods and compared to 

corresponding enzyme activities using the DSA assay (3). 

To reveal gene expression responses to environmental conditions in both wild-type and 

genetically engineered microbes, microarray analysis is underway and could become a valuable 

industrial tool for evaluation of new recombinant organisms (49). Mathematical molecular 

analysis is also being employed to gain greater understanding of structure-function relationships 

to complement the physiological understanding provided by proteomic and microarray analysis. 

Current work includes molecular mechanics efforts by Brady and colleagues at Cornell 

University as well as Palma and colleagues; cellulase crystallization work is also in progress by a 

number of groups (50-53). 

2.4 Fermentation Technologies 

While fermentation of glucose into ethanol is a well-understood process that occurs 

widely among microorganisms, the fermentation of pentoses such as xylose, which are abundant 

in biomass, has posed significant challenges. Recently, however, this challenge has been 

addressed by creating recombinant yeasts and bacteria (21, 22), although the solution may not 

yet have been fully optimized. 

A second important aspect limiting commercialization is the fermentation efficiency of 

the microorganisms: in typical fermentation pathways for glucose and xylose to ethanol, one 

contributor to the high cost of ethanol production is the loss of half of the fixed carbon to 

products other than ethanol (5). 

The ideal bioethanol-fermenting microorganism would therefore readily ferment all 

biomass sugars, resist toxic effects of aromatic lignin subunits and other inhibitory byproducts 

such as acetate, be thermostable and acid-tolerant, and produce a highly active cellulase 

multienzyme complex (54). 

2.4.1 Pentose fermentation. As mentioned above, Escherichia, Klebsiella, and 

Zymomonas have now been engineered to ferment not only glucose but also xylose and arabinose 

sugars (5, 54, 55). Some of these are already experiencing commercial use as well: BC 

International Corporation (www.bcintl-corp.com) is using genetically engineered Escherichia 

coli to produce ethanol from biomass sugars, and Arkenol Inc. (www.arkenol.com) is using 

Zymomonas in its concentrated-acid process. 

In another example, Zymomonas mobilis has been transformed with Escherichia coli 

xylose isomerase, xylulokinase, transaldolase, and transketolase genes. Expression of the added 

genes are under the control of Zymomonas mobilis promoters. This genetically modified 

microorganism, patented by the Midwest Research Institute, is now able to ferment mixtures of 

xylose, arabinose, and glucose to produce ethanol (56, 57). 

2.4.2 Combined cellulolysis and fermentation. Consolidated bioprocessing 

(CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation

of resulting sugars to desired products occur in one step, is currently envisioned as the most 

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promising and eminently achievable path toward optimally efficient bioethanol production (54). 

Efforts to develop such a culture through engineering fermentative capacity into cellulolytic 

organisms, as well as the alternative, engineering cellulolytic capacity into fermentative 

organisms, are both underway and have been reviewed extensively (54). 

In one example, Ingram and colleagues cloned two Erwinia endoglucanase genes into an 

ethanol-producing Klebsiella species, producing a new microbe that produced up to 22 percent 

more ethanol when fermenting crystalline cellulose synergistically with added fungal cellulases 

(58). Cellulase genes have also been introduced into Lactobacillus, although not necessarily for 

biomass utilization (59), and cellobiose utilization capability has been engineered into 

Saccharomyces cerevisiae (60). In an alternative example, with the additional goal of offering 

improved relief from product inhibition in SSF, in which cellobiose inhibition of exoglucanase is 

problematic, ethanol-producing genes have been successfully introduced into native cellobioseutilizing bacteria (61, 62). 

2.4.3 Synergistic co-cultures. In experiments involving the cellobiose-fermenting 

recombinant, Klebsiella oxytoca P2, in co-cultures with ethanol-tolerant strains of 

Saccharomyces pastorianus, Kluyveromyces marxianus, and Zymomonas mobilis, the 

combinations produced more ethanol, more rapidly, than any of the constituent strains. This was 

accomplished by early ethanol production by K. oxytoca, while ethanol produced in the later 

stages was primarily by the more ethanol-tolerant strain (18). 

2.4.4 Improved thermotolerance. Ethanol fermentation at elevated temperatures 

(>55°C) would facilitate product recovery, but thermophilic bacteria are poor ethanol producers. 

In addition, thermophilic Clostridium and Thermoanaerobium species have been investigated for 

potential as ethanol producers, but were consistently limited by end-product inhibition and 

solvent-induced membrane damage (63). 

In addition, efforts are underway to eliminate acid production during fermentation 

through genetic engineering, enabling use of salt-intolerant thermophilic strains like 

Thermoanaerobacterium thermosaccharolyticum, a microbe tolerant to high levels of ethanol but 

intolerant of salt accumulation during pH-controlled fermentations. If such a thermotolerant 

organism could be improved further to produce high-activity cellulases, a highly productive, 

anaerobic, ethanol-producing strain could result. Cellulase production could, however, pose an 

insurmountable energy burden to a fermentative organism; the energetic considerations of this 

combination are being evaluated (5). 

2.4.5 Fermentation of synthesis gas. Rajagopalan and coworkers report the 

discovery of a clostridial bacterium, P7, that converts mixtures of CO, CO2, and N2 into ethanol, 

butanol and acetic acid, with high ethanol production and selectivity compared to previous 

isolates. The authors report process parameters and consider options for improving ethanol yield 

(64). 

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2.5 Coproduct Development 

Finally, the investigation of potential ethanol coproducts is underway. Biomass sugars 

can support the production of many other products along with ethanol, including organic acids 

and other organic alcohols, 1,2-propanediol, and aromatic chemical intermediates. If these 

coproducts were sufficiently valuable, they could help greatly offset costs of ethanol production. 

However, such coproducts must be chosen carefully to ensure that sufficient markets are 

available (65). 

Additional coproducts may be available from lignin: this material is present at 15–30 

percent by weight in all lignocellulosic biomass, and any bioethanol production process will have 

lignin as a residue. A team of researchers from the NREL, the University of Utah, and Sandia 

National Laboratories is working to develop a process for making oxygenate fuel additives from 

lignin; these processes are chemical in nature and are detailed in other materials referenced (66-

68). 

3. Research Priorities 

The consensus among researchers and supporters of bioethanol research, in addition to 

those engaged in commercial projects, is that the improvement of cellulase enzyme activity and 

cellulase production, both to increase the efficiency of release of fermentable sugars from 

biomass and to reduce cellulase cost, are two of the greatest advances needed in the effort to 

commercialize fuel ethanol production (19, 5). In addition is the development of enzymatic 

pretreatment processes to release lignin from carbohydrate components (42, 9) and further 

improvement of fermentative organisms (69, 64), with the particular goal of designing microbes 

capable of consolidated bioprocessing (54). 

4. Commercialization 

4.1 Cellulases 

Although many commercial preparations of cellulase exist, costs have remained high 

because present applications are in higher-value markets (food and clothing) than fuels. In 

addition, these applications typically require much less than 100 percent cellulose hydrolysis, in 

contrast to ethanol production; much improvement is therefore needed to advance the current 

cellulase enzyme industry to the point at which it can support the fuel ethanol industry. As a 

result, many fuel ethanol commercialization efforts are choosing to use acid hydrolysis 

techniques for cellulose hydrolysis until cellulase preparations become less expensive or until 

recombinant microbes capable of combined cellulolysis and fermentation are perfected (3). 

Toward this goal, the DOE biofuels program is working with the two largest global enzyme 

producers, Genencor International and Novozymes Biotech Incorporated, to achieve a 10-fold 

reduction in cost of cellulases (3). 

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4.2 Ethanol Plants 

The first dedicated large-scale plants for the conversion of waste biomass to ethanol are 

now in planning and/or construction phases by BCI and the Masada Resource Group 

(www.masada.com), while Iogen Corporation (www.iogen.com) is currently operating a 50 ton 

per week pilot plant. BCI and the DOE Office of Fuels Development have formed a cost-shared 

partnership to develop a biomass-to-ethanol plant intended to produce 20 million liters of ethanol 

per year initially from sugar cane waste (bagasse) and other biomass, utilizing an existing 

ethanol plant in Jennings, LA. Dilute acid hydrolysis will be used to recover sugar from bagasse 

initially, allowing for addition of enzyme hydrolysis when cellulases become less expensive. A 

proprietary genetically-engineered microbe will ferment the sugars to ethanol. BCI is also 

planning to operate a plant in Gridley, CA, in which cellulases will be used in conversion of 

commercial rice straw to ethanol, again with partial DOE support. The Masada plant is expected 

to produce 9.5 million gallons from municipal solid waste using Masada’s patented CES 

OxyNolTM concentrated acid hydrolysis technology in New York (3). 

Petro-Canada, the second largest petroleum refining company in Canada, began to cofund research and development on biomass-to-ethanol technology with Iogen in 1997. PetroCanada, Iogen, and the Canadian government then began plans to fund construction of a 

demonstration plant based on Iogen’s cellulase enzyme technology in an SSF process (3). The 

plant of Iogen, a leading producer of cellulases, has completed a 40 ton per day biomass-toethanol demonstration facility that is now in its start-up phase (5). 

In the pulp and paper industry, Tembec and Georgia Pacific are using dilute acid 

hydrolysis to dissolve hemicellulose and lignin from wood, producing a cellulose pulp that can 

be fermented to ethanol. The lignin is then used to generate energy, through combustion, or 

converted to other products such as concrete additives and soil stabilizers (3). 

Pursuing the gasification and syngas-to-ethanol fermentation, BioEngineering Resources, 

Inc. (BRI) has developed syngas technology to the extent that plans are underway to pilot the 

technology as a first step toward commercialization. BRI has developed bioreactor systems for 

fermentation that result in retention times of minutes or less, yielding low equipment costs (3). 

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hydrocarbon gasoline, U.S. Patent 5,959,167. 

(68) Shabtai, J. S., W. Zmierczak, E. Chornet, and D. K. Johnson (1999). Lignin conversion 

to high-octane fuel additives, Fourth Biomass Conference of the Americas, Oakland, CA. 

(69) Wyman, C. E. (2001). Twenty years of trials, tribulations, and research progress in 

bioethanol technology: Selected key events along the way, Appl Biochem Biotechnol 91-

93:5-21. 

B. BIODIESEL 

1. Introduction 

Among biofuels (biomass, biohydrogen, bioethanol, and biodiesel), biodiesel is currently 

the most fully developed and widely used, with many countries producing in excess of 100,000 

tons per year, including Belgium, France, Germany, Italy, and the United States. Japan is also a 

leading entity in biodiesel research and in production of biodiesel from waste oils (1, 2). The 

current status of biodiesel is understandable given its many convenient features, such as the 

following: it can be used in common compression-injection engines (CIE), it poses no particular 

difficulties compared to fossil fuels in handling, transport, or storage, and it can be mixed in any 

proportion with conventional diesel fuel. Because it is oxygenated and typically lacks sulfur 

contamination, it also burns quite cleanly, greatly reducing the output of ash, particulate matter, 

CO, and sulfur oxides in comparison to conventional diesel fuels (1). 

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The primary obstacle to its more widespread adoption is simply its cost—to date, the raw 

material plant oils (typically rapeseed, soybean, canola, etc.) have been much more expensive 

than petroleum (3). Diminishing accessibility of petroleum, however, as well as new technology 

allowing recycling of waste vegetable oils into biodiesel, are eroding this obstacle, with the result 

that biodiesel is emerging as one of the most promising examples of the use of biotechnology for 

economic sustainability and pollution prevention (4). 

1.1 History and Development 

Conventional diesel fuel is the portion of crude oil that is distilled between approximately 

200°C (392°F) and 370°C (698°F), higher than the boiling range of gasoline and consistent with 

its heavier, oilier composition. Diesel fuel is ignited in a CIE cylinder by the heat of air under 

high compression, in contrast to motor gasoline, which is ignited by an electrical spark. Because 

of the mode of ignition, a high cetane number (cetane is the hydrocarbon C16H34, or 

1-hexadecane, that ignites very easily under compression and is therefore used as a standard in 

determining diesel fuel ignition performance) is required in a good diesel fuel. Two grades of 

diesel fuel have been established by the ASTM: Diesel 1 and Diesel 2. Diesel 1 is a kerosenetype fuel, which is lighter, more volatile, and cleaner than Diesel 2, and is used in engine 

applications with more frequent changes in speed and load. Diesel 2 is used in industrial and 

heavy mobile service (5, 6). 

The term Biodiesel, in turn, is collective describing fuels comprised of esterified plant 

oils or animal fats. These biological lipids originate as mixtures of triglycerides and free fatty 

acids that are derivatized through transesterification (also known as alcoholysis) with acid, base, 

or enzymatic catalysis to form, most commonly, methyl or ethyl esters (Figure 14) (7, 1, 3). 

Figure 14. Transesterification of a triglyceride with an alcohol, showing the 

production of fatty acid esters and glycerol. Adapted from (1). 

H2C

HC

H2C

O

O

R1

O

O

R2

O

O

R3

+ 3 R'OH

O

O

R1

O

O

R2

O

O

R3

R'

R'

R'

H2C

HC

H2C

OH

OH

OH

+

catalyst

triglyceride alcohol fatty acid esters glycerol

Before the advent of biodiesel, biological oils were investigated extensively in their 

native forms for use in diesel engines. Indeed, Rudolf Diesel himself experimented with 

vegetable oils in his engine (Figure 15) over 100 years ago (7). Unfortunately, however, plant 

oils and especially animal fats typically have sufficiently high viscosities, and sufficiently low 

cetane numbers, flash points, and combustibility, that they show numerous undesirable properties 

in CIEs. Principal among these are carbon deposition on engine parts, gelling and 

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polymerization during storage and in cool temperatures, and contamination of engine lubricating 

oils leading to deterioration of lubricant and ultimately engine performance (8, 7). 

Figure 15. The diesel engine, invented by Rudolf Diesel in 1894 and characterized 

by its ignition of hydrocarbon fuels by compression with heated air, rather than 

by ignition with an electrical spark (41). 

As a result, the availability of inexpensive petroleum led quickly to the nearly exclusive 

use of diesel fuel, which lacked the undesirable performance properties but brought with it its 

own undesirable, if not initially appreciated, environmental consequences. Nevertheless, 

experimentation with soybean, canola, and other vegetable oils, as well as beef tallow and other 

animal fats, continued for many decades as researchers attempted to discover mixtures with 

petroleum fuels that minimized the problems of incomplete combustion posed by the biolipids. 

While these measures successfully diminished the rate of engine and lubricating oil deterioration, 

most problems persisted to some extent, and at present the use of and experimentation with 

unmodified biolipids has effectively ceased (7). 

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2. State of the Science 

2.1 Feedstocks and Coproducts 

An important challenge faced by biodiesel in competing with petroleum diesel is the 

comparatively high price of its plant oil feedstocks. In June, 2004, diesel prices ranged from 

$1.50 per gallon in Oklahoma to $2.20 per gallon in Seattle, Washington (9), while the cost of 

soybean oil, the primary biodiesel feedstock in the United States, was reported at $2.19 per 

gallon (27.34 cents per lpound) before conversion to biodiesel even occurred (10). 

The margin is narrowing, however (in May 2005, diesel prices ranged from $1.98 per 

gallon in Knoxville, Tennessee to $2.53 per gallon in Seattle, Washington (11), while the cost of 

soybean oil dropped to 22.31 cents per pound (12)), and on April 25, 2005, Blue Sun 100 percent 

pure biodiesel (B100) reached a low of $2.39 per gallon in Denver, Colorado, compared to local 

petroleum diesel prices of $2.29 per gallon (www.boulderbiodiesel.org). 

 In addition, tax incentives are becoming popular internationally as governments attempt 

to reduce their dependence on foreign oil. Even in the United States, which has traditionally 

subsidized petroleum use substantially in comparison to Europe (diesel fuel cost is greater by a 

factor of 1.5–2.5 across Europe [13]), bills have been recently introduced to extend the federal 

biodiesel excise tax credit that provides one cent credit per percent of biodiesel per gallon. 

Under this incentive, taken by the producer and passed on to the consumer, B100 therefore 

realizes a $1.00 per gallon-cost reduction, and the price of 20 percent biodiesel combined with 

80 percent petroleum diesel (B20) becomes comparable to petroleum diesel (14). In other 

countries, biodiesel production and use is facilitated by higher petroleum prices (diesel fuel cost 

is greater by a factor of 1.5-2.5 across Europe [13]), by use of less-expensive feedstocks, and by 

a variety of creative pro-biodiesel incentives. In Europe, biodiesel production has been 

encouraged by EU farm production programs: much of the biodiesel expansion in the 1990s 

occurred as a result of EU policies that allowed farmers to grow crops for industrial uses, 

including oilseeds, on set-aside land. Tax benefits from Germany, Austria, and France have also 

encouraged biodiesel production and use, allowing biodiesel to find far greater success in these 

countries than is possible in the United States with its lack of government incentives (15). 

Rapeseed oil, the predominant feedstock in Europe, is also priced lower than soybean oil (16). 

Nevertheless, the cost of feedstock oils is expected to continue to be a concern in 

biodiesel success, promoting investigation of alternative oils. 

Waste cooking oil, tallow, and lard are quite inexpensive feedstocks, for example, that 

are currently used for biodiesel production in Japan and are promising as well for other areas of 

Asia that have limited agricultural land and where vegetable oils are fairly expensive (7, 17). 

Another approach is the development of processes that yield valuable coproducts: 

glycerol is a clear candidate coproduct in biodiesel production, especially if the 

transesterification of plant oils is accomplished enzymatically. Enzyme use eliminates the need 

for an alcohol evaporation process, necessary for glycerol recovery from alkali 

transesterifications, and also minimizes saponification (soap formation) of glycerol and its 

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attendant purification difficulties. Because of the favorable commodity market for glycerol, the 

cost of biodiesel could be lowered significantly if biodiesel plants incorporated glycerol recovery 

facilities (7) or provisions for production of other high-value coproducts, such as caproic or 

propionic acids (15). 

