DOE/SC-0095
 

Breakng the Bologcal Barrers 


to Cellulosc Ethanol:
 

A Jont Research Agenda
 

A Research Roadmap Resultng from the
 

Bomass to Bofuels Workshop
 

December 7–9, 2005
 

Rockvlle, Maryland
 

Workshop Sponsored 

by the 

U.S. Department of Energy 

Office of Energy Efficency Office of Scence 

and Renewable Energy Office of Bologcal and Envronmental Research 

Office of the Bomass Program Genomcs:GTL Program 

Publcaton Date: June 2006
 


 
 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Executve Summary 

“We’ll also fund additional research in cutting-edge methods of producing 

ethanol, not just from corn, but from wood chips and stalks or switchgrass.”

— President George W. Bush, State of the Union Address, January 2006* 

Arobust fusion of the agricultural, industrial biotechnology, and 

energy industries can create a new strategic national capability for 

energy independence and climate protection. In his State of the 

Union Address (*Bush 2006), President George W. Bush outlined the 

Advanced Energy Initiative, which seeks to reduce our national dependence on imported oil by accelerating the development of domestic,

renewable alternatives to gasoline and diesel fuels. The president has set a 

national goal of developing cleaner, cheaper, and more reliable alternative 

energy sources to substantially replace oil imports in the coming years.

Fuels derived from cellulosic biomass—the fibrous, woody, and generally 

inedible portions of plant matter—offer one such alternative to conventional energy sources that can dramatically impact national economic 

growth, national energy security, and environmental goals. Cellulosic 

biomass is an attractive energy feedstock because it is an abundant, domestic, renewable source that can be converted to liquid transportation fuels.

These fuels can be used readily by current-generation vehicles and distributed through the existing transportation-fuel infrastructure. 

The Biomass to Biofuels Workshop, held December 7–9, 2005, was 

convened by the Department of Energy’s Office of Biological and Environmental Research in the Office of Science; and the Office of the Biomass Program in the Office of Energy Efficiency and Renewable Energy.

The purpose was to define barriers and challenges to a rapid expansion of 

cellulosic-ethanol production and determine ways to speed solutions 

through concerted application of modern biology tools as part of a joint 

research agenda. Although the focus was ethanol, the science applies to 

additional fuels that include biodiesel and other bioproducts or coproducts 

having critical roles in any deployment scheme. 

The core barrier is cellulosic-biomass recalcitrance to processing to ethanol.

Biomass is composed of nature’s most ready energy source, sugars, but they 

are locked in a complex polymer composite exquisitely created to resist 

biological and chemical degradation. Key to energizing a new biofuel 

industry based on conversion of cellulose (and hemicelluloses) to ethanol is 

to understand plant cell-wall chemical and physical structures—how they are 

synthesized and can be deconstructed. With this knowledge, innovative 

energy crops—plants specifically designed for industrial processing to 

biofuel—can be developed concurrently with new biology-based treatment 

and conversion methods. Recent advances in science and technological 

capabilities, especially those from the nascent discipline of systems biology,

promise to accelerate and enhance this development. Resulting technologies 

*www.whitehouse.gov/stateoftheunion/2006/ 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy  
 

 

 

 

 

Executive Summary 

will create a fundamentally new process and biorefinery paradigm that will 

enable an efficient and economic industry for converting plant biomass to 

liquid fuels.These key barriers and suggested research strategies to address 

them are described in this report. 

As technologies mature for accomplishing this task, the technical strategy 

proceeds through three phases: In the research phase, within 5 years, an 

understanding of existing feedstocks must be gained to devise sustainable,

effective, and economical methods for their harvest, deconstruction, and 

conversion to ethanol. Research is centered on enzymatic breakdown of 

cellulosic biomass to component 5- and 6-carbon sugars and lignin, using a 

combination of thermochemical and biological processes, followed by 

cofermentation of sugars to specified endproducts such as ethanol. Processes will be integrated and consolidated to reduce costs, improve efficacy,

reduce generation of and sensitivity to inhibitors, and improve overall 

yields and viability in biorefinery environments. 

The technology deployment phase, within 10 years, will include creation of 

a new generation of energy crops with enhanced sustainability, yield, and 

composition, coupled with processes for simultaneous breakdown of 

biomass to sugars and cofermentation of sugars via new biological systems.

These processes will have enhanced substrate range, temperature and 

inhibitor tolerance, and the capability to function in complex biorefining 

environments and over time scales that are economically viable. 

The systems-integration phase, within 15 years, will incorporate concurrently engineered energy crops and biorefineries tailored for specific 

agroecosystems. Employing new and improved enzymes for breaking 

biomass down to sugars as well as robust fermentation processes jointly 

consolidated into plants or microbes, these highly integrated systems will 

accelerate and simplify the end-to-end production of fuel ethanol. In many 

ways, these final-phase technologies will strive to approach theoretical 

conversion limits. The new generation of biotechnologies will spur engineering of flexible biorefineries operable in different agricultural regions of 

the country and the world. 

The success of this program for effectively converting cellulosic biomass to 

ethanol will be based on coupling sophisticated engineering with fundamental biological research. The new generation of biological research—

systems biology—is built on the national investment in genomics. Systems 

biology involves new technologies for increasingly detailed high-throughput analyses and computing to make transparent the complexities of 

biology and allow predictive understanding and rational design. Multidisciplinary team research approaches will accelerate scientific progress and its 

translation to new biorefinery processes. Comprehensive suites of technologies, some in researchers’ laboratories and some consolidated in 

facilities, will enhance technical performance, improve productivity, and 

reduce costs to allow affordable and timely progress toward these goals.

New research capabilities and facilities will serve as an engine for fundamental research, technology development, and commercialization. 

v Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Contents
 

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

Introduction..............................................................................................................................................................................................1
 

Joint Workshop Challenges Biofuel Science and Technology Communities..........................................................................................1
 

America’s Energy Challenges....................................................................................................................................................................2
 

The.Promise.of.Biofuels..........................................................................................................................................................................2
 

A Growing Mandate for Biofuels: Policy, Legislative, and Other Drivers...............................................................................................3
 

Benefits of Biofuels...................................................................................................................................................................................5
 

National.Energy.Security.Benefits..........................................................................................................................................................6
 

Economic.Benefits..................................................................................................................................................................................6
 

Environmental.Benefits..........................................................................................................................................................................7
 

Feasibility of Biofuels................................................................................................................................................................................8
 

Land.Availability.....................................................................................................................................................................................8
 

Agricultural.Sustainability.of.Biomass.Production................................................................................................................................12
 

Today.–.Fuel.Ethanol.Production.from.Corn.Grain.(Starch.Ethanol)......................................................................................................12
 

Tomorrow.–.Biorefinery.Concept.to.Produce.Fuel.Ethanol.from.Cellulosic.Biomass..............................................................................13
 

Ethical,.Legal,.and.Social.Issues.(ELSI)..................................................................................................................................................17
 

EERE OBP Platform for Integrated Biorefineries...................................................................................................................................17
 

DOE Office of Science Programs..............................................................................................................................................................19
 

Biomass to Biofuels Workshop: Creating a Common Research Agenda to Overcome Technology Barriers......................................22
 

Cited References......................................................................................................................................................................................24
 

Background Reading...............................................................................................................................................................................28
 

Technical Strategy: Development of a Viable Cellulosic Biomass to Biofuel Industry....................................................29
 

Research Phase (within 5 years).............................................................................................................................................................29
 

Feedstock.Use.and.Optimization..........................................................................................................................................................29
 

Deconstruction.....................................................................................................................................................................................30
 

Fermentation.and.Recovery.................................................................................................................................................................31
 

Technology Deployment Phase (within 10 years).................................................................................................................................32
 

Feedstocks............................................................................................................................................................................................34
 

Deconstruction.....................................................................................................................................................................................34
 

Fermentation.and.Recovery.................................................................................................................................................................34
 

Systems Integration Phase (within 15 years)........................................................................................................................................34
 

Integration.and.Consolidation..............................................................................................................................................................35
 

SYSTEMS BIOLOGY TO OVERCOME BARRIERS TO CELLULOSIC ETHANOL.................................................................................37
 

Lignocellulosic Biomass Characteristics........................................................................................................................................39
 

Structure and Assembly of Cell Walls.....................................................................................................................................................41
 

Factors in Recalcitrance of Lignocellulose Processing to Sugars..........................................................................................................44
 

Plant.Architecture................................................................................................................................................................................45
 

Cell-Wall.Architecture...........................................................................................................................................................................45
 

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CONTENTS 

Molecular.Structure..............................................................................................................................................................................45
 

Optimization of Plant Cell Walls.............................................................................................................................................................47
 

Understanding.Cell-Wall.Structure.and.Function..................................................................................................................................47
 

Control.of.Lignin.Synthesis.and.Structure.............................................................................................................................................49
 

Improved Methods, Tools, and Technologies.........................................................................................................................................50
 

Technical Milestones...............................................................................................................................................................................51
 

Cited References......................................................................................................................................................................................56
 

Feedstocks for Biofuels.......................................................................................................................................................................57
 

Creation of a New Generation of Lignocellulosic Energy Crops............................................................................................................60
 

Maximizing.Biomass.Productivity........................................................................................................................................................61
 

Technical.Milestones............................................................................................................................................................................67
 

Ensuring Sustainability and Environmental Quality.............................................................................................................................68
 

Technical.Milestones............................................................................................................................................................................70
 

Model Systems for Energy Crops.............................................................................................................................................................71
 

Technical.Milestones............................................................................................................................................................................73
 

The Role of GTL Capabilities for Systems Biology..................................................................................................................................74
 

Protein.Production.Capabilities............................................................................................................................................................74
 

Molecular.Machines.Capabilities..........................................................................................................................................................75
 

Proteomic.Capabilities..........................................................................................................................................................................75
 

Cellular.System.Capabilities..................................................................................................................................................................75
 

DOE.Joint.Genome.Institute..................................................................................................................................................................76
 

Other.Needed.Capabilities....................................................................................................................................................................76
 

Other Biofuel Opportunities: Development of High-Productivity Biodiesel Crops.............................................................................77
 

Cited References......................................................................................................................................................................................80
 

For Further Reading................................................................................................................................................................................81
 

Deconstructing Feedstocks to Sugars.............................................................................................................................................85
 

Determining Fundamental Physical and Chemical Factors in the Recalcitrance of Lignocellulosic Biomass to Processing............86
 

Research.Goals.....................................................................................................................................................................................88
 

Technical.Milestones............................................................................................................................................................................89
 

The.Role.of.GTL.and.OBP.Facilities.and.Capabilities..............................................................................................................................90
 

Crosscutting.Tools,.Technologies,.and.Science......................................................................................................................................91
 

Developing Better Enzymatic Systems for Biological Pretreatment: Ligninases and Hemicellulases..............................................91
 

Research.Goals.....................................................................................................................................................................................92
 

Technical.Milestones............................................................................................................................................................................94
 

Crosscutting.Tools,.Technologies,.and.Science......................................................................................................................................97
 

Understanding the Molecular Machinery Underpinning Cellulose Saccharification: Cellulases and Cellulosomes........................97
 

Research.Goals.....................................................................................................................................................................................98
 

Technical.Milestones..........................................................................................................................................................................101
 

Crosscutting.Tools,.Technologies,.and.Science....................................................................................................................................103
 

The.Role.of.GTL.and.OBP.Facilities.and.Capabilities............................................................................................................................104
 

Harvesting the Biochemical Potential of Microorganisms Through Metagenomics ........................................................................105
 

Research.Goals...................................................................................................................................................................................106
 

v Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

Technical.Milestones..........................................................................................................................................................................106
 

The.Role.of.GTL.and.OBP.Facilities.and.Capabilities............................................................................................................................107
 

Characterizing Cell Walls Using High-Throughput Methods...............................................................................................................108
 

Technical.Milestones..........................................................................................................................................................................110
 

The.Role.of.GTL.and.OBP.Facilities.and.Capabilities............................................................................................................................111
 

Crosscutting.Tools,.Technologies,.and.Science....................................................................................................................................112
 

Breakthrough, High-Payoff Opportunity: Simplifying the Bioconversion Process by Understanding Cell-Wall 

Deconstruction Enzymes Expressed in Plants.....................................................................................................................................113
 

Scientific.Challenges.and.Opportunities.............................................................................................................................................113
 

Research.Goals...................................................................................................................................................................................113
 

References.............................................................................................................................................................................................115
 

Cellulosome References........................................................................................................................................................................117
 

Cellulosome.Reviews..........................................................................................................................................................................117
 

Designer.Cellulosomes.......................................................................................................................................................................117
 

Sugar Fermentation to Ethanol......................................................................................................................................................119
 

Optimizing Microbial Strains for Ethanol Production: Pushing the Limits of Biology......................................................................122
 

Science.Challenges.and.Strategy........................................................................................................................................................124
 

Technical.Milestones..........................................................................................................................................................................130
 

The.Role.of.GTL.Capabilities................................................................................................................................................................131
 

Advanced Microorganisms for Process Simplification........................................................................................................................132
 

Science.Challenges.and.Strategies.for.Process.Simplification.............................................................................................................135
 

Technical.Milestones..........................................................................................................................................................................137
 

The.Role.of.GTL.Capabilities................................................................................................................................................................138
 

Enabling Microbiological Tools and Technologies that Must be Developed......................................................................................138
 

Breakthrough, High-Payoff Opportunities..........................................................................................................................................140
 

Microbial.Communities.for.Robust.Energy.Production........................................................................................................................140
 

Model-Driven.Design.of.Cellular.Biocatalytic.Systems.Using.Systems.Biology....................................................................................142
 

Direct.Bioproduction.of.Energy-Rich.Fuels..........................................................................................................................................147
 

Optimal.Strains:.Fermentative.Production.of.40%.Ethanol.from.Biomass.Sugars...............................................................................149
 

An.Alternative.Route.for.Biomass.to.Ethanol:.Microbial.Conversion.of.Syngas....................................................................................152
 

Cited References....................................................................................................................................................................................154
 

Crosscutting 21st Century Science, Technology, and Infrastructure for a New Generation 

of Biofuel Research.............................................................................................................................................................................155
 

Opportunities and Challenges .............................................................................................................................................................155
 

Analytical Tools to Meet the Challenges of Biofuel Research.............................................................................................................156
 

Genomics...........................................................................................................................................................................................156
 

Transcriptomics:.High-Throughput.Expression.Analyses.....................................................................................................................158
 

Proteomics.........................................................................................................................................................................................158
 

Metabolomics....................................................................................................................................................................................159
 

Glycomics.and.Lignomics...................................................................................................................................................................160
 

Fluxomics...........................................................................................................................................................................................161
 

Enzyme.Structure.and.Function..........................................................................................................................................................162
 

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CONTENTS 

Imaging Technologies...........................................................................................................................................................................163
 

Imaging.Needs.for.Feedstock.Research...............................................................................................................................................163
 

Imaging.Needs.for.Microbial.Communities.in.Deconstruction.and.Conversion.of.Biomass.to.Ethanol................................................164
 

Microbial Cultivation.............................................................................................................................................................................165
 

Data Infrastructure................................................................................................................................................................................166
 

Computational Modeling......................................................................................................................................................................168
 

Modeling:.Genome.Sequence.Analysis...............................................................................................................................................168
 

Modeling:.Molecular..........................................................................................................................................................................170
 

Modeling:.Pathways.and.Networks....................................................................................................................................................171
 

Modeling:.Biorefinery.Process............................................................................................................................................................171
 

Capability Suites for Bioenergy Research and Facility Infrastructure...............................................................................................172
 

DOE.Joint.Genome.Institute................................................................................................................................................................173
 

Production.and.Characterization.of.Proteins.and.Molecular.Tags........................................................................................................173
 

Characterization.and.Imaging.of.Molecular.Machines........................................................................................................................175
 

Analysis.of.Genome.Expression:.The.Omics.........................................................................................................................................176
 

Analysis.and.Modeling.of.Cellular.Systems.........................................................................................................................................178
 

Cited References....................................................................................................................................................................................180
 

Bioprocess Systems Engineering and Economic Analysis......................................................................................................181
 

Research Goals.......................................................................................................................................................................................181
 

Milestones..........................................................................................................................................................................................182
 

The.Role.of.GTL.and.OBP.Facilities.and.Capabilities............................................................................................................................183
 

APPENDICES..........................................................................................................................................................................................185
 

Appendix A. Provisions for Biofuels and Biobased Products in the Energy Policy Act of 2005...................................186
 

Appendix B. Workshop Participants..............................................................................................................................................188
 

Appendix C. Workshop Participant Biosketches.......................................................................................................................192
 

v Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Introducton
 

I

n his 2006 State of the Union address (Bush 2006), the president 

outlined the new Advanced Energy Initiative (AEI) to help overcome 

America’s dependence on foreign sources of energy (AEI 2006) and 

the American Competitiveness Initiative to increase R&D investments 

and strengthen education (ACI 2006). He seeks to reduce our national 

dependence on imported oil by accelerating the development of domestic,

renewable alternatives to gasoline and diesel fuels. 

“With America on the verge of breakthroughs in advanced energy technologies, the best way to break the addiction to foreign oil is through 

new technologies.” —White House Press Release on the State of the Union 

Address and AEI ( January 31, 2006) 

Breakthrough technologies to realize the potential of cellulosic biofuels 

can be expedited by application of a new generation of biological research 

created by the genome revolution. Overcoming barriers to development of 

these fuels on an industrial scale will require high-performance energy feedstocks and microbial processes, both to break down feedstocks to sugars and 

to ferment sugars to ethanol. A focused set of investments linking revolutionary biofuel technologies with advances from the biological, physical,

computational, and engineering sciences will quickly remove barriers to an 

efficient, economic, and sustainable biofuel industry. 

Jont Workshop Challenges Bofuel Scence and 

Technology Communtes 

Two Department of Energy (DOE) offices are teaming to advance biofuel 

development and use: The Office of Biological and Environmental Research 

(OBER) within the Office of Science (SC) and the Office of the Biomass 

Program (OBP) within the Office of Energy Efficiency and Renewable 

Energy (EERE) (see descriptions of the two DOE programs, pp. 17 and 

19).These offices are challenging their communities to identify critical science needs to support a substantial and sustainable expansion of biomassderived fuels, specifically cellulosic ethanol. In the jointly sponsored 

Biomass to Biofuels Workshop held December 7–9, 2005, in Rockville,

Maryland, more than 50 scientists representing a wide range of expertise 

convened to define barriers and challenges to this new biofuel industry. The 

workshop concentrated on improvement of biomass crops and their processing to transportation fuels. Although the focus was ethanol, the science 

applies to additional fuels that include biodiesel and to other bioproducts or 

coproducts having critical roles in any deployment scheme. References: p. 24 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy  
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTRODUCTION 

The current approach to introducing biofuels relies on an “evolutionary”

business and economic driver for a steady but moderate entry into the 

market. Technologies for implementing this new industry are being tested 

either by producing higher-value products from renewables (such as lactic 

acid) or as incremental additions to current corn-ethanol refineries (such as 

the conversion of residual corn-kernel fibers to ethanol). 

This report is a workshop-produced roadmap for accelerating cellulosic 

ethanol research, helping make biofuels practical and cost-competitive by 

2012 ($1.07/gal ethanol) and offering the potential to displace up to 30% 

of the nation’s current gasoline use by 2030. It argues that rapidly incorporating new systems biology approaches via significant R&D investment will 

spur use of these technologies for expanded processing of energy crops and 

residues. Furthermore, this strategy will decrease industrial risk from use 

of a first-of-a-kind technology, allowing faster deployment with improved 

methods. Ultimately, these approaches foster setting more aggressive goals 

for biofuels and enhance the strategy’s sustainability. 

Amerca’s Energy Challenges 

The triple energy-related challenges of the 21st Century are economic and 

energy growth, energy security, and climate protection. The United States 

imports about 60% of the petroleum it consumes, and that dependency is 

increasing.* Since the U.S. economy is tied so closely to petroleum products 

and oil imports, disruptions in oil supplies can result in severe economic and 

social impacts. Conventional oil production will peak in the near future, and 

the resulting energy transition will require a portfolio of responses, including unconventional fossil resources and biofuels. Environmental quality and 

climate change due to energy emissions are additional concerns. Annual 

U.S. transportation emissions of the greenhouse gas (GHG) carbon dioxide 

(CO2

) are projected to increase from about 1.9 billion metric tons in 2004 

to about 2.7 billion metric tons in 2030 (EIA 2006). 

The Promse of Bofuels 

Fuels derived from cellulosic biomass**—the fibrous, woody, and generally 

inedible portions of plant matter—offer an alternative to conventional 

energy sources that supports national economic growth, national energy 

security, and environmental goals. Cellulosic biomass is an attractive energy 

feedstock because supplies are abundant domestically and globally. It is a 

renewable source of liquid transportation fuels that can be used readily by 

current-generation vehicles and distributed through the existing transportation-fuel infrastructure. Ethanol from corn grain is an increasingly 

important additive fuel source, but it has limited growth potential as a 

primary transportation fuel.*** The U.S. “starch-based” ethanol industry 

will jump start a greatly expanded ethanol industry that includes cellulosic 

ethanol as a major transportation fuel. 

Cellulose and hemicelluloses, found in plant cell walls, are the primary 

component of biomass and the most plentiful form of biological material on 

earth. They are polysaccharides made up of energy-rich sugars that can be 

converted to ethanol (see sidebar, Understanding Biomass, p. 53). Current 

2 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

methods to break down biomass into simple sugars and convert them into 

ethanol are inefficient and constitute the core barrier to producing ethanol 

at quantities and costs competitive with gasoline. 

Biological research is undergoing a major transformation. The systems 

biology paradigm—born of the genome revolution and based on highthroughput advanced technologies, computational modeling, and scientificteam approaches—can facilitate rapid progress and is a readily applicable 

model for biofuel technology. Systems biology is the core of the OBER 

Genomics:GTL program, whose goal is to achieve a predictive understanding of the complex network of interactions that underpin the biological 

processes related to biofuel production. Biological challenges to which GTL 

can apply systems biology approaches include enhancing the productivity 

of biomass crops optimized for industrial processing, improving enzyme 

systems that deconstruct plant cell walls, and increasing the yield of ethanol-producing microorganisms. Systems biology tools and knowledge will 

enable rational engineering of a new generation of bioenergy systems made 

up of sustainable energy crops for widely varying agroecosystems and tailored industrial processes. This research approach will encourage the critical 

fusion of the agriculture, industrial biotechnology, and energy sectors. 

A Growng Mandate for Bofuels: Polcy, Legslatve, 

and Other Drvers 

A primary goal of the president’s 2001 National Energy Policy (NEP) 

is to increase U.S. energy supplies, incorporating a more diverse mix of 

domestic resources to support growth in demand and to reduce national 

dependence on imported oil (NEPDG 2001). AEI accelerates and 

expands on several policy and legislative mandates (AEI 2006). It aims to 

reduce the nation’s reliance on foreign oil in the near term and provides a 

22% increase in clean-energy research at DOE for FY 2007, accelerating 

progress in renewable energy. 

According to AEI, the United States must move beyond a petroleumbased economy and devise new ways to power automobiles. The country 

needs to facilitate domestic, renewable alternatives to gasoline and diesel 

fuels. The administration will accelerate research in cutting-edge methods 

of producing such “homegrown” renewable biobased transportation fuels 

as ethanol from agricultural and forestry feedstocks including wood chips, 

*Gasoline and diesel constituted 98% of domestic transportation motor fuels in 2004,

with ethanol from corn grain supplying most of the remaining 2%. Annual gasoline 

consumption in 2004 was about 139 billion gallons, and 3.4 billion gallons of ethanol 

were used primarily as a fuel extender to boost gasoline octane levels and improve 

vehicle emissions. 

**Cellulosic biomass, also called lignocellulosic biomass, is a complex composite material 

consisting primarily of cellulose and hemicellulose (structural carbohydrates) bonded to 

lignin in plant cell walls. For simplification, we use the term cellulosic biomass. 

***In 2004, 11% of the U.S. corn harvest yielded 3.4 billion gallons of ethanol (NRDC 

2006), roughly 1.7% of the 2004 fuel demand. Thus if all corn grain now grown in the 

United States were converted to ethanol, it would satisfy about 15% of current transportation needs. 

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INTRODUCTION 

stalks, and switchgrass. AEI would foster the early commercialization of 

advanced biofuel technologies, enabling U.S. industry to lead in deploying 

biofuels and chemicals internationally. 

Achieving the goal of displacing 30% of the nation’s current gasoline use by 

2030 would require production levels equal to roughly 60 billion gallons a 

year (Bgal/year) of ethanol (see Table 1. Comparisons of 2004 Gasoline and 

Ethanol Equivalents, this page). An annual supply of roughly a billion dry 

tons of biomass will be needed to support this level of ethanol production.

A recent report by the U.S. Department of Agriculture (USDA) and DOE 

finds potential to sustainably harvest more than 1.3 billion metric tons of 

biomass from U.S. forest and agricultural lands by mid-21st Century 

(Perlack et al. 2005). Investments in R&D and infrastructure are needed to 

realize this feedstock potential. 

The U.S. Energy Policy Act of 2005 (EPAct; Appendix A, Provisions for 

Biofuels and Biobased Products in the Energy Policy Act of 2005, p. 186) 

has established aggressive near-term targets for ethanol production. A key 

provision requires mixing 4 Bgal of renewable fuel with gasoline in 2006.

This requirement increases annually to 7.5 Bgal of renewable fuel by 2012.

For 2013 and beyond, the required volume will include a minimum of 250 

million gallons (Mgal) of cellulosic ethanol. Another section of the EPAct 

authorizes funds for an incentive program to ensure the annual production 

of 1 Bgal of cellulosic biomass-based fuels by 2015. Ethanol is the most 

common biofuel produced from cellulose, but other possible biofuel compounds can be produced as well. 

Other important legislative drivers supporting biofuels are the Biomass R&D 

Act of 2000 and Title IX of the Farm Bill 2002 (U.S. Congress 2000; U.S.

Congress 2002).The Biomass R&D Act directed the departments of Energy 

and Agriculture to integrate their biomass R&D and established the Biomass 

Research and Development Technical Advisory Committee (BTAC), which 

advises the Secretary of Energy and 

the Secretary of Agriculture on stra- Table . Comparsons of 200 Gasolne and Ethanol Equvalents 

2004 Gasoline 

(billion gallons) 

Ethanol Equivalents 

(billion gallons) 

U.S. consumption, 2004 139 200 

About 60% from imports 83 120 

Requirements to displace 30% of 2004 U.S. 

consumption 

42 60 

• Biomass requirements at 80 gal/ton • 750 Mton 

• Land requirements at 10 ton/acre and 

80 gal/ton 

• 75 Macre 

• Numbers of refineries at 100 Mgal/

refinery 

• 600 (each 

requiring 

160 miles2 net or 

125,000 acres) 

tegic planning for biomass R&D. As 

a precedent to the current presidential 

initiative, in 2002 BTAC set a goal 

requiring biofuels to meet 20% of U.S.

transportation fuel consumption by 

2030 as part of its vision for biomass 

technologies (BTAC 2002).Title IX 

supports increased use of biobased fuels 

and products and incentives and grants 

for biofuel and biorefinery R&D. 

In addition to legislative mandates, several independent studies have acknowledged the great potential of biofuels 

in achieving a more diverse domestic 

energy supply (NCEP 2004; Greene et 

al. 2004; Lovins et al. 2005). Growing 

 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

support for developing biomass as a key energy feedstock is coming from a 

variety of national and international organizations (GEC 2005; Ag Energy 

Working Group 2004; IEA 2004). Although these reports differ in the 

amounts of gasoline that could be replaced by ethanol from biomass, they all 

agree on three key issues: (1) Current trends in energy use are not sustainable 

and are a security risk; (2) No single solution will secure the energy future—a 

diverse portfolio of energy options will be required; and (3) Biofuels can be a 

significant part of the transportation sector’s energy solution. 

In its evaluation of options for domestic production of motor fuels, the 

National Commission on Energy Policy (NCEP) recommended cellulosic 

biomass as an important topic for near-term federal research, development,

and demonstration and found that “cellulosic ethanol has the potential to 

make a meaningful contribution to the nation’s transportation fuel supply 

in the next two to three decades” (NCEP 2004). 

The Natural Resources Defense Council (NRDC) has projected that an 

aggressive plan to develop cellulosic biofuels in the United States could 

“produce the equivalent of nearly 7.9 million barrels of oil per day by 2050 

... more than 50 percent of our current total oil use in the transportation 

sector and more than three times as much as we import from the Persian 

Gulf alone” (Greene et al. 2004). This corresponds to roughly 100 Bgal/year 

ethanol. NRDC also recommends $1.1 billion in funding between 2006 

and 2012 for biomass research, development, and demonstration with 45% 

of this funding focused on overcoming biomass recalcitrance to ethanol 

processing. This level of funding is expected to stimulate a regular flow of 

advances needed to make ethanol cost-competitive with gasoline and diesel. 

An independent analysis from the Rocky Mountain Institute found that 

significant gains in energy efficiency and the large-scale displacement of 

oil with biofuels, mainly cellulosic ethanol, would be key components of 

its strategy to reduce American oil dependence over the next few decades 

(Lovins et al. 2005). 

To illustrate the widespread support for fuel ethanol, the Governors’ Ethanol Coalition, an organization devoted to the promotion and increased use 

of ethanol, now includes 32 member states as well as international representatives from Brazil, Canada, Mexico, Sweden, and Thailand. In a recent 

report, the coalition called for rapid expansion of ethanol to meet at least 

10% of transportation fuel needs “as soon as practicable” and for development of “lignocellulosic-based” fuels for expansion beyond those levels 

(GEC 2005). “The use of ethanol, particularly biomass-derived ethanol,

can produce significant savings in carbon dioxide emissions. This approach 

offers a no-regrets policy that reduces the potential future risks associated 

with climate change and has the added benefit of economic development.” 

