United States
Environmental Protection
Agency
Bioengineering for Pollution Prevention
Through Development of Biobased Materials and Energy
S T A
OF THE SCIENCE REPORT
Office of Research and Development
National Center for Environmental Research
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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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Ill
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 petroleum-
based 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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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 non-
photolytic 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 CC>2 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 USD A/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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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 Ill
4. Commercialization Ill
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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IX
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-l
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XI
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
CC>2 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 l,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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Xll
HA
IPTG
ISO
JGI
LCA
LCIA
LDPE
MF
MFA
Mg
MG
Mg2+
Mn2+
mRNA
MTBE
MW
NADPH
NADP
NCBI
NIH
NMR
NREL
NRMRL
NSF
PCR
PE
PEA
PET
PHA
PHB
PLAs
Poly (3HB)
PP
PPM
ppm
PS
PS I
PS II
PVC
[(R)-3HAs]
RISC
RNA
SEM
siRNA
Sn
S02
Hydroxyalkonate
Isopropyl-p-D-thiogalactoside
International Organization for Standardization
Joint Genome Institute
Life-cycle Analysis
Life-cycle Impact Assessment
Low-density Polyethelyne
Microfiltration
Metabolic Flux Analysis
Magnesium
D, L-3-methyl glycolide
Magnesium (II) Ion
Manganese (II) Ion
messenger RNA
Methyl Tertiary Butyl Ether
Molecular Weight
Reduced form of Nicotinamide Adenine Dinucleotide Phosphate
Nicotinamide Adenine Dinucleotide Phosphate
National Center for Biotechnology Information
National Institutes of Health
Nuclear Magnetic Resonance
National Renewable Energy Laboratory
National Risk Management Research Laboratory
National Science Foundation
Polymerase Chain Reaction
Polyethylene
Polyester Amide
Poly(ethylene terephthalate)
Polyhydroxyalkanoate
Polyhydroxybutyrate
Polylactic Acids
Poly [(R)-3-hydroxybutyrate]
Polypropylene
Primary Packaging Material
parts per million
Polystyrene
Photosystem I
Photosystem II
Polyvinyl Chloride
(R)-3 -hy droxy alkanoates
RNA-induced Silencing Complex
Ribonucleic Acid
Scanning Electron Microscope
short interfering RNA
Tin
Sulfur Dioxide
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Xlll
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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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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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
(CH/t), as well as sulfurous gases such as sulfur dioxide (802). 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 (USD A) 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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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 sustainably-
produced 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, CC>2, 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.
Crystalline array Cellulose
of molecules in microfibril
a micelle i
Individual cellulose
molecules j-
Cellulose
molecules
Polysaccharides
(other than
cellulose)
Reprinted from Purves et al., Life: The Science of Biology, 4th Edition, by Sinauer Associates
(www.sinauer.com) and WH Freeman {www.whfreeman.com), with permission.
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2. Engineering Optimal Organisms
Genetic and metabolic engineering techniques are now being used to address the
microbial and enzymatic problems of biocommodity production from hundreds of different
angles. The central goal, in virtually all cases, is the development of organisms that can use low-
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 well
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 technologies must play important roles in the
development of economically feasible biorefmeries, 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 biorefmeries 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. Important considerations in the overall sustainability of both
biofuels 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 biofuel
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 energy
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-waste-
generating 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 biologically-
modified 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 37percent 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/woiidoilsupplv/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 LoomingPVC Waste Crisis, Amsterdam,
The Netherlands, http://archive.greenpeace.org/comms/pvctoys/reports/
1 oomingcontents. html.
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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/Resource s/F act_Sheets/Key_Stage_4 /Waste/0 5. 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). Biohydrogenproduction: prospects and
limitations to practical application, Intl J Hydrogen Energy 29:173-185.
(18) Salehizadeh, H., and M. C. van Loosdrecht (2004). Production ofpolyhydroxyalkanoates
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 ofbiotech 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. Wiley-
VCH.
(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, genome-
scale, 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 GenBankฎ, 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 Ptoc. 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 represser and is inducible by addition of isopropyl-B-D-
thiogalactoside (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 1.28xl.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 reverse-
transcribed 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 adduct-
producing 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 non-
proofreading 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/Oea5cbe470/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 one-
dimensional 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 Swiss-
Prot 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.embl-
heidelberg. 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 site-
directed 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 ofde 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 Upases 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
insert!onal 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, biomass-
derived 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 (CC) 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.