2.2 Abiotic Processing 

2.2.1 Pyrolysis. Pyrolysis, the use of heat or heat plus a catalyst in the absence of 

oxygen to convert one substance into another, has been investigated worldwide for much of the 

last century for biofuel production. In this process, triglyceride fatty acids are “cracked” from 

the glycerol backbone and further decomposed, yielding a mixture comprised primarily of 

alkanes and alkenes with smaller proportions of carboxylic acids and aromatics. While this 

approach effectively diminishes the viscosity of the oils and fats, a number of new problems 

arise: the process tends to generate a greater proportion of lower molecular-weight products 

(gasoline), the equipment required is expensive for modest throughputs, and the removal of 

oxygen during the thermal processing also removes the environmental benefits of using an 

oxygenated fuel (1, 7). 

2.2.2 Microemulsions. Another approach to minimize vegetable oil viscosity is the 

formation of water-oil microemulsions, in which an oil is stably dispersed in a solvent such as 

methanol, ethanol, or butanol in 1-150 nanometer micelles by association with ionic or nonionic 

amphiphiles. While viscosity has been successfully diminished, and spray performance has been 

successfully enhanced by this technique, the problems of incomplete combustion leading to 

carbon deposition, clogging, and sticking of moving parts inherent to use of unmodified 

vegetable oils persisted (1, 3, 7). 

2.2.3 Transesterification. The transesterification of triglyceride fatty acids with 

alcohols, yielding esters (Figure 14), has emerged as the technology that yields products most 

similar to conventional diesel fuel in chemical and combustion characteristics (Table 3). This is 

a stepwise process, esterifying one fatty acid at a time, that ultimately yields fatty acid esters as 

well as glycerol. Because each step is reversible, the product yield is enhanced greatly by 

providing an excess of alcohols; in fact, the molar ratio of alcohols to triglycerides is one of the 

most important parameters in the process. While greater proportions of alcohol favor more rapid 

and extensive reaction, they also create a greater volume of alkali waste to be treated; optimal 

ratios have therefore been reported ranging from 6:1 to 30:1, varying with the nature of the fat or 

oil and alcohols in use. The optimization of the alcohol : triglyceride molar ratio is also a key 

challenge for enzymatic catalysis (1, 7, 18). 

Transesterification can be accomplished at a variety of temperatures, with temperature 

optima depending on the oils involved; process temperatures of 25–100 ̊C are common (7). 

Catalysis of the process is essential (except in supercritical solvents) and may be accomplished 

by acids, bases, or enzymes: alkali catalysis employing NaOH, KOH, or a corresponding 

alkoxide is typically the most rapid and efficient in fats and oils with extremely low-water 

contents and low concentrations of free fatty acids (1, 7). While this process is the one currently 

predominant in industry, it does have important limitations in the energy required to recover the 

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glycerol by-product and in treating the alkali waste, as well as in the interference of water and 

free fatty acids with reaction progress (18). 

Table 3. Physical and chemical properties of biodiesel in comparison to 

petroleum-derived diesel fuel. Adapted from (1). 

Vegetable oil 

methyl ester 

Kinematic 

viscosity 

(mm2

/s) 

Cetane 

number 

Lower heating 

value (MJ/l) 

Cloud 

point 

( ̊C) 

Flash 

point 

( ̊C) 

Density 

(g/l) 

Sulfur 

(wt 

percent) 

Peanut 4.9 (37.8°C) 54 33.6 5 176 0.883 -- 

Soybean 4.5 (37.8°C) 45 33.5 1 178 0.885 -- 

Soybean 4.0 (40°C) 45.7-56 32.7 -- -- 0.880 

(15°C) 

-- 

Babassu 3.6 (37.8°C) 63 31.8 4 127 0.879 -- 

Palm 5.7 (37.8°C) 62 33.5 13 164 0.880 -- 

Palm 4.3-4.5 (4O°C) 64.3-70 32.4 -- -- 0.872-0.877 

(15°C) 

-- 

Sunflower 4.6 (37.8°C) 49 33.5 1 183 0.860 -- 

Tallow -- -- -- 12 96 -- -- 

Rapeseed 4.2 (40°C) 51-59.7 32.8 -- -- 0.882 

(15°C) 

-- 

Used rapeseed 9.48 (30°C) 53 36.7 -- 192 0.895 0.002 

Used corn oil 6.23 (30°C) 63.9 42.3 -- 166 0.884 0.0013 

Diesel fuel 12-3.5 (40°C) 51 35.5 -- -- 0.830-0.840 

(15°C) 

-- 

JIS-2D (Gas oil) 2.8 (30°C) 58 42.7 -- 59 0.833 0.05 

If either water or free fatty acids are present in unacceptably high amounts, acid catalysis 

is preferable, typically employing hydrochloric, sulfuric, or phosphoric acids. While this process 

proceeds several thousand-fold more slowly than the alkali-catalyzed process, it does allow use 

of lower-quality oils (1). 

2.2.4 Supercritical methanol catalysis. Recently, researchers have attempted 

transesterification in supercritical methanol, where supercritical fluids are solvent phases 

obtained under high temperature and pressure that have both vapor and liquid characteristics and 

in which many reactions have been found to proceed more readily than in subcritical solvents 

(19). Since supercritical methanol has a hydrophobic nature with a lower dielectric constant than 

subcritical methanol, nonpolar triglycerides were well-solvated with supercritical methanol to 

form a single-phase oil-methanol mixture. As a result, the oil to methyl ester conversion rate 

increased dramatically in the supercritical state. Added advantages were that free fatty acids in 

the oil could also be converted efficiently to the methyl esters, improving product yield, and that 

purification of products following transesterification was much simpler and more 

environmentally friendly due to the absence of alkali (or acid) catalysts. Unfortunately, 

however, the process required a temperature of 350°C and pressure of 45MPa, as well as large 

quantities of methanol, with the result that much optimization will be required before this 

approach is commercially competitive (20–22). 

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2.3 Enzymatic (Lipase-based) Transesterification 

In efforts to improve upon the abiotic transesterification methods, considerable effort has 

been devoted to the investigation of lipases, carboxylesterases that catalyze the hydrolysis and 

synthesis of long-chain acylglycerols, for the synthesis of biodiesel (23). These remarkable 

enzymes currently constitute the single most widely-used biocatalyst class in biotechnology: 

they often possess high chemoselectivity, regioselectivity, and stereoselectivity; they are 

naturally extracellular enzymes and many are secreted in great quantity by fungi and bacteria, 

allowing relatively simple purification from culture media; they require no cofactors; they 

typically catalyze no undesirable side reactions; and the crystal structures of a number of 

representatives have been solved, facilitating rational design approaches in optimizing enzyme 

activity. The commercial lipase market is currently approximately 1 billion dollars per year, 

involving applications in detergents and in the production of food ingredients and enantiopure 

pharmaceuticals (24); this status is fortunate for biodiesel applications because it provides preexisting incentives for improvements in lipase production and activity. A chart comparing the 

relative merits of the alkali-catalyzed and enzyme-catalyzed transesterification processes for 

biodiesel production is shown in Table 4. 

Table 4. Comparison of alkali- and enzymatically-catalyzed transesterification for 

biodiesel production. Adapted from (1). 

Parameter Alkali-catalyzed process Lipase-catalyzed process 

Reaction temperature 60-70°C 30-40°C 

Free fatty acids in raw materials Formation of undesired 

saponified products 

Formation of desired methyl esters 

Water in raw materials Interference with the reaction No influence 

Yield of methyl esters Normal Higher 

Recovery of glycerol Difficult Easy 

Purification of methyl esters Repeated washing required No washing required 

Production cost of catalyst Low Relatively high 

Commercial lipases on which much biodiesel research has been based include enzymes 

from Mucor miehei (Lipozyme IM-20) and Candida antarctica (Novozym 435), as well as from 

Pseudomonas cepacia, Candida rugosa, and Rhizopus delemar. Both extracellular and 

intracellular lipases effectively catalyze a variety of triglyceride transesterification reactions, and 

they have the potential to overcome all of the problems of chemical catalysis (Table 4). On the 

other hand, the cost of lipase catalysts is significantly greater than that of alkali catalysts (1). 

Studies from the mid-1990s to present showed that, in general, lipase-catalyzed reactions 

with longer-chain fatty alcohols proceeded far more readily than those with methanol or ethanol. 

The reactions tolerated the presence of up to 20 percent water but were favored by the presence 

of organic solvents, which presented problems in that organic solvents were not suitable for fuel 

production due to the risk of explosion as well as the difficulty of removing the solvent. In 

addition, scientists found that even immobilized lipase preparations could not be re-used, a 

problem that would have to be solved to promote commercialization (18). The greatest obstacle 

to this technology is the cost of the lipase enzymes. Two primary approaches are in progress to 

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address this difficulty: first, genetic engineering of microorganisms to produce lipases in greater 

quantities and with greater activities, and second, the investigation of whole-cell systems to 

allow in situ regeneration of the catalytic units. 

2.3.1 Lipase engineering: production. The cost of lipase production is the 

primary impediment to commercialization of lipase-catalyzed systems. This is a general 

problem of enzyme-catalyzed processes, which industrial biotechnology has addressed primarily 

by the development of high-enzyme-expression systems and of whole-cell biocatalysts. 

Overexpression of lipases requires accurate protein folding and translocation across the 

cytoplasmic and, in gram-negative bacteria, outer membranes, processes estimated to involve up 

to 30 cytoplasmic proteins in some organisms. Through careful manipulation of lipase signal 

sequences and secretory pathways, however, several successes have been achieved: lipases from 

various Bacillus species have been overexpressed in E. coli systems, for example (24); Rhizopus 

oryzae lipases have been produced in extracellular, functional form in Saccharomyces cerevisiae

(25, 26); and the Candida antarctica lipase B has been similarly secreted in functional form from 

the yeast Pichia pastoris (27). Because lipase folding and secretion are highly specific processes 

that normally do not function properly in heterologous hosts, these successes represent 

breakthroughs that provide wonderful opportunities for further optimization and increases of 

lipase overexpression in heterologous hosts. 

Work directed toward modifying and accelerating the secretion pathways in native lipase 

hosts has also led to great increases in extracellular lipase yield within Pseudomonas fluorescens

and Serratia marcescens, showing that pathway manipulation within native hosts may be another 

promising avenue for further improvement (24). 

2.3.2 Lipase engineering: activity. Another important challenge faced by lipase 

use in biodiesel production has been the diminished efficiency of lipases in transesterifications 

with methanol and ethanol in comparison to longer-chain alcohols. Lipases typically accomplish 

most rapid catalysis when the substrates are freely soluble in one another; methanol and ethanol, 

however, were found to be only soluble in vegetable (soybean and rapeseed) oils at molar ratios 

of 1:2 and 2:3 (alcohol : triglyceride fatty acid), respectively. Moreover, insoluble methanol 

caused rapid inactivation of the lipases. These realizations led to the development of a system in 

which alcohols were added in discrete doses, below their solubility limits, to batch reaction 

mixtures, improving the reaction yield to near-completion (>97 percent) as well as extending the 

lifetime of the lipases to greater than 100 days (1, 18). 

Improving the tolerance of lipases to methanol or ethanol is also possible: certain species 

of Fusarium, Pseudomonas, and Bacillus produce solvent-tolerant lipases (1), and directed 

evolution has the potential to effect further desired changes in solvent tolerance, catalytic rate, 

and substrate specificity in these or other enzymes (24), reviewed in (28). 

Immobilization of lipases within gels may extend lipase activity, as well; work of 

Noureddini and colleagues in screening a number of extracellular lipases for methanoloysis and 

ethanolysis of soybean oil revealed that several of the enzymes were more active, and retained 

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their activities for longer periods of time, when subjected to such immobilization (29), building 

on previous work investigating lipase immobilization for transesterification (30, 31). 

2.3.3 Whole-cell systems. To diminish the time and energy requirements of enzyme 

purification, even of a relatively simple purification such as that required with lipases, as well as 

problems presented by instability of extracellular enzymes, significant effort has addressed the 

direct use of whole cells as biocatalysts (32–34). For example, lipases can be overexpressed 

within biotechnologically tractable hosts in which they are not secreted, followed by 

permeabilization of the host to allow catalysis to occur within the (compromised) cytoplasm. 

Cytoplasmic overexpression of the Rhizopus oryzae lipase in Saccharomyces cerevisiae, 

followed by freeze-thawing and air drying of the yeast cells, resulted in a whole-cell biocatalyst 

that effectively catalyzed triglyceride methanolysis (35); optimization of the membrane fatty acid 

composition further improved lipase activity and stability (36). 

In addition, cells producing their native lipases have been recruited. In work directed 

toward whole-cell lipase optimization by Ban and colleagues, cells were immobilized using 

porous biomass support particles (BSPs) made of polyurethane foam by introducing the BSPs 

during batch cultivation and allowing the cells to colonize them spontaneously. Cells were then 

cross-linked onto the support with glutaraldehyde, a procedure that greatly extended the 

enzymatic activity longevity. Once immobilized in this form, the particles could be treated much 

as conventional solid-phase catalysts: aseptic handling of the particles was unnecessary, the 

particles could withstand mechanical shear, and they could be reused for up to 6 batches. Mass 

transfer rates were also sufficiently rapid within the BSPs that conversion rates approached those 

obtained with extracellular lipases: methanolysis of soybean oil by immobilized Rhizopus 

oryzae cells, in the presence of 10–20 percent water, reached 80–90 percent without any organic 

solvent pretreatment (37, 1, 38); additional improvements in catalytic rate and durability were 

reported recently with this system in an air-lift reactor configuration (39). Further development 

of whole-cell biocatalysts is thus positioned to make important contributions to biodiesel 

production. 

3. Research Priorities 

 The cost, quality, and performance of biodiesel, as well as its overall environmental 

profile, could be improved by further efforts in several areas. First, feedstocks other than virgin 

plant oils, most of which are cultivated by non-sustainable, pesticide and energy intensive 

agricultural practices, would ideally be explored and developed; alternatively, sustainable 

cultivation of oil crops should be developed. Waste oil processing technology also deserves 

developmental effort to allow recovery of its intrinsic energy, and microbial and algal lipid 

production should be investigated to determine whether they might provide feedstocks at lower 

cost. 

Second, the further development of lipase technology will facilitate efficient enzymatic 

transesterifications of feedstock oils and fats and production of benign wastes with easilyrecoverable coproducts, principally glycerol. Specifically, genetic engineering of lipases for 

greater activity and durability, as well as metabolic engineering of lipase-production pathways to 

understand lipase synthesis and regulation and to facilitate extracellular production, are wellbioengineering report_5_x1a.pdf 139 5/25/07 6:45:51 PM
126 

positioned to offer valuable advances in enzymatic transesterification of oils and should be 

pursued. Research in these areas could have potentially great impacts within relatively short 

timescales and should be encouraged to the greatest extent possible. 

4. Commercialization 

International commercialization of biodiesel is well underway in both the United States 

and in many European and Asian countries. Recent news announcements by the U.S. National 

Biodiesel Board, revealing the growing trend in adoption of biodiesel fuels even in this country, 

include the following: the announcement on May 28, 2004, by World Energy Alternatives LLC 

of the re-opening and upgrading of the largest multi-feedstock biodiesel production facility in the 

United States, with a capacity of 18 million gallons, in Florida; the opening of Canada’s first 

retail biodiesel pump in Toronto by Topia Energy, Inc. on March 2, 2004; the opening of 

10 biodiesel pumps in Denver, Colorado in May 2004 by Blue Sun Biodiesel as part of a citywide pilot program; and the adoption of biodiesel for its maintenance vehicles by the Big South 

Fork National River and Recreation Area in Tennessee, joining dozens of other U.S. national 

parks (40). 

 Biodiesel is most readily available in B20, B50, and B100 blends, representing 20, 50, 

and 100 percent biodiesel mixed with a balance of petroleum diesel, respectively. These fuels 

are widely available in the United States, particularly in coastal areas and in midwest agricultural 

regions (a current map of U.S. retail biodiesel locations is provided at http://biodiesel.org/ 

buyingbiodiesel/retailfuelingsites/default.shtm/) and can be used in conventional diesel engines 

such as those found in the Volkswagen Golf, Jetta, Jetta Wagon, New Beetle, and 2004 Passat; 

2004 Mercedes E-320 Sedan; 2004 Chrysler Jeep Liberty and 2004 Volkswagen Touareg SUV; 

Chevy Silverado; GMC Sierra; Dodge Ram; and Ford E-series and F-series trucks. 

 Biodiesel production capacity exists and is expanding, and technology for storage and 

usage is in place. The primary deterrent to even more widespread commercialization is simply 

its price, which is increasingly comparable to petroleum diesel as oil prices rise and tax 

incentives are enacted. 

In contrast to other biofuels, biodiesel is already an established product with an 

established set of technologies (both for synthesis and for consumer use) supporting it. Because 

of this, further improvements in the cost-effectiveness of its production, in combination with 

rising emissions standards, will have significant impacts on its attractiveness to consumers. 

5. References 

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transesterification of oils, J Biosci Bioeng 92:405-416. 

(2) Pearl, G. G. (2001). Biodiesel Production in the US, Render Magazine, 

http://www.rendermagazine.com/August2001/TechTopics.html. 

(3) Dmirbas, A. (2003). Biodiesel fuels from vegetable oils via catalytic and non-catalytic 

supercritical alcohol transesterifications and other methods: A survey, Energy Conv 

Mgmt 44:2093-2109. 

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(4) National Biodiesel Board (2004). Biodiesel FAQs, National Biodiesel Board, 

http://www.biodiesel.org/resources/faqs/. 

(5) Chevron Corporation (2004). Dictionary of Lubricant Terms, http://www.chevron.com/

oronite/reference_materials/dictionary_of_lubricant_terms/li_dictionary_d.asp. 

(6) Wikipedia (2004). Cetane, Wikipedia, the Free Encylopedia, 

http://www.riograndesoftware.com/encyclopedia/c/ce/cetane.html. 

(7) Ma, F., and M. A. Hanna (1999). Biodiesel production: A review, Bioresource Technol 

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(8) Allen, C. A. W., K. C. Watts, R. G. Ackman, and M. J. Pegg (1999). Predicting the 

viscosity of biodiesel fuels from their fatty acid ester composition, Fuel 78:1319-1326. 

(9) TA Travel Centers (2004). Diesel Fuel Prices, http://www.tatravelcenters.com/ta/

display_diesel_fuel_prices.phtml. 

(10) Chicago Board of Trade (2004). Real-time quotes, charts, and news, 

http://www.unitedsoybean.org/. 

(11) TA Travel Centers (2005). Diesel Fuel Prices, http://www.tatravelcenters.com/ta/

display_diesel_fuel_prices.phtml. 