Benefits of Bofuels 

Biofuels, especially corn-derived and cellulosic ethanol, constitute the only 

renewable liquid transportation fuel option that can be integrated readily 

with petroleum-based fuels, fleets, and infrastructure. Production and use 

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INTRODUCTION 

of biofuels can provide substantial benefits to national energy security,

economic growth, and environmental quality. 

Natonal Energy Securty Benefits 

“National security is linked to energy through the dependence of this 

country and many others on imported oil—much of it located in 

politically troubled parts of the globe. As such, the potential for largescale failures in the global production and distribution system presents 

a real threat.” — Governors’ Ethanol Coalition (GEC 2005) 

Today the United States is dependent on oil for transportation. Alternative, domestically based, and sustainable fuel-development strategies,

therefore, are essential to ensuring national security. America accounts for 

25% of global oil consumption yet holds only 3% of the world’s known 

oil reserves. About 60% of known oil reserves are found in sensitive and 

volatile regions of the globe. Increasing strain on world oil supply is 

expected as developing countries become more industrialized and use 

more energy. Any strategy to reduce U.S. reliance on imported oil will 

involve a mix of energy technologies including conservation. Biofuels are 

an attractive option to be part of that mix because biomass is a domestic,

secure, and abundant feedstock. Global availability of biomass feedstocks 

also would provide an international alternative to dependence on an 

increasingly strained oil-distribution system as well as a ready market for 

biofuel-production technologies. 

Economc Benefits 

A biofuel industry would create jobs and ensure growing energy supplies 

to support national and global prosperity. In 2004, the ethanol industry 

created 147,000 jobs in all sectors of the economy and provided more than 

$2 billion of additional tax revenue to federal, state, and local governments 

(RFA 2005). Conservative projections of future growth estimate the addition of 10,000 to 20,000 jobs for every billion gallons of ethanol production (Petrulis 1993). 

In 2005 the United States spent more than $250 billion on oil imports,

and the total trade deficit has grown to more than $725 billion (U.S. Commerce Dept. 2006). Oil imports, which make up 35% of the total, could 

rise to 70% over the next 20 years (Ethanol Across America 2005). 

Among national economic benefits, a biofuel industry could revitalize 

struggling rural economies. Bioenergy crops and agricultural residues can 

provide farmers with an important new source of revenue and reduce reliance on government funds for agricultural support. An economic analysis 

jointly sponsored by USDA and DOE found that the conversion of some 

cropland to bioenergy crops could raise depressed traditional crop prices by 

up to 14%. Higher prices for traditional crops and new revenue from bioenergy crops could increase net farm income by $6 billion annually (De La 

Torre Ugarte 2003). 

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Fig. 1. Reduced Carbon 

Dioxide Emissions of Ethanol 

from Biomass. When compared with gasoline, ethanol 

from cellulosic biomass could 

dramatically reduce emissions 

of the greenhouse gas, carbon 

dioxide (CO ). Although burn­ 2

ing gasoline and other fossil 

fuels increases atmospheric 

CO concentrations, the pho­ 2

tosynthetic production of new 

biomass takes up most of the

carbon dioxide released when 

bioethanol is burned. [Source:

Adapted from ORNL Review 

(www.ornl.gov/info/ornlreview/

v33_2_00/bioenergy.htm)] 

Envronmental Benefits 

Clmate Change 

When fossil fuels are consumed, carbon sequestered from the global 

carbon cycle for millions of years is released into the atmosphere, where 

it accumulates. Biofuel consumption can release considerably less CO2

,

depending on how it is produced. The photosynthetic production of new 

generations of biomass takes up the CO released from biofuel production 2

and use (see Fig. 1. Reduced Carbon Dioxide Emissions of Ethanol from 

Biomass, this page). A life-cycle analysis shows fossil CO2

 emissions from 

cellulosic ethanol to be 85% lower than those from gasoline (Wang 2005).

These emissions arise from the use of fossil energy in producing cellulosic 

ethanol. Nonbiological sequestration of CO2

 produced by the fermentation 

process can make the biofuel enterprise net carbon negative. 

A recent report (Farrell et al. 2006) finds that ethanol from cellulosic 

biomass reduces substantially both GHG emissions and nonrenewable 

energy inputs when compared with gasoline. The low quantity of fossil 

fuel required to produce cellulosic ethanol (and thus reduce fossil GHG 

emissions) is due largely to three key factors. First is the yield of cellulosic biomass per acre. Current corn-grain yields are about 4.5 tons/acre.

Starch is 66% by weight, yielding 3 tons to produce 416 gal of ethanol,

compared to an experimental yield of 10 dry tons of biomass/acre for 

switchgrass hybrids in research environments (10 dry tons at a future 

yield of 80 gal/ton = 800 gal ethanol). Use of corn grain, the remaining 

solids (distillers’ dried grains), and stover could yield ethanol at roughly 

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INTRODUCTION 

700 gal/acre. Current yield for nonenergy-crop biomass resources is 

about 5 dry tons/acre and roughly 65 gal/ton. The goal for energy crops is 

10 tons/acre at 80 to 100 gal/ton during implementation. Second, perennial biomass crops will take far less energy to plant and cultivate and will 

require less nutrient, herbicide, and fertilizer. Third, biomass contains 

lignin and other recalcitrant residues that can be burned to produce heat 

or electricity consumed by the ethanol-production process. 

Energy crops require energy inputs for production, transportation, and 

processing—a viable bioenergy industry will require a substantial positive 

energy balance. Figure 2. Comparison of Energy Yields with Energy Expenditures, this page, compares results for cellulosic and corn ethanol, gasoline,

and electricity, demonstrating a substantially higher yield for cellulosic ethanol. Over time a mature bioenergy economy will substitute biomass-derived 

energy sources for fossil fuel, further reducing net emissions. 

Fig. 2. Comparison of Energy Yields with Energy Expenditures. The fossil energy–replacement ratio (FER) compares 

energy yield from four energy sources with the amount of fossil fuel used to produce each source. Note that the cellulosic 

ethanol biorefinery’s projected yield assumes future technological improvements in conversion efficiencies and advances 

that make extensive use of a biomass crop’s noncellulosic 

portions for cogeneration of electricity. Similar assumptions 

would raise corn ethanol’s FER if, for example, corn stover 

were to replace current natural gas usage. The corn ethanol 

industry, already producing ethanol as an important additive 

and fuel extender, is providing a foundation for expansion 

to cellulosic ethanol. [Source: Figure, based on the Argonne 

National Laboratory GREET model, is derived from Brinkman et al. 2005. Other papers that support this study include 

Farrell et al. 2006 and Hammerschlag 2006.] 

Other Envronmental Benefits 

Perennial grasses and other bioenergy crops have 

many significant environmental benefits over traditional row crops (see Fig. 3. Miscanthus Growth over 

a Single Growing Season in Illinois, p. 9). Perennial energy crops provide a better environment for 

more-diverse wildlife habitation. Their extensive 

root systems increase nutrient capture, improve soil 

quality, sequester carbon, and reduce erosion. Ethanol, when used as a transportation fuel, emits less 

sulfur, carbon monoxide, particulates, and GHGs 

(Greene et al. 2004). 

Feasblty of Bofuels 

The United States could benefit substantially by 

increasing its use of domestic, renewable fuels 

in the transportation sector, but can biofuels be 

produced at the scale needed to make a real difference in transportation consumption of fossil fuels? 

More specifically, is there enough land to provide 

the needed large-scale supply of biomass, is the use 

of biofuels sustainable agriculturally, can biofuels 

become cost-competitive with gasoline, and is cellulosic-biofuel production technically feasible for 

energy? The short answer to all these questions is 

yes, and this section summarizes recent reports that 

support this view. 

Land Avalablty 

A major factor influencing the extent to which 

biofuels will contribute to America’s energy future is 

the amount of land available for biomass harvesting. 

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Are biomass resources sufficient 

to meet a significant portion of 

transportation-fuel consumption, and how would harvesting biomass for energy affect 

current agricultural and forestry 

practices? 

In 2005, a study jointly supported by DOE and USDA 

examined whether land 

resources in the United States 

are sufficient to sustain production of over 1 billion dry tons 

of biomass annually, enough 

to displace 30% or more of the 

nation’s current consumption 

of liquid transportation fuels.

By assuming relatively modest 

changes in agricultural and forestry practices, this study projects that 1.366 billion dry tons 

of biomass could be available 

for large-scale bioenergy and 

biorefinery industries by mid21st Century while still meeting 

demand for forestry products,

food, and fiber (Perlack et al.

2005) (see sidebar, A Billion-Ton Annual Supply of Biomass, p. 10). This 

supply of biomass would be a sevenfold increase over the 190 million dry 

tons of biomass per year currently used for bioenergy and bioproducts.

Most of this biomass is burned for energy, with only 18 million dry tons 

used for biofuels (primarily corn-grain ethanol) and 6 million dry tons 

used for bioproducts. 

The biomass potential determined by the “billion-ton” study is one scenario 

based on a set of conservative assumptions derived from current practices and 

should not be considered an upper limit. Crop-yield increases assumed in 

this study follow business-as-usual expectations. With more aggressive commitments to research on improving energy crops and productivity, the biomass potential could be much greater. Energy-crop yield is a critical factor in 

estimating how much land will be needed for large-scale biofuel production,

and this factor can be influenced significantly by biotechnology and systems 

biology strategies used in modern plant breeding and biomass processing. 

Many potential energy crops (e.g., switchgrass, poplar, and willow) are 

essentially unimproved or have been bred only recently for biomass,

compared to corn and other commercial food crops that have undergone 

substantial improvements in yield, disease resistance, and other agronomic traits. A more complete understanding of biological systems and 

Fig. 3. Miscanthus Growth over 

a Single Growing Season in 

Illinois. Miscanthus has been 

explored extensively as a potential energy crop in Europe and 

now is being tested in the United 

States. The scale is in feet. These 

experiments demonstrate results 

that are feasible in development 

of energy crops. [Image source:

S. Long, University of Illinois] 

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INTRODUCTION 

A Bllon-Ton Annual Supply of Bomass: Summary of Potental Forest and 

Agrcultural Resources 

I

n 2005, a study jointly supported by DOE and USDA examined whether land resources in the United 

States are sufficient to sustain production of over 1 billion dry tons of biomass annually, enough to displace 30% or more of the nation’s current consumption of liquid transportation fuels (Perlack et al. 2005).

Assuming relatively modest changes in agricultural and forestry practices, this study projects that 1.366 billion 

dry tons of biomass (368 million dry tons from forest and 998 million dry tons from agriculture) could be 

available for large-scale bioenergy and biorefinery industries by mid-21st Century while still meeting demand 

for forestry products, food, and fiber (see Fig. A. Potential Biomass Resources, below). This supply of biomass 

would be a sevenfold increase over the 190 million dry tons of biomass per year currently used for bioenergy 

and bioproducts. Most of this biomass is burned for energy, with only 18 million dry tons used for biofuels 

(primarily corn-grain ethanol) and 6 million dry tons used for bioproducts. 

Land area in the United States is about 2 billion acres, with 33% forestlands and 46% agricultural lands consisting of grasslands or pasture (26%) and croplands (20%). Of the estimated 368 million dry tons of forest 

biomass, 142 million dry tons already are used by the forest products industry for bioenergy and bioproducts.

Several different types of biomass were considered in this study. Residues from the forest products industry 

include tree bark, woodchips, shavings, sawdust, miscellaneous scrap wood, and black liquor, a by-product of 

pulp and paper processing. Logging and site-clearing residues consist mainly of unmerchantable tree tops 

and small branches that currently are left onsite or burned. Forest thinning involves removing excess woody 

materials to reduce fire hazards and improve forest health. Fuelwood includes roundwood or logs burned for 

space heating or other energy uses. Urban wood residues consist primarily of municipal solid waste (MSW,

e.g., organic food scraps, yard trimmings, discarded furniture, containers, and packing materials) and construction and demolition debris (see Table A. Potential Biomass Resources, this page, and Fig. B. Biomass Analysis 

for the Billion-Ton Study, p. 11). 

Fig. A. Potential Biomass Resources: A Total of 

More than 1.3 Billion Dry Tons a Year from Agricultural and Forest Resources. 

Biomass Resources Million 

Dry Tons per Year 

Forest Biomass 

Forest products industry residues 145 

Logging and site-clearing residues 64 

Forest thinning 60 

Fuelwood 52 

Urban wood residues 47 

Subtotal for Forest Resources 368 

Agricultural Biomass 

Annual crop residues 428 

Perennial crops 377 

Miscellaneous process residues, manure 106 

Grains 87 

Subtotal for Agricultural Resources 998 

Total Biomass Resource Potential 1366 

Table A. Potental Bomass Resources 

0 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Fig. B. Biomass Analysis for the Billion-Ton Study [Source: Multi Year Program Plan, 2007–2012, OBP, EERE, 

U.S. DOE (2005)] 

Several assumptions were made to estimate potential forest biomass availability. Environmentally sensitive 

areas, lands without road access, and regions reserved for nontimber uses (e.g., parks and wilderness) were 

excluded, and equipment-recovery limitations were considered. As annual forest growth is projected to continue to exceed annual harvests, continued expansion of standing forest inventory is assumed. 

Among agricultural biomass resources, annual crop residues are mostly stems and leaves (e.g., corn stover and 

wheat straw) from corn, wheat, soybeans, and other crops grown for food and fiber. Perennial crops considered 

in the study include grasses or fast-growing trees grown specifically for bioenergy. Grain primarily is corn used 

for ethanol production, and miscellaneous process residues include MSW and other by-products of agricultural resource processing. 

A total of 448 million acres of agricultural lands, largely active and idle croplands, were included in this study;

lands used permanently for pasture were not considered. Other assumptions for agricultural biomass resources 

include a 50% increase in corn, wheat, and small-grain yield; doubling the residue-to-grain ratio for soybeans; recovery of 75% of annual crop residues with more efficient harvesting technologies; management of all 

cropland with no-till methods; 55 million acres dedicated to production of perennial bioenergy crops; average 

biomass yield for perennial grasses and woody plants estimated at 8 dry tons per acre; conversion of all manure 

not used for on-farm soil improvement to biofuel; and use of all other available residues. 

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INTRODUCTION 

application of the latest biotechnological advances will accelerate the 

development of new biomass crops having desirable attributes. These 

attributes include increased yields and processability, optimal growth in 

specific microclimates, better pest resistance, efficient nutrient use, and 

greater tolerance to moisture deficits and other sources of stress. Furthermore, many biotechnological advances for growing better biomass 

crops will be used to improve food crops, easing the pressure on land 

area needed to grow food. Joint development of these biotechnological 

advances with other countries will help moderate the global demand for 

crude oil. In an idealized future scenario with greater per-acre productivity in energy, food, and fiber crops and decreased demand for transportation fuels resulting from more efficient vehicles, the United States could 

have sufficient land resources to produce enough biomass to meet all its 

transportation-fuel needs. 

Agrcultural Sustanablty of Bomass Producton 

Sustainable practices for growing and harvesting biomass from dedicated 

crops will be essential to the success of large-scale biofuel production.

Capital costs of refineries and associated facilities to convert biomass 

to fuels will be amortized over several decades. These capital assets will 

require a steady annual supply of biomass from a large proportion of 

surrounding land. Therefore, a thorough understanding of the conversion 

pathway and of biomass harvesting’s long-term impacts on soil fertility 

is needed to ensure sustainability. Vital nutrients contained in process 

residues must be returned to the soil. Perennial crops expected to be 

used for biofuels improve soil carbon content and make highly efficient 

use of mineral nutrients (see sidebar, The Argument for Perennial Biomass Crops, p. 59). Additional information about the composition and 

population dynamics of soil microbial communities is needed, however,

to determine how microbes contribute to sustaining soil productivity (see section, Ensuring Sustainability and Environmental Quality, p.

68). Mixed cultivars of genetically diverse perennial energy crops may 

be needed to increase productivity and preserve soil quality. Because 

conventional annual food and fiber crops are grown as monocultures,

relatively little research has been carried out on issues associated with 

growing mixed stands. 

Today – Fuel Ethanol Producton from Corn Gran (Starch Ethanol) 

In 2004, 3.41 Bgal of starch ethanol fuel were produced from 1.26 billion 

bushels of corn—11% of all corn grain harvested in the United States.

This record level of production was made possible by 81 ethanol plants 

located in 20 states. Completion of 16 additional plants and other expansions increased ethanol-production capacity to 4.4 Bgal by the end of 

2005; additional planned capacity is on record for another 1 Bgal from 

2006 to 2007 (RFA 2005). Although demand for fuel ethanol more than 

doubled between 2000 and 2004, ethanol satisfied less than 2% of U.S.

transportation-energy demand in 2004. 

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In the United States, ethanol is produced in corn wet or dry mills. Corn 

wet mills fractionate the corn grain for products like germ and oil before 

converting the clean starch to sugars for fermentation or for such valuable food products as high-fructose corn syrup and maltodextrins. The 

corn fiber by-product usually is sold as animal feed. In corn dry mills,

the grain is ground, broken into sugar monomers (saccharified), and 

fermented. Since the grain is not fractionated, the only by-product is 

the remaining solids, called distillers’ dried grains with solubles, a highly 

nutritious protein source used in livestock feed. A bushel of corn yields 

about 2.5 gal ethanol from wet-mill processing and about 2.8 gal from 

dry grind (Bothast and Schlicher 2005). Some 75% of corn ethanol production is from dry-mill facilities and 25% from wet mills. 

Tomorrow – Borefinery Concept to Produce Fuel Ethanol from 

Cellulosc Bomass 

Cellulosic ethanol has the potential to meet most, if not all, transportationfuel needs. However, due to the complex structure of plant cell walls, cellulosic biomass is more difficult than starch to break down into sugars. Three 

key biomass polymers found in plant cell walls are cellulose, hemicellulose,

and lignin (see Lignocellulosic Biomass Characteristics chapter, p. 39).

These polymers are assembled into a complex nanoscale composite, not 

unlike reinforced concrete but with the capability to flex and grow much 

like a liquid crystal. The composite provides plant cell walls with strength 

and resistance to degradation and carries out many plant functions. Their 

robustness, however, makes these materials a challenge to use as substrates 

for biofuel production. 

Traditional cellulosic biorefineries have numerous complex, costly, and 

energy-intensive steps that may be incompatible or reduce overall process efficiency.The current strategy for biochemical conversion of biomass to ethanol 

has its roots in the early days of wood chemistry. Developed in the 1930s for 

wartime use in Germany, it is used in Russia today.This process involves three 

basic steps, each element of which can be impacted by cellulosic biomass 

research (see Fig. 4.Traditional Cellulosic Biomass Conversion to Ethanol 

Based on Concentrated Acid Pretreatment Followed by Hydrolysis and 

Fermentation, p. 14). After acquisition of suitable cellulosic biomass, biorefining begins with size reduction and thermochemical pretreatment of raw 

cellulosic biomass to make cellulose polymers more accessible to enzymatic 

breakdown and to free up hemicellulosic sugars, followed by production and 

application of special enzyme preparations (cellulases) for hydrolysis of plant 

cell-wall polysaccharides to produce simple sugars. Final steps in the process 

include fermentation, mediated by bacteria or yeast, to convert these sugars to 

ethanol and other coproducts that must be recovered from the resulting 

aqueous mixture. Recent research and development has reduced dramatically 

the cost of enzymes and has improved fermentation strains to enable simultaneous saccharification and fermentation (SSF), in which hydrolysis of 

cellulose and fermentation of glucose are combined in one step. 

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INTRODUCTION 

Figure 5. A Biorefinery Concept Incorporating Advanced Pretreatment 

and Consolidated Processing of Cellulose to Ethanol, p. 15, depicts key 

targets for simplifying and improving the biorefinery concept. Feedstock 

research seeks first to increase biomass yields and enhance biomass characteristics to enable more efficient processing. Advanced biocatalysts will 

augment or replace thermochemical methods to reduce the severity and 

increase the yield of pretreatment. More robust processes and reduction 

of inhibitors would allow elimination of the detoxification and separation 

steps. Developing modified enzymes and fermentation organisms ultimately will allow incorporation of hydrolysis enzyme production, hydrolysis, and fermentation into a single organism or a functionally versatile 

but stable mixed culture with multiple enzymatic capabilities. Termed 

consolidated bioprocessing (CBP), this could enable four components 

comprising steps 2 and 3 (green boxes) in Fig. 4 to be combined into 

one, which in Fig. 5 is called direct conversion of cellulose and hemicellulosic sugars. Further refinement would introduce pretreatment enzymes 

(ligninases and hemicellulases) into the CBP microbial systems as well, 

Fig. 4. Traditional Cellulosic Biomass Conversion to 

Ethanol Based on Concentrated Acid Pretreatment Followed 

by Hydrolysis and Fermentation. Three steps in the process are 

(1) size reduction and thermochemical pretreatment of raw cellulosic biomass to make cellulose polymers more accessible to enzymatic breakdown and free up hemicellulosic sugars (blue boxes on left);

(2) production and application of special enzyme preparations (cellulases) 

that hydrolyze plant cell-wall polysaccharides, producing a mixture of simple 

sugars (green boxes); and (3) fermentation, mediated by bacteria or yeast, to 

convert these sugars to ethanol and other coproducts (yellow diamonds). Recent 

research and development has reduced dramatically the cost of enzymes and has improved fermentation strains 

to enable simultaneous saccharification and fermentation (SSF, green boxes surrounded by orange), in which hydrolysis of cellulose and fermentation of glucose are combined in one step. Cellulosic biomass research is targeting these 

steps to simplify and increase the yield of biomass production and processing (see Fig. 5. p. 15). [Source: Adapted from 

M. Himmel and J. Sheehan, National Renewable Energy Laboratory] 

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Fig 5. A Biorefinery Concept Incorporating Advanced Pretreatment 

and Consolidated Processing of Cellulose to Ethanol. The strategies 

discussed in this roadmap are based on first developing technologies to 

allow more energy-efficient and chemically benign enzymatic pretreatment. Saccharification and fermentation would be consolidated into a simple step and ultimately into a single organism or stable mixed culture (consolidated bioprocessing), thus removing multiple whole steps in converting biomass 

to ethanol. Also see Fig. 6. p. 16. [Source: Adapted from M. Himmel and J. Sheehan, National Renewable Energy 

Laboratory] 

reducing to one step the entire biocatalytic processing system (pretreatment, hydrolysis, and fermentation). These process simplifications and 

improvements will lessen the complexity, cost, and energy intensity of the 

cellulosic biorefinery. 

In addition to polysaccharides that can be converted to ethanol, the lignin 

in plant cell walls is a complex polymer of phenylpropanoid subunits 

that must be separated from carbohydrates during biomass conversion.

Energy-rich lignin can be burned for heat, converted to electricity consumable by other steps in the ethanol-production pathway, or gasified 

and converted to Fischer-Tropsch (FT) fuels (see Fig. 6. Mature Biomass 

Refining Energy Flows: Example Scenario, p. 16, and Table A. Summary of Energy Flows in Mature Biorefinery Concept, p. 16). For more 

information, see Deconstructing Feedstocks to Sugars, p. 85, and Sugar 

Fermentation to Ethanol, p. 119. For an overview of how genomics can 

be applied to developing new energy resources, see megasidebar, From 

Biomass to Cellulosic Ethanol, p. 26. 

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INTRODUCTION 

Fig. 6. Mature Biomass Refining Energy Flows: Example Scenario. A mature integrated cellulosic biomass biorefinery encompasses biological and thermochemical processes, demonstrating the 

efficiencies possible with a fully integrated design. This scenario incorporates 

the consolidated bioprocessing (CBP) concept, in which all biological processes 

are incorporated into a single microbe or microbial community. Energy derived 

from feedstocks is chemically and physically partitioned to ethanol and other 

products. Dotted arrows from above indicate energy inputs needed to run 

machinery. The thermochemical portion releases energy that can be used, for 

example, to sustain necessary temperatures, both heating and cooling, and to 

power pumps and other ancillary equipment. Table A is a summary of energy 

flows in this biorefinery concept. [Source: Adapted from L. Lynd et al., “Envisioning Mature Biomass Refineries,” presented at First International Biorefinery Symposium, Washington, D.C. ( July 20, 2005).] 

Table A. Summary of Energy 

Flows n Mature Borefinery 

Concept 

Products 

54% ethanol 

5% power (electricity) 

10% diesel 

6% gasoline 

• 

• 

• 

• 

Production Inputs 

21% captured for process energy 

or lost 

<5% agricultural inputs (e.g., 

farming costs, feedstock transport) 

• 

• 

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Ethcal, Legal, and Socal Issues (ELSI) 

Using biomass to produce biofuels holds much promise 

for providing a renewable, domestically produced liquid 

energy source that can be a viable alternative to petroleumbased fuels. Biofuel R&D, therefore, aims to achieve more 

than just scientific and technological advances per se. It is 

conducted to accomplish important societal needs, with 

the broader goals of bolstering national energy security,

economic growth, and the environment. Analyzing and 

assessing the societal implications of, and responses to, this 

research likewise should continue to be framed within the 

context of social systems and not simply in terms of technological advances and their efficacy (see sidebar, Ethical,

Legal, and Social Issues for Widespread Development of 

Cellulosic Biofuels, this page). 

EERE OBP Platform for Integrated 

Borefineres 

The Department of Energy’s strategic plan identifies 

its energy goal: “To protect our national and economic 

security by promoting a diverse supply and delivery of 

reliable, affordable, and environmentally sound energy.”

One of several strategies identified to achieve this goal is 

to “research renewable energy technologies—wind, hydropower, biomass, solar, and geothermal—and work with the 

private sector in developing these domestic resources.” 

The department’s Office of Energy Efficiency and Renewable Energy (EERE) Office of the Biomass Program 

(OBP) elaborates on that goal: “Improve energy security 

by developing technologies that foster a diverse supply of 

reliable, affordable, and environmentally sound energy by 

providing for reliable delivery of energy, guarding against 

energy emergencies, exploring advanced technologies that 

make a fundamental improvement in our mix of energy 

options, and improving energy efficiency.” 

Major outcomes sought include the following. 

•	 By 2012, complete technology development necessary to enable startup demonstration of a biorefinery 

producing fuels, chemicals, and power, possibly at an 

existing or new corn dry mill modified to process corn 

stover through a side stream. 

•	 By 2012 (based on AEI), complete technology integration to demonstrate a minimum sugar selling price of 

$.064/lb, resulting in a minimum ethanol selling price of 

$1.07/gal. Ethanol would be produced from agricultural 

residues or dedicated perennial energy crops. 

Ethcal, Legal, and Socal Issues 

for Wdespread Development of 

Cellulosc Bofuels 

Societal questions, concerns, and implications 

clearly may vary according to the evolutionary stage of biofuel development. Acceptance 

and support from diverse communities will be 

needed. Further, societal and technological interactions at earlier phases of research, development,

demonstration, deployment, and decommissioning (RDDD&D) will affect interactions at later 

phases. Within the context of social systems, three 

overarching questions emerge. 

•	 What are the possible long-term implications 

of biofuel development and deployment for 

social institutions and systems if the strategy 

“works” as anticipated and if it does not? 

•	 How are individuals, organizations, and institutions likely to respond over time to this 

development and the changes integral to its 

deployment? 

•	 What actions or interventions (e.g., regulations) 

associated with biofuel development and its use 

and deployment will probably or should be taken 

at local, regional, and national levels to promote 

socially determined benefits and to avoid, minimize, or mitigate any adverse impacts? 

Broad topics raised at the workshop included the 

following: 

•	 Sustainability of the total integrated cycle. 

•	 Competing interests for land use. 

•	 Creation and use of genetically modified 

plants. Who creates and uses them, who 

decides based on what criteria, and how might 

or should they be regulated? 

•	 Creation and use of genetically modified microbial organisms in a controlled industrial setting. 

•	 Individuals and groups that have the authority 

to promote or inhibit R&D, demonstration,

and use. 

•	 Groups most likely to be affected (positively 

or negatively) by biofuels at all evolutionary 

stages of RDDD&D on the local, national,

and global levels. 

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INTRODUCTION 

•	 By 2030, help enable the production of 60 billion gallons of ethanol 

per year in the United States. A report elaborating on this goal will be 

released soon. 

The Biomass Program also is aligned with recommendations in the May 

2001 NEP to expand the use of biomass for wide-ranging energy applications. NEP outlines a long-term strategy for developing and using 

leading-edge technology within the context of an integrated national 

energy, environmental, and economic policy. 

Fig. 7. DOE Energy Efficiency and Renewable Energy Strategic Goals as They Relate to Development of Biofuels.

[Source: Multi Year Program Plan 2007–2012, OBP, EERE, U.S. DOE (2005)] 

 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The program’s overarching strategic goal is to develop biorefinery-related 

technologies to the point that they are cost- and performance-competitive 

and are used by the nation’s transportation, energy, chemical, and power 

industries to meet their market objectives.The nation will benefit by expanding clean, sustainable energy supplies while also improving its energy infrastructure and reducing GHGs and dependence on foreign oil.This goal is in 

alignment with DOE and EERE strategic goals as shown in Fig. 7. DOE 

Energy Efficiency and Renewable Energy Strategic Goals as They Relate to 

Development of Biofuels, p. 18. 