4. References
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(9) Baneyx, F., Ed. (2004). Protein Expression Technologies: Current Status and Future
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(10) Manoharan, M. (2004). RNA interference and chemically modified small interfering
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(12) Cummings, C. A., and D. A. Relman (2000). UsingDNA microarrays to study host-
microbe interactions, Emerg Infect Dis 6:513-525.
(13) Synthegen (2005). Table of Fluorescent Dyes, Synthegen Corporation,
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(14) Gershon, D. (2002). Microarray technology: an array of opportunities, Nature
416:885-891.
(15) Goldsmith, Z. G., and N. Dhanasekaran (2004). The microrevolution: applications and
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(16) Friedberg, E. C., and G. C. Walker (1995). DNA Repair andMutagenesis. ASM Press,
Washington, D.C.
(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) 0stergaard, 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:el76.
(24) Crameri, A., S.-A. Raillard, E. Bermudez, and W. P. C. Stemmer (1998). DNA shuffling
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 ofthymidine kinase for AZTphosphorylation 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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(28) Pain, R. H. (2000). Mechanisms of Protein Folding, 2nd Edition. Oxford University
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(29) Schlick, T. (2002). Molecular Modeling and Simulation. Springer.
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Press, Totowa, NJ.
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http://www.rsc.org/chemistryworld/.
(33) Lehmann, M., and M. Wyss (2001). Engineering proteins for thermostability: the use of
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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design, Proc Natl Acad Sci 98:14274-14279.
(36) Bolon, D. N., C. A. Voigt, and S. L. Mayo (2002). De novo design ofbiocatalysts, Curr
Opin Chem Biol 6:125-129.
(37) McKnight, S. L., and R. Kingsbury (1982). Transcriptional control signals of a
eukaryotic protein-coding gene, Science 217:316-324.
(38) Wikipedia (2005). Proteomics, http://en.wikipedia.org/wiki/Proteomics.
(39) Liebler, D. C. (2001). Introduction to Proteomics: Tools for the New Biology. Humana
Press, Totowa, NJ.
(40) Twyman, R. M. (2004). Principles of Proteomics. BIOS Scientific Publishers, New
York.
(41) Westermeier, R., and T. Naven (2002). Proteomics in Practice: A Laboratory Manual of
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 tofluxomics and
physiomicspathway engineering, Curr Opin Microbiol 5:318-322.
(44) De Boer, A. L., and C. Schmidt-Dannert (2003). Recent efforts in engineering microbial
cells to produce new chemical compounds, Curr Opin Chem Biol 7:273-278.
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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 ofbiotech indigo, J Ind Microbiol
Biotechnol28:127-133.
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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24
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 by-
products 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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25
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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26
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, u,
is related to the substrate concentration, S, in a nonlinear form (Equation 1). The parameters Ks
and |j,max, describing the half-saturation constant and maximum specific growth rate, respectively,
are determined experimentally (16).
Is]
(Equation 1 )
'max
Ks
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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27
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).
:
:
,-.
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]
ilt
h
,i1tU1tIUปHi
I.ttMittlirmn
,,,,,
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.iiMttlllllirf,.
.iiintimiM,,
Instantaneous results are shown on the left, time averages on the right.
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28
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 non-
Newtonian 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 perfectly-
designed 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).
500
250
Pyridoxin
Tryptophan
300 350 400 450 500
Emission [n m]
550
600
the processing of sugar beet molasses at an industrial scale (35). Glucose, fructose, glutamine,
ammonia, CC>2, 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 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.
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 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
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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).
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2002
5. References
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(6) Liao, J. C., and E. N. Lightfoot (1988). Characteristic reaction paths of biochemical
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(7) Delgado, J., and J. C. Liao (1992). Determination of flux control coefficients from
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(12) Varma, A., and B. O. Palsson (1993). Metabolic capabilities ofE. coli //. Optimal
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(13) Varma, A., and B. O. Palsson (1994). Stochiometric flux balance models quantitatively
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Environ Microbiol 60:3724-3731.