(12) Chicago Board of Trade (2005). Historical Data: Soybean Oil, www.cbot.com. 

(13) See-Search Engines (2004). Fuel/diesel/petrol prices across Europe, http://www.seesearch.com/business/fuelandpetrolpriceseurope.htm. 

(14) National Biodiesel Board (2005). Tax Incentive Fact Sheet, 

http://www.biodiesel.org/members/membersonly/files/pdf/fedreg/20041022_Tax_Incenti

ve_Fact_Sheet.pdf. 

(15) Raneses, A. R., L. K. Glaser, J. M. Price, and J. A. Duffield (1999). Potential biodiesel 

markets and their economic effects on the agricultural sector of the United States, Indust 

Crops Prod 9:151-162. 

(16) de Guzman, D. (2001). Biodiesel market strengthens as alternative to diesel fuel, 

http://www.worldenergy.net/pdfs/newsstories/chemical_marker.pdf. 

(17) Zhang, M. A., M. A. Dube, D. D. McLean, and M. Kates (2003). Biodiesel production 

from waste cooking oil: 1. Process design and technological assessment, Bioresource 

Technol 89:1-16. 

(18) Shimada, Y., Y. Watanabe, A. Sugihara, and Y. Tominaga (2002). Enzymatic 

alcoholysis for biodiesel fuel production and application of the reaction to oil processing, 

J Molec Catalysis B: Enzymatic 17:133-142. 

(19) Rayner, C. (2004). What are supercritical fluids?, University of Leeds, 

http://www.chem.leeds.ac.uk/People/CMR/whatarescf.html. 

(20) Kusdiana, D., and S. Saka (2001). Kinetics of transesterification in rapeseed oil to 

biodiesel fuel as treated in supercritical methanol, Fuel 80:693-698. 

(21) Kusdiana, D., and S. Saka (2001). Methyl esterification of free fatty acids of rapeseed oil 

as treated in supercritical methanol, J Chem Eng Jpn 34:383-387. 

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(22) Saka, S., and D. Kusdiana (2001). Biodiesel fuel from rapeseed oil as prepared in 

supercritical methanol, Fuel 80:225-231. 

(23) King, M. W. (2003). Fatty Acid Oxidation, Indiana University School of Medicine, 

http://www.med.unibs.it/~marchesi/fatox.html. 

(24) Jaeger, K.-E., and T. Eggert (2002). Lipases for biotechnology, Curr Opin Biotechnol 

13:390-397. 

(25) Takahashi, S., M. Ueda, H. Atomi, H. D. Beer, U. T. Bornscheuer, R. D. Schmid, and A. 

Tanaka (1998). Extracellular production of active Rhizopus oryzae lipase by 

Saccharomyces cerevisiae, J Ferment Bioeng 86:164-168. 

(26) Ueda, M., S. Takahashi, M. Washida, S. Shiraga, and A. Tanaka (2002). Expression of 

Rhizopus oryzae lipase gene in Saccharomyces cerevisiae, J Molec Catal B: Enz 17:113-

124. 

(27) Rotticci-Mulder, J. C., M. Gustavsson, M. Holmquist, K. Hult, and M. Martinelle (2001). 

Expression in Pichia pastoris of Candida antarctica lipase B and lipase B fused to a 

cellulose-binding domain, Protein Expression and Purification 21:386-392. 

(28) Powell, K. A., S. W. Ramer, S. B. del Cardayre, W. P. C. Stemmer, M. B. Tobin, P. F. 

Longchamp, and G. W. Huisman (2001). Directed evolution and biocatalysis, Angew 

Chem Int Ed Engl 40:3948-3959. 

(29) Noureddini, H., X. Gao and R.S. Philkana (2005). Immobilized Pseudomonas cepacia

lipase for biodiesel fuel production from soybean oil, Bioresource Technol 96:769-777. 

(30) Samukawa, T., M. Kaieda, T. Matsumoto, K. Ban, A. Kondo, Y. Shimada, H. Noda, and 

H. Fukuda (2000). Pretreatment of immobilized Candida antarctica lipase for biodiesel 

fuel production from plant oil, J Biosci Bioeng 90:180-183. 

(31) Iso, M., B. Chen, M. Eguchi, T. Kudo, and S. Shrestha (2001). Production of biodiesel 

fuel from triglycerides and alcohol using immobilized lipase, J Molec Catal B: Enzym 

16:53-58. 

(32) Liu, Y., H. Hama, Y. Fujita, A. Kondo, Y. Inoue, A. Kimura, and H. Fukuda (1999). 

Production of S-lactoyl-glutathione by high activity whole cell biocatalysts prepared by 

permeabilization of recombinant Saccharomyces cerevisiae with alcohols, Biotechnol 

Bioeng 64:54-60. 

(33) Kondo, A., Y. Liu, M. Furuta, Y. Fujita, T. Matsumoto, and H. Fukuda (2000). 

Preparation of high activity whole cell biocatalyst by permeabilization of recombinant 

flocculent yeast with alcohol, Enzyme Microb Technol 27:806-811. 

(34) Liu, Y., Y. Fujita, A. Kondo, and H. Fukuda (2000). Preparation of high-activity whole 

cell biocatalysts by permeabilization of recombinant yeasts with alcohol, J Biosci Bioeng 

89:554-558. 

(35) Matsumoto, T., S. Takahashi, M. Kaieda, M. Ueda, A. Tanaka, H. Fukuda, and A. Kondo 

(2001). Yeast whole-cell biocatalyst constructed by intracellular overproduction of

Rhizopus oryzae lipase is applicable to biodiesel fuel production, Appl Microbiol 

Biotechnol 57:515-520. 

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(36) Hama, S., H. Yamaji, M. Kaieda, M. Oda, A. Kondo, and H. Fukuda (2004). Effect of 

fatty acid membrane composition on whole-cell biocatalysts for biodiesel-fuel 

production, Biochem Eng J 21:155-160. 

(37) Ban, K., M. Kaieda, T. Matsumoto, A. Kondo, and H. Fukuda (2001). Whole cell 

biocatalyst for biodiesel fuel production utilizing Rhizopus oryzae cells immobilized 

within biomass support particles, Biochem Eng J 8:39-43. 

(38) Ban, K., S. Hama, K. Nishizuka, M. Kaieda, T. Matsumoto, A. Kondo, H. Noda, and H. 

Fukuda (2002). Repeated use of whole-cell biocatalysts immobilized within biomass 

support particles for biodiesel fuel production, J Molec Catal B: Enz 17:157-165. 

(39) Oda, M., M. Kaieda, H. Hama, H. Yamaji, A. Kondo, E. Izumoto, and H. Fukuda (2005). 

Facilitatory effect of immobilized lipase-producing Rhizopus oryzae cells on acyl 

migration in biodiesel-fuel production, Biochem Eng J 23:45-51. 

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http://www.biodiesel.org/. 

(41) United States Department of Energy (2004). Diesel Fuel Ignition, Office of Energy 

Efficiency and Renewable Energy, http://www.ott.doe.gov/images/dieselpiston.jpg. 

C. BIOHYDROGEN 

1. Introduction 

Molecular hydrogen (H2) is a promising future energy source due to its clean combustion 

and to its potential for sustainable production (1, 2). While the challenges in converting 

commercial processes based on hydrocarbon fuels to ones powered by hydrogen fuel cells are 

great, including major modifications in fuel storage and transport infrastructure, the potential 

advantages of hydrogen are sufficiently great to have drawn extensive attention to the research 

and development of H2 production and utilization technologies (3, 4). 

Currently, H2 is produced primarily by electrolysis of water, requiring a source of 

electricity, and steam reforming of natural gas, requiring both a nonrenewable fossil fuel 

feedstock and additional energy to create the necessary heat and pressure (2, 5). To improve the 

environmental profile of H2 production, numerous other technologies are being developed as 

well. Among these, microbial mechanisms that obtain energy either through photosynthesis (via 

photosynthetic, nitrogenase-mediated, or photo-fermentative pathways) or through consumption 

of organic substrates, potentially including organic wastes (water-gas shift and dark fermentative 

pathways), are of particular interest because of their potentially low requirements for expensive 

and non-renewable energy sources (6). 

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2. State of the Science 

2.1 Microorganisms 

Several types of microorganisms, undergoing different, yet related metabolisms, are 

involved in H2 production. To aid in clarifying and distinguishing these mechanisms, let us first 

consider the microorganisms involved. 

2.1.1 Oxygenic phototrophs. Oxygenic phototrophs include both aerobic 

eukaryotes (plants and green algae) and aerobic prokaryotes (cyanobacteria) that are able to use 

photons to energize electrons from water, thus yielding O2, for the ultimate purpose of generating 

the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) for CO2 fixation 

into organic carbon (Figure 16). The electrons are energized by passage through two successive 

photosystems, photosystems II (PS II) and I (PS I), each of which harvests photons with 

networks of chlorophyll and other accessory pigments and uses the photon energy to impart 

greater reducing power to the electrons. After nicotinamide adenine dinucleotide phosphate 

(NADP) is reduced to NADPH, and CO2 is fixed into organic carbon, phototrophs typically 

respire this substrate aerobically to generate adenosine triphosphate (ATP). Alternatively, 

electrons may also be returned by photosystem I to the plastoquinone pool in a process called 

cyclic photophosphorylation (Figure 16). This process allows additional protons to be pumped 

through the thylakoid membrane, contributing to the chemiosmotic gradient that drives ATP 

production (7). 

2.1.1.1 Green algae. Green algae are unicellular eukaryotes in which the majority of 

H2-producing capability lies in the use of photosynthetically-activated electrons by Fehydrogenase enzymes, requiring anaerobiosis in light. This process is known as direct 

photolysis. Green algae typically also have significant H2 uptake activity, although the enzymes 

responsible have not yet been identified. In general terms, therefore, the primary enzymes of 

concern for H2 production in green algae are Fe-hydrogenases, known to produce H2, and uptake 

hydrogenases of unknown composition. 

Green algae, as well as cyanobacteria (below), must withstand periods of anaerobiosis 

during darkness and therefore are also capable of fermentation (Figure 17). While reducing 

power generated by fermentation of endogenous substrates can enter the plastoquinone pool, 

undergo activation by PS I, and lead to H2 production, H2 generation through fermentation is 

estimated to have only 1/100 of the potential of direct photolysis in green algae (9, 10). 

Nevertheless, the presence of this fermentative metabolism is physiologically significant for H2

production by direct photolysis, as it appears that the ability of these organisms to evolve H2 by 

means of hydrogenases originated with the necessity of disposing of excess reducing power 

during fermentation (11). 

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Figure 16. The Z-scheme for electron transport in the oxygenic photosynthesis of 

cyanobacteria and green algae. 

cytb6

cyt b6

hv

Photosystem II 

OEC

Fd

H2O

P680*

P680

Z

Ph

QA

PQ 

pool

QB

PC P700

P700*

A0

FeSx

A1

FeSA,B

H2ase H2

FNR

NADP

0.0

-1.5

1.0

-1.0

-0.5

0.5

1.5

PS II antenna

FeS

cyt f

hv

O2

cytb6

cyt b6

hv

Em (Volts)

Fd

2

P680*

P

Ph

QA

PQ 

pool

QB

PC P700 PS I antenna

P700*

A0

A1

H2ase H2

FNR

NADP

CO2

fixation

Photosystem I 

-

-

-

FeS

cyt f

hv

2

NADPH

H2ase

H2

(cyanobacteria)

(green algae)

cyclic 

photophos-phorylation

 

Dashed arrows represent light reactions that extract electrons from H2O and deliver them to 

ferredoxin (Fd), from which they can be used to fix CO2 via NADPH or to produce H2 via hydrogenases. 

Abbreviations represent common electron carriers, arranged to show their relative redox potentials, Em. 

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Figure 17. The mixed-acid fermentation of Escherichia coli, an example of a 

common H2-producing fermentative pathway, showing H2 production from 

formate (8).

2.1.1.2 Cyanobacteria. Cyanobacteria or blue-green algae, in contrast to green algae, 

are unicellular or multi-cellular (filamentous) prokaryotes that generate H2 by three distinct 

mechanisms. These pathways can, under certain conditions, operate simultaneously. Among 

N2-fixing cyanobacteria, the majority of H2-producing capability lies in the activity of 

nitrogenase enzymes that use photosynthetically-generated ATP to reduce or “fix” molecular 

nitrogen and simultaneously to generate H2 (12); this process is known as indirect photolysis 

(Figure 18). 

The cyanobacterium Synechocystis sp. strain PCC 6803 M55, lacking genetic evidence of 

both nitrogenase and uptake hydrogenases, has been found recently to exhibit direct photolysis 

(13), opening the possibility that direct photolysis may be found in other cyanobacteria as well. 

Cyanobacteria can also metabolize endogenous substrate anaerobically through a number of 

fermentation pathways (e.g., Figure 17), several of which produce H2 by means of bidirectional 

hydrogenases (14, 15). 

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Figure 18. Nitrogenase reaction equation, showing electron donors, the role of 

ATP, and H2 production from reduced protons (16). 

2.1.2 Anoxygenic phototrophs. Anoxygenic phototrophs, in contrast to oxygenic 

phototrophs, are exclusively prokaryotic and often obligately anaerobic. These microbes each 

possess only one photosystem: some bacterial reaction centers are analogous to PS I, and some 

are analogous to PS II, but none extract electrons from water, and thus O2 is not produced 

(Figure 19). Instead of water, the reaction centers must energize electrons from organic or 

inorganic substrates found in their environments. Although many photosynthetic bacteria 

depend on Rubisco and the Calvin cycle for the reduction of CO2, some are able to fix 

atmospheric CO2 by other biochemical pathways. Despite these differences, energy transduction 

is carried out by mechanisms quite similar to those found in oxygenic phototrophs: the lightharvesting centers contain bacteriochlorophylls, analogous to the chlorophylls but with strongest 

absorption in the infrared (700–1000 nm), as well as carotenoids, and electron transport proteins 

are also analogous. As in oxygenic photosynthesis, electron transfer is coupled to the generation 

of an electrochemical potential that drives phosphorylation by ATP synthase, and the energy 

required for the reduction of CO2 is provided by ATP and NADH, a molecule similar to NADPH 

(17). 

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Figure 19. Relationships among photosystems of oxygenic (plants, algae, and 

cyanobacteria) and anoxygenic (purple bacteria, green filamentous bacteria, 

green sulfur bacteria, and heliobacteria) (17). 

 Anoxygenic phototrophs fall into several categories, based on pigmentation and electron 

donor. Of greatest interest to biohydrogen production are the purple non-sulfur bacteria, e.g., 

members of the genera Rhodobacter and Rhodopseudomonas, which use organic compounds or 

H2 as electron donors (17). These microbes prefer to grow photoheterotrophically, using simple 

fatty acids such as succinate and malate to supply electrons for photosynthesis as well as carbon 

for biosynthesis. In these organisms, most of which are able to fix nitrogen (18), H2 production 

is associated primarily with nitrogenase activity (19). 

2.1.3 Fermenters. Fermentations are energy-yielding metabolic processes in which 

an organic substrate is decomposed into smaller molecules, some of which are oxidized and 

some of which are reduced relative to the original substrate. Fermentations occur in the absence 

of an electron transport chain or exogenous terminal electron acceptors such as O2 (8). 

Fermentations are therefore anaerobic processes, but they may be carried out by either anaerobic 

or aerobic microorganisms within sufficiently anoxic microenvironments (20). Numerous 

fermentation pathways are known, many of which, like the mixed acid fermentation pathway 

(Figure 17), produce H2, and all of which generate multiple products that typically must be 

separated and purified for commercial use (8, 14, 5). While members of both the oxygenic and 

anoxygenic phototrophs possess H2-producing fermentation pathways, only bacteria appear to 

produce H2 from fermentation in appreciable quantities. These bacteria include members of the 

Bacillus, Clostridia, and Enterobacter, as well as the extreme thermophiles Caldicellulosiruptor 

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and Thermotoga, all heterotrophs that thrive on carbohydrates and typically are not capable of 

anaerobic respirations (5). 

2.2 Enzymes 

Two categories of enzymes, the hydrogenases and the nitrogenases, are responsible for 

H2 production in the microorganisms discussed above. 

2.2.1 Hydrogenases. Hydrogenases catalyze the redox interconversion of protons 

with H2 gas: 

2H+

 + 2e- ⇔ H2

They occur in two primary forms, known as uptake and bidirectional hydrogenases. 

Uptake hydrogenases oxidize H2 to H+

 to provide reducing power to many anaerobic respiratory 

organisms (21), while bidirectional hydrogenases may catalyze either H2 oxidation or H+

reduction, although in vivo they typically function in only one of the two capacities (22, 23). 

Hydrogenases may also be classified according to differences in their organometallic catalytic 

sites: NiFe(Se)-hydrogenases are characterized by catalytic sites possessing coordinated nickel, 

iron, sulfur, and in some cases selenium (21), while Fe-hydrogenases contain only Fe-S centers 

in their active sites (23). Metal-free hydrogenases also exist (24). Many of the known 

bidirectional hydrogenases are Fe-hydrogenases (23), while the majority of uptake hydrogenases 

are NiFe-hydrogenases; Fe-hydrogenases functioning to produce H2 are thus of greatest interest 

in microbial H2 production. Nevertheless, uptake hydrogenases are of equal or perhaps greater 

concern in some systems, because they frequently co-exist with H2-producing hydrogenases and 

can recycle H2 within the microbe, greatly diminishing overall H2 yield (12). 

The physiological role of hydrogenase-based H2 production appears to be the discharge 

of excess reducing power, necessary when other suitable electron acceptors such as O2 are absent 

(25-27). It is not surprising, therefore, that Fe-hydrogenases are rapidly inhibited both 

transcriptionally and post-translationally by molecular oxygen (28, 23). This feature has 

significant implications for biological H2 production, requiring anoxic cellular environments to 

be maintained for both induction of hydrogenase synthesis as well as for continued hydrogenase 

activity. For fermentative H2 production, this is no obstacle, as the entire microbial metabolism 

takes place anaerobically. However, oxygenic phototrophs provide the majority of activated 

electrons to Fe-hydrogenases through the O2-generating photosynthetic process (29). Because 

Fe-hydrogenases are inhibited both transcriptionally and post-translationally by O2, 

photosynthetic electron production and hydrogenase-based H2 production cannot occur 

simultaneously in a wild-type organism. These processes therefore must be separated either 

temporally or spatially, and much research in biohydrogen production is directed toward the 

accomplishment of these goals. 