Planning documents of EERE’s OBP describe advances the program seeks 

for four critical objectives: (1) Alter feedstocks for greater yield and for converting larger portions of raw biomass feedstocks to fuel ethanol and other 

chemicals; (2) decrease costs and improve enzyme activities that convert 

complex biomass polymers into fermentable sugars; (3) develop microbes that 

can efficiently convert all 5- and 6-carbon sugars released from the breakdown of complex biomass polymers; and (4) consolidate all saccharification 

and fermentation capabilities into a single microbe or mixed, stable culture. 

A commercial industry based on cellulosic biomass bioconversion to 

ethanol does not yet exist in the United States, but several precommercial 

facilities are in development. The Canadian company, Iogen Corporation, a 

leading producer of cellulase enzymes, operates the largest demonstration 

biomass-to-ethanol facility, with a capacity of 1 Mgal/year; production of 

cellulosic ethanol from wheat straw began at Iogen in April 2004. OBP 

has issued a solicitation for demonstration of cellulosic biorefineries (U.S.

Congress 2005, Section 932) as part of the presidential Biofuels Initiative. 

DOE Office of Scence Programs 

The DOE Office of Science (SC) plays key roles in U.S. research, including the contribution of essential scientific foundations to DOE’s national 

energy, environment, and economic security missions (see Fig. 8. DOE 

Office of Science Programs and Goals as They Relate to Development 

of Biofuels, p. 20). Other roles are to build and operate major research 

facilities with open access by the scientific community and to support core 

capabilities, theories, experiments, and simulations at the extreme limits 

of science. An SC goal for the Office of Biological and Environmental 

Research (OBER) is to “harness the power of our living world and provide 

the biological and environmental discoveries necessary to clean and protect 

our environment and offer new energy alternatives.” SC’s goal for its Office 

of Advanced Scientific Computing Research (OASCR) is “to deliver computing for the frontiers of science” (U.S. DOE 2004). 

To address these priorities, OBER and OASCR are sponsoring the 

Genomics:Genomes to Life (GTL) program. Established in 2002, GTL 

uses genome data as the underpinnings for investigations of biological systems with capabilities relevant to DOE energy and environmental 

missions. The GTL scientific program was developed with input from 

hundreds of scientists from universities, private industry, other federal 

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INTRODUCTION 

Fig. 8. DOE Office of Science Programs and Goals as They Relate to Development of Biofuels. [Derived from Office 

of Science Strategic Plan and Genomics:GTL Roadmap] 

agencies, and DOE national laboratories. Providing solutions to major 

national problems, biology and industrial biotechnology will serve as an 

engine for economic competitiveness in the 21st Century. DOE missions 

in energy security are grand challenges for a new generation of biological 

research. SC will work with EERE to bring together biology, computing,

physical sciences, bioprocess engineering, and technology development for 

the focused and large-scale research effort needed—from scientific investigations to commercialization in the marketplace. Research conducted by 

the biofuel R&D community using SC programs and research facilities 

will play a critical role in developing future biorefineries and ensuring the 

success of EERE OBP’s plans. 

The nation’s investment in genomics over the past 20 years now enables 

rapid determination and subsequent interpretation of the complete DNA 

sequence of any organism. Because it reveals the blueprint for life, genomics is the launching point for an integrated and mechanistic systems 

understanding of biological function. It is a new link between biological 

research and biotechnology. 

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Fig. 9. Understanding Biological Capabilities at All Scales Needed to Support Systems Biology Investigations of Cellulosic Biomass. Capabilities are needed to bring together the biological, physical, computational, and engineering sciences 

to create a new infrastructure for biology and the industrial biotechnology in the 21st Century. This figure depicts the focus 

of GTL on building an integrated body of knowledge about behavior, from 

ing. DOE

genomic interactions through ecosystem changes. Simultaneously studying 

multiple systems related to various aspects of the biofuel problem is powerfully synergistic because enduring biological themes are shared and general 

principles governing response, structure, and function apply 

throughout. Accumulating data as they are produced, the GTL 

Knowledgebase and GTL computational environment will 

interactively link the capabilities and research efforts, allowing 

this information to be integrated into a predictive understand-

’s technology programs can work 

with industry to apply such capabilities 

and knowledge to a new generation of processes, products, and industries. 

GTL’s goal is simple in concept but challenging in practice—to reveal how 

the static information in genome sequences drives the intricate and dynamic 

processes of life.Through predictive models of these life processes and supporting research infrastructure, GTL seeks to harness the capabilities of 

living systems. GTL will study critical properties and processes on four systems levels—molecular, cellular, organismal, and community—each requiring 

advances in fundamental capabilities and concepts.These same concepts and 

capabilities can be employed by bioprocess engineers to bring new technologies rapidly to the marketplace. 

Achieving GTL goals requires major advances in the ability to measure the 

phenomenology of living systems and to incorporate their operating principles into computational models and simulations that accurately represent 

biological systems.To make GTL science and biological research more 

broadly tractable, timely, and affordable, GTL will develop comprehensive 

suites of capabilities delivering economies of scale and enhanced performance 

(see Fig. 9. Understanding Biological Capabilities at All Scales Needed to 

Support Systems Biology Investigations of Cellulosic Biomass, this page). In 

vertically integrated bioenergy research centers, these capabilities will include 

the advanced technologies and state-of-the-art computing needed to better 

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INTRODUCTION 

Fig. 10. Creating a 

Common Research 

Agenda. The EERE 

Office of the Biomass Program’s Multi 

Year Program Plan 

2007–2012 contains 

a roadmap for biofuel 

development that 

identifies technological 

barriers to achieving 

goals defined in Fig. 7,

p. 18. These challenges include the need 

for new feedstocks, 

their deconstruction to fermentable 

sugars, and fermentation of all sugars 

to ethanol. Within 

the DOE Office of 

Science, OBER and 

OASCR’s roadmap for 

the Genomics:GTL 

program outlines scientific goals, technologies, computing needs, and a resource strategy to achieve the GTL goal of 

a predictive understanding of biological systems. This document is a roadmap that links the two plans. 

understand genomic potential, cellular responses, regulation, and behaviors of 

biological systems. Computing and information technologies are central to 

the GTL program’s success because they will allow scientists to surmount the 

barrier of complexity now preventing them from deducing biological function 

directly from genome sequence. GTL will create an integrated computational 

environment that will link experimental data of unprecedented quantity and 

dimensionality with theory, modeling, and simulation to uncover fundamental 

biological principles and to develop and test systems theory for biology. 

Bomass to Bofuels Workshop: Creatng a Common 

Research Agenda to Overcome Technology Barrers 

A product of the Biomass to Biofuels Workshop, this roadmap analyzes 

barriers to achieving OBP goals (as described herein) and determines 

fundamental research and capabilities (as described in the GTL Roadmap) that could both accelerate progress in removing barriers and allow a 

more robust set of endpoints (see Fig. 10. Creating a Common Research 

Agenda, this page). Relating high-level topical areas and their goals to key 

scientific milestones identified by workshop participants could help achieve 

progress toward OBP goals in collaboration with SC (see Table 2. Overcoming Barriers to Cellulosic Ethanol: OBP Biological and Technological 

Research Milestones, p. 23). 

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Table 2. Overcomng Barrers to Cellulosc Ethanol: OBP Bologcal and Technologcal Research Mlestones
 

Office of the Biomass 

Program (OBP) Barrier Topic Technology Goals Science Research Milestones 

Feedstocks Better compositions and structures for sugars Cell-wall architecture and makeup relative to processability 

Develop sustainable production Genome sequence for energy crops 

technologies to 

supply biomass to 

biorefineries 

Domestication: Yield, tolerance 

Better agronomics 

Sustainability 

Domestication traits: Yield, tolerance 

Cell-wall genes, principles, factors 

New model systems to apply modern biology tools 

Soil microbial community dynamics for determining 

sustainability 

Feedstock Pretreatment Enzymes Cell-wall structure with respect to degradation

Deconstruction to 

Sugars Reduced severity 

Reduced waste 

Modification of the chemical backbone of hemicellulose 

materials to reduce the number of nonfermentable and 

derivatized enzymes Develop biochemical 

conversion Higher sugar yields Cell-wall component response to pretreatments 

technologies to Reduced inhibitors Principles for improved cellulases, ligninases, produce low-cost 

sugars from Reduction in nonfermentable sugars hemicellulases 

lignocellulosic 

biomass Enzyme Hydrolysis to Sugars 

Higher specific activity 

Higher thermal tolerance 

Reduced product inhibition 

Broader substrate range 

Cellulases and cellulosomes 

Understanding of cellulosome regulation and activity 

Action of enzymes on insoluble substrates (fundamental 

limits) 

Fungal enzyme-production factors 

Nonspecific adsorption of enzymes 

Origin of inhibitors 

Sugar Fermentation to Cofermentation of Sugars Full microbial system regulation and control 

Ethanol C-5 and C-6 sugar microbes Rapid tools for manipulation of novel microbes 

Develop technologies 

to produce fuels, 

chemicals, and 

Robust process tolerance 

Resistance to inhibitors 

Utilization of all sugars 

Sugar transporters 

power from biobased 

sugars and chemical Marketable by-products Response of microorganisms to stress 

building blocks New microbial platforms 

Microbial community dynamics and control 

Consolidated Processing Enzyme Production, Hydrolysis, and Fundamentals of microbial cellulose utilization 

Reduce process steps Cofermentation Combined in One Reactor Understanding and control of regulatory processes 

and complexity by 

integrating multiple 

processes in single 

Production of hydrolytic enzymes 

Fermentation of needed products (ethanol) 

Engineering of multigenic traits 

Process tolerance 

reactors Process tolerance 

Stable integrated traits 

All processes combined in a single microbe or 

stable culture 

Improved gene-transfer systems for microbial engineering 

Understanding of transgenic hydrolysis and fermentation 

enzymes and pathways 

The workshop was organized under the following topical areas: Feedstocks 

for Biofuels (p. 57); Deconstructing Feedstocks to Sugars (p. 85); Sugar 

Fermentation to Ethanol (p. 119); and Crosscutting 21st Century Science,

Technology, and Infrastructure for a New Generation of Biofuel Research 

(p. 155). A critical topic discussed in several workshop groups was Lignocellulosic Biomass Characteristics (p. 39).These five topics and plans would 

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INTRODUCTION 

tie the two offices’ roadmaps together and also serve as a key driver for 

implementing the combined roadmaps in pursuit of a high-level national 

goal: Create a viable cellulosic-biofuel industry as an alternative to oil for 

transportation. These topics and their relationships are discussed in subsequent chapters outlining technical strategy and detailed research plans 

developed in the workshop. 

Cted References 

ACI. 2006. American Competitiveness Initiative: Leading the World in Innovation, Domestic Policy Council, Office of Science and Technology Policy 

(www.ostp.gov/html/ACIBooklet.pdf ). 

AEI. 2006. Advanced Energy Initiative, The White House, National 

Economic Council (www.whitehouse.gov/stateoftheunion/2006/energy/

energy_booklet.pdf ). 

Ag Energy Working Group. 2004. 25 by 25: Agriculture’s Role in Ensuring 

U.S. Energy Independence (www.bio.org/ind/25x25.pdf ). 

Bothast, R. J., and M. A. Schlicher. 2005. “Biotechnological Processes for 


Conversion of Corn into Ethanol,” Appl. Microbiol. Biotechnol. 67, 19–25.
 

Brinkman, N., et al. 2005. “Well-to-Wheels Analysis of Advanced Fuel/


Vehicle Systems—A North American Study of Energy Use, Greenhouse 


Gas Emissions, and Criteria Pollutant Emissions,” General Motors Corp.


and Argonne National Laboratory, www.transportation.anl.gov/pdfs/
 

TA/339.pdf; www.anl.gov/Media_Center/News/2005/news050823.html;


www.biorefineryworkshop.com/presentations/Wang.pdf
 

Bush, George W. 2006. “State of the Union Address,” Washington, D.C.,


Jan. 31, 2006 (www.whitehouse.gov/stateoftheunion/2006/).
 

BTAC. 2002. Roadmap for Biomass Technologies in the United States, Biomass Research and Development Technical Advisory Committee (www.


biomass.govtools.us/pdfs/FinalBiomassRoadmap.pdf ).
 

De La Torre Ugarte, D. G., et al. 2003. The Economic Impacts of Bioenergy 


Crop Production on U.S. Agriculture, U.S. Department of Agriculture and 


Department of Energy, Agricultural Economic Report No. 816 (www.


usda.gov/oce/reports/energy/AER816Bi.pdf ).
 

EIA. 2006. Annual Energy Outlook 2006 with Projections to 2030, DOE/


EIA-0383, Energy Information Administration, U.S. Department of 


Energy (www.eia.doe.gov/oiaf/aeo/).
 

Ethanol Across America. 2005. “Issue Brief: Energy Security” (www.ethanol.


org/documents/EnergySecurityIssueBrief_000.pdf).
 

Farrell, A. E., et al. 2006. “Ethanol Can Contribute to Energy and Environmental Goals,” Science 311, 506–8.
 

GEC. 2005. “Ethanol From Biomass: America’s 21st Century Transportation Fuel, Recommendations,” Governors’ Ethanol Coalition 

(www.ethanol-gec.org/coalitionstudies.htm). 

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Greene, N., et al. 2004. Growing Energy: How Biofuels Can Help End 

America’s Oil Dependence, Natural Resources Defense Council, New York 

(www.nrdc.org/air/energy/biofuels/biofuels.pdf ). 

Hammerschlag, R. 2006. “Ethanol’s Energy Return on Investment: A Survey of the Literature 1999–Present,” Environ. Sci. Technol. 40, 1744–50. 

IEA. 2004. Biofuels for Transport: An International Perspective, International 

Energy Agency (www.iea.org/textbase/nppdf/free/2004/biofuels2004.pdf ). 

Lovins, A. B., et al. 2005. Winning the Oil Endgame: Innovation for Profits, 

Jobs, and Security, Rocky Mountain Institute, Snowmass, Colorado. 

Multi Year Program Plan 2007–2012. 2005. Office of the Biomass Program,

Energy Efficiency and Renewable Energy, U.S. Department of Energy 

(www.eere.energy.gov/biomass/pdfs/mypp.pdf ). 

NCEP. 2004. Ending the Energy Stalemate: A Bipartisan Strategy to Meet 

America’s Energy Challenges, National Commission on Energy Policy (www.

energycommission.org/site/page.php?report=13). 

NEPDG. 2001. National Energy Policy: Reliable, Affordable, and Environmentally Sound Energy for America’s Future, National Energy Policy Development Group (www.whitehouse.gov/energy/National-Energy-Policy.pdf). 

NRDC. 2006. Ethanol: Energy Well Spent. A Survey of Studies Published 

since 1990, Natural Resources Defense Council and Climate Solutions,

New York City (www.nrdc.org/air/transportation/ethanol/ethanol.pdf ). 

Perlack, R. D., et al. 2005. Biomass as Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply, DOE/

GO-102005-2135, Oak Ridge National Laboratory, Oak Ridge, Tennessee 

(http://feedstockreview.ornl.gov/pdf/billion_ton_vision.pdf). 

Petrulis, M., J. Sommer, and F. Hines. 1993. Ethanol Production and 

Employment, Agriculture Information Bulletin No. 678, U.S. Department 

of Agriculture Economic Research Service. 

RFA. 2005. Homegrown for the Homeland: Ethanol Industry Outlook 2005,

Renewable Fuels Association (www.ethanolrfa.org/resource/outlook/). 

U.S. Commerce Dept. 2006. “U.S. International Trade in Goods and Services,” U.S. Census Bureau U.S. Bureau of Economic Analysis News, April 2006. 

U.S. Congress. 2005. Energy Policy Act of 2005, Pub. L. 109–58. 

U.S. Congress. 2002. Farm Security and Rural Investment Act of 2002, Pub. 

L. 107–71, Title IX, Sections 9001–10. 

U.S. Congress. 2000. Biomass Research and Development Act of 2000, Pub. L. 

106–224, Sections 301–10. 

U.S. DOE. 2005. Genomics:GTL Roadmap: Systems Biology for Energy and 

Environment, U.S. Department of Energy Office of Science 

(doegenomestolife.org/roadmap/). 

U.S. DOE. 2004. Office of Science Strategic Plan, U.S. Department of Energy 

Office of Science (www.er.doe.gov/Sub/Mission/Mission_Strategic.htm). References continued on p. 28. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 25 
 

INTRODUCTION 

link to From 

Biomass to Biofuelds 

26 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
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INTRODUCTION 

Wang, M. 2005. “Energy and Greenhouse Gas Emissions Impacts of Fuel 

Ethanol,” Ethanol Open Energy Forum, Sponsored by the National Corn 

Growers Association, National Press Club, Washington, D.C. (www.anl.

gov/Media_Center/News/2005/NCGA_Ethanol_Meeting_050823.html). 

Background Readng 

Yergin, D. 1992. The Prize: The Epic Quest for Oil, Money, and Power, Simon 

& Schuster, New York. 

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Techncal Strategy: Development 

of a Vable Cellulosc Bomass 

to Bofuel Industry 

I

nnovative energy crops—plants specifically designed for industrial processing to biofuels—can be developed concurrently with new biorefinery 

treatment and conversion processes. Recent advances in science and 

capabilities, especially those from the nascent discipline of systems biology,

promise to accelerate and enhance this paradigm. Resulting technologies 

will allow the fusion of agriculture, industrial biotechnology, and energy 

value chains to enable an efficient and economically viable industry for conversion of plant biomass to liquid fuels. Displacing up to 30% of the nation’s 

current transportation fuel by 2030 will require significant policy support 

and technical advancement over the next 5 to 15 years. Research and technology development described in this roadmap will occur in three phases to 

enable industry to meet the 2025 and 2030 goals (see Fig. 1. Phased 

Development of Bioenergy Systems, p. 32). In the Research Phase (this 

page), application of genome-based systems biology will provide the basis 

of knowledge, concepts, and tools for concerted research and deployment 

of technology modules in the Technology Deployment Phase (p. 32). In 

the Systems Integration Phase (p. 34), both fundamental and applied 

research and technology development will support multiple bioenergy systems through concurrent development of crops and biorefinery processes 

for various U.S. agroecosystems. Drivers for each phase and the research 

targets and goals for Feedstocks, Feedstock Deconstruction, and Fermentation to Ethanol and Recovery are outlined in Table 1. Technical Strategy 

Timeline, p. 33. 

Research Phase (wthn 5 years) 

Optimizing cellulose processing by refining biomass pretreatment and 

converting crop residues, first-generation energy crops, and other sources 

to liquid fuels will be the near-term focus. This will entail reducing cost,

enhancing feedstock deconstruction, improving enzyme action and stability, and developing fermentation technologies to more efficiently use sugars 

resulting from cellulose breakdown. One goal is to decrease industrial risk 

from a first-of-a-kind technology, allowing more rapid deployment of 

improved methods. 

Feedstock Use and Optmzaton 

A range of plant materials (e.g., corn stover and hard woods) with widely 

varying physical and chemical characteristics could be made available as 

feedstocks for conversion to ethanol in biorefineries. These legacy feedstocks 

are expected to satisfy one-fourth to one-third of the nation’s anticipated 

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TECHNICAL STRATEGY 

transportation biofuel needs. To achieve higher production goals, new 

energy crops with greater yield per acre and improved processability are 

needed. Advanced genome-based capabilities will help determine how soil 

microbial communities function and how much carbon from crop residues 

and dedicated energy crops, as well as other nutrients, is needed to sustain 

soil ecosystem function and productivity. 

To establish a new generation of plants as energy crops and develop stable 

agroecosystems, biological and chemical tools are needed to provide 

detailed understanding of plant cell walls, their roles in plant function, and 

factors controlling recalcitrance and optimization of processes for fermentation of sugars. Genome-based capabilities will identify genes involved in the 

synthesis of cell-wall polymers and higher structures; reactions performed 

by the multitude of enzymes involved; design principles of cell walls; and 

factors controlling the amounts, composition, and structure of polymers 

and polymer matrices. The complex structures of plant cell walls perform 

numerous critical functions in the plant’s growth and maintenance. Only 

some of these functions are now understood. Plant engineering’s end goal 

is to use rational design for preserving critical plant functions to maximize 

yield and agroeconomic factors, while optimizing plant biomass makeup 

and structure for creating biofuels and other products. Once desirable 

cell-wall traits are established for energy crops, modified varieties must be 

domesticated for robustness and yield, and bioprocessing steps must be 

adapted to the superior properties of these varieties. 

Plant design, bioprocess engineering, and biomass-processing strategies 

are intimately linked. Plants have evolved complex mechanisms for resisting assault on their structural sugars (wall polymers) from the microbial 

and animal kingdoms. Cell-wall polymer organization and interactions 

are formidable barriers to access by depolymerizing enzymes and must be 

deconstructed in the pretreatment step to obtain adequate rates of release 

and sugar yields. 

Deconstructon 

Understanding factors governing plant cell-wall recalcitrance to processing and deconstruction-enzyme interactions with the cell-wall matrix is 

critical to achieving the integrated biorefinery concept. Current technologies for biomass pretreatment (breaking down lignin and hemicelluloses 

and freeing crystalline cellulose) rely on thermochemical processing, which 

degrades the resulting biomass and results in accumulation of inhibitors 

that are toxic to ensuing biorefining processes. These harsh and energyintensive pretreatments must be replaced by more benign procedures,

including those that will enhance existing hydrothermal and mechanical 

methods and free cellulose microfibrils. Economical enzymatic procedures 

that would allow greater potential for integrated biorefinery strategies also 

will be incorporated. 

Genomics and the application of such relatively new tools as proteomics,

metabolomics, and imaging will aid investigators in mining burgeoning 

genomic databases to understand how microorganisms attack biomass 

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and take advantage of natural enzyme diversity. Available enzymes have 

relatively low specific activities, so enzyme interactions, mechanisms of 

action, and fundamental limits must be explored. We need to determine 

whether low activities reflect a fundamental limit to the hydrolysis rate of 

certain substrates or if rates can be improved by rational design through 

experimentation and modeling. New classes of ligninases and hemicellulases will be identified, their mechanisms of action understood, and their 

performance refined to allow introduction of enzymatic pretreatment 

that will free cellulose microfibrils for enzymatic saccharification (breakdown to sugars). 

Better methods will produce inexpensive and more robust cellulases with 

higher activity and longer lifetimes for breakdown of cellulose microfibrils to sugars. Mechanistic principles of cellulose-degrading enzymes will 

be evaluated using a range of genes and proteins found in the biosphere 

to understand basic design principles and allow rational redesign for 

enhancing properties. The diversity of cellulase and cellulosome functional 

schemes will be modeled and optimized for specific biomass substrates 

(feedstocks). The microbial cellulosome is a unique type of molecular 

machine that can efficiently solubilize native and pretreated lignocellulose 

substrates. Cellulosomes can contain the full repertoire of plant cell-wall 

polysaccharide-degrading enzymes, and a single cellulose-binding module 

presents the entire complement of enzymes to the substrate surface (see 

sidebar, The Cellulosome, p. 102). 

Fermentaton and Recovery 

Use of microbes for fermentation is the most common method for converting sugars produced from biomass into liquid fuels. To develop 

commercially viable processes for cellulose bioconversion to ethanol, an 

organism is needed that uses all sugars (cofermentation of C-5 and C-6 

sugars) produced from biomass saccharification at rates and in highalcohol concentrations that match or surpass current yeast-based glucose 

fermentations. These capabilities, along with process-tolerant traits, involve 

multiple genes and pathways that are not readily resolved. Today, the capability to introduce and control multiple gene changes simultaneously in an 

organism is limited. 

New classes of fermentation organisms and enzymes capable of metabolizing both C-5 and C-6 sugars resulting from biomass deconstruction are 

required to advance bioprocessing. Vast and largely untapped biochemical 

potential in the microbial world may be accessible through the sequencing of new microbial genomes and community “metagenomes.” As firstgeneration organisms are being tested and improved, the focus will be on 

advances that allow elimination of whole steps in the conversion process.

For example, thermophilic microorganisms will be examined for their ability to ferment biomass sugars at elevated temperatures, allowing development of optimal and simultaneous saccharification and cofermentation 

(SSCF) and thus increasing overall process efficiency. Metabolic engineering with advanced biological diagnostics will be used to develop strains 

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TECHNICAL STRATEGY 

Fig. 1. Phased Development of Bioenergy 

Systems. Over the next 5 

to 15 years, research and 

technology advancement 

will occur in three general 

phases. In the Research 

Phase, application of 

genome-based systems 

biology will provide 

the basis of knowledge,

concepts, and tools for 

implementation in the 

Technology Deployment 

Phase. In the Systems 

Integration Phase, both 

fundamental and applied 

research and technology will support multiple bioenergy systems 

through the concurrrent 

development of crops and 

biorefinery processes for 

the various U.S. agroecosystems. Details are 

outlined in Table 1, p. 33. 

with high tolerance to process stresses, inhibitors created in pretreatment,

and high-alcohol concentrations. Genomic, proteomic, metabolomic, and 

imaging technologies, coupled with modeling and simulation, will elucidate 

the regulation and control of microbial metabolism and provide a predictive understanding of cell-design principles to support system engineering 

of integrated bioprocessing (see Fig. 1. Phased Development of Bioenergy 

Systems, this page, and Table 1. Technical Strategy Timeline, p. 33). 

Technology Deployment Phase (wthn 0 years) 

A new generation of dedicated energy crops with composition and structure better suited for breakdown to sugars for fermentation, high yield, and 

robustness will be essential in achieving energy security. Newly engineered 

deconstruction enzymes will enhance or supplant thermochemical processing to deconstruct more efficiently a broad range of biomass feedstocks.

Ultimately, this will lead to enhanced energy-efficient and environmentally 

attractive processes for carrying out traditional pulping processes and other 

wood-processing techniques. 

2 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table . Techncal Strategy Tmelne
 

Research Phase (0 to 5 years) Technology Deployment Phase (5 to 10 years) Systems Integration Phase (10 to 15 years) 

Legacy Resources and Early Energy Crops (E-Crops) Transition: Modular Technology Deployment Integration and Consolidation 

Drivers Drivers Fusion of economic value chains 

Expand biomass resource base and increase • Need for new energy crops to reach or Drivers 

utilization exceed 1 billion tons of biomass • Consolidated bioenergy systems 

Early E-crops 

Cellulose-processing viability 

• 

• 

Process simplification and improvement 

in modules 

• Technologies tailored for various 

regions 

• 

• Cost reduction Use of systems biology, chemistry 

Bioprocess engineering 

Rational systems design 

• 

• 

• 

Buildout • 

Feedstocks 

E-crop sustainability 

Impacts on soil ecosystems, nutrients 

E-crop model development 

Genes, principles, E-crop subsystem 

controls 

Cell-wall makeup and structure 

Ties to deconstruction and 

fermentation 

• 

• 

• 

 

 

Feedstocks 

Understand plant as system 

Domesticate E-crops 

Enhance sugars, minimize lignin and 

toxic inhibitors 

Increase yield and soil sustainability 

• 

• 

• 

• 

End-to-End Concurrently Engineered 

Biofuel Systems (Biome-E Crop Processing) 

Systems tailored to regions and fully 

consolidated processing 

Includes E-crops with enhanced 

composition 

Tool kits for plant engineering 

Consolidated processing tied to 

biofuel systems 

Tailored deconstruction enzyme 

mix 

Engineered microbial metabolic 

systems

Stress and process tolerance 

Full system control

• 

• 

• 

• 

 

 

 

 

Feedstock Deconstruction 

Reduce enzyme costs 

Understand enzyme-lignocellulose 

interactions 

• 

• 

Deconstruction 

Deploy 

Improved enzymes (rate, specificity) 

Broadened substrate range 

• 

 

Cell-wall recalcitrance 

Survey natural enzyme diversity 

Establish fundamental enzyme limits 

Develop ligninases and hemicellulases 

Develop gene-transfer systems for 

cellulolytic machines and entire 

pathways 

 

• 

• 

• 

• 

Reduced inhibition 

E-crop concept 

Tools to diagnose and manipulate enzyme 

substrate interactions 

Tools to design and improve enzymes 

 

 

• 

• 

Tool kits for rapid manipulation and 

diagnosis 

• 

Fermentation to Ethanol and Recovery Fermentation and Recovery 

• Study use of all sugars, including direct Deploy 

cellulose utilization • Cofermentation of C-5 and C-6 sugars 

• Study stress response and inhibitors • New strains (multiple) 

High-alcohol and -sugar 

concentrations 

  Stress tolerance 

• Understand regulation and control  High temperature

• Survey natural diversity Tools for full regulatory control 

Tools for rapid analysis and manipulation 

Testing of consolidated organisms 

possessing celluloytic and ethanologenic 

properties 

• 

• 

• 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy  
 

TECHNICAL STRATEGY 

Mid-term technology will begin the consolidation of process steps. For 

example, improved organisms will be engineered with the ability to 

ferment mixed sugars, demonstrate resistance to toxic substances, and 

produce effective deconstruction enzymes. Systems biology and a new 

generation of synthetic and analytical organic chemistry will be critical for 

understanding these bioenergy systems and for predicting and modifying 

their function. 

Feedstocks 

Plants intended for biomass production and downstream processes involving conversion to sugars and, ultimately, ethanol will be understood and 

designed as a system. New breeds of energy crops will be introduced with 

enhanced sugar content and optimized cell-wall structures for processing,

including minimization of lignin and inhibitor precursors. Plant domestication and sustainable agroecosystems based on perennials engineered to 

increase yield, productivity, and tolerance to such stressors as drought and 

salinity will reach a mature state. Multiple crops will be developed for various regional and global agroecosystems. 