(14) Sonnleitner, B., and O. Kappeli (1986). Growth ofS. cerevisiae is controlled by its
limited respiratory capacity: formulation and verification of a hypothesis, Biotechnol
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modeling of the microbial cell for computer simulation, J Biotechnol 113:281 -294.
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(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., andR. 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 Coprocessing
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 ofphysiochemical 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 ofbiomass 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. 1, 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-cell-
density E. coli cultures using GFP-fusion monitoring, Biotechnol Bioeng 69:275-285.
(41) Li, 1, 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). Bioreactorperformance: a more scientific
approach for practice, J Biotechnol 85:187-212.
(43) Agger, T., A. B. Spohr, and J. Nielsen (2001). Alpha-amylaseproduction in high cell
density submerged cultivations o/"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).
Bioreactor
Intracellular products
^
Cell harvesting
Centrifugation
Microfiltration
Ultrafiltration
1
Cell disruption
Homogenization
Bead milling
Osmotic shock
Cell debris removal
Centrifugation
Microfiltration
Vacuum filtration
Press filtration
^
Product extr
Aqueous tw
Organic sol
Batch absor
Supercritica
Reverse mi
Extractive c
fc-
IBs
i '
Renaturation
Dissolution
Refolding
High purity required i
^
Final Purification
Chromotography
(affinity, reversed
phase, ion exchange,
size exclusion, etc.)
Crystallization
Fractional
precipitation
Diafiltration
Extracellular Products
r
iction by
o phases
vents
ed absorption
ption
1 fluids
Belles
istillation
+
Biomass removal
Vacuum filtration
Centrifugation
Microfiltration
Ultrafiltration
Press filtration
Candle filtration
Flotation
r
Concentration
Ultrafiltration
Evaporation
Reverse osmosis
Precipitation
Extraction
Adsorption
Distillation
' Low purity required
For solid final form ^
I
^
Dehydration or
solvent removal
Spray drying
Freeze drying
Tray drying
Fluid bed drying
Drum drying
Primary
Recovery
Stages
Intermediate
Recovery
Stages
Final
Purification
Stages
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, PHA-
containing 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)J and poly(3HB-co-3 V) by Alcaligenes eutrophus. In this process, cells are brought
to 80ฐC 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 non-
solvent based methods of cell lysis, including modern developments in enzymatic, chemical, and
mechanical cell disruption, are considered in detail in the recent book, Bioseparatiom 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 size-
based 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 jam-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 virus-
like 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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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 bio-
based 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 ultra filtration. Microfiltration and ultrafiltration promise to
become major unit operations in the emerging biorefmery 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 CC>2, a highly compressed phase of CC>2 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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(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 ofthepoly-
beta-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 organic-
organic 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 cross/low 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 ofsoluable 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, counter current
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 ofplasmidDNA by tangential flow filtration, Biotechnol Bioeng 69:101-106.
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(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-like-
particles 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, andBioseparation (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 ultrafiltrationprocess 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 10year review, J Membr Sci 179:1-27.
(29) Laffend, L. A. (1997). Byconversion 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 lower-
grade 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 (l)-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 promisenamely 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 CC>2 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 Natureworks (http://www.packexpo.com/ve/82489/main.html); the production of
Sorona 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 ฐC 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 + H2O
Plants Bacteria Catalyst
> Sugars > Lactic acid > Polylactide
Photosynthesis
Fermentation
Chemosynthesis
2-Stage Production ( Biopolyesters)
CO2 + H2O
Photosynthesis
3. 1-Stage Production ( Biopolyesters)
Plants Bacteria
> Sugars, Plant oils > Biopolyesters
Fermentation
CO2 + H2O
Plants, Bacteria, Algae
> Biopolyesters
Photosynthesis
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 CCh 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 polyolefms 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 CtL; rather than pure CO2. Because CtL; 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/industrv/impact.htm.
(2) Dreffeyes, K. S. (2001). Hubberfs 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). Modem 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.
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(11) Skraly, F. (2002). Bioplastics,m 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 AssessmentPrinciples 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/
Iibrary/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 NatureWorkspolylactide (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 PL A 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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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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Figure 7. Commercial route to biobased PLA plastics.