Fe-hydrogenases exist in monomeric, dimeric, and at least one trimeric form (30, 23). 

The two C. reinhardtii Fe-hydrogenase enzymes cloned and sequenced to date (31) encode 

enzymes that are among the smallest hydrogenases known. Consistent with other algal 

hydrogenases, they contain only the single catalytic Fe-S center, or H-cluster, and ferredoxin is 

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the only putative electron donor. In contrast, bacterial hydrogenases typically contain several 

additional iron-sulfur centers and accept electrons from a variety of donors. Clostridial 

hydrogenases, involved in hydrogen evolution during the fermentation of carbohydrates, can 

accept electrons from flavodoxins, for example (24).

2.2.2 Nitrogenases. Nitrogenases are tetrameric organometallic enzymes that 

catalyze reductive cleavage of the second-strongest chemical bond known:the triple bond of 

dinitrogen gas, N2. To supply the tremendous energy needed for this process, the enzyme uses 

ATP; additional activated electrons are also required, however, which may be delivered through 

NADH, pyruvate, photoactivation, or H2 itself (Figure 18). Nitrogenases share many 

characteristics with hydrogenases: they employ metallic catalytic centers to facilitate the redox 

reaction (Fe-Mo-Co and Fe-S clusters); they are rapidly inactivated by exposure to molecular 

oxygen; and their synthesis is tightly regulated, requiring N deficiency as well as anaerobiosis. 

Nitrogenases are found in many cyanobacteria, most of the purple non-sulfur bacteria, and 

numerous symbiotic and free-living eubacteria. They evolve H2 rapidly during active nitrogen 

fixation; however, this H2 is produced at the cost of 16 ATP per H2 (32). Nitrogenases appear to 

produce the majority of H2 in nitrogen-fixing cyanobacteria (indirect photolysis) and purple nonsulfur bacteria (photofermentation) (19). 

2.3 Direct Photolysis 

Direct photolysis is the process in which bidirectional hydrogenases use 

photosynthetically-activated electrons, via reduced Fd and/or NADPH, to reduce the hydrogen 

ion (H+

)to H2 (28) (Figure 16). This process is carried out by green algae such as 

Chlamydomonas reinhardtii, Scenedesmus, and Chlorella (22, 33, 28), as well as by 

cyanobacteria such as Synechocystis (13). Light-dependent H2 production by cyanobacteria 

utilizing nitrogenases and photosynthetically-generated ATP is known as indirect photolysis, and 

light-dependent H2 production by anoxygenic phototrophs utilizing organic electron donors, also 

making use of nitrogenases, is known instead as photofermentation. 

2.3.1 Photosynthetic efficiency. Energy efficiency, defined as the ratio of energy 

produced as H2 to the resources consumed by the microorganism (including its requirements for 

space as well as nutrients), is a central consideration guiding research directions and assessment 

of commercial applicability of biohydrogen systems. The rate of H2 production is an equally 

important concern, however, and systems of lower efficiency but higher H2 production rate may 

be competitive with those of higher efficiency in cases where substrate and space costs are low 

(34, 5). 

For photochemical processes, Einstein’s law of photochemistry states that a primary 

photochemical process is caused by the action of one absorbed photon acting on another 

molecule, emphasizing the important fact that a photon must first be absorbed before it can carry 

out photochemistry. The next important parameter is the photochemical quantum yield, Φ, 

defined as the ratio of the number of photochemical products to the number of absorbed protons. 

The quantum yield is a measure of the efficiency of the photochemical proces: the primary 

quantum yield ranges from one for a process in which every absorbed photon leads to products, 

to zero when no products are formed. In photosynthetic systems, the primary quantum yields are 

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often close to one under optimal conditions, indicating that almost all absorbed photons are 

effective in forming initial products. However, more than one photon is usually needed to 

produce a stable final product such as H2, so overall quantum yields are usually much less than 

one (7). 

As a consequence in part of the high primary quantum yield, direct photolysis has a very 

high potential energetic efficiency compared to other known systems (35–39). This efficiency is 

estimated to be as high as 10 percent for microalgal photosynthesis resulting in CO2 fixation (34) 

and as high as 24 percent for H2 production, under ideal conditions (35). For comparison, a 

typical commercial steam turbine generator is about 30 percent efficient, photovoltaic cells also 

approach 30 percent efficiency, automobiles are ~20 percent efficient, and a well-tuned bicycle 

rates about 75 percent (40). 

The important limitation to photosynthetic conversion efficiency lies in the difficulty of 

providing so-called ideal conditions to photosynthetic cultures, resulting in reported conversion 

efficiencies that are usually less than 1 percent (34). 

2.3.2 Light saturation effect. A salient difficulty in providing ideal conditions for 

phototroph culture involves provision of the light itself. The microbial aspect of this problem 

lies in the ability of both algae and cyanobacteria to absorb many more photons with the lightharvesting antennae, and therefore to generate many more excited electrons, than the 

photosynthetic electron transport chain can accommodate. Although these microbes have 

evolved sensitive regulatory systems to optimize the size of their photon-gathering antennae 

(chlorophylls a and b, xanthophylls, phycobilins, etc.), diminishing them considerably under 

high light and increasing them under low light, phototrophs nevertheless typically absorb many 

more photons than they are able to use productively. Wild-type algae exposed to full sunlight 

absorb up to nine times more photons than they can accommodate, wasting the remainder in the 

form of heat or fluorescence and shading cells below them quite effectively. This phenomenon 

is known as the light-saturation effect and represents one of the prominent challenges facing 

commercialization of direct photolysis (34). 

One promising approach to this problem is the creation of algal mutants with diminished 

light-harvesting antennae, with the logic that a photosynthetic apparatus with less lightharvesting capacity absorbs fewer photons at high light intensities and therefore wastes fewer 

photons. Japanese researchers working with microalgal mutants with reduced antenna sizes 

found increases in productivity of up to 50 percent under high light intensity, compared to the 

wild-type (41, 42); antenna mutants isolated in the United States also showed efficiency 

improvements, further supporting the potential of this approach (43, 44). Extensive cost analysis 

by modelers at the NREL confirmed the value of increased light transmission: increasing 

incident light transmission by a factor of 10, thought to be well within the capability of modern 

genetic techniques, reduced the cost of H2 production 57 percent (45). 

2.3.3 Land use. The land-use aspect of the light provision problem is the relatively 

low density of solar energy, estimated at a maximum of 5 kilowatt hours per square meter per 

day or 6.6 gigajoules per square meter per year in the most favorable locations (34, 46). 

Assuming a conversion of 10 percent of the solar energy into H2 and a price for H2 of $15 per 

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gigajoule (the benchmark price set by DOE for H2), this computes to ~0.66 gigajoules or ~$10 

H2 per square meter per year (34). For comparison, an average electrically-heated house in 

North America consumes approximately 55–70 gigajoules of energy per year (5, 47, 48). While 

this discrepancy seems large, it is worth noting that energy-efficient construction and practices, 

including higher-density housing, can reduce residential energy requirements substantially. Even 

without such measures, it is instructive to realize that, given a 10 percent solar conversion 

efficiency, the annual energy needs of the United States could be met by a square ~100 miles on 

a side located in a virtually unoccupied area of southern Nevada (49). 

To address the problem presented by the limits of solar irradiance, potentially requiring 

large land areas for photobioreactors, efforts have been made to optimize vertical 

photobioreactor arrays (34), to investigate thin-layer bioreactors (0.5-5 centimeters in depth) 

with corresponding low masses that could be installed on rooftops (45), and most futuristically, 

to design solar collectors to transmit solar energy to bioreactors through optical fibers (50). 

2.3.4 Oxygen sensitivity. The second primary difficulty in providing ideal 

conditions for direct photolysis lies in the extreme sensitivity of hydrogenases to the molecular 

oxygen produced by photosynthesis. As a result, cultures maintained under dark, anoxic 

conditions to induce hydrogenase synthesis are able to sustain H2 production for only a few 

minutes following exposure to light and consequent photosynthetic O2 generation (51). In 

response to this problem, two primary approaches are being taken: the first is the development 

of specialized bioreactors to separate O2 generation from H2 generation temporally, and the 

second is the effort to generate O2-tolerant hydrogenases through various mutagenic methods. 

The most successful approach to date, to minimize the problem of O2 inhibition of the 

algal Fe hydrogenase, has been the development of the two-stage photobioreactor. In this 

system, algal cultures experience two alternating growth conditions: the first supports 

photoautotrophic growth, in the presence of all essential nutrients as well as O2, while the second 

deprives the cultures of both O2 and sulfur. Reduced sulfur is essential to synthesis of cysteine 

and methionine and is therefore essential to the synthesis of proteins as well, including the 

rapidly-recycled D1 protein of PS II. In the absence of reduced sulfur, PS II function diminishes 

significantly, resulting in decline of O2 production as well, even under light exposure. Algal 

aerobic respiration is not strongly affected by the sulfur deprivation over relatively short time 

periods (<100 hours) and therefore proceeds, diminishing O2 concentrations sufficiently to 

induce the Fe-hydrogenase activity. Because PS I and the electron-transport proteins, 

cytochromes b6 and f, are not significantly affected, the cell can maintain an ATP-generating 

proton gradient across the thylakoid membrane by using PS I to activate electrons released to the 

plastoquinone pool (Figure 16) by the degradation of starch, proteins, and lipids via a NADH 

reductase complex. H2 production then follows, as a means of removing the spent electrons from 

the photosynthetic electron transport chain (11), for approximately 100h (52). 

Commercialization of this process is being actively investigated (44). 

Significant effort has also been directed toward the modification of Fe-hydrogenases to 

yield varieties with greater O2 tolerance. This work has been encouraged by the discoveries of 

greater O2 tolerance among some hydrogenases, particularly those of the bacterium 

Desulfovibrio vulgaris and the alga Pandorina morum (37), as well as the discovery of an Febioengineering report_5_x1a.pdf 152 5/25/07 6:45:58 PM
139 

hydrogenase sequence in the genome of the bacterium Shewanella oneidensis, a facultative 

rather than strict anaerobe (53). In addition, the solution of the crystal structures of both 

monomeric and dimeric Fe-hydrogenases (54, 55). Flynn, Ghirardi, and colleagues employed 

traditional chemical mutagenesis and screening with H2-sensitive tungsten oxide films to isolate 

mutants with up to 10 times greater O2-tolerance of H2 production (51, 29, 56–59) as well as 

increased rates of H2 production (60). This success has inspired further efforts to alter one of the 

Chlamydomonas Fe-hydrogenases to diminish substantially its sensitivity to O2. Toward this 

end, further random chemical mutagenesis, error-prone PCR-mediated mutagenesis, and sitedirected mutagenesis are currently underway (61). 

At least two other research groups are also investigating directed evolution as an 

approach for generating greater O2 tolerance in algal Fe-hydrogenases. Because gene shuffling 

requires a diverse pool of parental Fe-hydrogenase genes, it is fortunate that numerous potential 

parental genes exist, representing genera among the archaea, eubacteria, fungi, algae, protists, 

and higher eukaryotes as well as monomeric and dimeric forms. Even among the multimeric 

enzymes, genes for subunits that show significant homology to the monomeric forms are 

considered valid potential parents in generating the monomeric Fe-hydrogenase mutants in 

Chlamydomonas. 

While most known Fe-hydrogenases are highly O2-sensitive, experiencing irreversible 

inactivation, that of Desulfovibrio vulgaris (Hildenborough) appears to be only reversibly 

inactivated by O2, with the result that this sequence is highly attractive as a parent (62, 63), as is 

that of Pandorina morum (37). Fe-hydrogenases in general show very highly conserved active 

site structures and sequences, such that the three-dimensional active site structures of the 

enzymes from the distantly related Clostridium and Desulfovibrio bacteria can be superimposed 

with a calculated deviation of only approximately 1 angstrom (23). This feature indicates that a 

relatively high diversity of parental origins for the Fe-hydrogenases may result in functional 

progeny. 

The rate at which photosynthetically-generated O2 must be removed from an H2-

producing algal bioreactor depends directly on the O2-tolerance of the algal H2 production 

pathway. A thorough cost-benefit analysis of algal H2-producing bioreactor operation revealed 

that the viability of a commercial system depends heavily on obtaining algal mutants that can 

produce H2 under near-atmospheric concentrations of O2 (64). This level of oxygen tolerance 

represents an approximate 70–fold improvement over wild-type Fe-hydrogenases in the 

concentration of oxygen tolerated (from 0.3–21 percent), or a 720–fold increase in the half-life of 

Fe-hydrogenase activity (from ~1 minute to ~12 hours) in the presence of atmospheric oxygen 

levels. In either case, it is clear that such an improvement is well within the range of 

improvements that have been reported for a variety of other enzymes (65–72). 

2.3.5 H2 recycling. A third minimizer of photon-to-H2 conversion efficiency is the 

presence in many green algae, including those most well-studied for H2 production, 

Chlamydomonas reinhardtii, Scenedesmus obliquus, and Chlorella fusca, of H2 uptake activity 

(73, 44). However, the enzymes responsible for this activity have not yet been positively 

identified; in fact, the possibility remains that some of the same Fe-hydrogenases may be 

responsible for both H2 uptake and H2 production activity. The investigation of mechanisms 

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regulating hydrogen production, involving mutants deficient in individual functions of the 

pathway, remains an important area of research in the development of H2 production by direct 

biophotolysis (11). 

2.3.6 Rate and cost estimate. To date, the two-stage bioreactors making use of 

wild-type green algae are the only systems that have been developed for sustained production of 

H2 by means of direct photolysis. Rates of H2 production achieved in this way are reported as 

0.07–0.08 millimole per liter culture per hour (5) as seen in Table 5, the lowest rate reported for 

existing biohydrogen production systems. However, the substrate costs for direct photolysis are 

also extremely low, improving its relative efficiency in comparison with higher-rate, higherinput requiring systems. In addition, the potential benefit of realistic research accomplishments 

must be taken into account. In the Thorough-cost analysis by modelers at the NREL, the cost of 

H2 generated by direct photolysis was estimated under current laboratory conditions, as well as 

with improvements available in the near-term (incorporating measures definitely possible), in the 

long-term (incorporating reasonable research targets), and in the best case (incorporating 

improvements that are theoretically possible but which would require major accomplishments in 

several areas). The H2 selling prices were estimated at $5,300 per gigajoule, $930 per gigajoule, 

$110 per gigajoule, and as low as $9 per gigajoule, respectively, for these scenarios (45). 

Table 5. Comparison of rates of H2 biosynthesis (5).

BioH2 System H2 synthesis rate 

(reported units) 

H2 synthesis rate 

(converted units) 

Direct photolysis 4.67 mmol H2/l/80 h 0.07 mmol H2/(l x h) 

Indirect photolysis 12.6 nmol H2/μg protein/h 0.355 mmol H2/(l x h) 

Photo-fermentation 4.0 ml H2/ml/h 0.16 mmol H2/(l x h) 

CO-Oxidation by R. gelatinosus 0.8 mmol H2/g cdw/min 96.0 mmol H2/(l x h) 

Dark-fermentations 

Mesophilic, pure straina 21.0 mmol H2/l 1/h 21.0 mmol H2/(l x h) 

Mesophilic, undefinedb 1,600.0 l H2/m3

/h 64.5 mmol H2/(l x h) 

Mesophilic, undefined 3.0 l H2/l/h 121.0 mmol H2/(l x h) 

Thermophilic, undefined 198.0 mmol H2/l/24 h 8.2 mmol H2/(l x h) 

Extreme thermophilic, pure strainc 8.4 mmol H2/l/h 8.4 mmol H2/(l x h) 

a Clostridium species #2. b

 A consortium of unknown microorganisms cultured from a natural substrate and selected by the bioreactor culture 

conditions. 

c Caldicellulosiruptor saccharolyticus 

2.3.7 Research priorities. Direct photolysis is not yet able to compete with other 

mechanisms of biohydrogen production in rate or cost, but the necessary advances are well 

within reach of modern biotechnology. To achieve practicality, the most important challenges to 

be overcome are the light-saturation effect and the strong repression of the Fe hydrogenases, 

both transcriptionally and post-translationally, by molecular oxygen. In addition, improvements 

to specific H2 yield will be important, achievable through either modification of production 

hydrogenases or possibly through elimination of H2 uptake activity. More ambitious ideas 

foresee advances in the light-harvesting capability of the algae, possibly by adding lightharvesting pigments to cover additional portions of the solar spectrum. The land-use issue is also 

extremely important, which in turn presumes that effective scale-up is achieved. While direct 

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photolysis is a long-term prospect, its potentially low-energy requirements and the 

approachability of its challenges by established molecular techniques cause it to be well worth 

the continued research investment. 

2.4 Indirect Photolysis 

Indirect photolysis is the light-dependent evolution of H2 by nitrogenases that occurs in 

cyanobacteria. In this process, nitrogenases evolve H2 as they fix N2 into NH3, simultaneously 

consuming ATP supplied by respiration of organic carbon fixed through photosynthesis at the 

rate of 16 ATP per H2 produced (74, 5). Although nitrogenases are highly oxygen-sensitive, like 

hydrogenases, they are protected to a great extent from O2, in filamentous cyanobacteria such as 

Anabaena and Nostoc, in specialized cells known as heterocysts. As a result, cyanobacteria 

undergoing indirect photolysis can evolve H2 quite rapidly and continuously under oxic 

conditions (12), in contrast to microbes using direct photolysis. Because N2 fixation is an 

inducible process that requires an absence of fixed nitrogen, cultivation in nitrate-free media is 

required to induce H2 production. In addition, published H2 evolution rates are typically 

acquired under anoxic, light-saturated conditions that follow aerobic and frequently lower-light 

cultivation conditions (12). 

The nitrogenase complex consists of two proteins—the dinitrogenase and the 

dinitrogenase reductase. The former is an alpha2beta2 heterotetramer, with alpha and beta 

subunits encoded by the genes nifD and nifK, respectively. The dinitrogenase reductase, encoded 

by nifH, is a homodimer and mediates the transfer of electrons from the external electron donor, 

typically a ferredoxin or flavodoxin, to the dinitrogenase (75); mechanistic understanding of 

nitrogenase action is well-understood (76). Several distinct variations of the nitrogenase 

structural genes exist, although they are highly conserved, and many cyanobacteria possess 

multiple nitrogenases. Interestingly, the unusual non-molybdenum-containing variants appear to 

allocate more electrons to H2 production (75). Numerous additional genes have been identified 

and characterized that are involved with nitrogen fixation and regulation, recently reviewed in 

(77). 