Deconstructon 

This phase will result in deployment of improved pretreatment procedures 

and saccharifying enzymes with enhanced catalytic rate and substrate 

specificity, a broader range of applications, and reduced inhibitor production. Improved understanding of cell-wall recalcitrance and enzyme action 

will provide design specifications for new energy crops. Advanced highthroughput biological and chemical tools will be available to diagnose and 

manipulate enzyme-substrate interactions. Improved biocatalysts with 

desirable traits can be rationally designed for specific feedstocks and incorporated into molecular machines such as cellulosomes. 

Fermentaton and Recovery 

New strains of industrial-processing organisms with such novel capabilities 

as cofermentation of C-5 and C-6 sugars and high tolerance to inhibitors,

alcohol end product, and temperature will contribute to a more energy 

and product efficient bioprocess. Systems biology investigations will have 

produced a predictive understanding of cellular metabolism and regulatory 

controls in key fermentation microbes. This knowledge will serve as a foundation for rational development of new strains with consolidated subsets of 

pretreatment, hydrolysis, and fermentation capabilities. High-throughput 

biological and chemical tools, including computational modeling for rapid 

analysis and manipulation in the laboratory and in production environments, will be available. 

Systems Integraton Phase (wthn 5 years) 

Bioenergy systems will include a concurrently engineered set of designer 

energy crops for specific agroecosystems, deconstruction and saccharification enzymes, and robust fermentation. Incorporated as multiple processes 

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in plants or microbes, these methods will accelerate and simplify the endto-end production of fuel ethanol, enabling flexible biorefineries that can 

operate on a regional scale. 

Integraton and Consoldaton 

Creation of fully consolidated bioenergy systems tailored for specific 

regional climate and soil characteristics will allow buildout of these sustainable bioenergy economies. These systems will meld feedstocks, biomass deconstruction, bioprocess engineering, and fermentation research 

and development, yielding optimal two-step processes. The first step is 

based on consolidated feedstock traits, and the second is based on consolidated microbial traits. A concurrently engineered end-to-end biofuel 

system using advanced systems biology and chemical-analysis capabilities will be practicable. Toolkits for plant and agroecosystem engineering will support systems tailored to regions and consolidated processing.

Companion consolidated bioprocess engineering will be tied to agroecosystems; with tailored enzyme mixes, engineered microbial metabolic systems will incorporate stress and process tolerance and permit full system 

control. Instrumentation available both in facilities and in the field will 

enable rapid diagnosis and manipulation of all critical aspects of the 

integrated biorefinery. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 5 
 

TECHNICAL STRATEGY 

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SYSTEMS BIOLOGY TO OVERCOME 

BARRIERS TO CELLULOSIC ETHANOL 

Lignocellulosic Biomass Characteristics: Makeup, Structure, 


and Processability .............................................................................39
 

Sidebar
 

Understanding Biomass: Plant Cell Walls .................................................... 53
 

Feedstocks for Biofuels ......................................................................57
 

Deconstructing Feedstocks to Sugars .................................................85
 

Sugar Fermentation to Ethanol........................................................119
 

Crosscutting Science, Technology, and Infrastructure........................155
 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 7 
 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

Lgnocellulosc Bomass 

Characterstcs 

Makeup, Structure, and Processability 

Lignocellulosic biomass has long been recognized as a potential lowcost source of mixed sugars for fermentation to fuel ethanol. Plant 

biomass has evolved effective mechanisms for resisting assault on its 

structural sugars from the microbial and animal kingdoms. This property 

underlies a natural recalcitrance, creating technical barriers to the costeffective transformation of lignocellulosic biomass to fermentable sugars.

Moderate yields and the resulting complex composition of sugars and 

inhibitory compounds lead to high processing costs. Several technologies 

have been developed over the past 80 years, often in wartime, that allow 

this conversion process to occur, yet the clear objective now is to make the 

process cost-competitive in today’s markets. 

Cell walls in lignocellulosic biomass can be converted to mixed-sugar solutions plus lignin-rich solid residues by sequential use of a range of thermochemical pretreatments and enzymatic saccharification. The low rate at 

which biomass is converted to sugars and the coproduction of fermentation inhibitors increase equipment size and result in high pretreatment and 

enzyme costs. New approaches for designing improved energy feedstocks,

deconstructing plant cell walls, and transforming their polysaccharides 

to fermentable sugars are needed. A systematic understanding of enzyme 

interactions with plant cell architecture and hierarchy, as well as cellulose,

hemicellulose, and lignin structure during chemical and enzymatic hydrolysis, will allow the prediction of plant-tissue response to hydrolytic attack 

and the creation of new systems. 

Significant technology development will be needed for creation of largescale bioenergy and biorefinery industries that can handle a billion tons 

made up of a variety of biomass each year. In the DOE-USDA BillionTon Study, corn stover and perennial crops such as switchgrass and hybrid 

poplar make up about half the potential 1.3 billion tons of biomass that 

could be available by the mid-21st Century (Perlack et al. 2005). Understanding the structure and function of these and other biomass resources 

will be critical to enhancing their processability. 

The result of analysis and research described here will be to increase the 

efficiency with which the solid (substrate) interacts with large protein 

macromolecules (enzymes) at its surface while the surface itself is being 

eroded into soluble oligosaccharides [see sidebar, Image Analysis of Bioenergy Plant Cell Surfaces at the OBP Biomass Surface Characterization 

Lab (BSCL), p. 40]. This knowledge, combined with development of new 

proteins that catalyze these transformations as well as microbial systems 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

LIGNOCELLULOSIC BIOMASS 

Image Analyss of Boenergy Plant Cell Surfaces at the OBP Bomass Surface 

Characterzaton Lab (BSCL) 

Many aspects of current biomass conversion technology are becoming better understood, and a nascent 

biomass processing industry is emerging for some niche markets. To reach the mid- and long-term 

goals stated in the DOE Office of the Biomass Program’s Multi Year Program Plan: 2007-2012,

however, enhanced fundamental understanding of feedstocks and all biorefinery processes is critical. For example, detailed knowledge about plant 

cell-wall ultrastructure and function to formulate improved enzyme 

mixtures and pretreatments will 

reduce the cost of producing sugars. (See images, right.) 

In many cases, we know how to 

describe biomass compositionally.

That is, we can conduct chemical or spectroscopic analyses and 

determine the percentages of 

individual sugars, protein, uronic 

acids, and lignin. When we study 

biomass conversion of corn stover, hardwoods, or rice straw, for 

example, we are in fact working 

primarily with the plant’s structural parts, most of which are cell 

wall. Therefore, more knowledge is 

needed about the natural organization and structure of polymers and 

chemicals in plant tissue that affect 

chemical pretreatment, enzymatic 

digestibility, and the generation of 

compounds inhibiting fermentative 

microorganisms used to produce 

the final fuel or chemical. The study 

of plant cell walls at the submicron or macromolecular scale is 

challenging. Imaging and image 

analysis are at the cutting edge of 

botany, molecular biology, biochemistry, chemistry, and material 

and computer sciences. Descriptions of microscopies important 

for ultrastructure imaging are in 

the Imaging Technologies section 

of the Crosscutting Technologies 

chapter, p. 163, and sidebar, Some 

Imaging Technologies Relevant to 

Feedstock Characterization, p. 163. 

Fig. A. Collage of Scanning Electron Microscopy Images Showing a 

Rind and Adjacent Pith Section Cut from a Field-Dried Corn-Stem 

Cross Section. The rind shows a higher density of vascular elements made 

from thick-walled cells. The pith section (shown longitudinally) shows a 

greater number of thin-walled parenchyma cells. Overall, most cellulose 

needed for biomass conversion is located in the rind, although the pith represents most of the stem volume. Closeups of a cell-wall pit also are shown 

(~150,000×). These structures are thought to aid transfer of chemicals and 

enzymes used in processing within the biomass bulk. 

Fig. B. AFM: Corn Parenchyma Cell Wall. 

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for fermentation and consolidation, will enable the design of procedures 

and hardware that dramatically speed up the process, improve yield, and 

lower costs. 

Structure and Assembly of Cell Walls 

Plant cell walls are a complex and dynamic mixture of components that 

perform many functions (see Fig. 1. Simplified Cell Wall and Fig. 2.

Conceptual Illustration of Cell-Wall Biogenesis, p. 42; and sidebar, Understanding Biomass, pp. 53 to 55). The cell walls are intricate assemblages of 

celluloses, hemicelluloses (i.e., xyloglucans, arabinoxylans, and glucomannans), pectins (i.e., homogalaturonans, rhamnogalacturonan I and II, and 

xylogalacturonans), lignins, and proteoglycans (e.g., arabinogalactan-proteins, extensins, and proline-rich proteins). Most mass in the plant cell 

wall is in the form of polysaccharides (cellulose and hemicelluloses). The 

next most abundant polymer is lignin, which is composed predominantly 

of phenylpropane building blocks. Lignins perform an important role in 

strengthening cell walls by cross-linking polysaccharides, thus providing 

support to structural elements in the overall plant body. This also helps the 

plant resist moisture and biological attack. These properties of lignin, however, interfere with enzymatic conversion of polysaccharide components.

Additionally, since lignin is not converted readily to ethanol, we must find 

other uses in the process if we are to maximize energy yield from biomass. 

Several thousand gene products are estimated to participate in synthesis,

deposition, and function of cell walls, but very few associated genes have 

been identified and very little is known about their corresponding enzymes.

Many questions remain, for example, regarding how polysaccharides and 

lignin are synthesized, how wall composition is regulated, and how composition relates to cell-wall biological functions. To answer these questions,

we need to discover the functions of many hundreds of enzymes, where 

proteins are located within cells, whether or not they are in complexes,

where and when corresponding genes are expressed, and what factors and 

genes control expression and activities of the proteins involved. Application of new or improved biological, physical, analytical, and mathematical tools will facilitate a detailed mechanistic understanding of cell walls.

That knowledge will permit optimization of various processes involved in 

producing biomass and converting it to fuels. 

Productivity and conversion-process efficiencies can be increased by altering fundamental aspects of plant growth, development, and response to 

biotic and abiotic stress. Altering cell-wall composition to increase the 

relative amount of cellulose and decrease lignin, for example, could have 

significant effects (see sidebar, Optimizing Lignin Composition for More 

Efficient Bioethanol Production, p. 43). Eventually, a systems cell-wall 

model incorporating biophysical aspects with structural properties and 

knowledge of proteins involved in synthesis will aid in rational development of highly productive feedstock species whose cell walls are optimized for conversion. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy  
 

LIGNOCELLULOSIC BIOMASS 

Fig. 2. Conceptual Illustration of Cell-Wall Biogenesis. The Golgi apparatus participates in hemicellulose and lignin 

biosynthesis. Cellulose microfibrils (CMF) are laid separately in the swollen gel of hemicelluloses. As lignin is deposited,

the cell wall becomes hydrophobic. Water removal from the swollen gel, together with peroxidase (PO) and calcium,

causes anisotropic shrinkage perpendicular to the CMFs. This shrinkage drives further oligolignol polymerization by 

orienting the lignin aromatic ring parallel to the cell-wall surface. [Adapted from N. Terashima et al., “Comprehensive 

Model of the Lignified Plant Cell Wall,” pp. 247–70 in Forage Cell Wall Structure and Digestibility, ed. H. G. Jung et al.,

American 

Society of 

Agronomy,

Crop Science Society 

of America, 

and Soil Science Society 

of America 

(1993).] 

Fig. 1. Simplified Cell 

Wall. For more details, see 

sidebar, Understanding 

Biomass, p. 53. [Adapted 

with permission from 

C. Somerville et al., 

Science 306, 2206–11 

(2004); © 2004 AAAS.] 

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Plants can have two types of cell walls, primary and secondary. Primary 

cell walls contain cellulose, which consists of hydrogen-bonded chains of 

thousands of β-1,4-linked glucose molecules, in addition to hemicelluloses 

and other materials woven into a nanoscale network with the cellulose. 

Cellulose in higher plants is organized into microfibrils, each measuring 

about 3 to 6 nm in diameter and containing up to 36 cellulose chains. Each 

Optmzng Lgnn Composton for More Efficent Boethanol Producton 

Plant lignin (guaiacyl and syringyl) interferes with the release and hydrolysis of cell-wall polysaccharides. Metabolic engineering of the lignin biosynthetic pathway has been suggested as a method for modifying lignin content in feedstocks. Studies in Arabidopsis demonstrated that overexpression of the enzyme ferulate 5-hydroxylase 

(F5H) increases lignin syringyl monomer content and abolishes the tissue specificity of its deposition (Fig. A). 

To determine whether or not this enzyme has a similar regulatory role in woody plants, F5H was overexpressed 

in poplar trees using a cinnamate 4-hydroxylase promoter to drive F5H expression. Transgenic trees displayed 

enhanced lignin syringyl monomer content, indicating that F5H overexpression is a viable metabolic engineering strategy for modifying lignin biosynthesis. These high-syringyl lignin poplars demonstrated a significant 

increase in chemical pulping efficiency. [R. Franke et al., “Modified Lignin in Tobacco and Poplar Plants OverExpressing the Arabidopsis Gene Encoding Ferulate 5-Hydrosylase,” Plant J. 22(3), 223–34 (2000).] Similar 

metabolic engineering strategies hold promise for developing improved feedstocks for bioethanol production. 

Many aspects of lignin biosynthesis remain matters of debate. Although most genes involved in the biosynthetic pathway have been cloned and functions assigned, mechanisms that regulate the pathway still are largely 

unknown, as is its relationship with other cell-wall biochemical pathways and plant development. Topics to 

be studied include regulation of lignin deposition and tissue specificity, identity of proteins involved in monolignol transport and polymerization, and ways in which lignin content and composition can be modified (see 

Fig. 4. Phenylpropanoid Pathway Leading to Lignin Biosynthesis in Plants, p. 49). Also needed is a detailed 

understanding of lignin-biodegradation mechanisms, including that accomplished by white rot fungi, which 

break down yellow lignin and leave behind crystalline white cellulose (see sidebar, White Rot Fungus, p. 93).

Comprehensive explorations of lignin biosynthesis and degradation are required to maximize energy yield 

from biomass crops. 

Fig. A. Lignin Composition Controlled by Genetic Manipulation and Monitored via 

Histochemical Staining for Lignin Monomer Composition in Arabidopsis Stem Cross 

Sections. In the lignified cells of wild-type Arabidopsis stems, the presence of syringyl (S) 

or guaiacyl (G) monomers can be visualized by histochemical staining of S lignin (red) 

and G lignin (yellow). 

Histochemical staining allows a diagnosis of the effects of experiments to manipulate 

lignin composition. For example, eliminating one enzyme of the lignin biosynthetic pathway in an Arabidopsis mutant (fah1-2) leads to a pure G lignin, and overexpression of the 

same enzyme leads to a homogeneous deposition of S lignin (35S-F5H). The fah1 gene 

encodes ferulate-5-hydroxylase, a cytochrome P450–dependent monooxygenase that 

catalyzes hydroxylation of coniferaldehyde and coniferyl alcohol in the pathway leading to 

syringyl lignin. [Figures published in C. Chapple et al., “Lignin Monomer Composition 

is Determined by the Expression of a Cytochrome P450–Dependent Monooxygenase in 

Arabidopsis,” Proc. Natl. Acad. Sci. USA 95, 6619–23 (1998); ©1998 National Academy of 

Sciences, U.S.A.] 

S lignin 

S lignin 

G lignin 

G lignin 

S lignin 

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LIGNOCELLULOSIC BIOMASS 

cellulose chain is a linear collection of thousands of glucose residues. Pairs 

of glucose residues (cellobiose) make up the repeating unit of cellulose. 

Like steel girders stabilizing a skyscraper’s structure, the mechanical 

strength of the primary cell wall is due mainly to the microfibril scaffold.

A microfibril’s crystalline and paracrystalline (amorphous) cellulose core 

is surrounded by hemicellulose, a branched polymer composed of pentose 

(5-carbon) and, in some cases, hexose (6-carbon) sugars. In addition to 

cross-linking individual microfibrils, hemicelluloses in secondary cell walls 

also form covalent associations with lignin, a complex aromatic polymer 

whose structure and organization within the cell wall are not completely 

understood (see Fig. 3. Association of Lignin with Polysaccharides, this 

page). The crystallinity of cellulose and its association with hemicellulose 

and lignin are two key challenges that prevent the efficient breakdown of 

cellulose into glucose molecules that can be fermented to ethanol. 

Fig. 3. Association of Lignin with Polysaccharides. The schematic diagram shows possible covalent cross-links between 

polysaccharides and lignin in cell walls. Lignin is bonded to the 

cellulose and hemicellulose polysaccharides and serves as a stiffening and hydrophobic agent, complicating biomass breakdown.

[Source: Adapted from K. Iiyama, T. Lam, and B. Stone, “Covalent Cross-Links in the Cell Wall,” Plant Physiol. 104(2), 318 

(1994). Reprinted with permission of American Society of Plant 

Biologists, ©ASPB 1994.] 

Many enzymes involved in cell-wall synthesis 

or modification are thought to be located in 

protein complexes. Within the plasma membrane are rosettes composed of the enzyme 

cellulose synthase; these protein complexes 

move laterally along the membrane to synthesize cellulose molecular chains (36 per rosette),

which crystallize into microfibrils. Movement 

of the rosette molecular machine is associated with cortical microtubules that underlie 

the membrane, but that linkage also is poorly 

understood. The interaction of cellulose synthase with the cytoskeleton has an impact on 

cellulose fibril orientation and perhaps length.

Understanding the function of these complexes and their interactions with metabolic 

pathways that produce sugars will be important for eventually controlling cell-wall composition. A number of cellulose-synthase genes 

have been cloned for a variety of plants. (See 

sidebar, Understanding Biomass, beginning on 

p. 53, for an illustrated explanation.) 

Factors n Recalctrance of 

Lgnocellulose Processng to Sugars 

Organization and interactions among polymers 

of the cell wall—constructed for strength and 

resistance to biological, physical, and chemical attack—constitute a barrier to access by 

depolymerizing enzymes and must be partially 

deconstructed in the bioconversion pretreatment 

step before saccharification can occur. Although 

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various pretreatments have been developed, we still do not have a detailed 

understanding of fundamental physical and chemical features of lignocellulosic biomass that limit its breakdown to sugars and ultimate bioconversion 

efficiency. Improved methods must be developed to characterize biomass and 

its interaction with various chemical treatments, as well as with deconstruction and saccharification enzymes. 

Natural factors believed to contribute to lignocellulosic feedstock’s recalcitrance to chemicals or enzymes include plant and cell-wall architecture and 

molecular structure. 

Plant Archtecture 

The organs of a plant (leaf, stem or trunk, and root) are composed of 

myriads of cells with different functions in the plant’s economy. Each has 

its own particular type of cell wall whose composition is related directly to 

cell function [e.g., support (fibers), protection (epidermis), and transport 

(xylem and phloem)]. Leaf, stem, and root tissues invariably contain cells 

of more than one type. In tissues, individual cells are closely associated at 

their cell-wall interfaces to give a compact tissue structure. This structure 

must be disassembled by milling (comminution) to allow liquid access to 

cell walls. 

•	 The waxy barrier comprising grass cuticle and tree bark impedes penetration of enzymes. 

•	 Even milled plant stems and woody tissues limit liquid penetration by 

their nature. 

Cell-Wall Archtecture 

The nanoscale composite nature of the plant cell wall restricts penetration 

of chemicals and enzymes to their substrates. The lignin-hemicellulose 

coating on the cell wall’s cellulosic microfibrils affects the following: 

•	 Conformation of cellulose and noncellulosic polysaccharides making 

up the microfibril limits accessibility of hydrolytic enzymes to their 

substrates. 

•	 Lignin-carbohydrate complexes limit enzymatic hydrolysis of biomass 

polysaccharides. 

Molecular Structure 

Cellulose crystallinity severely restricts cellulase attacks. Cellulases must 

physically release individual cellulose chains from microfibril crystals for 

subsequent catalytic hydrolysis to sugars. Limiting factors: 

•	 Inherent difficulty of enzymes in acting on poorly hydrated cellulose surfaces. 

•	 Amount and composition (including heterogeneity) of lignin. 

•	 Chemical heterogeneity and strength of covalent interactions between 

lignin and noncellulosic polysaccharides. 

•	 Robustness of hydrogen bonding in cellulose microfibrils arising from 

extended hydrogen-bond periodicity. 

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LIGNOCELLULOSIC BIOMASS 

In addition, all native celluloses undergo physical modifications that can 

inhibit saccharification as they are dehydrated in traditional methods of 

isolation or storage after harvest and in pretreatment processes. Characterizing the effects of conditions and storage environments would point 

to modifications in harvesting and storage for biomass resources. Understanding physicochemical characteristics of the cell-wall polysaccharide 

system would guide genomic modifications of bioenergy crops to facilitate 

processing and resist deleterious physical modification as much as possible.

For example, structural elements of many lignocellulosic materials react to 

pretreatment in ways that reduce enzymatic digestibility. 

•	 High mechanical pressure such as that from plug feeders collapses the 

natural vascular structure. 

•	 Dilute-acid chemical pretreatments may permit cellulose to reanneal,

leading to “hornification” of cellulose in microfibrils. 

•	 Ambient or elevated temperatures may accelerate denaturation (e.g., the 

tendency of most (beta-1,4)-pentosans and hexosans to have inverse 

water-solubility relationships with temperature). 

•	 Some pretreatments may permit lignin to become soluble and “plate 

out” on cellulose surfaces during the cool-down phase. 

These “process-induced” causes of recalcitrance must be understood and 

overcome through process modification or biomass design. 

After cellulose, hemicellulose is 

Optmzng Hemcellulose Acetylaton n Cell Walls the next most abundant polysaccharide in native biomass feedHemicellulose Acetylation Degradation Products Are Toxic to Microbes stocks. Structural information 

on these polymeric substrates is Acetyl side groups from hemicellulose biomass polymers are released 

during current pretreatment steps. These small acetyl molecules necessary, and mechanistic models 

often are toxic and inhibit the microbial activity that converts sug- must be developed to identify 

ars to ethanol. Hemicellulosic components such as xyloglucan and gluc- “bottlenecks” in hemicellulose 

uronoarabinoxylan and pectic cell-wall components often are O-acetylated. bioconversion (see sidebar, OptiFor instance, O-acetyl groups may be present on the glucan backbone of mizing Hemicellulose Acetylation 

xyloglucan or on galactose or arabinose residues of side chains. The degree in Cell Walls, this page). 

of sugar-residue O-acetylation of pectins varies from 0 to 90% depend- A systematic approach to under­ ing on the tissue, species, and method of preparation. The role of O-acetyl standing these factors will substituents in vivo is not known, but in vitro experiments suggest that one promote more effective use of function may be their involvement in hindering enzymatic polysaccharide lignocellulosic biomass in biocon­ breakdown. O-acetyl substituents also affect polysaccharide solubility and version systems. Fortunately, we pectin’s gelation properties (Pauly and Scheller 2000). now have new biological, physiPlant genes exhibit weak sequence similarity to putative bacterial acetyl- cal, analytical, and mathematical 

transferase genes. Genetic tools in plants such as Arabidopsis will enable tools that can help in reliably 

the identification of gene products catalyzing polysaccharide acetylation identifying and quantifying the 

and the determination of acetylation’s role in cell-wall structure and func- relative importance of various 

tion. Such studies will provide insights into the possibility of developing potentially limiting factors. We 

biomass crop varieties with significantly reduced polysaccharide acetyla- also have tools to identify and 

tion and thus improving the fermentation process. optimize facilitating factors, for 

example, through plant breeding. 

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The goal is to provide a rational basis for design of practical, effective, and 

economical pretreatments, including controlling the physical modification 

of native celluloses and related cell-wall polysaccharides during thermal 

and chemical treatments. Current thermochemical treatments ultimately 

will be replaced with more benign enzymatic treatments to the degree 

feasible. Necessary detailed analyses are discussed in the chapter, Deconstructing Feedstocks to Sugars, p. 85. 

Optmzaton of Plant Cell Walls 

Optimal efficiency of biofuel production depends on maximizing fuel yield 

from a unit of biomass and minimizing energy inputs. The plant cell wall, a 

complex assembly that plays a primarily, but not exclusively, structural role 

during plant growth and development, may be particularly amenable to the 

application of engineering principles in redesigning the cell wall to meet 

energy needs. To breed plants in which cell-wall composition is optimized 

for conversion efficiency, understanding how cell walls are made, how 

composition is regulated, and the roles of various polymers in supporting 

plant growth and development will be necessary. The long-term goal is to 

develop a systems-level understanding to facilitate rational improvement 

of plant cell-wall composition in dedicated energy crops. Such knowledge 

of plant cell walls is in a very primitive stage because of scientific and 

technical challenges that have impeded scientific progress. Future research 

on cell-wall synthesis and function requires interdisciplinary approaches 

ranging from genomics to synthetic carbohydrate chemistry and biophysics. Model organisms are important in facilitating advances in basic biology 

and in bringing the most sophisticated biological tools to the problem.

Several new plant models closely related to species selected for energy 

crops are advocated. A powerful first step is to obtain comprehensive DNA 

sequences for these organisms. 

Understandng Cell-Wall Structure and Functon 

Increasing the production of biofuels begins with increasing biomass productivity, either by making more cell walls or making cell walls with more 

carbon. In addition, changes in cell-wall composition could have major 

effects on the efficiency with which biomass can be converted to fuels; relative amounts of certain sugars could be increased or wall polymers could be 

made more amenable to enzymatic hydrolysis, thus improving the yield of 

sugars delivered to the biorefinery as raw feedstock. 

Important questions remain about the structures of cell-wall polymers, how 

they are made, and their functions in plant growth and development.To 

optimize the amount, composition, and structure of walls for biofuel production, we must identify the genes involved in synthesis of cell-wall polymers,

the design principles for cell walls, and factors that control the amounts and 

organization of various types of enzymes and resultant polymers. Preliminary evidence suggests that cell-wall biophysical properties important to 

plant growth and development may be achieved in many different ways with 

regard to chemical composition.Thus, cell-wall composition of energy crops 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 7 
 

 

 

 

 

LIGNOCELLULOSIC BIOMASS 

probably can be altered so they are better suited for fuel production. Desirable improvements include increasing the amount of such useful polysaccharides as cellulose or certain hemicelluloses and minimizing the content of 

such undesirable components as lignin or acetyl groups. 

Evidence indicates that photosynthetic CO2 fixation is regulated by plants 

in response to demand for fixed carbon, so understanding photosynthate 

flux into cell-wall polymers relative to other pathways of primary carbon 

metabolism and storage is important. Understanding mechanisms that 

regulate carbon flux and synthesis of various polysaccharides may make 

possible the development of plants that accumulate significantly more 

polysaccharide per cell. Expected significant increases in the ratio of 

carbon to nitrogen and mineral nutrients would have a beneficial effect on 

agricultural inputs (e.g., planting, fertilizing, cultivating, and harvesting),

costs, and sustainability. 

Progress in this area requires broad approaches to achieve a foundation 

of knowledge about cell-wall structure and function that will be the basis 

for a systems approach to predicting and controlling biomass composition. Before a systems approach can be implemented, a comprehensive 

understanding is needed about what reactions are performed by the many 

hundreds of enzymes involved in cell-wall synthesis and deposition, where 

and when relevant genes are expressed, and what genes control expression 

and activity of proteins involved in polysaccharide and lignin synthesis 

and modification. Indeed, one “grand challenge” in systems biology may 

be understanding how to engineer cell walls that meet the need of chemical biorefineries for optimized feedstocks yet still meet the plant’s need for 

development, robustness, and maximal rates of growth. 

GTL capabilities could provide extensive support for research on cell-wall 

synthesis, structure, and function. Sequencing support for model organisms 

(see below) and for identifying relevant genes in energy crops is an immediate goal. The development of populations of transgenic experimental 

plants with epitope-tagged proteins would greatly facilitate the determination of subcellular protein localization and the application of proteomic 

techniques to identify protein complexes. DNA chips, in conjunction with 

advanced genetic technologies, can be used for a systems-level understanding of transcriptional control of cell-wall synthesis and modification pathways. Epitope tagging also may be used to facilitate mRNA purification 

from single cells, facilitating insights into processes specific to cell types.

Ultimately, GTL capabilities in systems analysis will permit an integrated 

systems model that can be used to support directed modification of cell 

walls for specific applications. 

Efforts to understand and modify cell walls need to be coordinated with 

bioconversion and plant cell-wall deconstruction initiatives to optimize 

feedstock composition based on pretreatment and conversion methods 

and effects. These objectives also need coordination to develop analytical 

and visualization methods, computational facilities, and organic-chemistry 

methods for production of enzyme substrates and standards used in phenotyping and gene characterization. 

 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

Control of Lgnn Synthess and Structure 

Although lignin is not converted readily to ethanol, lignin biomass may be 

amenable to chemical or thermal processing to achieve such liquid fuels as 

low-grade diesel or fuel oil. One aspect of optimizing biomass composition for ethanol production is minimizing lignin content. Alternatively,

developing plants with modified lignin that can be removed easily during 

biomass processing may be possible. 