Corn
Starch
(65%
Starch
Unrefined
Dextrose
Fermentation
Lactic Acid
Starch & Gluten
(5.3%)
__ & Fiber
(23%)
(6.7%)
Applications
Modified
for customer
needs
Poly lactic.
Acid
Monomer
Product/on
Lactide
I
Polymer
Production
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).
Nylon 66 Nylon 6
HIPS Ceซopliane GPPS LDPE PETSSP PP
Fossil fuels 13 Fossil feedstock
PET AM PLA1
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 PL A 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 PL A 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 CC>2 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 one-
third of the gross energy requirement in PLA production; in addition, processing of oil into
conventional plastics releases even greater amounts of CC>2 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.
Poly(L-lactide)
H .CH-ฃ>
H
H3Cฐ "H
Poly(D-lactide)
H,
ฐ\^
6 M CH3o H3C" "H
Poly(D,L-lactide)
M
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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 145-186ฐC (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 230ฐC (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 non-
woven 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 > 150ฐC) 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 molecular-
weight 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 molecular-
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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 = 97ฐC, or Meso-lactide with melting point = 52ฐC,
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 126-127ฐC (16).
Figure 10. Types of lactide: Meso-lactide, L-lactide, and D-lactide.
o o o
H
O T"CH3 O-
H
H3C II H
O O O
Meso-lactide L-lactide D-lactide
O ii CH3n o H CH3n O H3C n
poly(Meso-lactide) poly(L-lactide) poly(o-lactide)
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, coordination-
insertation is the most prevalent and industrially most important. Polymerization reactions may
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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).
Tin(ll) bis(2-ethyl-hexanoate)
Sn(Oct)2
H
R
Oct2-Sn--O
H
.R
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/?-, d-, or/- 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 180ฐC. 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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57
Both kinetics and mechanisms of tin octoate polymerizations have been well
characterized at both low (64) and high (>180ฐC) 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 Mark-
Houwink 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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59
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 non-
renewable 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 CC>2, 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, CC>2, and CFL; in very thin (5 jim) 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 HID.) 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 HID.) 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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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 CC>2 than do aerobic processes (210). The success of the DuPont
Sorona 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
(p-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, p-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 P-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 P-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 ฐC containing depolymerases (18).
P. stutzeri PHB depolymerase (Type A)
A. faeca/is PHB depolymerase (Type A)
C. acidovorans PHB depolymerase (Type B)
0.05 0.10 0.15 0.20 0.25
Rate of erosion (mg/cm'/h)
0.30
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-co-
3HHx) (25). Feeding 4-hydroxybutyric acid, 1,4-butanediol, or butyrolactone as the carbon
source produces a random copolyester of (R)-3FฃB and 4-hydroxybutyrate, poly(3FฃB-co-4FฃB)
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 p-oxidation mR. eutropha, such that the microbe accumulated a copolymer of both short-
and 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,
Pseudomonasputida, Pseudomonas oleovorans, and Escherichia coll, 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 Aeromonaspunctata 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-CoAan
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 Ambidopsis thaliona 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 ultra-
high 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
poly(3HB),
amorphous
poly(3HB),
crystalline
ultra-high Mw
poly(3HB)
PP
Density
g/cm
1 18
1.26
Tg
ฐC
4
Tm
ฐC
180
Crystal
structure
ortho-
rhombic
Confor-
mation
left-handed
2i helix
Morph-
ology
lamellar
lamellar
Young's
modulus
GPa
3.5
1.1
Tensile
strength
MPa
43
62
Extension
to break
percent
5
58
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
poly(3HB-co-3HV)a
poly(3HB-co-3HHx)b
poly(3HB-co-4HB)c
non-HB mole
fraction
percent
various
0
17
25
0
16
49
64
100
Crystallinity
percent
50-70
60
18
60
14
Tm
ฐC
177
52
Tg
ฐC
4
-4
Tensile
strength
MPa
43
20
43
26
17
104
Elongation to
break
percent
6
850
5
444
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(s-caprolactone-co-lactide) (53),
poly(butylene succinate-co-butylene adipate) and poly(butylene succinate-co-s-caprolactone)
(54). Immiscible blends are formed in mixtures of poly(3HB) with poly(p-propiolactone)
(55, 13), poly(ethylene adipate) (55), poly(butylene adipate) (56), and poly(s-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(s-caprolactone) with poly(3FฃB), 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
Biopol by ICI, Zeneca, and Monsanto. Metabolix recently acquired the Biopol 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 chain-
length monomers under the trade name Nodax 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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hydroxbutyrate-co-3-hydroxyproprionate), Polymer 34:4782-4786.