Indirect photolysis requires a great deal of energy—16 ATP per H2 molecule generated 

(34). Furthermore, it is limited by the H2-recycling activity of uptake hydrogenase found in all 

known N2-fixing cyanobacteria (13). Another limitation is the vulnerability of filamentous 

cyanobacteria to fragmentation by mixing, which greatly diminishes H2 evolution capability 

(12). Nevertheless, the O2-tolerance of the process is uniquely attractive. 

2.4.1 H2 recycling. Uptake hydrogenases are found in apparently all nitrogen-fixing 

cyanobacteria, where they efficiently recover H2 energy, and they thus present one of the primary 

limitations of indirect photolysis (75, 13). Cyanobacterial uptake hydrogenases appear to be 

conserved, encoded by hupL (large subunit) and hupS (small subunit) genes; the recombinase 

xisC is also essential to uptake hydrogenase expression because it facilitates excision by sitespecific recombination of a 10.5 kilobit element within the hupL structural gene during 

heterocyst differentiation (75). In addition, bidirectional hydrogenases are present in many but 

not all N2-fixing cyanobacteria. These are heterotetrameric enzymes consisting of a hydrogenase 

part encoded by hoxYH and a diaphorase part encoded by hoxFU, typically function in the H2-

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uptake direction, and can oxidize nitrogenase-generated H2 in the absence of uptake 

hydrogenases. 

In attempts to improve H2 evolution by indirect photolysis, several mutants have been 

generated in which the activity of uptake hydrogenases has been impaired (78) or in which the 

hupSL structural genes have been inactivated (79, 80). These have shown significantly improved 

H2 production (32, 80–81), with H2 evolution rates enhanced by 3- to 10-fold over those 

achieved with the corresponding wild-type strains (12). 

2.4.2 Cultivation conditions. Photobioreactors for hydrogen production are 

undergoing rapid development in attempts to provide light evenly, abundantly, and efficiently to 

photosynthetic cultures while facilitating gas removal (e.g., [50, 83–86]). At the same time, 

experimental work in indirect photolysis is investigating effects of variations in dissolved 

oxygen, light, temperature, nutrients and waste products, and suspension versus immobilization 

of cells, not to mention cell morphologies and genetic compositions (32, 75, 80). It is not 

surprising, therefore, that an optimal range of conditions has not emerged, and indeed, such 

optimization may need to follow the identification and/or engineering of a few outstanding H2-

producing organisms. 

2.4.3 Rate and efficiency. Reports of photosynthetic conversion efficiency (energy 

in H2 produced divided by energy in photosynthetically active radiation [400–700 nm 

wavelengths]) vary, with the highly active strain Synechococcus sp. Miami BG043511 yielding a 

performance of 3.5 percent (82). H2 synthesis rates achievable by indirect photolysis, however, 

compare quite favorably to those presently achievable by direct photolysis and by 

photofermentation (Table 5), at 0.355 millimoles H2 per liter culture per hour, compared to 0.07 

millimoles per liter per hour for direct photolysis and 0.16 millimoles per liter per hour for 

photo-fermentation (5). 

2.4.4 Research priorities. While the conversion efficiency of indirect photolysis is 

low compared to the theoretical efficiency of direct photolysis, it nevertheless presents the 

advantages of sustained H2 production over longer periods of time, great tolerance of aerobic 

conditions, and requirement for little input other than sunlight, minerals, and CO2. In addition, 

several promising avenues are available for the improvement of H2 production. 

The engineering of cyanobacteria to include alternative, non-molybdenum-containing 

nitrogenases, as well as to inactivate uptake hydrogenases, are paramount, and overexpression of 

nitrogenase as well as nitrogenase engineering to improve its catalytic rate have also been 

suggested (75). Engineering efforts directed toward antenna improvements, minimizing lightsaturation effects as well as enabling photon collection from wider regions of the solar spectrum, 

would also be applicable to indirect photolysis. 

Among experimental organisms, the filamentous cyanobacteria have received the 

majority of investigation to date, but non-filamentous cyanobacteria such as Oscillatoria that 

protect nitrogenases from O2 by temporal rather than physical separation of photosynthesis and 

N2 fixation also deserve further examination. In addition, a great diversity of cyanobacteria 

adapted to a wide variety of environments exists that should be investigated for potential 

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contributions to the H2 production effort. The ability of some cyanobacteria to grow 

heterotrophically is especially interesting in light of the possibility it presents to convert organic 

wastes into H2 (75). 

Finally, further work directed toward optimizing cultivation conditions for highly 

desirable organisms, particularly outdoor and marine cultivation (80), is essential to the 

realization of commercially viable indirect photolysis. 

2.5 Photofermentation 

Photofermentation is the light-dependent process carried out by anoxygenic phototrophic 

bacteria, particularly purple non-sulfur bacteria such as Rhodobacter sphaeroides and 

Rhodobacter capsulatus, in which H2 is evolved by nitrogenase under nitrogen-deficient 

conditions, using ATP supplied by photoheterotrophic growth (19). Although photosynthetic, 

this process does not split water and does not evolve O2 as in the oxygenic photosynthesis of 

green algae and cyanobacteria (Figure 19). It requires an anoxic atmosphere, because 

anoxygenic phototrophs do not protect their nitrogenases from O2 intracellularly in the way that 

cyanobacterial heterocysts do; however, the absence of O2 production causes the continuous 

production of H2 for >100h to be relatively uncomplicated, in contrast to the case with direct 

photolysis. 

2.5.1 Cultivation conditions. Rhodobacter and other anoxygenic phototrophs are 

versatile organisms capable of a wide variety of growth modes, including aerobic respiration, 

anaerobic respiration, fermentation, and photoautotrophy, enabling them to withstand the varying 

culture conditions that would necessarily accompany outdoor cultivation. In addition, the 

nitrogenase activity of these organisms is more stable under diurnal illumination. At the same 

time, significant H2 production only occurs during photoheterotrophic growth, with the result 

that an important aspect of cultivation design will be the balance between conditions that favor 

nitrogenase stability and those that favor H2 production (19, 87). 

 Another important challenge in cultivation of all phototrophs is the self-shading effect 

that intensifies as cultivation volumes become larger or as cell densities become greater. This 

problem is already being addressed through genetic modification of the photosynthetic apparati, 

as it is in green algae, and a mutant of R. sphaeroides with less than half of the pigment of wildtype algae has already shown a 50 percent increase in H2 production (88). 

 Finally, the scale-up of photobioreactors with retention of optimal culture conditions 

presents several challenges of its own. Several studies have shown that H2 production is 

increased when cells are immobilized, and immobilization of cells on a large scale will require 

discovery of gels or solid supports that can minimize the problems of inhomogeneous 

distribution of cells and nutrients and difficulty of control, while maximizing the benefits of 

higher H2 production rates and a cell-free effluent. In addition, efficient mixing also poses 

special problems:while mixing is necessary to distribute substrates and collect gases, mechanical 

mixing is difficult in high surface-area-to-volume reactors, and gas sparging risks diluting the H2

produced (19). 

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2.5.2 Substrate range. An attractive feature of photofermentation is the high 

substrate-conversion efficiencies of many anoxygenic phototrophic bacteria, as well as their 

abilities to use a wide variety of substrates for growth and H2 production. Malate, lactate, other 

organic acids, sugars, and even some alcohols are used readily. The greatest potential value of 

photofermentation for H2 production, however, depends on the use of complex substrates such as 

those found in mixed organic wastes. In pursuit of this goal, numerous approaches have been 

attempted, and early success has been achieved with use of dairy wastewater blended with 

malate, sugar refinery wastewater also blended with malate, tofu wastewater, wastewater of a 

lactic acid fermentation plant, and olive mill wastewater. An especially promising approach for 

H2 production from wastewater involves the fermentative pretreatment of wastes to generate 

small organic acids such as lactate and malate favorable for H2 production (19). 

2.5.3 Rate and efficiency. In general, H2 production by photofermentation occurs 

more rapidly when cells are immobilized or on a solid matrix; currently, the most rapid rates 

have been obtained when the cells are immobilized in porous activated glass. In addition, some 

substrates support more rapid H2 production than others. If an ideal laboratory system could be 

scaled up without diminishing the rate of H2 synthesis, rates of 3.6–4.0 liters H2 per liter 

immobilized culture per hour would result, corresponding to 0.145–0.161 millimoles H2 per liter 

per hour (Table 5) (5). 

Photofermentation generates H2 at the expense not only of sunlight energy but also of 

organic substrates. Furthermore, the H2 is produced by nitrogenase, with the result that much of 

the input energy is diverted to N2 fixation. The result is that photofermentation has a calculated 

efficiency that is significantly lower than the theoretical efficiency obtainable by direct 

photolysis. However, great hope lies in the possibility that photofermentation may be adapted to 

the use of organic wastes, raising the practical efficiency a great deal (34). 

In calculating efficiency, two components are typically considered: the substrate 

conversion efficiency and the sunlight conversion efficiency. The substrate conversion 

efficiency describes the percentage of a substrate utilized for H2 production rather than 

biosynthesis or growth, according to the equation: 

CxHyOz + (2x – z)H2O Æ (y/2 + 2x – 2)H2 + xCO2

Although purple non-sulfur bacteria can use a wide variety of substrates for photoheterotrophic 

growth, only some of these are suitable for H2 production. Substrate conversion efficiencies vary 

by strain; those with the highest values for the well-studied Rhodobacter sphaeroides include 

malate, lactate, and butyrate, ranging from 50–100 percent (19). 

The light conversion efficiency, in turn, is the ratio of the total energy (heat of 

combustion) value of the H2 that has been obtained to the total light energy input to the 

photobioreactor. For photofermentative H2 production, light energy conversion efficiencies 

range from 1–5 percent on average (19). 

2.5.4 Research priorities. The most immediate priority for photofermentative H2

production, given its high (nitrogenase-based) energy requirement as well as its need for organic 

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substrates, is the development of cultivation techniques and/or organisms that allow the use of 

organic wastes. Improved understanding of the energy flow within the photofermentative H2-

producing metabolism, including the mechanisms by which organic substrates improve H2-

production activity, would complement these efforts greatly and should be quite achievable 

through available metabolic engineering techniques. The nature of the wastes used will then 

inform the design of cultivation conditions, which form the next priority. Genetic modifications 

of antennae to limit self-shading and to allow utilization of a greater portion of the solar 

spectrum remain a high priority for all photobiological H2-production techniques. 

2.6 Water-gas Shift Mediated H2 Production 

The water-gas shift reaction describes the oxidation of CO to CO2 with the release of H2: 

CO(g) + H2O(l) ⇔ CO2(g) + H2(g) 

This process is a light-independent reaction carried out by certain photoheterotrophic bacteria 

within the Rhodospirillaceae such as Rhodospirillum rubrum and Rubrivivax gelatinosus CBS, 

using an enzyme known as carbon monoxide dehydrogenase (CODH) in combination with a 

hydrogenase (89, 90). Although light does not affect the CO oxidation rate, in light an uptake 

hydrogenase is able to oxidize the H2 to support light-dependent CO2 fixation, whereas H2

accumulates during incubation in darkness (90). 

 During this metabolism, the bacteria are able to use CO as the sole source of carbon and 

energy for ATP production. This has two important implications. First, it enables the microbes 

to produce additional H2 from synthesis gas, which is a mixture composed primarily of CO and 

H2 that results from the gasification of biomass. As a result, H2 production from biomass that is 

not readily fermented, such as lignin, becomes possible. Second, the water-gas shift process is 

highly thermodynamically favorable, with the result that enzymatic catalysis allows CO partial 

pressures to diminish rapidly and to very low levels (approaching 100 percent removal from 

synthesis gas). This process thus offers the possibility of a low-cost purification step for H2

before use in fuel cells, in which catalysts are rapidly poisoned by CO (5, 90). 

2.6.1 Genetics. The regulation and structure of the CO dehydrogenase system of 

Rhodospirillum rubrum is well-characterized and is presumably similar to that of Rubrivivax 

gelatinosus. In R. rubrum, in the presence of CO, the CooA regulatory protein binds to the 

promoters of the cooFSCTJ and cooMKLXUH operons, initiating expression of the CO oxidation 

system. CODH, which oxidizes CO to CO2, is encoded by the cooS gene. Electrons released by 

the oxidation of CO are transferred to a ferredoxin-like protein, CooF, and then through an 

undefined path to the CO-tolerant hydrogenase, CooH, which uses them to reduce H+

 to yield H2. 

Functions of the other genes in the two operons have been studied as well (91, 92). While the 

CODH-associated hydrogenase of R. rubrum is highly O2-sensitive, that of R. gelatinosus is 

surprisingly O2-tolerant, exhibiting a half-life near 20 hours when whole cells were stirred in air 

(90). 

2.6.2 Rate and efficiency. H2 production by the water-gas shift reaction is one of 

the most rapid processes known, achieving 96.0 millimoles H2 per liter culture per hour under 

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laboratory conditions (Table 5); estimated substrate conversion efficiencies have not yet been 

reported (5). 

2.6.3 Research priorities. A major challenge for the water-gas shift reaction is the 

achievement of efficient transfer of synthesis gas into aqueous solution, as the CO must be 

available to the bacteria at sufficient concentrations to allow efficient metabolism (5). 

Genetically, in addition, greater understanding of the interaction between CODH and the 

hydrogenase might indicate features amenable to optimization, and investigation of the O2-

tolerant R. gelatinosus hydrogenase to understand the basis for its resilience would be valuable to 

all efforts to engineer improved O2-tolerance in hydrogenases. 

Further priorities, several of which the water-gas shift pathway shares with dark 

fermentation, are described in Section 2.7 below. 

2.7 Dark Fermentation 

Dark fermentation refers to the light-independent production of H2 by anaerobic 

heterotrophic bacteria. While the water-gas shift process is thus technically a dark fermentation, 

and green algae and cyanobacteria are also capable of light-independent fermentations as 

described above, in the context of H2 production this term most often refers to non-phototrophic 

bacteria unless otherwise specified. These microbes may be mesophilic (with optimal metabolic 

temperatures of 25–40°C), thermophilic (40–65°C), extremely thermophilic (65–80°C) or 

hyperthermophilic (>80°C). They typically ferment carbohydrates (glucose, starch, or cellulose) 

and generate a gas of mixed composition, including not only H2 but also CO2, CH4, and/or 

volatile fatty acids, depending on the fermentation pathway used (Figure 17). In practice, 

highest H2 yields are associated with fermentations yielding a mixture of acetate and butyrate, 

while lower yields are associated with more reduced end-products such as propionate, lactate, 

and alcohols. Most fermentative bacteria are capable of multiple fermentation pathways, and 

culture conditions strongly influence the pathway(s) used; in particular, H2 concentrations must 

be kept very low to avoid end-product inhibition (93, 5). 

2.7.1 Genetics. Fermentation pathways are numerous, diverse, and well-characterized 

genetically and physiologically in numerous organisms, particularly in gram-negative bacteria 

(93); their regulatory mechanisms have been thoroughly investigated and widely-modeled (94); 

they have been metabolically engineered for the synthesis of numerous organic products (e.g., 

[95]); and numerous fermentative enzymes have been structurally characterized (e.g., [96]). As a 

result, the genetic, genomic, proteomic, metabolomic, and physiological groundwork has been 

done to facilitate a vast array of genetic and metabolic engineering efforts directed to the 

improvement of H2 production by dark fermentations. 

2.7.2 Substrates. Since fermentations are, by definition, supported by organic 

substrates, an important question for dark fermentative H2 production is the availability of 

sufficiently inexpensive and abundant carbon sources that the resulting H2 could be 

commercially viable. Organic wastes are an attractive option, yet are typically complex and 

variable and therefore challenging to combine with sophisticated metabolic engineering 

approaches. In response to this problem, Logan and colleagues at the University of Pennsylvania 

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decided to attempt H2 production with a similarly complex microbial community obtained from 

soil and achieved promising success (97), indicating that highly diverse, low-cost substrates may 

be accommodated with appropriate microbial inocula. 

2.7.3 Rate and efficiency. A number of different dark fermentation systems have 

reported H2 synthesis rates well above 1 millimole H2 per liter culture per hour, using both pure 

and undefined cultures, with values reaching 121.0 millimoles H2 per liter culture per hour for 

undefined mesophilic cultures (Table 5). Dark fermentation, even by quite diverse systems, thus 

produces H2 at rates that are up to two orders of magnitude greater than those currently achieved 

by any of the phototrophic mechanisms. 

 At the same time, these high laboratory rates are achieved at the expense of purified 

organic substrates that would be prohibitively expensive at larger scales, especially with a 

substrate conversion efficiency of approximately 28 percent with readily-fermented sucrose and 

glucose (98, 99). Moreover, thermophilic systems are calculated to consume unacceptably great 

quantities of energy to maintain high temperatures (5). To become economically attractive, 

therefore, research in mesophilic dark fermentations must explore the feasibility of using organic 

waste streams and less-expensive organic substrates such as lignocellulose; pioneering efforts in 

this direction are few to date, but have already achieved early success and should be encouraged 

further (34).

2.7.4 Research priorities. Dark fermentation is a promising avenue for biohydrogen 

production that could benefit greatly from progress in a few key areas. Improvement in gas 

separation technology, as well as gas removal from cultures during fermentation, are uniquely 

crucial to economically feasible H2 production by dark fermentation, for fermentations are 

constrained to generate mixtures of gases and are typically subject to strong end-product 

inhibition (94). Investigation of this problem is underway using hollow fiber membrane 

technologies, resulting in a 15 percent improvement in H2 yield, as well as using other synthetic 

polymer membranes, but further advances are achievable (5). 

Interestingly, both of these problems may also be addressed through genetic engineering. 

Because most fermentation pathways are well-understood at both the genetic and physiological 

levels, and because many of the most attractive fermentative organisms are genetically tractable, 

great potential exists to enhance, diminish, or even to alter the products of specific fermentative 

pathways through metabolic engineering, as well as to diminish the sensitivity of crucial 

enzymes to end-product inhibition (34). 