Lignin is a complex aromatic polymer associated with polysaccharides in 

secondary cell walls (see Fig. 3, p. 44, and Fig. 4. Phenylpropanoid Pathway 

Leading to Lignin Biosynthesis in Plants, this page). Lignin constitutes 

a significant barrier in biomass conversion to fuels by inhibiting enzyme 

access to polysaccharides and by releasing toxins during degradation that 

inhibit organism growth during fermentation of cell-wall hydrolysates to 

ethanol. Genetic studies have indicated that lignin reductions may cause 

deleterious changes in plant growth and development. However, lignin 

possibly may be reduced with or without harmful effects on plant growth 

if compensating changes could be made in the amount of cell-wall polysaccharides. Some early experiments are under way. The degree to which 

Fig. 4. Phenylpropanoid Pathway 

Leading to Lignin 

Biosynthesis in 

Plants. Horizontal 

reactions are ring 

modifications; 

vertical reactions are 

side-chain modifications. [Figure 

source: C. Fraser 

and C. Chapple,

Purdue University] 

4CL,.4-(hydroxy)cinnamoyl.CoA.ligase;.C3'H,.p-coumaroyl.shikimate/quinate.3'-hydroxylase;.C4H,.cinnamate.4-hydroxylase;.CAD,.. 

cinnamyl.alcohol.dehydrogenase;.CCoAOMT,.caffeoyl.CoA.O-methyltransferase;.CCR,.cinnamoyl.CoA.reductase;.COMT,.caffeic.acid/. 

5-hydroxyferulic.acid.O-methyltransferase;.F5H,.ferulate.5-hydroxylase;.HCALDH,.hydroxycinnamaldehyde.dehydrogenase;.HCT,. 

hydroxycinnamoyl-CoA.shikimate/quinate.hydroxycinnamoyltransferase;.PAL,.phenylalanine.ammonia-lyase. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIGNOCELLULOSIC BIOMASS 

cellulose amount can be increased with or without simultaneous changes in 

hemicellulose content and composition must be ascertained. 

Exploring lignin biosynthesis and its regulation in a comprehensive fashion may make possible the formulation of methods for limiting and altering lignification to maximize biomass-to-energy conversion. For instance,

the gene for ferulate-5-hydroxylase has been used to increase the syringyl 

monomer content of poplar lignin (see sidebar, Optimizing Lignin Composition for More Efficient Bioethanol Production, p. 43). The resulting trees 

had normal growth and development, but the pulping time was reduced 

by more than 60% (Huntley et al. 2003). Similarly, opportunities exist to 

modify lignification cell specificity so its impact on energy conversion can 

be minimized. A goal is to create a lignification toolbox to manipulate 

polymer depositions genetically and analyze the impact of those manipulations with advanced analytical organic chemistry. Such detailed knowledge 

could create novel opportunities for fundamentally changing how biomass 

is synthesized and subsequently processed for biofuels. For instance, novel 

monomers might be incorporated to generate lignins with unique, useful 

chemistries—readily cleavable linkages that could facilitate lignin depolymerization under more benign conditions (i.e., with enzymes). 

Improved Methods, Tools, and Technologes 

New analytical methods, tools, and technologies will accelerate the understanding of cell-wall synthesis, makeup, structure, and function and will 

speed breeding or rational modification of energy crop varieties. 

At the basic research level, new and improved methods are needed to analyze wall composition and nanoscale structure. Ideally, these methods could 

be applied to analysis of a small number of cells. Molecules in cell walls 

range from 2 to 5 angstroms (0.2 to 0.5 nm) in diameter (i.e., a polysaccharide chain) and to many microns in length. Primary cell walls are from 

50 to 100 nm in thickness and, in some cells, are thought to be chemically 

differentiated from one side to another. New imaging modalities that take 

advantage of various chemically specific imaging tags will support the longterm vision of in situ images of living plant cell walls. Images will reveal key 

molecular processes occurring in real time during the full life cycle of cellwall formation, maturation, transformation, dehydration, and processing 

into simple feedstocks. The understanding obtained through research using 

such imaging is expected to result in quantitative, predictive modeling as a 

guide to formulating advanced feedstocks and their subsequent processing.

A systematic approach is required to identify plant biomarkers and specific 

antibodies or other molecular tags useful in feedstock improvement. 

Poorly understood now, the fine structure of intact walls must be studied to 

determine how the parts fit together to comprise the whole wall’s physical 

properties. Some aspects of the general problem may be resolved simply by 

encouraging the application of such existing methods as very high resolution 

electron and scanning probe microscopy (see sidebar, Image Analysis of Plant 

Cell Surfaces at the OBP Biomass Surface Characterization Lab, p. 40). 

50 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Similarly, greater use of nuclear magnetic resonance (NMR) and magnetic resonance imaging may allow the development of 2D and 3D maps 

of cell-wall composition from important experimental and production 

species such as Arabidopsis and poplar. Use of NMR may be expanded by 

isotopic labeling and further development of solvents capable of dissolving cell-wall components. Complete annotation of 2D maps could 

facilitate greatly the analysis of genetic variation in cell-wall composition 

and the assignment of function to genes implicated in wall biosynthesis 

and modification. 

Other approaches meriting investment include expanded collections of 

antibodies and aptamers to cell-wall components, the use of enzymebased polysaccharide fingerprinting, pyrolysis gas chromatography–mass 

spectrometry (GC-MS), and related methods. A challenge is associated 

with characterizing enzymes that synthesize cell-wall polysaccharides:

Many enzymes add sugars to preexisting polysaccharides (i.e., “acceptors” or “primers”) that are not readily available as standards and reagents.

Focused investments in carbohydrate chemistry will be required to 

construct substrates—including labeled substrates—for measuring the 

activity of many wall biosynthetic enzymes. Expertise in carbohydrate 

synthetic chemistry also would be a needed complement to proteomic 

and metabolomic capabilities envisioned in GTL capability suites.

Expanded capabilities in synthetic carbohydrate chemistry could open 

up new high-throughput methods for characterizing carbohydrate-active 

enzymes based on high-density and high-diversity “glycochips.” In this 

method, the activity or binding of a target protein could be evaluated 

simultaneously with hundreds or thousands of potential substrates and 

very small amounts of reagent. 

High-throughput methods of cell-wall analysis are needed for plant 

breeding and improvement, allowing timely analysis with the most 

sophisticated analytical techniques. Ultimately, infield characterization 

is required to support breeding, molecular marker mapping, and studies 

involving such environmental variables as fertilizers and various biotic 

and abiotic stresses. Methods must be accurate and relatively inexpensive for the large numbers of samples typically handled during a breeding program. Additionally, they should be applicable to a wide variety 

of materials, from corn stover to wood. In principle, a high-throughput 

sample analysis may be enabled by detailed analysis of the relationship 

between cell-wall composition and features of Fourier transform infrared 

spectroscopy spectra or pyrolysis GC-MS chromatograms, combined 

with computational methods. 

Techncal Mlestones 

Wthn 5 years 

•	 Develop rapid, accessible tools and methods for consistent biomass 

compositional analysis in bulk and fractions (see section, Characterizing 

Cell Walls Using High-Throughput Methods, p. 108). 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 5 
 

LIGNOCELLULOSIC BIOMASS 

•	 Identify genes for enzymes that catalyze synthesis of major polysaccharide backbones. 

•	 Identify a substantial fraction of enzymes that catalyze synthesis of 

polysaccharide sidechains and determine sidechain biological function 

in model plant species. 

•	 Identify enzymes that acetylate polysaccharides, and establish biological 

function for such modifications. 

•	 Identify genetic regulatory factors that control lignin synthesis and 

deposition. 

Wthn 0 years 

•	 Clarify regulation of polysaccharide biosynthesis, including key steps 

that regulate carbon flow from photosynthesis into cell-wall polymers. 

•	 Define mechanisms that control cellulose amount and fibril length and 

angle. 

•	 Modify celluloses with altered numbers of glycan chains in secondary 

walls, and produce and test them in model species. 

•	 Make available for testing biomass crop plants with decreased lignin 

and increased amounts of cellulose or other polysaccharides. 

•	 Develop new tools and methods to help us understand cell-wall structure, including highly parallel computational simulations and highsensitivity 2D NMR and MS instrumentation for analysis of lignin in 

small tissue samples. 

•	 Identify all genes that catalyze synthesis of polysaccharide sidechains. 

Wthn 5 years 

•	 Determine regulatory genes that control amounts of major polysaccharides, including cellulose. 

–	 Develop methods for manipulating polysaccharide composition of 

any particular cell type within a specific tissue. 

•	 Make available plants with improved wall composition. These plants will 

have increased yields of fermentable sugars, requiring less costly preprocessing; cell-wall degradation will result in insignificant levels of inhibitory compounds (in the fermentation process). 

•	 Develop a detailed model of lignin monomer transport, polymerization 

initiation, and the interactions of lignin polymers with polysaccharide 

components of the plant cell. 

52 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A First Step to Optimizing Feedstocks 

for Fuel Production 

Optimizing plant biomass for more efficient processing requires a better understanding of 

plant cell-wall structure and function (see next two pages). Plant cell walls contain long 

chains of sugars (polysaccharides) that can be converted to transportation fuels such as 

ethanol. The saccharification process involves using enzymes to break down (hydrolyze) the polysaccharides into their component sugars for fermentation by microbes to ethanol (see sidebar, From 

Biomass to Cellulosic Ethanol, p. 26). Significant challenges for efficient conversion are presented 

by both the large number of enzymes required to hydrolyze diverse sugar linkages and the physical 

inaccessibility of these compounds to enzymes because other cell-wall components are present. 

Plant cell walls contain four different polymer types—cellulose microfibrils, hemicelluloses, 

pectins, and lignins. Microfibrils perform an important role in strengthening cell walls, thus 

providing support to the overall plant body. Some properties of lignin, however, interfere with 

enzymatic conversion of polysaccharide components. Additionally, since lignin is not readily 

converted to ethanol, we must find other ways it can be used if we are to maximize the yield of 

energy from biomass. 

Several thousand genes are estimated to participate in cell-wall synthesis, deposition, and function, but very few genes have been identified and very little is known about their corresponding 

enzymes. Many questions remain, for example, regarding how polysaccharides and lignin are 

synthesized, how wall composition is regulated, and how composition relates to the biological 

functions of cell walls. To answer these questions, we need to discover the functions of many 

hundreds of enzymes, where proteins are located within cells, whether or not they are in complexes, where and when the corresponding genes are expressed, and which genes control the 

expression and activities of proteins involved. Application of new or improved biological, physical, analytical, and mathematical tools will facilitate a detailed mechanistic understanding of 

cell walls. That knowledge will permit optimization of various processes involved in producing 

biomass and converting it to fuels. 

Major opportunities exist to increase productivity and conversion-process efficiencies by altering fundamental aspects of plant growth, development, and response to biotic and abiotic stress. 

Altering cell-wall composition to increase the relative amount of cellulose and to decrease lignin, 

for example, could have significant effects (see sidebar, Optimizing Lignin Composition for 

More Efficient Bioethanol Production, p. 43). Eventual development of a comprehensive physiological cell-wall model incorporating biophysical aspects with structural properties and knowledge of proteins involved will aid in rational development of highly productive feedstock species 

whose cell walls are optimized for conversion. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 5 
5 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 55 
 

 

LIGNOCELLULOSIC BIOMASS 

Cted References 

Huntley, S. K., et al. 2003. “Significant Increases in Pulping Efficiency 

in C4H–F5H– Transformed Poplars: Improved Chemical Savings and 

Reduced Environmental Toxins,” J. Agric. Food Chem. 51(21), 6178–83. 

Pauly, M., and H. V. Scheller. 2000. “O-Acetylation of Plant Cell Wall 

Polysaccharides: Identification and Partial Characterization of a Rhamnogalacturonan O-Acetyl-Transferase from Potato Syspension-Cultured 

Cells,” Planta 210(4), 659-67. 

56 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Feedstocks for Bofuels
 

One critical foundation for developing bioenergy crops and their 

processing technologies is ameliorating cell-wall recalcitrance to 

breakdown. Understanding cell walls is essential for optimizing 

their synthesis and the processes used to deconstruct them to sugars for 

conversion to ethanol (as discussed in the previous chapter, Lignocellulosic 

Biomass Characteristics, p. 39). A prerequisite for a competitive biofuel 

industry is the development of crops that have both desirable cell-wall 

traits and high biomass productivity under sustainable low-input conditions. Major agricultural crops grown today for food, feed, and fiber in 

the United States have not been bred for biofuels. Thus, many carefully 

selected traits in food and feed crops, such as a high ratio of seed to straw 

production (harvest index), are disadvantageous in biofuel production. A 

suite of new crops and new varieties of existing crops specifically bred for 

biofuels and adapted to a range of different soil types and climatic conditions is required. 

During the past century, improvement of agricultural crops was supported 

by federal investment in many aspects of basic plant science, agronomy,

plant breeding, pathology, agricultural engineering, and soil science. However, many topics particularly important in biofuel production have not 

been emphasized and are poorly developed as a result. A recent editorial 

in Science noted: 

“There are major technological challenges in realizing these goals.

Genetic improvement of energy crops such as switchgrass, poplar, and 

jatropha has barely begun. It will be important to increase the yield 

and environmental range of energy crops while reducing agricultural 

inputs. Plant development, chemical composition, tolerance of biotic 

and abiotic stresses, and nutrient requirements are important traits to 

be manipulated. The combination of modern breeding and transgenic 

techniques should result in achievements greater than those of the 

Green Revolution in food crops, and in far less time.” (Koonin 2006) 

— Steven E. Koonin, Chief Scientist, BP, London 

The Department of Energy (DOE) mission-oriented research program 

envisioned herein is designed to supplement current investment in plants,

with the focus on facilitating rapid progress in formulating biomass feedstock crops, also referred to as “energy crops.” One workshop goal was to 

identify specific areas in which a focused research investment would speed 

progress toward an optimized feedstock supply for conversion to biofuels.

In general terms, the goal of feedstock development is to obtain maximum usable 

“Large and cost-effective 

energy production on a scale 

that signif icantly impacts 

petroleum use calls for new 

crops with yield and productivity not currently available....” (EERE 2003). 

References: p. 80 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 57 
 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

organic carbon per acre in an environmentally and economically sustainable 

way. Many previous studies have indicated that minimizing such inputs 

as annual field preparation and fertilization implies the use of such perennials as switchgrass and poplar, thus decreasing costs (see Fig. 1. Switchgrass Bales from a 5-Year-Old Field in Northeast South Dakota, this 

page). A joint document of DOE and the U.S. Department of Agriculture 

(USDA)—Biomass as a Feedstock for a Bioenergy and Bioproducts Industry: The Technical Feasibility of a Billion-Ton Annual Supply (Perlack et al.

2005)—called for perennial crops to provide about one-third of biomassderived fuels for the initial phase of bioethanol development. 

In addition, because transition to large-scale cultivation of dedicated 

energy crops may take years if not decades, research imperatives must be 

explored for optimizing the use of currently available agricultural crop 

and forestry residues. Sustainability will be a key issue in implementing 

the use of crop and forestry residues for biofuel, since the removal of crop 

residues can reduce organic carbon and nutrient levels in the soil and affect 

soil microbial community health (see section, Ensuring Sustainability and 

Environmental Quality, p. 68). More information about the composition 

and population dynamics of soil microbial communities is needed to facilitate modeling of long-term effects on soil fertility. 

Current knowledge indicates that perennial species expected to be used 

for biofuel production improve soil carbon content and make highly 

efficient use of mineral nutrients. Development of perennial energy crops 

also may facilitate use of genetically diverse mixed stands rather than 

monocultures of single cultivars. Because conventional crops are grown 

as monocultures, relatively little research has been carried out on issues 

associated with growing mixed stands (see sidebar, A Billion-Ton Annual 

Supply of Biomass, p. 10, and sidebar, The Argument for Perennial Biomass Crops, p. 59). 

Fig. 1. Switchgrass Bales from a 5-YearOld Field in Northeast South Dakota 

in 2005. Each 1200-lb. bale represents 48 

gallons of ethanol at a conversion rate of 

80 gallons per ton. The cultivar used in this 

field has a yield potential of 5 to 6 tons per 

acre (corresponding to 400 to 500 gallons 

per acre) because it was bred for use as a 

pasture grass. In experimental plots, 10 tons 

per acre have been achieved. Processing 

goals target 100 gallons per ton of biomass,

which would increase potential ethanol 

yield to 1000 gallons per acre. [Source:

K. Vogel, University of Nebraska] 

5 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

The Argument for Perennal Bomass Crops
 

Many major agricultural crops today are annual plants propagated from seed or cuttings at the beginning of each growing season. By contrast, crops developed and grown specifically for biofuel production are expected to be based on perennial species grown from roots or rhizomes that remain in 

the soil after harvesting the above-ground biomass. Perennial species are considered advantageous for several 

reasons (see Fig. 2. Attributes of an “Ideal” Biomass Crop, p. 61). First, input costs are lower than for annuals 

because costs of tillage are eliminated once a perennial crop is established. Additionally, long-lived roots of 

perennials may establish beneficial interactions with root symbionts that facilitate acquisition of mineral nutrients, thereby decreasing the amount of fertilizer needed. Some perennials also withdraw a substantial fraction 

of mineral nutrients from above-ground portions of the plant at the end of the season but before harvest (see 

Fig. A. Nitrogen Use Efficiency Theory for Perennials, below). 

Perennial plants in temperate zones also may have significantly higher total biomass yield per unit of land 

area than comparable annual species. Perennials establish a photosynthetically active canopy more quickly in 

the spring and may persist longer in the fall (see Fig. B. Comparing Net Photosynthesis of Corn and Several 

Perennial Species, below). Thus, their annual solar-energy conversion efficiency is higher than that of annual 

plants with similar capabilities. 

Perennial species have much lower fertilizer runoff than do annuals. For instance, comparing the native 

perennial switchgrass with corn indicates that switchgrass has about one-eighth the nitrogen runoff and onehundredth the soil erosion. Perennial grasses harvested for biomass exhibit increased soil-carbon levels and 

provide habitat for up to five times 

as many species of birds. Finally, in 

contrast to annual row crops that 

typically are monocultures, increasing habitat diversity by growing several intermixed species of 

perennials may prove more feasible. 

Fig. A. Nitrogen Use 


Efficiency Theory 


for Perennials.
 

[Source: S. Long,


University of Illinois]
 

Fig. B. Comparing Net Photosynthesis of Corn and 

Several Perennial Species. Annualized net photosynthesis is proportional to the area under the curve. Thus,

if maximal rates of photosynthesis are similar, the perennial crops (yellow) have much higher annualized net 

photosynthesis than the annual crop, corn (Zea mays,

blue). [Source: S. Long, University of Illinois] 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 59 
 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

Among many factors in plant productivity, several are thought to be of 

central importance in biofuel production. Because of evidence that most 

plants do not routinely achieve maximal photosynthetic CO fixation rates, 2

understanding factors limiting the overall process is important. Emphasis 

should be placed on determining how plants allocate recently fixed carbon 

to products such as storage polysaccharides (e.g., starch) and structural 

polysaccharides such as cellulose. Energy crops will be grown on marginal,

excess, or surplus agricultural lands, so identifying factors that facilitate 

tolerance and survival during exposure to drought, freezing, and other abiotic stresses will be vital. Issues for perennials may be quite different from 

those for annuals, which have been the subject of most research and crop 

experimentation. These issues may be particularly acute in regard to pressure from pests and pathogens that can be controlled to some extent by 

crop rotation in annual species. During the past 25 years, DOE’s offices of 

Energy Efficiency and Renewable Energy (EERE) and Basic Energy Sciences have been a primary source of research support on centrally important topics in developing bioenergy feedstocks. Research on key issues 

identified here constitutes a compelling opportunity. Unique capabilities 

developed in both the Biomass and Genomics:GTL (GTL) programs 

can be brought to bear on remaining practical and fundamental problems 

in producing feedstocks. The following sections outline these issues and 

opportunities in the context of tangible goals, timelines, and milestones. 

Creaton of a New Generaton of Lgnocellulosc 

Energy Crops 

Three distinct goals are associated with development of biofuel feedstocks: 

• Maximizing the total amount of biomass produced per acre per year, 

• Maintaining sustainability while minimizing inputs, and 

• Maximizing the amount of fuel that can be produced per unit of biomass. 

Exact values for each of these parameters will vary from one type of energy 

crop and one growing zone to another. A yield of 20 dry tons per acre per 

year may be considered a reasonable target in areas of the country with 

adequate rainfall and good soils, whereas 10 dry tons per acre per year may 

be acceptable in drier or colder zones. 

Thus, the overall objective of developing feedstocks must be focused on 

broadly useful insights applicable to a variety of plant species grown 

under various growing conditions and exhibiting beneficial attributes 

(see Fig. 2. Attributes of an “Ideal” Biomass Crop, p. 61). This is best 

accomplished by working toward systems-level predictive models that 

integrate deep knowledge of underlying mechanisms for guiding cultivar 

and process development. This ambitious goal is only now beginning to 

be realized by companies that have bred advanced cultivars for major 

agricultural commodities. 

This systems-level approach is feasible because of the last decade’s biology 

revolution in genomic sequencing of higher plants and microbes. Sequencing 

60 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

provides a means for connecting 

knowledge about all organisms 

into a common framework for 

understanding all forms of life.

Current and future DOE investments in plant DNA sequencing 

afford the opportunity to create 

the mechanistic knowledge of 

energy crops needed for costeffective and practical feedstocks.

This enabling information must 

be elaborated further by strategic 

investments in understanding 

aspects of basic biology specifically relevant to energy crops (see 

sidebar, Enhancing Poplar Traits 

for Energy Applications, p. 62). 

Maxmzng Bomass 

Productvty 

Domestcaton of Energy Crops 

The continental United States is 

composed of a number of growing zones or agroecoregions that 

vary with such factors as mean 

temperature, rainfall, and soil 

quality. No single plant species 

is optimal for all zones, so using 

different species as energy crops 

will be necessary. Previous DOE 

studies have identified a number 

of promising plant species, and 

academic studies have suggested 

additional ones (see Fig. 3. Geographic Distribution of Biomass 

Crops, this page). In general,

energy crops can be divided into 

two types: Those, such as maize,

which are used for agricultural 

food and feed production but 

produce substantial amounts of 

usable biomass as a by-product 

(Type I), and those used only for 

energy (Type II). Type I plants 

are highly developed from many 

decades of research and study for 

another purpose (food or fiber). 

Fig. 2. Attributes of an “Ideal” Biomass Crop. [Table adapted from S. Long, 

University of Illinois] 

Fig. 3. Geographic Distribution of Biomass Crops. Multiple types designed for 

various agroecosystems probably will be required to obtain enough biomass for 

large-scale production of liquid fuels. [Source: Adapted from ORNL Biomass 

Program] 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 6 
 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

Enhancng Poplar Trats for Energy Applcatons 

Gaining a better understanding of genes and regulatory mechanisms that control growth, carbon allocation, and other relevant traits in the poplar tree (Populus trichocarpa) may lead to its use as a major 

biomass feedstock for conversion to bioethanol. An international team led by the DOE Joint Genome 

Institute recently completed sequencing the poplar genome, making it the first tree (and fourth plant, after the 

mustard weed Arabidopsis thaliana, rice, and the alga Chlamydomonas) to have its complete genome sequenced 

(Tuskan et al., in press). These data now offer the molecular access needed to define, quantify, and understand—at a mechanistic level—basic biological processes that impact important traits. 

Poplar was chosen for sequencing because of its relatively compact genome (500 million bases), only 2% that 

of pine. Moreover, many species are available worldwide, and their rapid growth allows meaningful measures 

of important traits within a few years. Extensive genetic maps already available include initial identification of 

markers associated with such traits. 

Early comparative sequence analyses of poplar and Arabidopsis genomes are providing insights into genome 

structure and gene-family evolution; biosynthetic processes such as cell-wall formation, disease resistance, and 

adaptation to stress; and secondary metabolic pathways. Comparisons of gene-family sizes show substantial 

expansion of poplar genes involved in carbon to cellulose and lignin biosynthesis. 

Moving from a descriptive to predictive understanding of poplar growth, development, and complex function will require integration of sequence information with functional data. These data will be generated by 

such new tools and approaches as gene and proteome expression studies, metabolic profiling, high-throughput 

phenotyping and compositional analysis, and modeling and simulation. Ultimately, this information will lead 

to the engineering of faster-growing trees that produce more readily convertible biomass (see below, Fig A.

Vision for the Future). The International Populus Genome Consortium has produced a science plan to guide 

postsequencing activities 

(The Populus Genome 

Science Plan 2004–2009: 

From Draft Sequence to 

a Catalogue of All Genes 

Through the Advancement 

of Genomics Tools, www. 

ornl.gov/ipgc). 

Other areas to be 

addressed for poplar and 

other potential bioenergy crops include sustainability for harvesting 

biomass, harvesting 

technologies to remove 

biomass at low costs, and 

infrastructure technologies that allow biomass 

to be transported from 

harvest locations to conversion facilities. 

Fig. A. Vision for the Future: Desired Traits of the Domesticated Energy Poplar. 

62 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

They were bred as monocultures under intensive agriculture. As noted 

elsewhere in this document, research priorities regarding production of 

energy from these crops are concerned largely with three issues. These 

issues are sustainability following removal of previously underutilized 

straw or stover, processing the residues, and the possibility of making 

improvements in cell-wall composition for enhanced conversion to fuels. 

By contrast, Type II plants are relatively poorly developed as agronomic 

crops and generally are perennials. Perennial herbaceous and woody plants 

have several properties that make them better suited for biofuel production than are annual crops (see sidebar, The Argument for Perennial Biomass Crops, p. 59). First, because they typically retain a significant tissue 

mass below ground, they rapidly form a canopy in the spring and accumulate biomass when many annuals are still seedlings. Thus, they may exhibit 

higher rates of net photosynthetic CO2 fixation into sugars when measured annually, resulting in higher amounts of total biomass accumulation 

per acre per year. Second, perennials require little if any tillage, saving 

energy and labor and significantly reducing soil erosion and nutrient loss.

Perennials such as switchgrass and Miscanthus can be harvested annually 

without replanting. Third, perennials typically withdraw mineral nutrients 

into roots at the end of a growing season, thereby reducing fertilizer costs.

Perennial herbaceous and woody plants represent a critical component of 

our bioenergy future. 

Advances in conventional breeding, coupled with molecular tools and highthroughput transformation systems (Busov et al. 2005) will be required to 

accelerate domestication of species having promise as energy crops. Associating genotypes with phenotypes will require high-throughput technologies for genotyping (i.e., identifying genes and alleles) and phenotyping 

a wide array of relevant traits (e.g., biomass yield, cell-wall composition) 

with “molecular” techniques such as gene expression (transcriptomics),

proteomics, and metabolomics. Availability of whole-genome sequences 

and deep expressed sequence tag (EST) libraries of all potential energy 

crops will be necessary to provide genetic reagents for marker-aided 

selection, association genetics, and transformation studies (see sidebar,

Marker-Assisted Breeding, p. 64). Association genetics identifies allelic 

variants [e.g., single nucleotide polymorphisms (SNPs)] with empirically 

determined phenotypic effects on traits of interest and allows selection 

for favorable alleles using molecular markers (Rafalski 2002; Neale and 

Savolainen 2004; Remington et al. 2001). This form of marker-assisted 

selection is direct (as opposed to indirect on quantitative trait loci) and 

allows simultaneous selection of many genes. The result is a rational,

marker-aided breeding and selection approach with the expectation of 

significantly enhanced genetic gain and an accelerated development process (Brown et al. 2003). 

For most Type II biomass crops, significantly enhanced breeding, testing,

and selection populations will be necessary, along with appropriate infrastructure to breed for desired traits and adaptability across a wide array of 

environments in multiple physiographic regions. In addition to targeted 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 6 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

Marker-Asssted Breedng 

One of the ways in which DNA sequencing of biomass species impacts biofuel 

feedstock development is by enabling 

accelerated breeding methods to be applied 

to plants used for biomass production. The 

goal in plant breeding is to first identify useful genetic variation for traits of interest (e.g.,

disease resistance and drought tolerance) by 

screening natural or mutagenized populations of 

individuals. Many such traits are controlled by 

multiple genes. Individuals with useful variations 

are intercrossed to produce progeny with new 

combinations of the useful variation. Usually,

many traits are of interest and many sources of 

variation are used, making the overall breeding process very time consuming and expensive.

The availability of large amounts of sequence 

information facilitates identification of DNA 

polymorphisms—small differences in the DNA 

sequences of individuals within a species. Having 

complete genomic sequences also makes possible 

the identification of genes located near polymorphisms on chromosomes. 

This knowledge has practical applications in 

“marker-assisted breeding,” a method in which 

a DNA polymorphism, closely linked to a gene 

encoding a trait of interest, is used to track the 

trait among progeny of sexual crosses between 

plant lines. The method allows breeders to 

monitor plants for a trait that may be expressed 

only in certain tissues or developmental stages 

or may be obscured by environmental variation. Similarly, by correlating traits and DNA 

polymorphisms in individuals from genetically 

diverse natural populations, associating a particular chromosomal region with a trait of interest frequently is possible. If a large number of 

polymorphisms are available, the amount of time 

required to breed an improved plant cultivar is 

greatly reduced. 

In principle, plants with optimal combinations 

of parental genes may be identifiable within the 

first several generations following a sexual cross 

rather than eight generations or more following 

conventional breeding. In the case of species such 

as trees, marker-assisted breeding could eliminate many decades of expensive steps to develop 

more highly productive plants. 

breeding, many crops will require fertility control in the 

field, either to ensure parentage or prevent gene flow 

to wild populations. A holistic approach for improving 

feedstocks includes molecular resources, high-throughput 

screening tools, and well-characterized breeding populations. GTL resources and technologies are well suited to 

creating comprehensive sets of molecular markers (i.e.,

SNPs and single sequence repeats) and high-throughput, low-cost phenotyping tools. These efficient deliverables will be used to develop appropriate cultivars and 

varieties to meet target goals. GTL genomic and other 

resources will play critically important roles via implementation of deep EST sequencing, marker identification, high-throughput genotyping, and development and 

application of analytical tools.These tools will be used 

for high-throughput molecular phenotyping of biomass 

composition and plant structure and a high-throughput 

transformation process for major biomass energy species. Such capability suites also will provide centralized 

bioinformatics support for analysis and archiving of 

genome data. 