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Physical properties and biodegradability ofmicrobialpoly(3-hydroxybutyrate-co-3-
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co-4-hydroxybutyrate) by Alcaligenes latus, Biotechnol Lett 15:461-464.
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16:99-104.
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poly(b-hydroxyalkanoates) obtained from by Pseudomonas oleovorans grown with
mixtures of 5-phenylvaleric acid and n-alkanoic acids, Macromolecules 24:5256-5260.
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(33) Van der Leij, F. R., and B. Witholt (1995). Strategies for the sustainable production of
new biodegradable polyesters in plants: A review, Can J Microbiol(Suppl) 41:222-238.
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Polyhydroxybutyrate, a biodegradable thermoplastic produced in transgenic plants,
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biosyntheticpathway to the plasmids o/"Arabidopsis thaliana results in high levels of
polymer accumulation, Proc Natl Acad Sci USA 91:12760-12764.
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(36) Mittendorf, V., E. J. Robertson, R. M. Leech, N. Kruger, A. Steinbuchel, and Y. Poirier
(1998). Synthesis oj"medium-chain-lengthpolyhydroxyalkanoates in Arabidopsis
thaliana using intermediates of peroxisomalfatty acid b-oxidatioin, Proc Natl Acad Sci
USA 95:13397-13402.
(37) Snell, K. D., and O. P. Peoples (2002). Polyhydroxyalkanoate polymers and their
production in transgenic plants, Metab Eng 4:29-40.
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(39) Barnard, G. N., and J. K. M. Sanders (1989). Thepoly-b-hydroxybutyrate granule in
vivo: a new insight based on NMR spectroscopy of whole cells., J Biol Chem 264:3286-
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(40) Amor, S. R., T. M. Rayment, and J. K. M. Sanders (1991). Poly(hydroxybutyrate) in
vivo: NMR and x-ray characterization of the elastomeric state, Macromolecules
24:4583-4588.
(41) Lundgren, D. G., R. Alper, C. Schnaitman, and R. H. Marchessault (1965).
Characterization of poly-b-hydroxybutyrate extracted from different bacteria, J B acted ol
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(42) Iwata, T., and Y. Doi (1999). Crystal structure and biodegradation of aliphatic polyester
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(43) Khanna, S., and A. K. Srivastava (2005). Recent advances in microbial
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
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(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 inpoly(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 ofpoly(3-hydroxybutyrate)-
based blends: poly(3-hydroxybutyrate)/poly(ethylene oxide) blend, Polym Deg Stabil
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(50) Azuma, Y., N. Yoshie, M. Sakurai, Y. Inoue, and R. Chujo (1992). Thermal behaviour
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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 ofpoly[(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[e-
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(54) He, Y., T. Masuda, A. Cao, N. Yoshie, and Y. Doi (1999). Thermal, crystallization and
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(55) Kumagai, Y., and Y. Doi (1992). Enzymatic degradation of binary blends ofmicrobial
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256.
(56) Kumagai, Y., and Y. Doi (1992). Enzymatic degradation and morphologies of binary
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(57) Peoples, O. P., and A. J. Sinskey (1996). Polyhydroxybutyratepolymerase, U.S. Patent
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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 (IPS)
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 water-
soluble 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 (90-180ฐC), under pressure and shear stress, starch granules melt and flow to give
an amorphous material called IPS, 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(ethylene-
co-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 lattice-
fluid 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-a-l,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(l,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,
Biomaxฎ, 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 (3 A, 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]).