Further gains in fermentative H2 production will also require optimization of bioreactor 

designs; for example, the highest rate shown in Table 5 involved the use of activated carbon 

fixed-bed bioreactors that allowed retention of the H2-generating bacteria (5). 

3. Research Priorities 

 Because of the diversity of approaches to biohydrogen production (direct and indirect 

photolysis, photofermentation, the water-gas shift pathway, and dark fermentation), specific 

research priorities have been summarized at the end of each section above. These emerging 

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technologies have been carefully investigated for future practicability, and all face challenging 

problems that are nevertheless approachable by creative use of metabolic and chemical 

engineering. Given continued investment, each of the major biohydrogen pathways should be 

able to find a niche in a future sustainable-energy economy by delivering competitively-priced 

H2 at commercial scales. 

4. Commercialization 

While biohydrogen systems exist at the pilot scale that can produce H2 continuously from 

direct photolysis (44), indirect photolysis (12), photofermentation (19), the water-gas shift 

reaction (90), and dark fermentation (5), no commercial systems are yet available, and many 

questions regarding the practical applications of biohydrogen remain to be answered. In 

particular, it is not yet clear whether biohydrogen systems can be integrated with hydrogen fuel 

cell technologies to generate electricity at practical scales (5). A major limitation to 

commercialization efforts, cited by multiple researchers, is in fact the limited communication 

between scientists who study biohydrogen systems and engineers who develop hydrogen fuel 

cell technologies. Great advancements are potentially achievable by encouraging this form of 

collaboration, in particular (34, 5). 

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D. BIODESULFURIZATION OF FOSSIL FUELS 

1. Introduction 

Each day, more than 80 million barrels of oil are pumped from the Earth’s surface. Of 

this vast amount, the majority (~90 percent) is processed for use in fuels, which then are 

combusted and released to the atmosphere in gaseous products (1). Since sulfur constitutes up to 

5 percent of crude oils, usually as organosulfur compounds, these combustion products include 

sulfur oxides and dioxides (SOx) that dissolve in atmospheric water vapor and ultimately yield 

acid rain (2, 1, 3). In addition, SOx are also believed to reduce the efficiency of automobile 

catalytic converters, leading to increased tailpipe emissions of both nitrogen oxides (NOx) and 

CO2. 

Governments worldwide have responded by enacting laws to restrict the quantity of 

sulfur allowed in fossil fuels, primarily those intended for transportation. In the past 10 years, 

allowable levels of sulfur in transportation fuels have diminished from 2,000–5,000 parts per 

million (ppm) to less than 500 ppm; recent regulations proposed by the Directive of the 

European Parliament (4) and the EPA (5) will lower these levels to below 350 ppm. By 2010, 

even lower restrictions (less than 10–15 ppm in practice) are expected (1). 

1.1 Hydrodesulfurization 

The primary conventional technology used to remove sulfur from crude oil is 

hydrodesulfurization, or HDS. By subjecting crude oil to elevated temperatures and hydrogen 

partial pressure in the presence of a CoMo/Al2O3 or NiMo/Al2O3 catalyst, reactive sulfur 

components such as mercaptans, sulfides, and disulfides are converted to H2S and hydrocarbons. 

Lower boiling point fractions of crude oil contain primarily these aliphatic organosulfur 

compounds and are therefore desulfurized with great success by HDS. In higher boiling point 

fractions, however, the organosulfur compounds primarily contain thiophenic rings, including 

thiophenes, benzothiophenes, and their alkylated derivatives. Unfortunately, HDS is much less 

efficient in the desulfurization of these compounds (Figure 20), with the result that so-called 

deep desulfurization technologies are being actively explored (7). 

2. State of the Science 

2.1 Microbial Desulfurization 

A promising alternative biotechnological approach employs the unusual abilities of a 

microbial enzymatic pathway to oxidize thiophenic sulfur atoms and subsequently cleave them 

from carbonaceous rings, releasing the sulfur and leaving the carbon essentially untouched (7). 

This biodesulfurization (Dsz) pathway was first discovered in Rhodococcus erythropus strain 

IGTS8 in the late 1980s and has been studied in detail both in this organism and in analogous 

form in several others, including several other members of the Rhodococci as well as 

Agrobacterium MC501, Mycobacterium G3, Gordona CYKS1, Sphingomonas AD109, and 

strains of Klebsiella, Xanthomonas, Nocardia globelula, and thermophilic Paenibacillus and 

Bacillus (2, 7). Physiological studies of these organisms have been conducted primarily with the 

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model compound dibenzothiophene (DBT) and have made great progress in establishing the 

platform of understanding necessary to allow further improvements through enzyme and 

pathway engineering (8). 

2.2 Genes and Pathways 

2.2.1 DBT uptake. In the native rhodococcal transformation (Figure 21), the 

dibenzothiophene first gains access to the cell through apparently passive means that are 

nevertheless assisted greatly by the tendency of rhodococci, in contrast with many other bacteria, 

to collect at oil-water interfaces and even to partition into the oil phase in fine emulsions (1, 9). 

Figure 20. Organosulfur compounds present in fossil fuels (7). 

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Figure 21. Dsz pathway of R. erythropus (8). 

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Despite their tendency to gather at oil drop surfaces, Rhodococci nevertheless rank low in 

solvent tolerance (7), in contrast to Pseudomonas strains that are typically quite solvent-tolerant 

(2). To address this problem, as well as that of enzyme expression (below), researchers cloned 

the dszABC genes of R. erythropolis IGTS8 behind the constitutive tac promoter into P. putida 

and P. aeruginosa species. The resulting strains grew more rapidly than the rhodococci with 

DBT as the sole sulfur source and converted DBT to HBP quantitatively, showing that this 

approach could provide strains that are more useful commercially (2). 

2.2.2 Sulfur oxidation. Once within the cell, the DBT sulfur atom is first oxidized by 

a mono-oxygenase known as DszC. This enzyme makes use of a noncovalently-bound FMNH2, 

provided in reduced form by the flavin reductase DszD, to activate the molecular oxygen (8). 

Notably, the availability of FMNH2 is a crucial control on the rate of the desulfurization, as 

discussed below. A second oxidation of the sulfur atom is also catalyzed by DszC, again using 

FMNH2 provided by DszD, forming dibenzothiophene sulfone. A second mono-oxygenase, 

DszA, catalyzes the transformation of the sulfone to the sulfinate, again using FMNH2 and 

cleaving one carbon-sulfur bond. The remaining carbon-sulfur bond is then cleaved by a most 

unusual enzyme, the desulfinase DszB, releasing sulfite and hydroxybiphenyl, HBP (1, 2, 7, 8). 

The availability of FMNH2 for the two mono-oxygenases appears to be a crucial ratelimiting factor in microbial desulfurization. FMNH2 activity depends on the activity of DszD, 

the NADH-dependent FMN reductase, although DszD can be replaced in vitro by other FMN 

reductases (2), and FMN reduction in turn depends on the availability of NADH from cellular 

metabolism. This is an energy-intensive process, with ~4 NADH required per DBT desulfurized. 

The importance of this step has been illustrated by experiments in which flavin reductases, flavin 

mononucleotide reductases, or various oxidoreductases were added to the reaction mixture or 

over-expressed in recombinant constructs, leading to up to 100–fold increases in desulfurization 

rates (7). 

2.2.3 Substrate specificity. The enzymes of the rhodococcal Dsz pathway appear to 

have fairly relaxed specificities within the DBT family, desulfurizing derivatives with alkyl or 

aryl substitutions at rates depending on the position of the substitution: those with fewer 

substituents, and with substituents farther from the S atom, are generally desulfurized more 

slowly (10). However, these enzymes have little activity toward the smaller thiophenes and 

benzothiophenes, with the result that new catalysts must be developed for efficient 

desulfurization of gasoline. Development of catalysts with broad specificities, especially ones 

that can accommodate compounds with sterically obstructed S atoms, will be essential for 

commercial applications (1, 7). 

2.2.4 Regulation. The oxidized sulfur, released as sulfite, is used as a nutrient by the 

cell, and indeed the cell will not carry out desulfurization unless it is experiencing sulfur 

deprivation (2). The nutritional needs of the cells thus impose an additional limit on the rate of 

desulfurization. The HBP, all carbon atoms intact, then leaves the cell by an unknown 

mechanism to rejoin the nonaqueous phase (1). 

The DszA, B, and C enzymes of the pathway are encoded in the dsz operon, typically 

carried on a large plasmid and transcriptionally repressed in the presence of sulfate, cysteine, or 

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methionine (2) by means of a promoter responsive to sulfur-containing amino acids (1). DszD, 

in contrast, is encoded chromosomally. The genes for these enzymes from a variety of 

organisms have been cloned, sequenced, and engineered (8). 

The specificity, potential to proceed nearly to completion without loss of valuable carbon, 

and low capital and operating costs in comparison to HDS are highly attractive features of 

microbial desulfurization (7), while the rate at which whole microbial cells can remove sulfur 

remains the greatest challenge to commercialization (8). The throughput of substrates in this 

pathway may be hindered at several steps, including substrate acquisition, the supply of reducing 

equivalents, and enzyme turnover rates for specific substrates (Figure 22) (8). 

Figure 22. Conceptual diagram of biodesulfurization in R. erythropus (1). 

Although the Dsz operon is transcriptionally repressed in the presence of bioavailable 

sulfur, the enzymes themselves (Dsz A, B, C) are not post-translationally inhibited (7), opening 

the possibility of overexpressing the genes under control of strong constitutive promoters. The 

first report of this appeared in 1997, in which researchers cloned the dszABC genes of R. 

erythropolis IGTS8 behind the constitutive tac promoter into P. putida and P. aeruginosa

species (11). The resulting strains grew more rapidly than the rhodococci with DBT as the sole 

sulfur source and converted DBT to HBP quantitatively, showing that this approach could 

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provide strains that are quite useful commercially. The first patent on the incorporation of the 

Dsz genes into Pseudomonas was issued in 1999 (12), followed by another incorporating a flavin 

reductase (to fulfill the function of DszD) as well as the other Dsz genes into an artificial operon 

(13). 

2.2.5 Improvements in rate and extent. Despite advances in enzyme expression, 

further improvements were necessary to increase the rate and extent of desulfurization to levels 

sufficient for commercialization; sustained rates of >20 micromoles of substrate per minute per 

gram catalyst were needed, far in excess of the capability of the natural Rhodococcus Dsz system 

(7, 1). Between 1990 and 1998, however, optimization of biocatalyst production increased the 

activities of recombinant BDS catalysts 200–fold by increasing concentrations of DszA, B, and 

C, and optimizing conditions for DszD, bringing the catalysis rate to within an order of 

magnitude of that required for commercial operation (7). Work in chemostat selection for gainof-function mutants yielded R. erythropus strains that effectively utilized octyl sulfide and 5-

methyl benzothiophene (8), showing the utility of conventional approaches. At the same time, 

novel enzyme engineering was also required, and the combinatorial method known as RACHITT 

(see Chapter 2) was developed in the context of this problem. In the directed evolution of dszC

genes from Rhodococcus and Nocardia, new chimeric enzymes were generated that possessed 

higher activity, more extensive substrate oxidation, and broader substrate specificities than either 

of the parents. These activities were more than sufficient to meet industrial needs and no longer 

limited by nutritional needs of the microbes (14, 15).

2.2.6 Tolerance to industrial conditions. Microbial desulfurization is currently 

most attractive as a step following the conventional HDS, which in turn requires elevated 

temperatures. Ideally, therefore, the biocatalytic process would require as little cooling as 

possible, saving energy and time; in addition, higher temperatures could afford the advantages of 

increased enzymatic rates and diminished contamination by other bacteria. Advances in the 

development of thermotolerant Dsz pathways include the discovery of desulfurization in the 

thermotolerant Paenibacillus, able to desulfurize DBT at 55°C (7); the discovery of Bacillus 

subtilis WU-S2B, able to desulfurize DBT at 50°C (16); and Mycobacterium phlei GTIS10, also 

able to function at temperatures >50°C (8). An important note in these efforts is that the 

thermotolerance appears not to result from the DNA sequences, which are highly similar or even 

nearly identical to those of the mesophilic R. erythropus IGTS8, but from other factors in the 

whole-cell pathway. Thermophilic catalysis may therefore be constrained to occur within a 

thermophilic organism. 

2.3 Process Design 

An important consideration in BDS process design and in choice of the microbial host is 

the transfer of substrate from oil into cells. Close contact between cells and oil requires 

emulsification of the oil-water mix that must then be broken to recover the desulfurized oil, at 

cost of additional time and energy. Mass transfer has been shown to limit rates of DBT 

degradation in Pseudomonas Dsz systems (12, 17) but presents less of a problem during use of 

the comparatively hydrophobic Rhodococcus cells that tend to adhere to oil-water interfaces (1). 

Not only is the transfer of substrate into the cells improved, but the mixture is then also easily 

manipulated by means of patented devices known as hydrocyclones that readily and 

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inexpensively separate oil-water emulsions (18). These devices are ~1 meter long conical tubes 

that cause fluid to spin as it is pumped from the wide end to the narrow end, driving the denser 

fraction to the outside where it can be drawn off continuously. In a system where bacteria 

partition to the oil-water interface, the cells stay with the discontinuous phase. In a water-in-oil 

emulsion, cells associate with water droplets and allow separation of the clean oil phase; in an 

oil-in-water emulsion, cells can be concentrated with the oil for return to the reactor (1). 

Additional reactor design research has reduced the influence of mass transport limitations, and 

current BDS reactors use staging, air sparging, and media optimization with lower water-to-oil 

ratios to reduce reactor size, although these conditions increase the difficulty of downstream 

separations (7). 

 As with any biological technology, the maintenance of an active catalytic population is a 

challenge in the context of a conventional industrial system. An important advance in this area 

incorporated the production and regeneration of microbial cells within the BDS process, 

lengthening biocatalyst activity to 200–400 hours (14) and setting an example for other 

bioprocesses. 

3. Research Priorities 

 While a number of issues described above have been fully addressed, additional effort is 

still needed to expand the substrate specificity of the desulfurization process to include smaller 

compounds, allowing efficient desulfurization of gasoline, as well as to include compounds with 

sterically obstructed S atoms, allowing more complete desulfurization of all fossil fuels. In 

addition, metabolic engineering to increase the availability of FMNH2 to the mono-oxygenases 

has the potential to improve the rate of desulfurization further, while additional increases in 

thermotolerance and solvent tolerance of the catalytic microbes would improve the robustness of 

commercial-scale systems. 

4. Commercialization 

Commercialization efforts in the United States were initially spearheaded by Energy 

BioSystems Corporation of The Woodlands, Texas, which obtained a broad patent in 1999 

covering recombinant microbes based upon the Rhodococcus erythropolis IGTS3 genome and 

worked extensively to isolate, characterize, and manipulate desulfurization genes and develop 

bioprocess concepts (19). This company, later incorporated under the name Enchira 

Biotechnology Corporation, constructed and operated several small pilot plants, later entering an 

agreement with Petro Star, Inc. to design and build a 5,000 barrel-per-day BDS facility at their 

Valdez, Alaska refinery. The company also entered a number of technology development 

alliances with oil refiners, including TOTAL Raffinage Distribution S. A. of France, Koch 

Refining Company, Kellogg, Brown & Root, and the Exploration & Production Division of 

Texaco. These efforts were discontinued in 2000, however, when Enchira discontinued 

development of the BDS technology (20, 21). 

International government regulations are currently providing immense, annuallyincreasing pressure on oil refiners to diminish the sulfur content of fossil fuels below the levels 

attainable by current technologies. These incentives, combined with the many attractive features 

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of biodesulfurization, are creating a highly favorable environment in which further development 

of biodesulfurization can occur. Nevertheless, BDS is not yet fully optimized for pilot-scale 

work, and basic research to optimize the rate and extent to which the Dsz pathway can remove 

sulfur may still require governmental support. 

5. References 

(1) Monticello, D. J. (2000). Biodesulfurization and the upgrading of petroleum distillates, 

Curr Opin Biotechnol 11:540-546. 

(2) Kertesz, M. A. (1999). Riding the sulfur cycle -- metabolism of solfonates and sulfate 

esters in Gram-negative bacteria, FEMS Microbiol Rev 24:135-175. 

(3) Song, C., and X. Ma (2003). New design approaches to ultra-clean diesel fuels by deep 

desulfurization and deep dearomatization, Appl Catal B: Environ 41:207-238. 

(4) European Parliament (2003). Directive of the European Parliament Relating to the 

Quality of Petrol and Diesel Fuels, 2003/17/EC. 

(5) United States Environmental Protection Agency (2000). Control of Air Pollution from 

New Motor Vehicles: Tier 2 Motor Vehicle Emissions Standards and Gasoline Sulfur 

Control Requirements, Federal Register 65:6698-6746, AMS-FRL-6516-2. 

(6) Babich, I. V., and J. A. Moulijn (2003). Science and technology of novel processes for 

deep desulfurization of oil refinery streams: A review, Fuel 82:607-631. 

(7) McFarland, B. L. (1999). Biodesulfurization, Curr Opin Microbiol 2:257-264. 

(8) Gray, K. A., G. T. Mrachko, and C. H. Squires (2003). Biodesulfurization of fossil fuels, 

Curr Opin Microbiol 6:229-235. 

(9) Kaufman, E. N., J. B. Harkins, and A. P. Borole (1998). Comparison of batch-stirred 

and electrospray reactors for biodesulfurization of dibenzothiophene in crude oil and 

hydrocarbon feedstocks, Appl Biochem Biotechnol 73:127-144. 

(10) Folsom, B., D. Schieche, P. DiGrazia, J. Werner, and S. Palmer (1999). Microbial 

desulfurization of alkylated dibenzothiophenes from a hydrodesulfurized middle distillate 

by Rhodococcus erythropolis I-19, Appl Environ Microbiol 65:4967-4972. 

(11) Gallardo, M. E., A. Ferrandez, V. De Lorenzo, J. L. Garcia, and E. Diaz (1997). 

Designing recombinant Pseudomonas strains to enhance biodesulfurization, J Bacteriol 

179:7156-7160. 

(12) Darzins, A., L. Xi, J. Childs, D. J. Monticello, and C. H. Squires (1999). DSZ gene 

expression in Pseudomonas hosts, U.S. Patent 5952208. 