By definition cultivar development, field and plantation 

establishment and growth, and biomass conversion to 

biofuels necessitate a holistic, systems biology approach.

Integrating the smaller subcomponents will be challenging, requiring a coordinated and focused program to 

facilitate exchange of information and genetic materials 

across organizations and institutions. 

Enhancng the Yeld of Bomass Crops 

The yield of biomass crops can be defined as the amount 

of fixed carbon per acre per year. Achieving the maximal 

yield of a dedicated energy crop (Type II) is a significantly different goal from maximizing the yield of most 

existing crop species (Type I), where only the number of 

reproductive or storage organs is considered. The yield 

of a Type II species is a function of the total number 

of cells per acre multiplied by the mean amount of 

accumulated carbon per cell. Thus, biomass yield can be 

enhanced by increasing the number of cells per acre per 

year, the amount of carbon per cell, or both. Achieving 

either type of enhancement is a complex systems problem. At the core of the problem, however, is the need 

to maximize photosynthetic CO fixation to support 2 

carbon accumulation. Additionally, fixed carbon must be 

directed into either cell-wall polymers or storage carbohydrates or used to support extra cell division. Cell-wall 

6 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

polymers include cellulose, hemicellulose, and lignin; storage carbohydrates include sugars and starches. 

Plants are regulated to fix only the carbon needed for normal growth and 

development. This generally is referred to as “source-sink” regulation, a 

poorly understood phenomenon. Plants can fix considerably more carbon,

however; the actual photosynthetic CO2

 fixation rate of most or all plants 

is significantly below (i.e., ~50%) the rate observed following experimental 

partial defoliation. That is, plants appear to accumulate more carbon per 

unit of leaf area following defoliation than they normally would without 

any changes in architecture or photosynthetic electron transport. When 

mechanisms underlying this regulation are understood, plants can be 

developed that exhibit significantly higher rates of net photosynthetic CO2 

fixation and higher amounts of total carbon accumulation per acre per year.

Therefore, a high-priority research goal is to understand mechanisms that 

regulate net photosynthetic CO2

 fixation. A closely related priority is to 

identify factors that limit carbon flux into cell-wall polysaccharides and 

storage polymers. 

A complementary approach is to identify factors that regulate plant growth 

rate and duration. Different plant species vary widely in growth rates, suggesting that growth rates are under genetic control and, therefore, subject 

to modification. Recently, several genes have been identified in functional 

genomics screens that cause significant increases in growth rates of different types of plants. Identifying other genes that control growth and 

development and understanding gene action may create new opportunities 

to develop highly productive energy crops (see Fig. 4. Growth Rate Modification, this page). 

Enhancng Abotc Stress Tolerance of Bomass Speces 

Water availability is a major limitation to plant productivity worldwide,

generally in two ways. First, because water escapes from plant leaves 

through stomata when CO2

 enters, a certain amount of water is required 

to support a unit of photosynthetic CO fixation. Plants with C4 pho­ 2

tosynthesis (e.g., corn, sugarcane, switchgrass, and Miscanthus) typically 

require less water per unit of CO2

 fixed than do C3 species (e.g., wheat and 

soybean) because C4 

plants can achieve 

high rates of CO2 

fixation with partially closed stomata.

Other plants such 

as cacti close their 

stomata during the 

day to reduce water 

loss but open them

at night to take in

CO2

 for photosynthesis the next day.

This phenomenon 

Fig. 4. Growth Rate 

Modification. The 

Arabidopsis plant on

the right has been 

modified by altering the expression 

of regulatory genes 

controlling growth.

[Source: Mendel 

Biotechnology]

 Unmodified Modified 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 65 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

also inhibits carbon loss by photorespiration. Except for the possibility of 

enhancing these adaptations, current theory implies no other options for 

significantly reducing a plant’s water requirement to obtain maximal yields. 

The water problem’s second component, however, concerns the effects of 

temporal variation in soil-water content. In rain-fed agriculture, periods 

of low soil-water content are frequent because of irregularities in rainfall.

The ability of plants to survive extended periods of low soil water can be 

a critical factor in a crop’s overall yield (see Fig. 5. Corn Yield on a Missouri Experiment Station, this page). Furthermore, different plants exhibit 

widely different abilities to survive extended periods of drought, indicating 

that drought-tolerant energy crops may be possible (see Fig. 6. Modification in Drought-Stress Tolerance, this page). 

Currently, the most productive farmland is used for food production, with 

an amount held in reserve [Conservation Reserve Enhancement Program 

(CREP) lands]. While initial energy crops would be grown in highly 

productive CREP land, a substantial proportion of biomass crops will be 

grown on marginal land that is suboptimal in water availability, soil quality,

or both. Recent progress in understanding the mechanistic bases of plant 

drought, salt, and cold tolerance has raised the possibility of modifying 

plants to enhance productivity under these and other stress conditions. A 

priority in dedicated energy crops is to understand mechanisms by which 

plants survive drought and adapt this knowledge to improving energy crops. 

Fig. 5. Corn Yield on a Missouri Experiment Station.

This figure illustrates wide differences in yield observed 

in various annual growing conditions, including 

periods of drought. The strong upward trend in yield 

reflects advances in breeding and agronomical practices 

for corn. [Figure adapted from Q. Hu and G. Buyanovsky, “Climate Effects on Corn Yield in Missouri,”

J. Appl. Meteorol. 42(11), 1623–35 (2003).] 

Fig 6. Modification in 

Drought-Stress Tolerance.

The plants on the left of each 

photograph are unmodified,

and the plants on the right 

have been altered genetically 

for drought-stress tolerance.

All plants were subjected to 

a severe drought. [Photos 

©2003 Monsanto Company.

Used with permission.] 

66 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

Understandng and Elmnatng Undesrable Bomass Crop Characterstcs 

Research should be conducted to understand and eliminate such undesirable plant traits as inappropriate residues and invasiveness of non-native 

energy-crop species. Two examples are described here. 

Relatively large amounts of silica in plants such as rice lead to accumulations of ash when the plant biomass is burned for energy. When the biomass is converted to liquid fuels, unusable solids are produced. Since many 

plants do not accumulate silica, the trait appears to be dispensable; developing cultivars with reduced silica accumulation by genetic methods may 

be possible. The roles of silica in plant growth and development, however,

are poorly understood and need further investigation. 

Many features considered ideal for a biomass crop (see Fig. 2, p. 61) are 

characteristic of invasive weeds, particularly perennial C4 grasses. Thus,

a key consideration in adapting these grasses for use as dedicated energy 

crops is to ensure that the species can be contained and will not become 

a problem. Some highly productive perennial grasses, such as Miscanthus 

giganteus, have been studied intensively in Europe for more than a decade 

and are thought not to exhibit invasive characteristics. All candidate energy 

crops, however, should be studied directly for potential invasiveness at 

diverse locations within the United States. These studies also can provide 

insights into pests and pathogens that might pose a threat to productivity. 

Techncal Mlestones 

Wthn 5 years 

•	 Facilitate the initiation and advancement of biomass breeding programs 

in key U.S. geographic regions in cooperation with USDA, private companies, and universities. 

•	 Develop appropriate test populations for conducting association genetics and quantitative trait locus identification (QTL, direct and indirect 

marker-assisted selection). 

•	 Identify and target for selection and improvement key traits that affect 

biomass yield and conversion efficiency. 

•	 In support of marker development for gene discovery, carry out targeted 

sequencing (SNPs, SSRs, ESTs, and cDNAs) for potential biomass 

species having large and complex genomes; sequence whole genomes for 

species with modest genome sizes. 

•	 Develop markers that can differentiate superior parents and offspring. 

•	 Initiate and validate methods for high-throughput screening for specific 

traits such as improved cell-wall digestibility. 

•	 Formulate the infrastructure for functional screening of thousands of 

potentially useful genes in selected species. 

Wthn 0 years 

•	 Apply high-throughput phenotyping tools to integrated conventional 

and molecular breeding programs. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 67 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

•	 Gain new understanding of genome structure and gene expression in bioenergy crops, including the genetic basis of heterosis (hybrid vigor). Major 

barriers to facile transformation of select genotypes will be overcome. 

Wthn 5 years 

•	 Identify and integrate major new domestication genes into energy crops. 

•	 Integrate enabling technologies with breeding programs to allow for 

deployment of genetically superior materials over large land bases dedicated to biomass production.Time to commercial deployment will be 

shortened dramatically through highly reliable screening methods, genetic 

control of flowering and sexual reproduction, and effective early-selection 

models for predicting performance and yield. Genetically enhanced cultivars, hybrids, and varieties capable of meeting targeted goals for ethanol 

yield per acre per year will be available for major biomass crop species. 

Ensurng Sustanablty and Envronmental Qualty 

To ensure the viability of bioethanol to meet the large national need for 

transportation fuels, we must understand the effects of long-term biomass 

harvesting on soil fertility and other aspects of sustainability. Objectives 

for this work are to determine how to maintain soil ecosystem function 

and productivity. A further objective is to develop management practices 

that can optimize sustainability. Because very long periods of time may be 

required for directly testing the effects of biomass removal on soil quality,

a mechanistic understanding of this issue is essential to obtaining predictive models and monitoring procedures. The critical question in using crop 

residues and dedicated crops for biomass energy is, How much, if any, of 

above-ground biomass needs to be left on a field to protect soil from erosion and sustain soil function (soil quality)? 

In the past 30 years, crop and soil research has emphasized using crop 

residues in minimum and no-till farming operations to improve soil organic 

carbon (SOC) and soil quality. Research has demonstrated that corn stover 

removal in some production systems can reduce grain yield (Wilhelm et al.

2004).The amount of stover removal and lower yield were associated with 

the amount of SOC (Maskina et al. 1993). At any time, SOC content is the 

balance between the rates of input and decomposition (Albrecht 1938). If all 

other cultural practices are unchanged, removal of crop residue will further 

decrease carbon inputs and SOC will decline (Follett 2001) (see Fig. 7. Soil 

Carbon Alterations with Management Changes, p. 69). Loss of SOC 

typically has detrimental effects on soil productivity and quality, presumably because microbial communities are impacted negatively. For example,

considerable carbon is translocated to mycorrhizal fungus communities that 

play a significant role in soil nutrient cycling (Fitter et al. 2005). 

This research must be completed for each major agroecosystem in the 

United States where crop residues and dedicated crops are a feasible supply 

source for biomass energy. Sustainability analysis of both dedicated energy 

crops and of stover or straw removal from agricultural crops should be carried out since the effects may be significantly different. 

6 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

Soils are complex ecosystems composed of unknown numbers of organisms. The effects of crop-residue quantity and composition on soil ecosystems are largely unknown. Soil microbiologists need access to large-scale 

genomics and other analytical facilities to conduct soil microbial research.

Systems biology analyses of highly productive soils, including their ecosystem-scale genomic characterization, are accessible with current sequencing 

capabilities. Questions include the following. 

•	 What characteristics and functions of soil microbial communities are 

needed to maintain soil-ecosystem function and productivity? 

•	 How do soil microbial communities function? 

•	 What are the interactions, positive and negative, among microbes, fungi,

and roots in the rhizosphere? 

•	 How do nutrient levels affect microbial communities, and how do 

microbial communities affect nutrient availability? 

•	 How can management of microbial communities improve productivity? 

•	 How much carbon from crop residues and dedicated energy crops is 

needed to maintain soil ecosystem function and productivity? 

–	 Does the composition of residue have an effect? 

–	 What is happening both physically and biologically in soils as various levels of plant biomass are removed? 

•	 Can management factors, no tillage, modified minimum tillage, or special practices affect the amount of needed residue? 

•	 How does soil composition vary between annual and perennial crops in 

response to varying levels of biomass removal? 

•	 What are the microbial-community characteristics of marginal and 

severely depleted lands, and how can they be restored to support energy 

crops? 

•	 Can energy crops be used to 

restore lands for food and fiber 

crops by building up carbon and 

nutrients in marginal lands? 

Finally, how might these processes be utilized for reduction 

in greenhouse gas emissions or 

carbon sequestration? 

GTL facility capabilities can be 

used in many ways to answer these 

general questions. In particular,

GTL facilities could support ecosystem-analysis studies, including 

sequencing of microbial communities and genomics approaches for 

analysis of microbial-community 

functioning. GTL could support 

Fig. 7. Soil Carbon Alterations with Management Changes. Cultivation 

generally leads to reduction of soil organic carbon, which, without offsetting 

practices, is exacerbated by corn-stover removal. A mechanistic understanding 

of long-term harvesting effects on soil fertility and other aspects of sustainability is essential for predictive models and monitoring procedures.[Source:

W. W. Wilhelm, USDA Agricultural Research Service, Lincoln, Nebraska.] 

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FEEDSTOCKS FOR BIOFUELS 

technologies to evaluate how microbial communities function by, for example, developing gene chips with millions of diagnostic probes. 

Determination of microbial-community physiology must be linked with 

analysis of soil physicochemical states—facilitating analysis of soil emissions or carbon sequestration in soils. Improved methods of soil carbon 

analyses need to be developed and made available in high-throughput 

format. Current methods are extremely laborious. Improving analytical 

methods for analyzing composition of plant cell walls, described below,

also may facilitate analysis of soil carbon composition. 

Techncal Mlestones 

Wthn 5 years 

•	 Determine the effects of corn-stover and other crop-residue removal on 

soil productivity in each major agroecosystem of the United States. 

•	 Use existing study sites where stover has been removed for 5 or more 

years to initiate comprehensive analysis of microbial communities (and 

soil carbon and mineral content) in underlying soils, in comparison 

with land where stover has not been removed. 

•	 Establish long-term study sites for future sustainability studies on prospective energy crops in lands in all major agroecosystems. 

•	 Pursue long-term contracts with private or public providers. The 

European Miscanthus Productivity Network, supported by the European Commission, provides a useful model. Sequence analysis of DNA 

extracted from soils should be carried out to survey diversity (Handelsman et al. 1998). Sequence analysis should be used to develop diagnostic methods for examining the dynamics of soil microbial composition 

and abundance. 

Wthn 0 years 

•	 Conduct additional studies with varying harvest rates and management 

practices, including cropping systems and analyses of microbial communities and of carbon levels in the soils. 

•	 Perform comprehensive ecological system analyses of soils as a living 

medium. 

•	 Include not only microorganisms present but also how their complex 

interactions contribute to their net function as a community. Markers 

of optimally functioning microbial communities should be developed 

to enable identification of sustainability requirements for soils in each 

major agroecosystem. In addition to its value to the energy-production 

function of croplands, this research should enhance the sustainability of 

food production. 

Wthn 5 years 

•	 Formulate management guidelines on the amount of biomass that can be 

removed in each major U.S. agroecosystem. 

70 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Model Systems for Energy Crops 

Application of model systems toward the study of both basic and applied 

problems in plant biology has become routine and can quickly bring the 

techniques of 21st Century systems biology to bear on the complex problems associated with domesticating energy crops (see sidebar, Translational 

Research: The Path from Discovery to Applications, p. 72). Researchers 

using the model dicot Arabidopsis thaliana have made tremendous strides 

in understanding areas of plant biology ranging from nutrient uptake and 

metabolism to plant-pathogen interactions. Unfortunately, as an annual 

dicot, Arabidopsis is not an optimal model to study questions unique to 

potential woody and grassy perennial energy crops (e.g., wood formation 

in trees or cell-wall composition in grasses). Despite its sequenced genome 

and genetic resources, rice also is not an ideal model for grassy perennial 

energy crops because it is a specialized semiaquatic tropical grass. In addition, its large size, long generation time, and demanding growth requirements make experiments expensive. Using Brachypodium and Populus as 

model systems would provide researchers working to domesticate energy 

crops with some of the most powerful tools developed by the highly successful Arabidopsis community. These model systems would help identify 

genes controlling traits relevant to energy-crop productivity and quality,

including such global processes as cell-wall biosynthesis, nutrient uptake,

carbon flux, and plant architecture. The models can be used for rapid testing of strategies to improve the usefulness of grasses and trees as energy 

crops. Such tools would allow scientists to use both forward and reverse 

genetic approaches and modern molecular genetic methods to identify 

genes controlling traits relevant to the design of superior energy crops. This 

is important because of the problems (i.e., difficult transformation, large 

size, long generation time, and self-incompatibility) associated with working directly with energy crops. 

Brachypodium distachyon is a small temperate grass with all attributes 

needed to be a modern model organism, including simple growth requirements, fast generation time, small stature, small genome size, and selffertility (Draper et al. 2001). Brachypodium also is transformed readily by 

Agrobacterium (Vogel et al. 2006) or biolistics (Christiansen et al. 2005),

thus facilitating many biotechnological applications. Brachypodium is 

now scheduled to be sequenced by the DOE Joint Genome Institute.

The Populus genome has been sequenced (Brunner, Busov, and Strauss 

2004; Tuskan, DiFazio, and Teichmann 2004), assembled, and annotated 

through investments from DOE, NSF, and a large international consortium (Tuskan et al., in press). This resource is available to researchers working on a dedicated woody crop for biofuels and biomass energy 

applications. Despite these advances, many molecular tools and resources 

currently not available would greatly enhance the discovery and use of 

genes and gene families related to energy traits and the development of 

poplar as an energy crop (see sidebar, Enhancing Poplar Traits for Energy 

Applications, p. 62). As with other model organisms, research on poplar 

would be facilitated by organism-specific tools such as full-genome DNA 

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FEEDSTOCKS FOR BIOFUELS 

Translatonal Research: The Path from Dscovery to Applcatons 

Model organisms play an important role in both basic and applied research. The most highly developed plant model is Arabidopsis thaliana, a small plant in the mustard family (Fig. 4. Growth Rate 

Modification, p. 65). It was adopted as a model in the early 1980s because of its rapid life cycle,

simple genetics, small genome (125 Mb), easy transformability, and traits typical of flowering plants in most 

respects. Genome sequencing was completed in 2000 by an international consortium, and about 13,000 

researchers worldwide currently use the plant for studies of plant biology. 

The Arabidopsis community has developed a suite of very powerful experimental resources that include several 

hundred thousand sequenced insertion mutations available through stock centers, a full-genome DNA chip 

that can be used to measure the expression of most genes, a high-density polymorphism map, and a heavily curated database (arabidopsis.org) that provides access to genomic information. These and other resources 

have resulted in an explosion of fundamental discoveries about all aspects of plant growth and development.

In addition, several companies, founded to exploit knowledge gleaned from Arabidopsis, have translated the 

new information into such crop species as corn, soybean, and canola (see Fig. A. Drought Resistance in Canola 

Caused by Directed Modification of a Single Gene, below). Plant-improvement goals that were unattainable 

by applied research on crop plants have been realized by translating basic research on model species to crops. 

Much accumulated knowledge about crop species and model organisms will be applicable to improving species 

suited for use as dedicated biomass crops. However, some traits of interest in that regard have not been a priority in crop research and are poorly understood. For instance, knowledge about cell-wall structure, function, and 

synthesis is very underdeveloped and will need extensive research. One important species suited for use as a 

biofuel is poplar, a model woody species that has a relatively small genome. Poplar was sequenced recently at 

the DOE Joint Genome Institute (see sidebar, Enhancing Poplar Traits for Energy Applications, p. 62). 

The DNA sequence of many poplar genes is similar to that of corresponding Arabidopsis genes. This comparability will help link mechanistic knowledge about these species to a holistic understanding of common 

processes. Poplar is relatively easy to transform, which greatly facilitates experimental tests of theories about 

protein and gene function. Most important, poplar has several important traits, such as a long life cycle and the 

formation of wood, that cannot be studied in herbaceous plants. 

Brachypodium distachyon is another potentially important model for such highly productive grasses as switchgrass 

and Miscanthus. Interest in Brachypodium arises because its very small genome has a DNA content about 2.5 

times larger than that of Arabidopsis. Additionally, its simple growth requirements, small stature, self-fertility, and 

ready transformability make it well suited to become a model organism. Because Arabidopsis is distantly related 

to and differs from grasses in a number of important respects (e.g., cell-wall composition), Brachypodium could 

become a powerful new 

model for cell and molecular biological studies of 

grasses. A high priority in 

facilitating the development of Brachypodium is 

sequencing its genome. 

Fig. A. Drought Resistance in Canola Caused by 

Directed Modification of a 

Single Gene. [Photo ©2003 

Monsanto Company. Used 

with Permission.] 

72 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

chips, sequence-indexed mutations, facile transformation methods, and 

specialized libraries for methods including two-hybrid screens. 

Brachypodium and Populus must be accelerated into powerful model systems by first sequencing Brachypodium and then creating extensive EST 

databases (i.e., 1 to 2 million per species) from diverse (including fulllength) cDNA libraries for both organisms (Sterky et al. 2004). Agrobacterium-mediated transformation of both Brachypodium and Populus has 

been developed but must be optimized for high-throughput approaches to 

gene discovery. High-throughput capacity for transforming diverse genotypes would enable forward and reverse genetic approaches by generating 

collections of sequence-indexed insertional mutants, including but not 

limited to activation tagging (Groover et al. 2004, Busov et al. 2005), and 

by allowing targeted silencing or overexpression of large numbers of genes.

Transposon-based methods for generating insertional mutants also should 

be developed. High-throughput resequencing capacities should be used for 

identifying molecular markers (e.g., SNPs) associated with biomass and 

bioenergy traits. Similarly, phenotyping (transcript and protein profiling) 

capacities that identify genotypes associated with relevant cell-wall composition or agronomic traits would be useful. 

Using this suite of tools, the function of all Brachypodium and Populus genes 

will be evaluated for their contribution to biomass energy-relevant traits.

Genes deemed relevant will be used to create advanced genotypes in energy 

crops. Public organism-specific databases should be created and curated in 

parallel with advanced experimental capabilities for these organisms. 

To set research priorities, the DOE Office of Science’s GTL program is 

interacting with the conversion program managed through EERE’s Office 

of the Biomass Program and other interested parties (e.g., USDA, NSF,

and private industry). 

Techncal Mlestones 

Wthn 5 years 

•	 Complete Brachypodium genome sequencing, plus EST sequencing and 

molecular-marker identification for both Brachypodium and Populus. 

•	 Optimize transformation methods for diverse genotypes of Brachypodium and Populus, and generate sequence-indexed insertion mutant 

populations of >100,000 events for both organisms. 

•	 Develop tools for transcriptional and protein profiling and carry out 

first-stage profiling of key tissues (e.g., cambial development in Populus). 

•	 Develop organism-specific databases around annotated genome 

sequences. 

Wthn 0 years 

•	 Identify genes relevant to biomass production (e.g., those that control 

cell-wall composition, nutrient uptake, carbon partitioning, flowering, stress tolerance, and disease resistance) using forward and reverse 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 7 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

genetic screens and natural populations based on high-throughput 

phenotyping [e.g., use Fourier transform infrared spectroscopy (called 

FTIR) or other techniques that can be automated to identify genotypes with altered cell-wall composition and advanced image analysis to 

examine plant architecture]. 

•	 Develop and test strategies using these genes to improve energy crops in 

Brachypodium and Populus. 

Wthn 5 years 

•	 In breeding programs, use genes and strategies shown to affect biomass 

production and quality in Brachypodium and Populus to improve energy 

crops, either by transgenic approaches or by marker-assisted selection of 

naturally occurring variability in orthologous genes. 

•	 Transfer improved energy crops and genetic information to the conversion program managed through EERE and the Office of the Biomass 

Program and to other interested parties (e.g., USDA, NSF, and private 

industry). 

The Role of GTL Capabltes for Systems Bology 

Capabilities of GTL (within DOE’s Office of Biological and Environmental Research) will support or enable the attainment of lignocellulosic characteristics (see Lignocellulosic Biomass Characteristics chapter, p. 39) and 

feedstock objectives in numerous ways. Potential contributions of each of 

these, whether distributed or consolidated in a facility, are described below. 

Proten Producton Capabltes 

Most enzymes of interest in cell-wall biosynthesis are thought to be membrane associated and, therefore, difficult to purify and characterize by 

conventional methods. Based on preliminary experiments with membranelocalized glycosyltransferases, a potentially powerful tool in characterizing 

the function of all plant glycosyltransferases is to express them in suitable 

hosts such as insect cells, in which endogenous activities will not interfere 

with assays.This capability could undertake the high-throughput expression 

of all glycosyltransferases and other cell wall–active enzymes (e.g., peroxidases and laccases) from higher plants (e.g., Arabidopsis, Populus, Brachypodium, or rice) or specific biomass species in one or more suitable surrogate 

hosts. Such expression systems will greatly facilitate function identification 

of enzymes that catalyze synthesis of cell walls. Heterologously expressed 

proteins would be tested for their substrate specificity and catalytic activity 

and also will be used as a resource for generating antibodies and tags. 

Antibody resources would have multiple applications. A collection of 

fluorescent- or epitope-tagged proteins in a plant such as Arabidopsis or 

Brachypodium also would be very useful for a variety of reasons. First, the 

tagged proteins could be used to recover complexes for assay by mass spectrometric and imaging methods. Fluorescent tags could be utilized for cellular localization or collocation [e.g., confocal microscopy or fluorescence 

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resonance energy transfer (called FRET)]. Additionally, if certain proteins 

are not active in a surrogate, the tagged version in a plant host might be 

used to identify function by in vitro assays of affinity-purified proteins.

Developing such a resource would require a high-throughput transformation capability that also would be useful for generating insertion mutants 

in Brachypodium and Populus. 

Molecular Machnes Capabltes 

Cellulose is synthesized by an intricate multienzyme complex in the 

plasma membrane, and matrix polysaccharides probably are synthesized 

by multienzyme complexes in the Golgi. Understanding the function of 

these complexes and their interactions with metabolic pathways that produce sugar nucleotides in the cytosol will be important for understanding 

the control of polysaccharide biosynthesis. In addition, cellulose synthase 

interacts with the cytoskeleton to control the orientation of cellulose 

fibrils and possibly influence fibril length. Many questions about this and 

related processes will benefit from the application of new technologies 

for imaging and manipulating protein complexes. Since most or all such 

complexes are membrane bound, specialized methods must be developed 

to work with the complexes. Also, because plant cell walls are intricate 

mechanical assemblies, tools developed for investigating protein machines 

may be used to gain new insights into assembly and function of the large 

polysaccharide complexes that comprise cell walls. 

Proteomc Capabltes 

These capabilities will facilitate identification of proteins that make up 

complexes in such model plants as Arabidopsis, Populus, and other biomass 

crops. Since many complexes are membrane bound, innovative methods 

will be needed to purify membrane complexes using an array of capabilities. Also, because of poor correlation between mRNA and protein 

abundance, proteomic capabilities will enable documentation of the 

proteome of living cell types found in vascular and other tissues, including ray parenchyma and phloem, as a function of time and conditions.

Proteomic analysis of plants with fully sequenced genomes will identify 

post-translationally modified proteins. Preliminary evidence indicates 

that some key proteins involved in cell-wall synthesis are regulated by 

modifications such as phosphorylation. Finally, comprehensive proteomic 

analysis coupled with imaging analysis is required to identify proteins 

located in such compartments as the cell wall, nuclear membrane, Golgi,

and endoplasmic reticulum. 

Cellular System Capabltes 

These capabilities are envisioned to help investigators understand how 

complicated microbial communities in soils respond to various cropping 

regimes by developing baseline analyses of species composition and abundance in suitable experimental plots. Also needed are cost-effective tools 

(e.g., diagnostic DNA chips) for monitoring these and other plots over 

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FEEDSTOCKS FOR BIOFUELS 

long time periods and for integrating resulting data into a view of how 

biomass cropping alters soil ecology. 

In addition to analyzing soil microbial communities, acquisition and analysis of transcriptomic and proteomic data from model and crop plants will be 

a powerful tool for assigning probable function to genes implicated in cellwall synthesis. As a function for each enzyme emerges, the cellular systems 

capability through imaging and dynamical analyses will be used to develop 

a systems model to incorporate the wall’s biophysical aspects with structural properties and knowledge of the functions of proteins involved in wall 

synthesis. This model will facilitate the rational development of feedstock 

species based on “design principles,” in which the wall’s chemical composition and structure are optimized while, at the same time, not compromising 

maximal plant productivity. The cellular systems’ analytical and modeling 

resource and proteomic capabilities will carry out a systematic approach for 

identifying plant biomarkers to guide protein production and the generation of specific molecular tags utilizing protein production capabilities. 

The long-term vision is to make available in situ images of living plant cell 

walls that show all key molecular processes occurring in real time. Studies 

will cover the full life cycle of cell-wall formation, maturation, transformation, dehydration, and processing into simple feedstocks for conversion 

into ethanol or production of other fuels and chemicals. The understanding obtained through research that uses such imaging is expected to result 

in quantitative, predictive modeling as a guide to developing advanced 

feedstocks and processing them into fuels. These tools will take advantage 

of various chemically specific imaging tags created with protein production resources. 

DOE Jont Genome Insttute 

DOE JGI will sequence, assemble, and annotate the gene space or entire 

genomes of model and emerging bioenergy crops and will resequence 

additional cultivars or ecotypes for marker discovery. Identification of 

genes that control cell-wall polysaccharide synthesis or modifications 

in biomass species and the development of tools such as gene chips will 

depend on the availability of nucleotide sequences. Micro-RNAs are 

expected to play a role in expression control of many relevant genes, so 

DNA sequencing must be deep enough to identify them in these species.

JGI also will sequence community genomes, including those of the rhizosphere associated with key proposed energy crops and trees. 