93
Ring Opening
Polymerization
t
Free-Radical
Polymerization
3A
3B
Polycondensation
X i O
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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 300ฐC, with the consequence that its use is limited to
plastics that can be processed at lower temperatures. Fortunately, that is not an obstacle for soy-
and 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 sheet-
shaped 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 three-
fold 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 CC>2, 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-Bi
Mater-Bi 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-Bi 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-Bi biodegrade on
average, in the time of one composting cycle. Although Mater-Bi was developed only in the
late 1980s, and Novamont was founded only in 1990, Mater-Bi products have already become
internationally visible. The product called Green Pen, made of Mater-Bi 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 Mater-
Bi bags for separate waste collection began in Germany. Research collaborations with
Goodyear Tire began in 1995, and in 2001 production of the GTS tire began, using Biotred
technology that uses biofillers made of Mater-Bi. Production of Mater-Bi 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-Bi (www.materbi.com).
Mater-Bi 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-Bi
(www.polargruppen.com: www.biobag.ca) that are largely ASTM, DIN CERTCO, and EK
certified.
4.2 Ecoflexฎ
In 1998, the German company BASF introduced a family of biodegradable aliphatic-
aromatic copolyesters, including formulations consisting of thermoplastic starch, under the brand
name Ecoflexฎ. Ecoflexฎ 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 Ecoflexฎ, 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, Ecoflexฎ is water-
proof, 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 Ecoflexฎ with PLA under the tradename Ecovioฎ.
4.3 PPM
PPM was developed by Biosphere Industries from cellulose-reinforced starch to meet
rigid packaging needs and has two primary grades, PPM 100 and PPM200, which are currently
used primarily in food-service items and general packaging. PPM 100 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 SoyPlus. 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.
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(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 andpolysaccharide fillers, Recent Research Developments in Macromolecules
Research 3(2):455-474.
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microcrystalline starch reinforced thermoplastic, Macromolecules 29:7624-7626.
(45) Dufresne, A., M. B. Kellerhals, and B. Witholt (1999). Transcrystallization in mcl-
PHAs/Cellulose whisker composites, Macromolecules 32:7396-7401.
(46) Dufresne, A., and M. R. Vignon (1998). Improvement of starch film performances using
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2:476-482.
(48) Grunert, M., and W. Winter (2000). Progress in the development of cellulose reinforced
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Chapter IV
Biofuels
The worldwide depletion of fossil fuels and widespread concern over increasing
atmospheric CC>2 have sparked interest not only in biomaterials but also in sustainable, non-
fossil-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 CC>2, 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
CC>2 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 semi crystalline 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 enzyme-
producing 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, CC>2, 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 xylose-
fermenting 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 El 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
El; currently they are pursuing El 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 heat-
activated 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: p-l,4-endoglucanases, P-1,4-
cellobiohydrolases, xylanases, p-glucosidases, a-L-arabinofuranosidase, acetyl xylan esterase, P-
mannanase, and a-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 cellobiose-
utilizing 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
(>55ฐC) 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, CC>2, 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 logen 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
OxyNol concentrated acid hydrolysis technology in New York (3).
Petro-Canada, the second largest petroleum refining company in Canada, began to co-
fund research and development on biomass-to-ethanol technology with logen in 1997. Petro-
Canada, logen, and the Canadian government then began plans to fund construction of a
demonstration plant based on logen's cellulase enzyme technology in an SSF process (3). The
plant of logen, a leading producer of cellulases, has completed a 40 ton per day biomass-to-
ethanol 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).
5. References
(1) Turner,!. (1999). A realizable renewable energy future., Science 285:687-689.
(2) United States Department of Energy (2003). Biofuelsfor Sustainable Transportation.
Office of Energy Efficiency and Renewable Energy, http ://www. ott. doe, gov/biofuels/
advanced_bioethanol .html.
(3) United States Department of Energy (2003). AdvancedBioethanol Technology., Office of
Energy Efficiency and Renewable Energy, http://www.ott.doe.gov/biofuels/
advanced bioethanol .html.
(4) Kaltschmitt, M., G. Reinhardt, and T. Stelzer (1997). Life cycle analysis ofbiofuels
under different environmental aspects, Biomass Bioenergy 12:121-134.
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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 costto 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
200ฐC (392ฐF) and 370ฐC (698ฐF), 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 CieHs^ 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 lisa kerosene-
type 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).
catalyst
triglyceride
+ 3 R'OH
alcohol
fatty acid esters
H2COH
+ HCOH
H2COH
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).