(13) Squires, C. H., W. Ji, L. Xi, B. Ortego, O. Pogrebinsky, K. A. Gray, and J. Childs (1999). 

Method of desulfurization of fossil fuel with flavoprotein, U.S. Patent 5985650. 

(14) Pacheco, M. A., E. A. Lange, P. T. Pienkos, L. Q. Yu, M. P. Rouse, Q. Lin, and L. K. 

Linguist (1999). Recent advances in biodesulfurization of diesel fuel, National 

Petrochemical and Refiners Association Annual Meeting, San Antonio, TX. 

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163 

(15) Coco, W. M. (2000). A novel method for shuffling chimeragenesis of mutated genes or 

of gene families from divergent bacterial genera, 100th General Meeting of the American 

Society for Microbiology, Los Angeles, CA. 

(16) Kirimura, K., T. Furuya, Y. Nishii, Y. Ishii, K. Kino, and S. Usami (2001). 

Biodesulfurization of dibenzothiophene and its derivatives through the selective cleavage 

of carbon-sulfur bonds by a moderately thermophilic bacterium Bacillus subtilis WUS2B, J Biosci Bioeng 91:262-266. 

(17) Setti, L., P. Farinelli, S. Di Martino, S. Frassinetti, G. Lanzarini, and P. Pifferi (1999). 

Developments in destructive and non-destructive pathways for selective desulfurizations 

in oil biorefining processes, Appl Microbiol Biotechnol 52:111-117. 

(18) Yu, L., T. Meyer, and B. Folsom (1998) Oil/water/biocatalyst three-phase separation 

process, U.S. Patent 5772901. 

(19) Rambosek, J., C. Piddington, B. Kovacevich, K. Young, and S. Denome (1999). 

Recombinant DNA encoding a desulfurization biocatalyst, U.S. Patent 5,879,914. 

(20) Nasser, W. (1999). Testimony of William Nasser, CEO Energy BioSystems Corporation, 

before the Senate Environment and Public Works Subcommittee on Clean Air, Wetlands, 

Private Property, and Nuclear Safety, http://epw.senate.gov/107th/nas_5-18.htm. 

(21) OTC Bulletin Board (2002). ENBC - Enchira Biotechnology Corporation, 

http://www.otcbb.com/profiles/ENBC.htm. 

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Chapter V 

Summary of Future Research Priorities 

A. INTRODUCTION 

Petroleum-based fuels and related materials are finite and expected to enter a period of 

diminishing availability within the next several decades. As a result, economies based on 

petroleum and its feedstocks will have to move towards using fuels and materials that are 

renewable, environmentally friendly, and of greater availability. Science and engineering 

communities worldwide are exploring many options. Several promising areas for future 

exploration and development are identified in the following sections. 

B. GENETIC ENGINEERING 

Current research in genetic engineering platform technologies is proceeding at an almost 

incomprehensibly rapid pace under the impetus of medical and basic biological research goals. 

While environmental biotechnology has much to gain from advances in these research goals, the 

support in these technologies and pace of progress are already sufficiently great, but funding for 

environmental goals is limited such that agencies with primarily environmental goals are 

encouraged to direct their support toward research priorities in other areas. 

C. BIOREACTOR TECHNOLOGIES 

Since bioengineering for pollution prevention involves relatively low-value products, 

requiring optimal bioprocessing for commercial feasibility, improvements in bioreactor 

technology should be a high priority in general in this field. In addition, several areas are worthy 

of specific mention: 

• Sensing. Real-time sensing of gases and aqueous metabolites is of central 

importance because it allows or has the potential to facilitate model validation, 

development of descriptive kinetic expressions, and real-time process control based 

on sensor feedback alone and in combination with model predictions. Biosensors 

suitable for monitoring bioconversions in bioreactors have been previously identified 

as a bottleneck in the development of high-volume, low-cost processes, indicating 

that the development of promising emerging technologies should be encouraged in 

every instance possible. 

• More-detailed Structural Modeling. In process design and optimization, the utility 

of a mathematical model lies in its ability to predict the operating characteristics in 

regions for which experimental data do not exist. Present kinetic models generally do 

not allow such procedures in great detail. Detailed kinetic models are also useful for 

capturing the dynamic responses of bioreactors to external stimuli, a set of important 

concerns in process control. Accordingly, a promising area of investigation is the 

development of more-detailed structural models that capture the salient features of 

complete metabolic pathways through the integration of biochemistry, molecular 

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biology, and computational techniques. In particular, new approaches to structural 

kinetic modeling that include transcriptional and post-translational regulatory effects 

are needed. 

• Multi-scale Modeling. In addition, new developments are needed in computational 

methods to capture effects of turbulence in bioreactors, effects of shearing and 

mechanical stresses on cellular growth and death, and the non-Newtonian nature of 

cellular media. These must involve multi-scale modeling to capture details at small 

spatial scales and must also be able to transfer relevant information to lowerresolution models that describe greater volume and time scales. CFD models are now 

able to establish hydrodynamic profiles among different zones of a reactor and could 

serve as the basis for such models, describing concentration and temperature 

gradients both instantaneously and over time. Such models could be extended to 

bridge length, volume, and time scales, linking detailed calculations at smaller scales 

or in critical areas with lower-resolution models that track averaged quantities. Given 

recent and continuing increases in inexpensive computing power, such models could 

contribute greatly to reactor optimization. In addition, they have the potential to 

guide the scale-up of industrial processes by revealing important mass and heat 

transfer limitations as reactor configurations are changed. 

• Applications of Genetic Engineering. Certain challenges inherent in bioreactor 

operation can be greatly alleviated by the skillful application of genetic engineering 

technologies. For example, substrate and product-based inhibitions are common 

phenomena which occur when enzyme activities diminish in the presence of locally 

high concentrations of certain metabolites. Increasing reactor-mixing can often 

alleviate these problems, but diminishing the inhibitory mechanisms genetically can 

offer a more convenient solution. An example of this approach is presented by Agger 

and colleagues, who disrupted the gene responsible for glucose repression so that the 

glucose conversion rate did not decrease with increasing glucose concentrations that 

were, in turn, needed to work at high biomass concentrations. The ability of genetic 

engineering to solve bioreactor-based problems offers a set of great opportunities for 

improvements in reactor productivity. 

D. BIOSEPARATIONS AND BIOPROCESSING 

Separation technologies to facilitate commercial success of biomass conversions include 

those suitable for low-molecular-weight organic acids, organic esters, diacids, and alcohols; 

gases such as H2; and biobased oils such as biodiesel and biolubricants. Among these, advances 

in membrane technologies and in processes utilizing environmentally-benign solvents promise 

especially great benefits. 

• Membrane Techniques. The development of new and/or improved membrane 

materials that provide increased selectivity and specificity for the desired substances, 

as well as increased flux with stability and robustness, is of central importance to the 

membrane-based techniques discussed below: 

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- Pervaporation. The use of pervaporation to remove either water or bioproducts 

from bioreactor media appears promising. Continued support for new membrane 

materials, new module and process designs, and improved theoretical 

understanding and modeling of the pervaporation process should therefore be 

pursued. The work of Vane and colleagues at the EPA National Risk 

Management Research Laboratory (NRMRL) is a noteworthy example of efforts 

in the development of pervaporation modeling and performance prediction 

software. 

- Micro- and ultrafiltration. Microfiltration and ultrafiltration promise to become 

major unit operations in the emerging biorefinery arena. The development of new 

materials for UF and MF, including porous metals and ceramics as well as 

polymers, is therefore an important priority. Similarly, nanofiltration and reverse 

osmosis are becoming increasingly important, with recent developments in 

nanotechnology promising to yield new materials with significantly improved 

fluxes and selectivities. 

- Membrane chromatography. An improved understanding of the interactions 

between culture media components and synthetic polymers suitable for 

membranes would greatly facilitate the design of synthetic substrates for use in 

membrane chromatography. Among those, ligand-binding and stericallyinteracting species should be investigated closely to improve the selectivity of 

membrane chromatography while maintaining acceptably high throughput. 

- Antifouling techniques. Fouling is a persistent problem among membrane 

technologies, with the result that methods to diminish fouling of membranes and 

ion exchange materials, as well as to remove impurities such as salts or acids that 

cause complications in downstream processes, are high priorities in the 

advancement of bioseparations. 

• Environmentally Benign Solvents. New renewable, biodegradable solvents are 

needed to support environmentally-friendly extraction processes. Supercritical CO2, 

a highly compressed phase of CO2 possessing properties of both liquid and gas 

phases, is one benign solvent that has already achieved great popularity and that has 

the potential to contribute performance, cost-effectiveness, and sustainability to 

separations of both biofuels and biomaterials. 

• Integrated Modules. Combined- or hybrid-unit operations in which a bioreactor is 

integrated with a bioseparation module, as in two-phase reactor systems, are 

particularly attractive as means to overcome limitations inherent to bioprocessing. 

These are particularly desirable for their potential to remove products as they are 

synthesized, alleviating the nearly universal problem of product inhibition in culture 

media. 

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E. POLYACTIDES 

• Development of LCIA Tools. One of the greatest challenges facing the production 

of truly environmentally-benign plastic materials, PLA and otherwise, is the 

evaluation of net environmental impacts, beginning with feedstock production 

(agriculture or collection of biomass wastes), including processing steps (production 

of lactide and subsequent polymerization) and ending with the emissions resulting 

from biodegradation. Specific processes chosen at each stage, particularly concerning 

conventional vs. sustainable methods, are likely to have dramatic impacts on the net 

environmental profiles of individual materials, yet the tools with which to evaluate 

these differences are not yet fully refined. These metrics are needed urgently, both to 

guide research and development of bioplastics and to advocate use of the truly 

environmentally beneficial materials. Consequently, a top priority in the 

development of environmentally benign plastics is the continuation of efforts to 

develop tools and standards within the context of LCIA that will make comparisons 

transparent and meaningful. 

• Improvement of Physical Properties. Presently, PLA and other biopolyesters suffer 

from two important deficiencies that limit their use. The first of these is their 

relatively low heat distortion temperatures, and the second is their relatively high 

permeabilities toward a number of substances, particularly water. As current, bestavailable LCIA analyses have indicated that PLA is indeed environmentally benign, 

continued research into biological, chemical, and physical transformations of PLAbased materials to improve these properties is warranted. In particular, 

nanocomposite technologies (Chapter III.D.) hold promise of improving both 

temperature distortion and permeation characteristics, as they have in conventional 

plastics, and should be investigated. Microcomposite technologies are related, 

already well-established approaches to achieve similar improvements in conventional 

plastics. In addition, plant microparticles derived from waste agricultural residues 

and simple grasses can be used directly as microparticles, providing both economic 

and environmental advantages. Alternatively, blending and trans-reacting PLA-based 

plastics with starch- or triglyceride-based materials (Chapter III.D.) may improve 

performance while maintaining biodegradability, with the result that these techniques 

also deserve further investigation. Recently, copolymerization of cellulose acetate 

with PLA has demonstrated that the heat distortion temperature can be increased. In 

this interesting case, both constituents of the plastic material come from renewable 

resources. This suggests that copolymerization of PLA, especially with other 

polymers based on renewable resources, can provide a viable route towards improved 

performance.

• Exploration and Development of New Polyesters. A recent comprehensive study 

by the DOE has identified 12 promising low molecular-weight materials that can be 

produced by fermentation in commercial quantities from plant sugars (succinic, 

fumaric, malic, 2,5-furandicarboxylic, 3-hydroxypropionic, aspartic, glutaric, 

glutamic, itonic, and levulinic acids, and the alcohols 3-hydroxybutyrolactone, 

glycerol, sorbitol, and xylitol). Combination of these acids and alcohols can produce 

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polyesters by direct condensation. In particular, reactive intermediates that can be 

produced by anaerobic fermentations are desirable, because anaerobic processes 

typically lose much less of the feedstock carbon to CO2 than do aerobic processes. 

The success of the DuPont SoronaTM material, a polyester of such low molecularweight precursors (1,4-benzenedicarboxylic acid-dimethyl ester with 1,3-propanediol) 

shows that development of sustainable processes to take advantage of readily 

available, renewable substances to produce additional biodegradable plastics deserves 

high priority for its great potential to yield both homopolymeric and copolymeric 

materials with new ranges and combinations of desirable properties.

F. POLYHYDROXYAKANOATES 

PHA development is proceeding in a number of promising directions on both metabolic 

engineering and chemical engineering fronts. Fortunately, most of these have the potential for 

success both individually and in combination with others, such that no particular obstacle is 

currently forming a bottleneck to further progress. The range of physical and thermal properties 

achievable with PHAs is still expanding rapidly as new configurations of copolymers and blends 

are explored. An on-going challenge will be the ability of the metabolic engineers to keep pace 

with the discoveries of the materials scientists, enabling microbes to synthesize the desired 

polymers both conveniently and inexpensively. These efforts can be categorized as follows. 

• Investigation of Novel Polymers and Properties. Clearly, a number of 

modifications of PHA composition have the potential to improve the plasticity, 

moldability, heat tolerance, and durability of the resulting plastics to approach those 

of conventional thermoplastics. Because of the promising availability and flexibility 

of routes to PHA synthesis, and because of increasing oil prices that will enable PHA 

polymers to become increasingly cost-competitive, it is a valuable effort to explore 

the properties of new PHA-based homopolymers, copolymers, and blends even before 

microbial pathways to their syntheses are in place.

• Metabolic and Genetic Engineering. The increasing availability of mathematical 

modeling tools, genomic and proteomic data and techniques, and microarray and 

antisense RNA technology will allow increasingly accurate prediction of useful 

targets for metabolic engineering. At the same time, genetic manipulation within 

both microorganisms and plants is becoming increasingly possible and rapid. Several 

enzymes central to PHA synthesis are just beginning to be explored through 

combinatorial and rational design mutagenesis approaches, and efforts to understand 

their catalytic mechanisms, substrate specificities, modes of competition with other 

enzymes, and regulation are likely to contribute greatly to the microbial or plantbased syntheses of novel polymers.

• Reactor and Processing Technology. Gains in commercial feasibility are often 

found in improving bioreactor yield and in diminishing processing costs. Without 

reiterating topics addressed previously, it is nevertheless important to include these in 

the research priorities for PHA efforts, with special note that the transfer of PHA 

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synthesis to plants may circumvent many limitations of both reactor efficiency and 

processing costs.

G. STARCHES, PROTEINS, PLANT OILS, AND CELLULOSICS 

• Basic Biosciences. From a long-term perspective, continued support for basic 

biosciences that allow the manipulation of plants at the genetic level is absolutely 

essential. Only through continued development of genetic and physiological 

engineering techniques will the possibility of inducing organisms to produce 

polymers directly, and thereby reducing the cost of bioplastics by minimizing 

processing steps, be realized. In addition, the preference for anaerobic processes 

among microbial fermentations should be recognized due to the minimization of 

carbon loss from feedstocks. In the context of the plant-based plastics described in 

this section, the required tools include the genetic engineering of the cellulose-lignin 

and oil distribution in plants. Such basic genetic manipulation of plants is supported 

by the USDA. Specific strategies for pollution prevention are necessarily more 

narrow and should focus on developing cost-effective methods for producing plastics 

from plant-based matter. 

• Biodegradable Plastics. Within the realm of starch-based plastics, commercial 

interest and success is currently carrying the development of compostable disposable 

packaging, including garbage bags, food wraps, diapers, as well as disposable food 

service items such as plates, cups, and utensils. Commercial research and 

development is even leading to improvements in water resistance and durability of 

starch-based materials, with the result that these are not considered to be high 

priorities for federal research funding. In contrast, the possibility of vastly improved 

strength, lightness, durability, and heat and water resistance offered by natural fiberreinforced composites, particularly nanocomposites, cause this area to be highly 

attractive for additional effort. Low-cost polysaccharide-based plastic materials have 

the greatest potential to displace significant amounts of petroleum-based plastics. 

- Green chemical processes. Similarly, more benign greener chemical processes 

should be investigated for transforming the available renewable resources. Such 

activities should include enzymatic transformations when they are less energy 

intensive than existing chemical routes. 

- Plant proteins. Genetic engineering of plant proteins for specific functionality is 

a lower priority simply because the potential application, adhesives, is small 

relative to the packaging and structural uses for plastics. If plant proteins can be 

engineered for larger volume applications, for example, into thermoplastic film or 

sheet materials, the promise of coproducing both fuels and materials in an 

integrated biorefinery would be supported. Specialized materials produced 

through recombinant DNA technologies are expected to be of high commercial 

value but of relatively small volume. 

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H. BIOETHANOL 

The consensus among researchers and supporters of bioethanol research, in addition to 

those engaged in commercial projects, is that the improvement of cellulase enzyme activity and 

cellulase production, both to increase the efficiency of release of fermentable sugars from 

biomass and to reduce cellulase cost, are two of the greatest advances needed in the effort to 

commercialize fuel ethanol production. In addition is the development of enzymatic 

pretreatment processes to release lignin from carbohydrate components and further improvement 

of fermentative organisms, with the particular goal of designing microbes capable of 

consolidated bioprocessing. 

I. BIODEISEL 

The cost, quality, and performance of biodiesel, as well as its overall environmental 

profile, could be improved by further efforts in several areas. 

• Altenative Feedstocks. First, feedstocks other than virgin plant oils, most of which 

are cultivated by non-sustainable, pesticide and energy intensive agricultural 

practices, would ideally be explored and developed; alternatively, sustainable 

cultivation of oil crops should be developed. Waste oil processing technology also 

deserves developmental effort to allow recovery of its intrinsic energy, and microbial 

and algal lipid production should be investigated to determine whether they might 

provide feedstocks at lower cost. 

• Lipase Technology. The further development of lipase technology will facilitate 

efficient enzymatic transesterifications of feedstock oils and fats and production of 

benign wastes with easily-recoverable coproducts, principally glycerol. Specifically, 

genetic engineering of lipases for greater activity and durability, as well as metabolic 

engineering of lipase-production pathways to understand lipase synthesis and 

regulation and to facilitate extracellular production, are well-positioned to offer 

valuable advances in enzymatic transesterification of oils and should be pursued. 

Research in these areas could have potentially great impacts within relatively short 

timescales and should be encouraged to the greatest extent possible. 