Other Needed Capabltes 

GTL capabilities as currently envisioned do not encompass all resources 

needed for development of energy crops. To accomplish objectives associated with the energy mission, the current vision should be expanded to 

include the three capabilities described below. 

76 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Transformaton Servces 

Substantial capabilities will be needed to carry out transformation of model 

and applied species important to feedstock development on behalf of the 

R&D community and to warehouse genetic resources for these species. 

Chemcal Phenotypng Servces 

Feedstock improvement for increased yield and processing will require 

manipulation and assessment of multiple genes in candidate organisms 

or model systems. New tools are needed to detect, quantify, and compare 

changes in cell-wall composition and 3D architecture in primary and 

secondary walls during assembly and before and during processing. The 

tools would facilitate feedstock development for improved performance 

in biomass conversion. Detailed analysis of cell-wall composition by 

2D nuclear magnetic resonance (NMR) imaging or other methods is 

beyond the scope of routine processing in most research laboratories.

Capabilities for such analyses would broadly facilitate research. 

Synthetc Carbohydrate Chemstry 

Analyses of cell walls in their native state and as a function of treatment and processing require advanced synthetic carbohydrate chemistry capabilities to create standards and models. NMR and mass 

spectroscopy facilities will be necessary to support the synthetic chemistry work. 

Other Bofuel Opportuntes: Development 

of Hgh-Productvty Bodesel Crops 

To maximize solar energy use and storage, an ideal biomass crop 

would carry out photosynthesis at the theoretical maximum every day 

of the year and would store fixed carbon in a directly useful and easily 

harvestable form, even without mineral nutrients. In carrying out this 

process, many plants accumulate large amounts of oils or waxes in 

such specialized storage tissues as seeds or mesocarp tissues. In some 

cases, oil accounts for more than half the dry weight of these tissues.

Most oilseed species, however, are not as productive as others such as 

maize that store primarily starch rather than oil. In theory, obtaining higher yields of oil should be possible by genetically modifying 

already highly productive species (e.g., sugar beet, potato, and maize) 

so they will accumulate oils or waxes instead of carbohydrates in their 

storage and vegetative tissues. 

Other plants produced by genetic modifications of developmental processes accumulate oil in their roots (Ogas et al. 1997), suggesting the feasibility of this strategy. Acetyl-CoA carboxylase is the first committed 

step in fatty-acid biosynthesis in potato tubers, which accumulate starch 

almost exclusively. Overexpression of this enzyme resulted in a fivefold 

increase in triacylglycerol accumulation and provided an example of oil 

deposition in a carbohydrate-storage tissue (Klaus et al. 2004). However, 

Decodng the DNA 

of Soybean—A Source 

of Bodesel 

DOE and the U.S. Department of Agriculture will 

support genome sequencing of the soybean as the first 

project in an agreement to share 

resources and coordinate studies of 

plant and microbial genomics. The 

soybean, Glycine max, is the world’s 

most valuable legume crop and the 

principal U.S. source of biodiesel, a 

renewable alternative fuel. Diesel 

engines inherently are more 

thermodynamically efficient than 

combustion engines. Biodiesel has 

the highest energy content of any 

alternative fuel and is significantly 

more environmentally friendly than 

comparable petroleum-based fuels,

since it degrades rapidly in the 

environment. It also burns more 

cleanly than conventional fuels,

releasing only half the pollutants 

and reducing the production of 

carcinogenic compounds by more 

than 80%. Sequencing will take 

place at the DOE Joint Genome 

Institute, supported by DOE’s 

Office of Science. The soybean 

genome is about 1.1 billion base 

pairs in size, less than half the size 

of maize and human genomes. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 77 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FEEDSTOCKS FOR BIOFUELS 

understanding how to reprogram plant cells to store oil rather than carbohydrate is very challenging. It requires not only large-scale changes in the 

complement of expressed metabolic enzymes but also changes in cellular 

structure typically associated with cell identity. 

Because recovery of oil from plants is technically simple and efficient,

processing oil-accumulating plant material on the farm may be possible 

so mineral nutrients and soil adjuvants can be retained at the farm for 

return to the soil (see sidebar, Decoding the DNA of Soybean—A Source 

of Biodiesel, p. 77). In principle, oil could be recovered during harvest by 

inexpensive screw-press technology, greatly reducing transport and processing costs and enhancing sustainability. 

The lipids that comprise most plant oils are highly reduced forms of carbon and therefore represent the most energy-dense plant-storage compounds. Derivatives such as biodiesel can be produced easily by inexpensive 

and efficient conversion of plant-derived oil to fatty acid methyl esters.

Production of high-yielding, oil-accumulating plants could result in a new 

biofuel source that would reduce loss of carbon associated with fermenting 

sugars to ethanol as well as costs associated with converting lignocellulosic 

feedstocks to liquid fuels. The plant that most nearly meets this description 

is oil palm (see sidebar, Oil Palm, this page). 

Ol Palm: An Important Bofuel Plant 

Oil palm, Elaeis guineensis Jacq., is a tropical 

tree species that produces bunches of oilrich fruit resembling avocados. The plants 

grow in lowlands of the humid tropics (15°N to 

15°S), where rainfall of 1800 to 5000 mm is evenly 

distributed throughout the year. Oil palms mature 

slowly but, once established, yield as much as 10.6 

tonnes of oil per hectare (ha) per year, although the 

average is less than half that amount. They begin to 

bear fruit after about 3 years and remain in use for 

some 25 years, so annual maintenance costs are low. 

Palm oil from the mesocarp, which contains 45 to 

55% oil, is similar in composition to soy oil. Oil 

from palm kernels, extracted from the endosperm,

contains about 50% oil rich in medium-chain 

fatty acids and well suited for biodiesel applications. Malaysia is the major source of these oils,

producing 13.4 billion pounds of mesocarp oil 

and 3.5 billion pounds of kernel oil from about 

3.8 million ha. Plants are harvested by hand, but 

typical planting density is only 150 plants/ha, and 

labor costs are not a major factor in production.

Because of the relatively straightforward conversion of palm oils to diesel and food uses, palm 

acreage in the tropics is expected to expand significantly. [Picture source: C. Somerville, Stanford 

University] 

7 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Some questions to be addressed by basic research: 

•	 How is cell identity controlled? More specifically, what high-level, regulatory genetic controls program a cell to express genes involved in lipid 

synthesis and accumulation? 

•	 What regulates carbon flux from photosynthetic-source leaves to storage organs? 

•	 What regulates metabolic partitioning of carbon among proteins, starch,

and oil production? 

•	 What species are most promising for conversion to oil accumulation? 

For instance, is oil potato a better candidate than oil beet or very high 

oil maize, or should plant stems be engineered to deposit extremely 

thick wax-rich cuticles? 

•	 What are the opportunities for developing new high-yielding oilseed 

species tailored to energy production by uncoupling oil accumulation 

from seed carbohydrate or protein accumulation? 

•	 What is the impact of high lipid levels on plant cells? Most cellular 

mechanisms operate in aqueous environments. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 79 
 

 

 

 

 

 

 

 

 

 

 

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Deconstructng Feedstocks to Sugars
 

This chapter describing the challenges of deconstructing cellulosic 

biomass to ethanol is critically linked to both the feedstock and 

fermentation areas. Lignocellulose recalcitrance to bioprocessing 

will remain the core problem and will be the limiting factor in creating an 

economy based on lignocellulosic ethanol production. Understanding biomass recalcitrance will help to drive crop design. Knowledge about feedstock breakdown mechanisms and products will drive fermentation and 

consolidation strategies, which ultimately will include consolidated bioprocessing (CBP). CBP incorporates the traits for deconstruction and fermentation of sugars to ethanol into a single microbe or culture. The previous 

chapter describes how tools of modern genomics-based systems biology 

can provide tremendous opportunities to engineer energy-plant genomes 

for new varieties. Those engineered plants will grow more efficiently while 

also producing optimal polysaccharide compositions for deconstruction to 

sugars and fermentation to ethanol and other products. Further advancements in plant engineering also can generate new energy crops and trees 

harboring polysaccharide storage structures (principally in the cell walls) 

that are designed for deconstruction. This achievement will be an important 

outcome of scientific research needed to optimize deconstruction of native 

cell walls in such crop residues as corn stover and wheat straw and such 

energy crops as switchgrass and poplar. 

Once we understand more about the chemistry and ultrastructure of cell 

walls, improved thermochemical and biological means can be used to disassemble them. For example, this report will show that new and improved 

(existing) enzymes capable of depolymerizing cellulose in cell-wall microfibrils can significantly reduce the cost of deriving sugars from biomass—a 

critical factor in lowering the overall cost of cellulosic ethanol and making 

it cost-competitive with gasoline. Currently, the structure of cellulose itself 

is not adequately understood. For example, we do not know the actual faces 

of the cellulose crystal to which cellulases bind, and surface interactions 

among experimentally produced enzymes and substrates are not understood. In addition, such new findings about lignin as its synthesis, modification, and depolymerization are needed to develop conversion processes 

that are less energy intensive. 

Sugars produced in optimal cell-wall deconstruction will be used by 

microbes specializing in converting (fermenting) these compounds to 

ethanol. In the near term, biomass conversion will produce some quantities 

of fermentation inhibitors that can include acetate, polyphenolics (aromatic compounds derived from lignin breakdown), and cellobiose. Even References: p. 115 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 5 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

high concentrations of sugars from cell-wall deconstruction are inhibitory 

to many ethanologens. Thus the chapter, Sugar Fermentation to Ethanol,

p. 119, describes research to develop processes at the cellular and genomic 

levels (e.g., evolutionary engineering to modify microbial strains that 

tolerate elevated levels of toxins or sugars). Also, genomic and bioinformatic tools can assist in the design of new metabolic pathways that permit 

efficient simultaneous fermentation of mixed sugars. 

Looking ahead to longer-term improvements in biorefinery productivity, this report proposes that new organisms consolidating traits for both 

deconstruction and fermentation should be the subject of considerable 

research. For example, new ethanologen strains are needed that also 

are able to produce mixtures of highly competent cellulases and other 

hydrolytic enzymes. The application of advanced techniques in genetics,

molecular biology, high-throughput screening, imaging, and mathematical 

modeling will accelerate the pace at which viable processing can be accomplished for energy security. 

Determnng Fundamental Physcal and Chemcal 

Factors n the Recalctrance of Lgnocellulosc 

Bomass to Processng 

Lignocellulosic biomass is a complex structure with crystalline cellulose,

hydrated hemicellulose, and lignin as major components. To date, the best 

enzyme cocktails proposed for saccharification of this material are synergistic mixtures of enzymes with defined activities, primarily those that 

degrade cellulose. The substrate’s heterogeneous nature and enzyme cocktails’ complexity suggest that traditional studies of bulk properties will not 

provide the detailed understanding required for knowledge-based advancements in this field. 

The question that must be answered is, How will structural and chemical details of enzyme substrate–binding sites affect enzyme adsorption 

and reaction rates? From cellulase kinetics alone, we never would be able 

to answer that question. We must be able to improve cellulases using an 

informational approach, considering that 20 years of “mixing and testing”

individual cellulase proteins has yielded only modest progress toward an 

improved cellulase system. Enzyme kinetics always has been a measure of 

“ensemble average” results from experiments, and thus sugar-release values 

from biomass never can deliver information about the reactive site of the 

individual enzyme. Because of this dilemma, we do not know if cellulose 

recalcitrance is due to enzyme inadequacy, enzyme-substrate mismatch, or 

both. While in many cases we can perform bulk compositional analyses,

molecular and structural correlations that are key to processability remain 

a challenge. The science and technology for these analyses must come 

from frontier capabilities in many disciplines in the physical, biological,

and computational sciences [see sidebar, Image Analysis of Bioenergy 

Plant Cell Surfaces at the OBP Biomass Surface Characterization Lab 

(BSCL), p. 40]. 

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Such enzymes as cellulases, hemicellulases, and other glycosyl hydrolases 

(GH) synthesized by fungi and bacteria work synergistically to degrade 

structural polysaccharides in biomass. These enzyme systems, however, are 

as complex as the plant cell-wall substrates they attack. For example, commercial cellulase preparations are mixtures of several types of GH, each with 

distinctly different substrate specificities (cellulose and xylan) and action 

patterns (exoenzymes acting from the chain ends, endoenzymes cleaving within the chain, and GH cleaving side-chain branches, for example,

arabinose from arabinoxylans and xylose from xyloglucans). Optimization 

of these enzymes will require a more detailed understanding of their regulation and activity as a tightly controlled, highly organized system. 

Tools must be developed first to analyze biomass at the levels and specificities needed to describe, understand, predict, and control its behaviors.

These include molecular-level and nanoscale understanding of critical cellwall physical and chemical properties, how they are formed in the plant,

how they function, and how certain structural features inhibit or facilitate enzymatic interactions and subsequent saccharification. Due to the 

dynamic and interconnected nature of these conversion processes, we must 

be able to monitor them as they occur—from native structural biomass to 

free sugars. Critical interactions and reactions occur at many scales of time 

and length throughout multiple stages of intricately interlinked processing. All biomass structural and chemical properties must be analyzed in 

the native state, in various stages of dehydration and processing, and as the 

material is deconstructed into complex mixtures of reaction intermediates. These analyses must support research for biomass optimization for 

processing and ethanol yield, as well as investigations of robust growth and 

tolerance in the cultivar. Pretreatment optimization will both maximize 

fermentable sugar yield while minimizing inhibitors and other deleterious 

factors in subsequent steps. 

This problem further involves structure and function at both the plant and 

microbial levels; their interactions; and the functions of proteins, enzymes,

and living systems. They are studied under a very wide range of conditions,

from pretreatment temperatures and chemistry to the enzymatic breakdown of complex cellulosic microfibrils and other polymers into sugars.

To understand reaction pathways, we must be able to discern intermediate 

digestion and reaction products—physical structures as they degrade and 

chemical moieties as they are transformed and react. All these capabilities 

will enable the ultimate consolidation of all steps into one process. 

An important and necessary departure from traditional approaches stems 

from our appreciation that biological systems are incredibly complex 

and interconnected. Cellular systems of plants and biomass-degrading 

microbes consist of highly coupled subsystems. In these living systems,

each component (molecules and macromolecules) affects and is affected by 

other components. Resolution of research issues described in this section 

will advance biomass pretreatment and hydrolysis by developing a systemslevel, predictive, and quantitative new understanding. Such understanding 

will facilitate rational design of biomass-conversion systems, inform the 

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DECONSTRUCTING FEEDSTOCKS 

Fig. 1. Scanning Electron Microscopy.

(a) A corn stover particle shows a smooth surface 

with a few micron-sized pores after enzyme hydrolysis converted 11% of cellulose to glucose in 3 h.

(b) This corn stover particle has many more pores.

It was pretreated in water at 190°C for 15 min and 

hydrolyzed by enzymes at 50°C for 3 h, resulting 

in 40% cellulose conversion to glucose. 

The results illustrate that pretreatment changes 

lignocellulosic-structure susceptibility to attack 

by enzymes. Higher resolution in future imaging 

techniques will facilitate a deeper understanding 

of underlying molecular mechanisms. [Source:

Images and conditions from unpublished work of 

M. Zeng, N. Mosier, C. Huang, D. Sherman, and 

M. Ladisch, 2006.] 

(a) Enzyme Hydrolysis Only. 

(b) Enzyme Hydrolysis Following Pretreatment. 

design of a new generation of energy crops, and accelerate 

adoption of innovative biorefinery technologies. 

Research Goals 

Biological, mathematical, imaging, and other analytical tools 

must be developed and applied to identify and quantify the 

relative importance of potentially limiting (or facilitating) 

physical and chemical factors in bioconversion. All these 

methods will be used to characterize the effects of dehydration, heating, acidity, and cosolvents, among other biomass 

treatments. Detailed studies using these tools will help us 

better understand mechanisms of action and apply various 

pretreatments and enzyme systems to enhance lignocellulose 

bioconversion to ethanol. Such topics are discussed below. 

Measurement of Bomass Propertes 

The organization of polymer components in biomass structures needs to be noninvasively imaged in 3D to elucidate 

the material’s organization. Various experimental approaches 

including X-ray and neutron diffraction of crystals and 

drawn fibers, enhanced atomic force microscopy (AFM), and 

biochemical probe–enabled and chemically specific imaging 

will be used to characterize the different forms of lignocellulose in both “native” and treated (stored and processed) 

biomass cell walls. 

Analytical methods [e.g., nuclear magnetic resonance (NMR),

Fourier transform infrared (FTIR), and Raman] should 

provide information on chemical moieties, chemical bonds,

and conformation of wall polymers. Surfaces that are enzymebinding sites should be characterized by such tools as AFM,

scanning electron microscopy, transmission electron microscopy, and electron spectroscopy for chemical analysis (see 

Fig. 1. Scanning Electron Microscopy, this page). 

This information can be correlated using statistical-analysis 

packages based on principal components to reliably relate 

changes in lignin carbohydrate complexes (LCC) and lignin 

profiles, degrees of cellulose dehydration, and pretreatment 

chemistries to the abilities of hydrolytic enzymes to convert 

the resultant material biomass. 

Models for Drect Enzymatc Interactons, Acton 

Mechanistic models of enzyme-system substrate relationships, based on new knowledge about plant cell-wall architecture and enzyme structure and function, need to be 

developed. The action of individual and combinations of 

enzymes on native and model substrates should be examined using single-molecule spectroscopy and imaging. 

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Mechanistic characterization of relevant enzymes, with attention to combined chemical and biological conversion processes of cell-wall materials, then will support models for interaction. Finally, new computational 

models for microfibrils and cell walls using improved codes and petascale 

leadership-class computers will provide the needed fidelity to support 

reaction-pathway calculations. 

Techncal Mlestones 

Wthn 5 years 

•	 Using integrated analytical and mathematical methods, quantitatively 

analyze effects of selected biomass properties and pretreatment chemistries on enzymatic hydrolysis. 

•	 Identify principles, genes, and controlling factors that influence biomass 

makeup, assembly, and processability, in conjunction with plant design. 

•	 Perfect and extend existing spectroscopic, surface, and imaging characterization methods for biomass. 

•	 Develop methods for monitoring the progress of physical modification 

during dehydration under various conditions of storage or pretreatment 

at elevated temperatures. Develop improved protocols to be tested in 

field trials. 

•	 Define the primary structure of lignins and LCCs and the shape,

dimensions, and heterogeneity of their domains. 

•	 Develop models connecting lignocellulosic properties with deconstruction and hydrolysis processes. 

•	 Definitively characterize the detailed organizational structures of 

principal types of plant cellulose and their relative energies and interrelationships. 

–	 Understand the energetics of different cellulose forms and ways in 

which these structures give rise to characteristics of the next organization level in the mesoscopic range of sizes, and 

–	 Determine how microfibrils interact with other principal structural 

components (lignins and noncellulosic polysaccharides). 

Wthn 0 years 

•	 Develop an understanding of the physicochemical basis of polysaccharide interactions with water. This information can be used to rationally 

design genomic variations for cell-wall polysaccharide composites with 

more desirable properties. 

•	 Apply new biological and chemical tools to alter LCC and other relevant properties of selected biomass species. 

•	 Subject these altered biomass species and controls to various pretreatment chemistries and temperatures. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

•	 Hydrolyze the resulting pretreated biomass with various specific 

enzymes and correlate spectroscopic, surface, and image data with 

hydrolysis of structural carbohydrates. 

•	 Quantitatively relate key biomass and pretreatment properties with 

hydrolysis yields for different biomass species. 

•	 Improve enzymes based on the knowledge of substrate-imposed limitations and an understanding of optimized cell walls. 

•	 Develop molecular models for enzyme-substrate structure-function 

relationships. 

Wthn 5 years 

•	 Develop mathematical models of cell walls and the transport of water,

pretreatment chemicals, and enzymes through the walls. Such models 

should help in designing cell walls that can be digested more efficiently 

by developed enzyme systems. Designs optimized by modeling can then 

help guide genomic engineering for plant and enzyme optimization. 

•	 Establish the foundation for integrated biomass processing by predicting and then verifying overall hydrolysis yields for native and modified 

biomass species using different pretreatment chemistries and severities 

(time, temperature, acidity) and a range of hydrolytic enzymes. 

•	 Perform rational engineering of enzymes to specific requirements 

based on new understandings about enzymes and their mechanisms of 

action and substrate cellulose-hemicellulose-lignin complexes. 

The Role of GTL and OBP Facltes and Capabltes 

The program outlined most likely will need all facility resources at one 

point or another. 

Proten Producton 

Protein production capabilities will be used for enzymes and tags (biomarkers). 

Molecular Machne and Cellular System Analyss 

These capabilities can image and diagnose interactions between enzymes 

and biomass substrates. In addition, BSCL, recently established at NREL,

certainly will prove essential. Imaging facilities would be used to correlate 

digestibility to biomass ultrastructure. The sidebar, Image Analysis of Bioenergy Plant Cell Surfaces at the OBP Biomass Surface Characterization 

Lab, p. 40, illustrates the cell wall’s highly coupled nature and how each 

component affects and is affected by other components, thereby highlighting the need to study the intact system. 

DOE Jont Genome Insttute 

DOE JGI will map and identify genes concerned with deconstruction 

barriers or facilitating factors. 

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Crosscuttng Tools, Technologes, and Scence
 

•	 Better characterization of cell-wall polysaccharide association profiles 

in hydrated and dehydrated states using spectroscopic and ultramicroscopic methods. 

•	 Larger-scale molecular mechanics modeling to explore the association of 

cell-wall polymers with each other and with water at various length scales. 

•	 Robust and rapid methods for quantifying cell-wall polymers and interpolymer linkages. 

•	 Genomic and chemical tools to identify genes involved in rate-limiting 

LCC linkages. 

•	 New biomass variants with enhanced accessibility for structural carbohydrate–digesting enzymes. 

•	 New methods for spectroscopic characterization of cell-wall polysaccharides in the living hydrated state. Most past work has been based on 

investigation of isolated dehydrated samples and very little on cell-wall 

constituents in their native state. 

•	 Coupled structure and processing models of cell walls and components. 

•	 Data-analysis software to identify and quantitatively describe the relative importance of different factors governing response to pretreatment 

and enzymatic hydrolysis of biomass. 

Developng Better Enzymatc Systems for Bologcal 

Pretreatment: Lgnnases and Hemcellulases 

The most efficient conditions for simultaneous saccharification and fermentation of lignocellulosic biomass will be those in which transformations now performed during and following thermochemical pretreatment 

are seamlessly integrated into the overall process. Effective enzymatic 

breakdown of LCC, to expose cellulose to enzyme action, represents an 

important step toward this objective. 

Lignocellulose, an extremely complex and widely varying nanoscale composite, is well designed to resist attack. Ligninases and hemicellulases, for 

which few examples are known, are inadequately understood. Technically,

understanding and optimizing these enzymes will enable the ultimate goal 

of consolidating pretreatment and saccharification. Consequently, research 

is needed to identify, characterize, improve, and economically produce the 

most effective enzyme systems for biomass preconditioning. These enzymes 

would be applied before or after traditional pretreatment to minimize and,

eventually, replace thermochemical processes, thus lessening the effects of 

overall pretreatment severity at the macromolecular level and simplifying 

processing. To support improvements, research must be focused on identifying more enzymes of this class and characterizing their principles of action.

A crucial component for ligninase identification will be genomic, functional genomic, and proteomic comparisons of white rot fungi known or 

presumed to express such activities (see sidebar, White Rot Fungus, p. 93). 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

The ultimate goal of this research is to produce a recombinant ligninasehemicellulase microbial system with enhanced catalytic activity and stability,

industrialized for biorefinery operations. 

Research Goals 

The chemical and structural nature of native lignin is poorly understood.

Developing robust ligninases requires a foundation of knowledge about the 

range of lignin chemistries and lignin associations that inhibit or facilitate 

freeing cellulose and hemicellulose for saccharification (see sidebar, Lignification: Random vs Template Directed, p. 94). Similarly, despite the importance of hemicelluloses and hemicellulases for saccharification, both are 

poorly understood. Due to the complex structure and compositional diversity 

of lignin-hemicellulose and -cellulose substrates that must be contemplated 

for feedstock biomass, a variety of enzymatic activities will be needed to 

catalyze deconstruction for conversion to monosaccharides (see Fig. 2. Complex Mixture of Enzymes for Degrading Hemicelluloses, p. 96). 

Hemcelluloses and Hemcellulases 

After cellulose, hemicelluloses (xylan polymers) are the next most abundant polysaccharides in native biomass feedstocks (see Table 1. Cellulose,

Hemicellulose, and Lignin Content in Various Sources of Biomass, p. 93).

Although considerable research has focused on converting biomass cellulose 

to fermentable glucose, less has been done on bioconverting other plant 

cell-wall components. A better understanding of the complex structure 

and composition of this polysaccharide group, which will differ in type and 

abundance among different feedstocks, will help to identify and optimize 

the mechanistic basis of required enzymatic activities. Structural information and mechanistic models must be developed to pinpoint “bottlenecks” in 

hemicellulose bioconversion. 

Lgnn and Lgnnases 

Despite its critical importance, the enzymatic basis of lignin depolymerization in vivo has remained elusive, if not controversial. Research during 

the past 20 years on putative “ligninases,” which can both polymerize and 

depolymerize lignin preparations, has not yielded reliable insights into the 

mechanisms of lignin cleavage.Thus the possibility of another entirely different class of lignin-degrading enzymes cannot be disregarded, and potential candidates have been identified in white rot fungi. Genes encoding a 

range of ligninases must be identified, and genomic sequences of white rot 

fungi that degrade lignin but do not express known peroxidases will be compared with the sequenced P. chrysosporium genome. A suitable host organism 

will be needed to produce heterologous ligninases in sufficient quantities;

possible hosts include E. coli, S. cerevisiae, P. pastoris, and A. oryzae. Heterologous ligninase expression and manipulation have had limited success. 

This research would include the following goals: 

• Detailed characterization of the range of lignin and hemicellulose structures encountered in available biomass. Special attention must be paid 

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to the nature of covalent bonds linking LCC.

Model systems will facilitate these analyses. 

Systems biology–style survey and molecular and 

functional characterization of the diversity and activity of relevant hemicellulases (glycoside hydrolases and 

esterases), including discovery of activities from such 

novel sources as soils, the rhizosphere, termite hindgut,

and decaying biomass. 

Similar discovery and molecular and functional characterization of new lignin-degrading enzymes and their 

activities. 

Reliable expression systems and hosts for these 

enzyme classes. 

Mechanistic characterization of enzymes with attention to properties relevant to combined chemical and biological conversion processes. 

Examination of individual and combination enzyme activities on native 

and model substrates. 

Development of mechanistic models of enzyme system substrate relationships, based on new knowledge about plant cell-wall architecture. 

Table . Cellulose, Hemcellulose, and Lgnn 

Content n Varous Sources of Bomass 

Feedstock Cellulose Hemicellulose Lignin 

Corn stover 36.4 22.6 16.6 

Wheat straw 38.2 24.7 23.4 

Rice straw 34.2 24.5 23.4 

Switchgrass 31.0 24.4 17.6 

Poplar 49.9 20.4 18.1 

Source: A. Wiselogel, S. Tyson, and D. Johnson, “Biomass 

Feedstock Resources and Composition,” pp. 105–18 in 

Handbook on Bioethanol: Production and Utilization (Applied 

Energy Technology Series), ed. C. E. Wyman, Taylor and 

Francis (1996). 

Whte Rot Fungus: Genome of Known Lgnn Degrader Sequenced 

Lignin degradation is the key to making polysaccharide components of cell walls available for breakdown. White rot fungi are the primary degraders of lignin, which is among the most abundant of natural materials on earth and plays a pivotal role in global carbon cycling. These organisms also degrade the 

cellulose and hemicellulose components of plant cell walls. 

To aid in understanding these processes, the DOE Joint Genome 

Institute sequenced the genome of the white rot fungus Phanerochaete 

chrysosporium. This fungus degrades brown lignin, the protective 

matrix surrounding cellulose microfibrils of plant cell walls, leaving behind crystalline white cellulose. [D. Martinez et al., “Genome 

Sequence of the Lignocellulose Degrading Fungus Phanerochaete 

chrysosporium strain RP78,” Nat. Biotechnol. 22, 695–700 (2004).] 

Analysis of the white rot fungus genome revealed genes encoding 

oxidases, peroxidases, and other enzymes that contribute to depolymerization of lignin, cellulose, and hemicellulose. Extensive genetic 

diversity was observed in gene families encoding these enzymes,

possibly reflecting that multiple specificities are needed for effective 

degradation of cell-wall polymers from different plant species. Elucidating the regulation of genes, proteins, and metabolites from this 

organism and others will enhance understanding of the individual 

and collective mechanisms of degradative enzymes as well as their 

interactions with other organisms in their ecosystems. Such advances 

are necessary for generating the framework to engineer large-scale 

processes for biomass utilization. 

Cover used with permission from Nature 

Biotechnology, www.nature.com/nbt/. 

• 

• 

• 

• 

• 

• 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

•	 Improvement of enzyme catalytic efficiency through protein engineering 

and of biomass through plant design. 

•	 Coordination with feedstocks and biological-conversion research to 

inform plant design and to support improved fermentation and eventual 

incorporation of pretreatment enzymatic processes into consolidated 

processing schemes. 

Lignin and hemicellulose investigations will follow similar research paths 

as described below. 

Techncal Mlestones 

Wthn 5 years 

•	 Initiate metagenomic surveys in environments known to break down biomass naturally, and identify and molecularly characterize ligninases and 

hemicellulases, including accessory enzymes. 

•	 Heterologously express enzymes in suitable protein production hosts. 