Diesel Fuel Ignition
Intake Fuel Exhaust
Valve Injector valve
Intake
Air
Cylinder
Mead -
Exhaust
Cy -idL-
'
Piston
Crankshaft
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 Ipound) 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 25-100ฐC 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
Peanut
Soybean
Soybean
Babassu
Palm
Palm
Sunflower
Tallow
Rapeseed
Used rapeseed
Used corn oil
Diesel fuel
JIS-2D (Gas oil)
Kinematic
viscosity
(mm2/s)
4.9 (37.8ฐC)
4.5 (37.8ฐC)
4.0 (40ฐC)
3.6 (37.8ฐC)
5.7 (37.8ฐC)
4.3-4.5 (4OฐC)
4.6 (37.8ฐC)
~
4.2 (40ฐC)
9.48 (30ฐC)
6.23 (30ฐC)
12-3.5 (40ฐC)
2.8 (30ฐC)
Cetane Lower heating
number value (MJ/1)
54
45
45.7-56
63
62
64.3-70
49
~
51-59.7
53
63.9
51
58
33.6
33.5
32.7
31.8
33.5
32.4
33.5
~
32.8
36.7
42.3
35.5
42.7
Cloud Flash
point point
5 176
1 178
~
4 127
13 164
~
1 183
12 96
~
192
166
~
59
Density
(g/1)
0.883
0.885
0.880
0.879
0.880
0.872-0.877
0.860
~
0.882
0.895
0.884
0.830-0.840
0.833
Sulfur
(wt
percent)
~
~
~
~
~
~
~
0.002
0.0013
~
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 350ฐC 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 Upases, 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 pre-
existing 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 60-70ฐC 30-40ฐC
Free fatty acids in raw materials Formation of undesired Formation of desired methyl esters
saponified products
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
fromMucor miehei (Lipozyme IM-20) and Candida antarctica (Novozym 435), as well as from
Pseudomonas cepacia, Candida mgosa, 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 Upases 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 product!on 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 pastor is (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 Pseudomonasfluorescens
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
ofFusarium, 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 Upases, 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, Upases 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 Upases 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 Upases: 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 easily-
recoverable coproducts, principally glycerol. Specifically, genetic engineering of Upases 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-
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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 city-
wide 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
(1) Fukuda, H., A. Kondo, and H. Noda (2001). Biodiesel fuel production by
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). Biodieselfuels 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). BiodieselFAQs, 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.eom/encyclopedia/c/ce/cetane.html.
(7) Ma, F., and M. A. Hanna (1999). Biodiesel production: A review., Bioresource Technol
70:1-15.
(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/petrolprices across Europe, http://www.see-
search.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,
JMolec 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 rapeseedoil 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 rapeseedoil
as treated in supercritical methanol, J Chem Eng Jpn 34:383-387.
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(22) Saka, S., and D. Kusdiana (2001). Biodieselfuelfrom rapeseed oil as prepared in
supercriticalmethanol, 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). Lipasesfor 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 andbiocatalysis, 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, JBiosci 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. Kama, 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) Kama, 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. Kama, 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. Kama, 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.
(40) National Biodiesel Board (2004). In the News, National Biodiesel Board,
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 Fe-
hydrogenase 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.
Photosystem II
Photosystem I
-1.5-I
-1.0-
-0.5-
Em (Volts)
0.5-
1.0-
1.5J
(green algae)
FNR
*NADP>NADPH
* ป
C02 H2ase
fixation \
H2
(cyanobacteria)
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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132
Figure 17. The mixed-acid fermentation of Escherichia coli, an example of a
common H2-producing fermentative pathway, showing H2 production from
formate (8).
K QJycose
NADU
NADH
PEP-
Qxatoaeetaie
ATP'
^>
-^
Aspaitaic
-> Fumarale -^ >
HADH
Pyruvate ^*
AcclyJ-P
V
Acetjl-CoA ,mm
AcetaJdehyde
ATP
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 Reproducing 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)
hv
NADH
H2
Pyruvate ^
Reduced
Ferredoxin
Nilrogeoase
16
16ADP
+ 16
N,
s, Nitrogenase f 8
2NH
H
Garret! & Grisham: Biochemistry, 2/e
Figure 26.6
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 Q^ 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 CC>2, some are able to fix
atmospheric CC>2 by other biochemical pathways. Despite these differences, energy transduction
is carried out by mechanisms quite similar to those found in oxygenic phototrophs: the light-
harvesting 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 CC>2 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).