J. BIOHYDROGEN 

Approaches to biohydrogen production (direct and indirect photolysis, 

photofermentation, the water-gas shift pathway, and dark fermentation), have been carefully 

investigated for future practicability, and all face challenging problems that are nevertheless 

approachable by creative use of metabolic and chemical engineering. Given continued 

investment, each of the major biohydrogen pathways should be able to find a niche in a future 

sustainable-energy economy by delivering competitively-priced H2 at commercial scales. 

• Direct Photolysis. Direct photolysis is not yet able to compete with other 

mechanisms of biohydrogen production in rate or cost, but the necessary advances are 

well within reach of modern biotechnology. To achieve practicality, the most 

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important challenges to be overcome are the light-saturation effect and the strong 

repression of the Fe hydrogenases, both transcriptionally and post-translationally, by 

molecular oxygen. In addition, improvements to specific H2 yield will be important, 

achievable through either modification of production hydrogenases or possibly 

through elimination of H2 uptake activity. More ambitious ideas foresee advances in 

the light-harvesting capability of the algae, possibly by adding light-harvesting 

pigments to cover additional portions of the solar spectrum. The land-use issue is 

also extremely important, which in turn presumes that effective scale-up is achieved. 

While direct photolysis is a long-term prospect, its potentially low-energy 

requirements and the approachability of its challenges by established molecular 

techniques cause it to be well worth the continued research investment. 

• Indirect Photolysis. While the conversion efficiency of indirect photolysis is low 

compared to the theoretical efficiency of direct photolysis, it nevertheless presents the 

advantages of sustained H2 production over longer periods of time, great tolerance of 

aerobic conditions, and requirement for little input other than sunlight, minerals, and 

CO2. In addition, several promising avenues are available for the improvement of H2

production: 

- Engineering of cyanobacteria. The engineering of cyanobacteria to include 

alternative, non-molybdenum-containing nitrogenases, as well as to inactivate 

uptake hydrogenases, are paramount, and overexpression of nitrogenase as well as 

nitrogenase engineering to improve its catalytic rate have also been suggested. 

Engineering efforts directed toward antenna improvements, minimizing lightsaturation effects as well as enabling photon collection from wider regions of the 

solar spectrum, would also be applicable to indirect photolysis. 

- Investigation of alternative forms of cyanobacteria. Among experimental 

organisms, the filamentous cyanobacteria have received the majority of 

investigation to date, but non-filamentous cyanobacteria such as Oscillatoria that 

protect nitrogenases from O2 by temporal rather than physical separation of 

photosynthesis and N2 fixation also deserve further examination. In addition, a 

great diversity of cyanobacteria adapted to a wide variety of environments exists 

that should be investigated for potential contributions to the H2 production effort. 

The ability of some cyanobacteria to grow heterotrophically is especially 

interesting in light of the possibility it presents to convert organic wastes into H2. 

- Optimizing cultivation conditions. Finally, further work directed toward 

optimizing cultivation conditions for highly desirable organisms, particularly 

outdoor and marine cultivation, is essential to the realization of commercially 

viable indirect photolysis. 

• Photofermentation. The most immediate priority for photofermentative H2 production, 

given its high (nitrogenase-based) energy requirement as well as its need for organic 

substrates, is the development of cultivation techniques and/or organisms that allow the 

use of organic wastes. Improved understanding of the energy flow within the 

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photofermentative H2-producing metabolism, including the mechanisms by which 

organic substrates improve H2-production activity, would complement these efforts 

greatly and should be quite achievable through available metabolic engineering 

techniques. The nature of the wastes used will then inform the design of cultivation 

conditions, which form the next priority. Genetic modifications of antennae to limit selfshading and to allow utilization of a greater portion of the solar spectrum remain a high 

priority for all photobiological H2-production techniques. 

• Water-Gas Shift Mediated H2 Production. A major challenge for the water-gas shift 

reaction is the achievement of efficient transfer of synthesis gas into aqueous solution, as 

the CO must be available to the bacteria at sufficient concentrations to allow efficient 

metabolism. Genetically, in addition, greater understanding of the interaction between 

CODH and the hydrogenase might indicate features amenable to optimization, and 

investigation of the O2-tolerant R. gelatinosus hydrogenase to understand the basis for its 

resilience would be valuable to all efforts to engineer improved O2-tolerance in 

hydrogenases. 

• Dark fermentation. Dark fermentation is a promising avenue for biohydrogen 

production that could benefit greatly from progress in a few key areas. Improvement in 

gas separation technology, as well as gas removal from cultures during fermentation, are 

uniquely crucial to economically feasible H2 production by dark fermentation, for 

fermentations are constrained to generate mixtures of gases and are typically subject to 

strong end-product inhibition. Investigation of this problem is underway using hollow 

fiber membrane technologies, resulting in a 15 percent improvement in H2 yield, as well 

as using other synthetic polymer membranes, but further advances are achievable. 

Interestingly, both of these problems may also be addressed through genetic engineering. 

Because most fermentation pathways are well-understood at both the genetic and 

physiological levels, and because many of the most attractive fermentative organisms are 

genetically tractable, great potential exists to enhance, diminish, or even to alter the 

products of specific fermentative pathways through metabolic engineering, as well as to 

diminish the sensitivity of crucial enzymes to end-product inhibition. Further gains in 

fermentative H2 production will also require optimization of bioreactor designs. 

K. BIODESULFURIZATION OF FOSSIL FUELS 

While a number of issues described above have been fully addressed, additional effort is 

still needed to expand the substrate specificity of the desulfurization process to include smaller 

compounds, allowing efficient desulfurization of gasoline, as well as to include compounds with 

sterically obstructed S atoms, allowing more complete desulfurization of all fossil fuels. In 

addition, metabolic engineering to increase the availability of FMNH2 to the mono-oxygenases 

has the potential to improve the rate of desulfurization further, while additional increases in 

thermotolerance and solvent tolerance of the catalytic microbes would improve the robustness of 

commercial-scale systems. 

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A-1 

APPENDIX 

Contributions of the NSF/EPA Technology for a 

Sustainable Environment Program 

1995–2004 

1. Introduction 

 Between the years of 1995 and 2004, the NSF and EPA jointly funded the Technology 

for a Sustainable Environment (TSE) Program as part of the NSF/EPA Partnership for 

Environmental Research. This program had the goal of supporting the investigation and 

development of pollution avoidance and prevention processes, especially those with the potential 

to have long-term impact on industrial applications. Specific areas of interest included 

chemistry- and reaction-based engineering, non-reaction-based engineering, green design, green 

manufacturing, and industrial ecology for the realization of sustainable products and services. 

While the TSE Program was equally open to chemical, physical, mathematical, and 

bioengineering technologies, the Program Review in May 2004 revealed that a significant 

proportion of TSE grants have supported biologically-relevant endeavors. Of these, the projects 

that directly addressed issues raised in this State of the Science Report are summarized below. 

2. TSE Contributions to Genetic and Metabolic Engineering 

 While the TSE program has appropriately refrained from funding projects intended 

primarily to advance genetic and metabolic engineering technologies, TSE-funded projects have 

nevertheless made excellent use of available technologies. They have also contributed 

substantially to the refinement of basic techniques for application in the syntheses of 

biomaterials and biofuels. 

 The following projects used metabolic engineering to develop biological pathways for the 

synthesis of industrially- and agriculturally-useful products, some of which are related to 

monomers that may be used in biomaterials. The use of biologically-generated and 

biodegradable starting materials to yield similarly environmentally benign products is a 

fundamental goal of bioengineering for pollution prevention. 

• EPA-R824726 Fermentation of Sugars to 1,2-Propanediol by Clostridium 

thermosaccharolyticum (PI: Cameron, University of Wisconsin-Madison) 

• EPA-R826116 Environmentally Benign Synthesis of Resorcinol from Glucose (PI: 

Frost, Michigan State University) 

• NSF-9819957; EPA-R826729 Metabolic Engineering of Methylotrophic Bacteria for 

Conversion of Methanol to Higher Value-Added Products (PI: Lidstrom, University 

of Washington) 

• NSF-9985421 Metabolic Engineering of Carbon Fixation and Utilization for 

Biopolymer Production by Cyanobacteria (PI: Stephanopoulos, MIT) 

• NSF-0118961 Metabolic Engineering of Bacillus for Enhanced Product Yield (PI: 

Ataai, University of Pittsburgh) 

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• NSF-0124401 Metabolic Engineering of Monooxygenases for 1-Naphthol and 

Styrene Epoxide Formation (PI: Wood, University of Connecticut) 

• EPA-R829589 Analysis and Management of Fluxes in Bacillus Pathways for 

Pesticide and Protein Production (PI: Grossmann, Carnegie-Mellon University) 

The engineering of microorganisms and enzymes to withstand side reactions or industrial 

bioreactor conditions, often facilitating more rapid and/or less expensive production, is another 

prominent goal of metabolic and genetic engineering that has been addressed. 

• NSF-9817621 Improving Resistance to Enzyme Alkylation During EnzymeCatalyzed Production of Acrylamide (PI: Oriel, Michigan State University) 

• NSF-9911231; EPA-R828562 Metabolic Engineering of Solvent Tolerance in 

Anaerobic Bacteria (PI: Papoutsakis, Northwestern University) 

3. TSE Contributions to Bioreactor Technology, Bioseparations, and 

Bioprocessing 

 A fundamental component of any bioreactor-based sustainable technology is the ability to 

use non-toxic, biodegradable and/or environmentally benign solvents for the separation and 

processing of the desired bioproducts. The TSE Program has contributed substantially to this 

effort, funding projects to develop supercritical CO2, supercritical and near-supercritical water, 

polyglycols, and ionic liquids as solvents, as well as processes that avoid the need for solvents 

altogether. 

• NSF-9613258 Coexisting Chemical-Biological Modifications of Chlorinated Solvents 

as a Basis for Waste Reduction in Pollution Prevention (PI: Watts, Washington State 

University) 

• NSF-9817069 Novel Compressed Solvent Extraction Processes for Enhanced 

Biomass Conversion by Thermophilic Bacteria (PI: Knutson, University of 

Kentucky) 

• EPA-R826113 Synthetic Methodology “Without Reagents” Tandem Enzymatic and 

Electrochemical Methods for the Manufacturing of Fine Chemicals (PI: Hudlicky, 

University of Florida) 

• EPA-R826117 Aqueous Processing of Biodegradable Materials from Renewable 

Resources (PI: McCarthy, University of Massachusetts – Lowell) 

• EPA-R828130 Nearcritical Water as a Reaction Solvent (PI: Eckert, Georgia 

Institute of Technology) 

• EPA-R828133 Aqueous Polyglycol Solutions as Environmentally Benign Solvents in 

Chemical Processing (PI: Kirwan, University of Virginia) 

• EPA-R828135 Homogeneous Catalysis in Supercritical Carbon Dioxide with 

Fluoroacrylate Copolymer Supported Catalysts (PI: Akgerman, Texas A&M 

University) 

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• EPA-R828206 Development of a Heterogeneous Catalyst for Hydroformylation in 

Supercritical CO2 (PI: Abraham, University of Toledo) 

• EPA-R828541 Investigation of Room Temperature Ionic Liquids as Environmentally 

Benign Solvents for Industrial Separations (PI: Rogers, University of Alabama – 

Tuscaloosa) 

Industrial bioprocess development also relies heavily upon the use of real-time sensors to 

identify promising targets for process improvements, especially in cases involving narrow profit 

margins. The following project addressed the development of a type of sensor that holds great 

promise for real-time bioreactor sensing. 

• NSF-9613556 Advanced Fluorescence-Based Environmental Sensors (PI: 

Thompson, University of Maryland - Baltimore County) 

4. TSE Contributions to the Development of Bioplastics and Biomaterials 

 A pressing need within the field of biomaterials is the development of consistent, reliable 

tools for the assessment of the total environmental impacts of particular products. The TSE 

Program has supported numerous mathematical modeling efforts directed toward the assessment 

of product and process environmental sustainability at both small and large scales; even those not 

specifically intended for biomaterials applications have contributed to the general development 

of environmental life cycle analysis thought and are therefore included. 

• EPA-R825345 Environmentally Conscious Design and Manufacturing with Input 

Output Analysis and Markovian Decision Making (PI: Olson, Michigan 

Technological University) 

• EPA-R826114 BESS, A System for Predicting the Biodegradability of New 

Compounds (PI: Punch, Michigan State University) 

• EPA-R826739 New Methods for Assessment of Pollution Prevention Technologies 

(PI: Frey, North Carolina State University) 

• EPA-R826740 Economic Input-Output Life Cycle Assessment: A Tool to Improve 

Analysis of Environmental Quality and Sustainability (PI: Lave, Carnegie-Mellon 

University) 

• EPA-R828128 Designing for Environment: A Multi-objective Optimization 

Framework Under Uncertainty (PI: Diwekar, Carnegie-Mellon University) 

• EPA-R829597 Computer-Aided Hybrid Models for Environmental and Economic 

Life-Cycle Assessment (PI: Horvath, University of California – Berkeley) 

• EPA-R829598 Material Selection in Green Design and Environmental Cost Analysis 

(PI: Lin, SUNY Buffalo) 

• NSF-9985554 A Systems Ecology Approach to Life-Cycle Product Assessment and 

Process Design (PI: Bakshi, Ohio State University) 

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• NSF-0124761 New Modeling Framework and Technology to Optimize Resource 

Utilization in the Plastics Supply Cycle (PI: Stuart, Purdue University) 

• EPA-R829576 Composite Resins and Adhesives from Plants (PI: Wool, University 

of Delaware) 

The development of metabolic pathways for the synthesis of bioplastic monomers, such 

as 1,2-propanediol used in DuPont SoronaTM, of other small molecules that could become useful 

in biopolymeric materials, and of biopolymers themselves, are central challenges for 

biomaterials engineering that have been supported through several TSE grants. 

• EPA-R824726 Fermentation of Sugars to 1,2-Propanediol by Clostridium 

thermosaccharolyticum (PI: Cameron, University of Wisconsin-Madison) 

• EPA-R826116 Environmentally Benign Synthesis of Resorcinol from Glucose (PI: 

Frost, Michigan State University) 

• NSF-9985421 Metabolic Engineering of Carbon Fixation and Utilization for 

Biopolymer Production by Cyanobacteria (PI: Stephanopoulos, MIT) 

In addition, the abiotic polymerization of biosynthetic monomers, as well as the 

biocatalysis of polymerization of other monomers, offer the potential to minimize pollution by 

creating biodegradable products or using non-toxic catalysts, respectively. 

• EPA-R826733 Environmentally Benign Polymeric Packaging from Renewable 

Resources (PI: Dorgan, Colorado School of Mines) 

• NSF-9613166; EPA-R825338 Biocatalytic Polymer Synthesis in and from Carbon 

Dioxide for Pollution Prevention (PI: Russell, University of Pittsburgh) 

• NSF-9728366; EPA-R826123 Development of Green Chemistry for Synthesis of 

Polysaccharide-Based Materials (PI: Wang, Wayne State University) 

• EPA-R828131 Biocatalytic Polyester Synthesis (PI: Russell, University of 

Pittsburgh) 

• EPA-831436 Plant-Derived Materials to Enhance the Performance of Polyurethane 

Materials (PI: Nelson, University of Massachusetts, Amherst) 

Efforts to develop enzymes for non-polluting production of pulp and paper, by far the 

most widely-used biomaterials in developed countries, have also been supported. 

• NSF-0328031; NSF-0328033 Collaborative Research: Production and Use of a 

Ligninolytic Enzyme for Environmentally Benign Paper Manufacturing (PIs: Kelly, 

Syracuse University and Scott, SUNY College of Environmental Science and 

Forestry) 

Finally, the sustainable production of biomass has been supported as well. 

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• EPA-R831421 Cost-effective Production of Baculovirus Insecticides (PIs: 

Murhammer, University of Iowa; Bonning, Iowa State University) 

5. TSE Contributions to the Development of Bioenergy and Biofuels 

Bioethanol is one of the most promising biofuels, and the TSE Program has supported 

numerous projects that have addressed its primary challenges in the development of novel 

feedstocks, biomass pretreatment technologies, metabolic pathways to allow simultaneous 

fermentation of xylose and glucose resulting from lignocellulose hydrolysis, and alcohol 

tolerance of fermentative microbes. 

• NSF-9613342 Conversion of Paper Sludge to Ethanol and Potentially Recyclable 

Minerals (PI: Lynd, Dartmouth College) 

• NSF-9727096 Cellulose Conversion Using Aqueous Pretreatment and Cellulose 

Enzyme Mimetic (PI: Ladisch, Purdue University) 

• NSF-9817069 Novel Compressed Solvent Extraction Processes for Enhanced 

Biomass Conversion by Thermophilic Bacteria (PI: Knutson, University of 

Kentucky) 

• EPA-R826118 Development of Biotechnology to Sustain the Production of 

Environmentally Friendly Transportation Fuel Ethanol from Cellulosic Biomass (PI: 

Ho, Purdue University) 

• NSF-9911231; EPA-R828562 Metabolic Engineering of Solvent Tolerance in 

Anaerobic Bacteria (PI: Papoutsakis, Northwestern University) 

• NSF-9985351 Dissolution and Kinetic Fundamentals Underlying Novel Cellulosic 

Biomass Pretreatment Technologies (PI: Wyman, Dartmouth College) 

• EPA-R831645 Pretreatment of Agricultural Residues Using Aqueous Ammonia for 

Fractionation and High Yield Saccharification (PI: Lee, Auburn University) 

In addition, the TSE Program has supported projects addressing both fermentative and 

photosynthesis-based H2 production, as well as one investigating whole-cell biocatalysis with 

relevance to biodiesel production. 

• NSF-0124674 Biological Hydrogen Production as a Sustainable Green Technology 

for Pollution Prevention (PI: Logan, Pennsylvania State University) 

• NSF-0124821 Combinatorial Mutagenesis of a Bidirectional Hydrogenase in 

Chlamydomonas reinhardtii (PI: Ahmann, Colorado School of Mines) 

• NSF-0327902 Modulating Cell Permeability for Whole-Cell Biocatalysis in Chemical 

Synthesis (PI: Chen, Virginia Commonwealth University) 

• NSF-0328187 Functional and Structural Analysis of Algal Hydrogenase 

Combinatorial Mutants (PI: Ahmann, Colorado School of Mines) 

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Office of Research 

and Development (8101R)

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January 2007

www.epa.gov

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