Pheophytin-Quinone Type
Reaction Centers
Iron-Sulfur Type
Reaction Centers
Purple & Filamentous
Green Bacteria
Plants, Algae and Cyanobacteria
-1.2 r
Green Sulfur Bacteria &
Heliobacteria
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135
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 non-
sulfur 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, O,
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 light-
harvesting 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 6, 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 light-
harvesting 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 Dl 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 lOOh (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 Fe-
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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 site-
directed 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 ofPandorina 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 Chlorellafusca, 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, higher-
input 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 H2 synthesis rate
(reported units) (converted units)
Direct photolysis 4.67 mmol H2/l/80 h 0.07 mmol H2/(l x h)
Indirect photolysis 12.6 nmol H2/ug protein/h 0.355 mmol H2/(lxh)
Photo-fermentation 4.0mlH2/ml/h 0.16 mmol H2/(lxh)
CO-Oxidation by R. gelatinosus 0.8 mmol H2/g cdw/min 96.0 mmol H2/(lxh)
Dark-fermentations
Mesophilic, pure strain8 21.0 mmol H2/l 1/h 21.0 mmol H2/(lxh)
Mesophilic, undefined15 1,600.0 1 H2/m3/h 64.5 mmol H2/(l x h)
Mesophilic, undefined 3.01H2/l/h 121.0 mmol H2/(lxh)
Thermophilic, undefined 198.0 mmol H2/l/24 h 8.2 mmol H2/(lxh)
Extreme thermophilic, pure strain0 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.
0 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 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
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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 proteinsthe dinitrogenase and the
dinitrogenase reductase. The former is an alpha2beta2 heterotetramer, with alpha and beta
subunits encoded by the genes nifD and ni/K, 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 energy16 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 site-
specific 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 light-
saturation 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 wild-
type 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 -ป (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:
C02(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 25-40ฐC), thermophilic (40-65ฐC), extremely thermophilic (65-80ฐC) or
hyperthermophilic (>80ฐC). 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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(99) Das, D., and T. Veziroglu (2001). Hydrogen production by biological processes: A
survey of literature, Intl J Hydrogen Energy 26:13-28.
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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
C02.
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 MC50l,Mycobacterium G3, Gordona CYKS1, Sphingomonas AD109, and
strains ofKlebsiella, 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).
SULFUR
FOSSIL FUEL COMPOUND CHEMICAL DISTILLATION
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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 sulfmate, again using FMNH2 and
cleaving one carbon-sulfur bond. The remaining carbon-sulfur bond is then cleaved by a most
unusual enzyme, the desulfmase DszB, releasing sulfite and hydroxybiphenyl, HBP (1, 2, 7, 8).
The availability of FMNH2 for the two mono-oxygenases appears to be a crucial rate-
limiting 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).
Ctitrenl Opinion in
Although the Dsz operon is transcriptonally 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 gain-
of-function mutants yielded,/?, 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 ofdszC
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 55ฐC (7); the discovery of Bacillus
subtilis WU-S2B, able to desulfurize DBT at 50ฐC (16); and Mycobacterium phlei GTIS10, also
able to function at temperatures >50ฐC (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 Raffmage 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, annually-
increasing 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 andsulfate
esters in Gram-negative bacteria., FEMS Microbiol Rev 24:135-175.
(3) Song, C., and X. Ma (2003). New design approaches to ultra-clean dieselfuels 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 1-19, 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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(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 WU-
S2B, 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 biorefiningprocesses, 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 de sulfur ization 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 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 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 biorefmery 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 sterically-
interacting 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 CC>2,
a highly compressed phase of CC>2 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, 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 HID.) 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 HID.) 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 CC>2 than do aerobic processes.
The success of the DuPont Sorona 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.
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 plant-
based 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 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.
- 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 biorefmery 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 Upases 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 light-
saturation 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 Reproducing 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.
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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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 Enzyme-
Catalyzed 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 CC>2, 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 Sorona, 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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