Lgnficaton: Random vs Template Drected 

I

n terms of energy content, lignins are 

thought to be the most abundant of

all biopolymers. They are composed 

of p-hydroxyphenylpropanoid units interconnected through 8-O-4, 8-5, 8-8, 8-1,

5-5, and 4-O-5 linkages. Corresponding 

substructures in the polymer include alkyl 

aryl ethers, phenylcoumarans, resinols,

tetrahydrofuran-spiro-cyclohexadienones,

biphenyls, dibenzodioxocins, and diaryl 

ethers (see Fig. A, at left). The primary 

precursors themselves—the three monolignols p-coumaryl, coniferyl, and sinapyl 

alcohols—differ only according to their aromatic methoxy substitution patterns. These 

monolignols are oxidized enzymatically 

through single-electron transfer to generate 

the respective phenoxy radicals. The actual 

coupling of a monolignol radical with the 

growing end of a lignin chain, however, may 

not fall under direct enzymatic control. 

Accordingly, many investigators have 

assumed that lignin primary structures 

must be “random” or combinatorial as far as 

sequences of interunit linkages are concerned. More recently, this theory has been 

reinforced by reports that certain kinds Fig. A. Contemporary View of Lignin Substructures.Theory proposed 

by G. Brunow and coworkers in 1998 (reproduced with permission). 

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•	 Determine catalytic properties of enzymes. 

•	 Use overproduced and commercial enzymes to digest native hemicelluloses, and identify intermediate products in hemicellulose decomposition. 

•	 Perform molecular evolution of enzymes, and select for improved 

kinetic efficiency and compatibility with other enzymes and with 

desired industrial applications. This is expected to result in the identification of factors limiting enzymatic conversion and the development of 

enzyme cocktails to overcome them efficiently. 

Wthn 0 years 

•	 Extend metagenomic surveys to discover novel ligninases and hemicellulases. 

•	 Concurrently improve enzymes in the cell walls of energy crops in parallel with knowledge about substrate-imposed limitations. 

•	 Develop molecular models for enzyme-substrate structure-function 

relationships. 

of non-native monolignols can be incorporated into macromolecular lignin structures. Lignins and lignin 

derivatives exhibit two fundamental characteristics that traditionally have been viewed as evidence in favor of 

randomness in their configurations: They are both noncrystalline and optically inactive.1 

Nevertheless, a number of observations are thought by some to point in the opposite direction. The individual 

molecular components in (nonpolyionic) lignin preparations tend to associate very strongly with one another 

in a well-defined way. These processes are thought to be governed by vital structural motifs derived from corresponding features disposed nonrandomly along the native biopolymer chain. Moreover, dimeric pinoresinol 

moieties are linked predominantly to the macromolecular lignin chain through at least one of their aromatic 

C-5 positions. 

We do not know whether such features can be explained through combinatorial mechanisms under simple 

chemical control or if higher-level control mechanisms are required. One hypothesis proposes a way to replicate specific sequences of interunit linkages through a direct template polymerization mechanism. According 

to this model, an antiparallel double-stranded lignin template, maintained in a dynamic state at the leading 

edge of each lignifying domain, determines the configuration of the daughter chain being assembled on the 

proximal strand’s exposed face. Furthermore, replication fidelity could be controlled by strong nonbonded 

orbital interactions between matching pairs of aromatic rings in the parent and the growing daughter chains.

The overall process seems to be consistent with the lack of both crystallinity and optical activity in macromolecular lignin domains. 

Finally, required sequence information may be encoded in polypeptide chains that embody arrays of adjacent 

lignol-binding sites analogous to those found in dirigent positioning proteins.2 

Cted References 

1. J. Ralph et al. 2004. “Lignins: Natural Polymers from Oxidative Coupling of 4-Hydroxyphenylpropanoids,”

Phytochemistry Rev. 3, 29–60. 

2. S. Sarkanen. 1998. “Template Polymerization in Lignin Biosynthesis,” pp. 194–208 in Lignin and Lignan

Biosynthesis 697, ed. N. G. Lewis and S. Sarkanen, American Chemical Society, Washington, D.C. 

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DECONSTRUCTING FEEDSTOCKS 

•	 Improve catalytic efficiency and stability of enzymes that degrade cell 

walls using protein engineering. 

Constraints imposed by the fairly limited set of industrial host organisms 

on expression of native or heterologous GH proteins must be understood 

(for example, the role and modification of various glycoforms). Greater 

understanding in these areas would allow more efficient improvements in 

enzyme performance in industrialized hosts and resultant production economics. Understanding biomass recalcitrance will enable enzyme preparations to be engineered for superior performance using proteomics and 

the systems biology approach. These challenges will be more pronounced 

when dealing with “foreign” proteins (such as ligninases) and multienzyme 

complexes (cellulosomes) in heterologous hosts. 

Wthn 5 years 

•	 Perform rational engineering of enzymes to specific requirements based 

on new understanding of enzymes and substrate cellulose-hemicelluloselignin complexes.The ability to design specific enzymes to attack specific 

substrates is expected by this time. 

Fig. 2. Complex Mixture of Enzymes for Degrading Hemicelluloses. The example depicted is cross-linked glucurono 

arabinoxylan. The complex composition and structure of hemicellulose require multiple enzymes to break down the polymer 

into sugar monomers—primarily xylose, but other pentose and hexose sugars also are present in hemicelluloses. A variety of debranching enzymes (red) act on diverse side chains hanging off the xylan backbone (blue).These debranching enzymes include 

arabinofuranosidase, feruloyl esterase, acetylxylan esterase, and alpha-glucuronidase (see table below). As the side chains are 

released, the xylan backbone is exposed and made more accessible to cleavage by xylanase. Beta-xylosidase cleaves xylobiose 

into two xylose monomers; this enzyme also can release xylose from the end of the xylan backbone or a xylo-oligosaccharide. 

The table shows that some of these enzymes are multifunctional, with catalytic domains belonging to different 

enzyme families. Their great diversity and that of other 

enzymes involved in hemicellulose degradation present a 

remarkably complicated enzymatic system whose morethorough analysis may yield greater understanding of 

hemicellulosic degradation. 

[Source: Molecular structure adapted from L. B. Selinger,

C. W. Forsberg, and K. J. Cheng, “The Rumen: A Unique 

Source of Enzymes for Enhancing Livestock Production,”

Anaerobe 2(5), 263–84 (1996).] 

Glycosde Hydrolase (GH) and Carbohydrate Esterase 

(CE) Enzyme Famles for Degradng Hemcelluloses 

Enzyme Enzyme Families 

Endoxylanase GH5, 8, 10, 11, 43 

Beta-xylosidase GH3, 39, 43, 52, 54 

Alpha-L-arabinofuranosidase GH3, 43, 51, 54, 62 

Alpha-glucuronidase GH4, 67 

Alpha-galactosidase GH4, 36 

Acetylxylan esterase CE1, 2, 3, 4, 5, 6, 7 

Feruloyl esterase CE1 

---Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4X--­

---Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4Xβ1–4X--­

β-xylosidase 

α-Glucuronidase Acetylxylan 

esterase 

Arabinofuranosidase 

Feruloyl esterase 

Lignin

Xylanase 

Xβ1–4X

2X

3

α

Af

5

Fer

Fer–O–Fer–

5

Af

α

3

Ac

3

3

Af

mGu

1

2

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•	 Greatly increase understanding of fundamental protein-secretion limitations in hyperproducing strains. Similar challenges must be met to 

enable consolidated bioprocessing microbes. 

•	 Similarly, the catalytic activity and thermal stability of ligninases must 

be enhanced through directed evolution strategies, which require identification of suitable model substrates for screening mutant enzymes in 

variant libraries. 

Crosscuttng Tools, Technologes, and Scence 

Computational science, advanced imaging, and high-throughput genomics 

tools must be incorporated to attain the objectives described here. Computational science is important in coordinating process engineering and 

in exploiting the products of protein engineering. Structural imaging of 

enzymes, substrates, and enzyme-substrate complexes will be needed to 

understand enzyme function at the molecular level; a library of model substrates for these kinetic and mechanistic studies is required. 

A better understanding of the structure and composition of specific xylans 

and lignins would greatly improve the ability to design optimal enzyme 

mixes. Methods to monitor the process of polysaccharide hydrolysis (e.g.,

NMR and mass spectroscopy) would be beneficial.Transgenic plants and 

trees with reduced lignin contents or modified lignin configurations could 

exhibit differences in lignin-depolymerization rate; understanding enzymatic 

mechanisms would be crucial in manipulating conditions to the best effect. 

Advanced imaging, spectroscopic, and enzymatic methods of characterizing cell walls before and after pretreatment also require new resources for 

producing and disseminating chemical standards. These new standards will 

be representative of the range of plant cell-wall chemistry and will permit 

improved cross-comparison research, not only on polysaccharide disassembly but also on subsequent sugar metabolism. 

Understandng the Molecular Machnery 

Underpnnng Cellulose Saccharficaton: 

Cellulases and Cellulosomes 

With new biology tools, we have the opportunity to understand enzyme 

structure-function relationships that govern the critical processes of bioethanol production. Given the importance of alternative renewable energy 

sources, the prospect of engineering improved cellulases is an exciting and 

universally appealing concept. Cellulosomes, large molecular machines that 

integrate numerous enzymes and functions to break down biomass, are key 

to reducing the enzyme loading required for processing. Understanding 

cellulosomes and learning how to manipulate and modify them for greater 

efficiency will be important in consolidated processing. Research first will 

allow understanding and then improvement in the performance of both free 

and complexed (cellulosomal) cellulases on biofeedstocks. The rate-limiting 

step in hydrolysis is not catalytic cleavage but disruption of a single substrate 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 97 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

chain from its native matrix, thereby rendering it accessible to the catalytically active cellulase site. Thus, we must be able to analyze and understand 

the processes and interactions that facilitate this disruption of insoluble 

cellulose.To approach this problem in a systematic and rational fashion,

detailed understanding of the structure and energetics of both the microcrystalline and noncrystalline portions of cellulose fibrils is first necessary. 

•	 How do soluble enzymes act on an insoluble crystalline substrate? 

Hydrolysis of crystalline cellulose is the rate-limiting step in biomass 

conversion to ethanol because aqueous enzyme solutions have difficulty 

acting on this insoluble, highly ordered structure. Cellulose molecules in 

their crystalline form are packed so tightly that enzymes and even small 

molecules such as water are unable to permeate the structure. 

•	 How do different biomass-degrading enzymes work together as a synergistic system? Cellulases and hemicellulases are secreted from cells as 

free enzymes or as extracellular cellulosomes. The collective activity of 

enzyme systems is believed to be much more efficient than the individual activity of any isolated enzyme; therefore, to truly understand how 

enzymes function, they must be studied as systems rather than individually or a few at a time. In addition, systems eventually must be analyzed 

under laboratory conditions more representative of real-world environments. For example, laboratories often use purified cellulose as the 

substrate for enzyme analysis rather than more heterogeneous, natural 

lignocellulosic materials; this can provide erroneous conclusions about 

natural enzyme activity. New analytical methods that are spatially and 

chemically sensitive will allow realistic understanding of the mechanisms 

of biomass degradation. 

Research Goals 

Dscoverng and Improvng Free Cellulases 

New generations of engineered cellulases will provide enhanced performance (activity) requiring lower protein loadings, so process cost will be 

reduced. Cellulase cost currently is estimated at 10 to 25 cents per gallon 

of ethanol produced. The new target will be similar to starch hydrolysis,

or 1 to 2 cents per gallon of ethanol. For example, loadings of about 25­

mg fungal cellulase proteins are required to hydrolyze about one gram 

of cellulose in pretreated biomass. Improved cellulases would convert 

an equivalent amount of cellulose to sugars using around one-tenth the 

enzyme loading. More efficient cellulase biomass digestion also may permit reduced-severity thermochemical pretreatment. 

Acquiring new scientific insights into cellulose structure and the function 

of cellulase cocktails is a critical objective (see sidebar, New Cellulase 

Enzymes Dramatically Reduce Costs of Plant Biomass Breakdown, p.

100). The initial research phase involves an exhaustive search for examples of important families of free cellulases (those secreted into the extracellular mixture) using high-througput genomic and enzyme-production 

and -characterization methods. Cellulose and cell-wall interaction with 

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degrading enzymes must be characterized. Biochemical analysis of family 

members will reveal much about the natural diversity of solutions to cellulase action. Combined mutational analysis and computational modeling then will be used to define structure-function relationships of these 

enzymes and newly engineered mutants. With this kinetic and thermodynamic understanding as a guide, enzyme-engineering approaches will 

be used to test novel hypotheses for improving cellulase performance on 

the cell walls of plants considered suitable biofeedstocks. 

Understandng and Utlzng Cellulosomes 

The cellulosome, a unique type of molecular machine, can efficiently 

solubilize native and pretreated lignocellulose substrates. The cellulosome serves as a more efficient way of enzyme loading and presentation 

to the substrate. In this case, native cellulosomes attached to the microbe 

contain the full repertoire of plant cell-wall polysaccharide-degrading 

enzymes, and a single carbohydrate-binding module (CBM) targets the 

entire complement of enzymes to the substrate surface (see sidebar, The 

Cellulosome, p. 102). 

•	 Artificial designer cellulosomes that exhibit a precise architecture and 

composition will help reveal various principles of cellulosome construction and action. In this context, 

–	 Structure and biochemical-biophysical aspects of assorted cellulosomal modules (i.e., cohesins, dockerins, CBMs, and catalytic 

domains) from different subunits and species will be examined. 

–	 Recombinant primary and adaptor chimeric scaffoldins will be constructed, designed to bear divergent cohesins for subsequent incorporation of native or hybrid (dockerin-tagged) cellulosomal enzymes. 

–	 Through this approach, improved high-performance (noncellulosomal) cellulases can be incorporated into a cellulosome to take 

advantage of enhanced synergistic properties inherent in the cellulosome complex. 

Genes encoding for the complement of designer cellulosomal components 

also can be cloned into a suitable host cell system for heterologous production, assembly, and secretion of active designer cellulosomes of desired 

composition and architecture. 

Several key scientific questions and issues are especially important for 

reaching the proposed goals. 

•	 We do not understand enough about substrate microcrystalline cellulose’s structure as it exists in the plant cell wall. Uncertainties about 

the crystal lattice structure in the microfibril and the crystal faces of 

cellulose targeted by enzyme diversity in the biosphere are examples of 

our lack of insight. Understanding the substrates’ molecular architecture would help in using rational design to produce improved hydrolytic molecular machines. With such knowledge it might be possible,

for example, to select specific mutations of cellulose-binding domains 

that tend to disrupt fibril packing or to use molecular dynamic simuBofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 99 
 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

lations to find environmental conditions (temperature, pressure, and 

molecular agents) that affect such disruption before the cellulase 

attacks the substrate. 

•	 The kinetic and thermodynamic mechanism of processive cellulases 

or exoglucanases (GH family 7) is not known. These enzymes, protein 

machines, conduct most cellulose hydrolysis, with endoglucanases playing a lesser role. To understand this mode of catalysis at the molecular 

level, a mathematical model of the functioning of GH family 7 celNew Cellulase Enzymes Dramatcally Reduce Costs of Plant Bomass 

Breakdown: R&D 00 Award 

Further Advances Needed to Improve Efficiency and Economics 

Cellulase enzymes are used to break down 

the cellulose of plant cell walls into simple 

sugars that can be transformed (fermented) 

by microbes to fuels, primarily ethanol, as well as to 

chemicals, plastics, fibers, detergents, pharmaceuticals, and many other products. 

Like starch and sugar, cellulose is a carbohydrate 

(compound of carbon, hydrogen, and oxygen) made 

up of simple sugars (glucose) linked together in 

long chains called polysaccharides. These polymers 

form the structural portion of plant cell walls, and 

unraveling them is the key to economical ethanol 

fermentation. Technical barriers to large-scale use 

of cellulose technology include the low specific 

enzyme activity and high enzyme-production costs,

as well as a general lack of understanding about enzyme biochemistry and mechanistic fundamentals. 

In 2004, the DOE National Renewable Energy Laboratory (NREL), working with two of the largest industrial enzyme producers (Genencor International and Novozymes Biotech), achieved a dramatic reduction in 

cellulase enzyme costs. Cellulases belong to a group of enzymes known as glycosyl hydrolases, which break 

(hydrolyze) bonds linking a carbohydrate to another molecule. The new technology involves a cocktail of three 

types of cellulases—endoglucanases, exoglucanases, and beta-glucosidases. These enzymes work together to 

attack cellulose chains, pulling them away from the crystalline structure and breaking off cellobiose molecules 

(two linked glucose residues), splitting them into individual glucose molecules, and making them available for 

further processing. This breakthrough work resulted in 20- to 30-fold cost reduction and earned NREL and 

collaborators an R&D 100 Award (press release: www.nrel.gov/awards/2004hrvtd.html). 

Further cost reductions are required, however, to support an economical and robust cellulose biorefinery industry. For example, costs of amylase enzymes for converting corn grain starch to ethanol are about 1 to 2 cents 

per gallon of ethanol produced, but the most optimistic cost estimates for cellulase preparations now are about 

tenfold higher than that. Routes to improving enzyme efficiencies include the development of enzymes with 

more heat tolerance and higher specific activities, better matching of enzymes and plant cell-wall polymers,

and development of high-solid enzymatic hydrolysis to lower capital costs. A comprehensive understanding of 

the structure and function of these enzymatic protein machines, how their production and activity are controlled, and changes they promote on plant cell-wall surfaces will be critical for success. 

(Also see sidebar, Image Analysis of Bioenergy Plant Cell Surfaces at the OBP Biomass Surface Characterization Lab, p. 40.) 

Cellulase image from M. Himmel et al., “Cellulase Animation,” run time 11 min., National Renewable Energy Laboratory (2000). 

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lulase must be developed and tested. Finally, the range of permissible 

modifications of these specific protein folds is not known, nor are 

constraints imposed by the fairly limited set of industrial host organisms on expression of wild-type members of GH families or newly 

engineered mutants. 

•	 The major objective of the designer-cellulosome concept is to reconstruct improved cellulosomes by linking essential and most efficient 

enzymes to desired substrates. Numerous scientific issues and opportunities are still “at large.” 

–	 Our capacity to control the final designer-cellulosome’s composition 

and architectural arrangement will enable us to pose new hypotheses 

regarding enhanced cellulosome performance in degradation of plant 

cell-wall material. 

–	 The best set of cellulosomal cellulases most appropriate (synergistic) 

for use on lignocellulosic substrates is still unknown. 

–	 Although hybrid enzymes derived from noncellulosomal species can 

be incorporated into designer cellulosomes, whether their observed 

synergistic activity is comparable to that of native cellulosomal 

enzymes is still unknown. 

–	 Many other scientific questions have to be addressed, including the 

optimal stoichiometry and arrangement of components and whether 

the combined action of cellulosomal and noncellulosomal systems 

results in an additional improvement in performance. This research 

offers a unique opportunity to explore the expanding universe of 

molecular machines that can be constructed by the designer-cellulosome approach. 

Techncal Mlestones 

Wthn 5 years 

•	 Near-term accomplishments in cellulase biochemistry should focus on 

improving the enzymes, primarily from fungi and actinomycetes, used 

in industrial preparations marketed for the biorefinery. This accomplishment would require searching for new enzymes from new sources 

(including metagenomic databases) and using directed evolution to 

probe mutational space for possible improvements over wild-type 

examples. The outcome would be better cocktails based on wild-type 

enzymes and enzymes improved by noninformational methods that 

could be used immediately by industry. 

•	 Construction of designer cellulosomes will concentrate on several fronts,

including incorporation of currently available enzymes; development of 

novel cellulosomal components—scaffoldins, cohesins, dockerins, CBMs,

and linker segments; improvement of hybrid enzymes and assessment 

of their enhanced synergistic action within designer cellulosomes; and 

analysis of the combined action of designer cellulosomes with highly 

active noncellulosomal enzymes, including ligninases and hemicellulases. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 0 
 

DECONSTRUCTING FEEDSTOCKS 

Wthn 0 years 

•	 The mid-term scope permits a more systematic approach to cellulase 

and cellulosome biochemistry. Such understanding will require combining classical biochemistry with computational science. 

•	 A particularly important goal is identifying thermodynamic limitations 

of improvement, which would impact protein engineering. 

•	 Initial studies to convert suitable host-cell microorganisms into cellulosome-producing strains will be pursued. The desired result will be 

improved biomass degradation and increased understanding of the 

structure-function relationship of cellulosome components. 

•	 To achieve heterologous production, assembly, and secretion of active 

designer cellulosomes, suitable host cells that can accommodate the 

genes for such large proteins must be identified and inherent cloning 

and expression barriers overcome. 

The Cellulosome: The “Swss Army Knfe” of Molecular Machnes 

The cellulosome is an extracellular supramolecular machine synthesized by some anaerobic microorganisms capable of degrading crystalline cellulose and other plant cell-wall polysaccharides. Each cellulosome contains many different complementary types of carbohydrate-active enzymes, including 

cellulases, hemicellulases, and carbohydrate esterases that are held together by a scaffoldin protein to form 

a single multienzyme complex (see Fig. A. Schematic of a Cellulosome, below). The cellulosome enhances 

cell-wall degradation by bringing several different enzymes into close proximity so they can work together to 

exploit enzyme-accessible regions of cellulose. The various product intermediates from one enzymatic subunit 

can be transferred readily to other enzymatic subunits for further hydrolysis (breakdown) of the cellulose. The 

cellulosome also promotes cell adhesion to the insoluble cellulose substrate, thus providing individual microbial 

cells with a direct competitive advantage in using soluble sugars produced in the hydrolysis. Cellulosomes need 

not be associated with cells for activity, and they function under both aerobic and anaerobic conditions. 

Each enzymatic subunit contains a definitive catalytic module and a dockerin domain that binds tightly to a 

scaffoldin cohesin. Thus, cohesin-dockerin interaction governs 

assembly of the complex, while 

cellulosome interaction with 

Fig. A. Schematic of a Cellulosome.

The scaffoldin subunit, to which the 

cellulosomal enzymatic subunits 

are bound, is made up of different 

functional modules that dictate its 

architecture and various activities. 

These include a single CBM and 

nine very similar repeating domains 

called cohesins, which bind the enzymatic subunits. 

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Wthn 5 years 

•	 HTP approaches and crystallographic and microscopic techniques will 

be used to elucidate the three-dimensional structure of cellulosomal 

components, the intact native and designer cellulosomes, and their 

action with pure (cellulose) and native (plant cell-wall) substrates. 

•	 Understanding mechanistic principles of the entire accessible range of 

glycoside hydrolases and their function in the biosphere will be evaluated. The diversity of cellulase and cellulosome functional schemes will 

be modeled and optimized for specific biomass substrates (feedstocks).

This improved understanding of cellulase action will provide new saccharification paradigms for the biorefinery. 

Crosscuttng Tools, Technologes, and Scence 

Computational science, advanced imaging, and HTP genomic tools must 

be incorporated to attain the objectives described here. 

cellulose is mediated by scaffoldin-borne cellulose-binding molecules (CBM). Some scaffoldins also bear a 

divergent type of dockerin that interacts with a matching cohesin on an anchoring protein, thereby mediating 

cellulosome attachment to the cell surface. 

The LEGO-like arrangement of cellulosomal modules offers an excellent opportunity to engineer new multienzyme complexes for desired purposes. The various cohesins (c), dockerins (d), and catalytic modules (A, B,

C) are functionally independent and can be tethered together in any combination via recombinant genetics.

The resulting complex can be applied either in the test tube (bioreactors) or in a cellular setting (fermentors). 

Native scaffoldin-borne cohesins in cellulosome-producing bacteria generally recognize all enzyme-bearing 

dockerins nonspecifically. Thus, reconstruction of a cellulosome from its component parts would result in 

random incorporation of different enzymes into cellulosome complexes, yielding a heterogeneous population 

of artificial cellulosomes (Fig. B1. Random Incorporation, below). 

To control the incorporation of desired enzymes into a precise position, distinctive cohesin-dockerin pairs 

must be used (Fig. B2. Controlled Incorporation, below.). Dockerin domains that bind a specific cohesin 

must be fused to the different 

catalytic modules, and a chimeric scaffoldin is engineered 

to contain complementary 

cohesins and a single CBM for 

substrate targeting. Subsequent 

self-assembly of the mature 

“designer” cellulosome complex 

can then occur selectively in 

vitro, resulting in a homogeneous population of cellulosome complexes. 

Cellulosome References: p. 117 

Fig. B1. Random Incorporation. Fig. B2. Controlled Incorporation. 

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DECONSTRUCTING FEEDSTOCKS 

•	 Computational science is needed for understanding cellulase function at 

a holistic level, specifically, new programs and codes to handle models of 

ultralarge biological molecules and systems of more than 1 million atoms. 

•	 New imaging methods are critical for elucidating cellulase action on the 

plant cell wall. 

•	 Finally, tools to acquire, archive, and interpret new cellulase structures 

(both native and modified) are needed to better understand their natural 

structural and functional diversity. 

We must develop new science and technical methodologies to explore 

protein-machine function at the molecular and atomic levels to attain these 

objectives. Solution physics and thermodynamics ruling protein-domain biological function at the nanometer scale are poorly understood. In this respect,

the new field of nanoscience may provide important insights. For example,

describing the mechanism of a processive cellulase at such level of detail will 

require more knowledge about the cellulose surface, water-protein-glycan 

dynamics, and biomolecular mechanics than is possible today. 

The Role of GTL and OBP Facltes and Capabltes 

Proten Producton 

Protein production capabilities will be critical for supplying samples of 

wild-type and recombinant proteins, including (1) new enzymes from 

new sources, (2) modified enzymes from directed evolution, (3) modified 

enzymes from site-directed mutation, and (4) large quantities of enzymes 

suitable for pilot testing. This resource will be critical in identifying the 

most effective new hemicellulases and ligninases without first having to 

solve high-level expression challenges for all candidates. 

High-throughput (HTP) biochemical and biophysical assays will be developed and carried out, and functional annotation will enrich the first-pass 

sequence-based annotations. This problem is more difficult and complex 

for GH, in which a single enzyme can have several comparable activities 

on different polysaccharide substrates. Thus, a microchip HTP assay facility is needed. 

Molecular Machne 

These capabilities can provide analytical and computational science to 

study cellulase protein-machine function and advanced techniques for isolating and reconstituting complex interactions within native or improved 

hemicellulase and ligninase systems. Synergistic interactors including 

chaperonins, excretion paths, and cofactors will be isolated and identified 

in novel cellulases, hemicellulases, and ligninases. 

Proteomcs 

Proteomic capabilities can be used to document how secretomes of 

selected white rot fungi and other organisms such as microorganisms in 

the termite hindgut respond to changing culture conditions and optimize 

biodegradation rates of lignocellulosic components. Correlations between 

0 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

ligninase activity and concentrations of enzymes responsible for cosubstrate production will be of particular interest and will assist researchers in 

designing more effective experimental plans for improving the energetics 

and carbon-allocation efficiency of cell-wall degradation. This also will be 

important in monitoring and controlling the effects of substrate inducers 

and responses to heterologous expression, especially of the most “foreign”

components. This resource can support studies aimed at improving production of ligninase and hemicellulase proteins from both near-term hosts 

such as T. reesei and longer-term systems including bacteria and yeast. 

Cellular System 

Cellular system capabilities will visualize cellulases, ligninases, cellulosomes,

and other molecular species directly on substrates in real time under varying conditions and from varying species. Models will be developed to 

optimize hydrolysis under different conditions. 

DOE Jont Genome Insttute 

DOE JGI could embark on sequencing new white rot fungal genomes,

including those that do not encode lignin and manganese-dependent 

peroxidases. This work may lead to the discovery of more-active and lesslabile ligninase variants. After that, more biological diversity from such 

bacterial populations as decaying biomass, termite hindgut, ruminants,

and the rhizosphere could be examined. 

Metagenomcs 

Metagenomic approaches will identify mechanisms and agents of effective 

hemicellulose and lignin conversion in communal or unculturable populations. They also can play a key role in sequencing new genomes thought 

to harbor cellulolytic components acquired by lateral gene transfer and in 

studying natural adaptations that allow (initially) heterologous expression. 

Harvestng the Bochemcal Potental 

of Mcroorgansms Through Metagenomcs 

The conceptual foundation for metagenomics is the realization that more 

than 99% of microbes in most habitats have not yet been cultured. By 

providing deep insight into biological capabilities and function without the 

need for culturing, metagenomics effectively expands our scientific capabilities and understanding beyond the small percentage of bacteria that currently can be cultivated in the laboratory. It further permits a more holistic 

and mechanistic analysis of microbial communities. Natural systems long 

ago learned how to attack lignocellulose and use the resultant sugars and 

other chemicals for their own purposes. The metagenomes of complex 

natural communities provide a fertile resource for data mining to search for 

new examples of relevant enzymes. The ability to construct and evaluate an 

engineered metagenome (from existing genome sequences) now enables 

creation of systems with superior capabilities. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 05 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DECONSTRUCTING FEEDSTOCKS 

This research has two objectives: (1) employ genetic and biochemical 

studies to characterize novel lignocellulose-degrading systems and (2) 

use metagenomics to augment these efforts by characterizing and recovering the genetic potential (including that from uncultured microbes) 

resident in microbial communities capable of rapid and extensive biomass 

degradation. Outcomes of these functional and comparative studies will 

include a repertoire of new enzymes and proteins available for engineering 

approaches (e.g., designer cellulosomes or free cellulase systems). In that 

context, knowledge and bioresources arising from this research are well 

integrated within the overall goal of improving plant cell-wall deconstruction and conversion of lignocellulosic products to bioethanol. 

Research Goals 

To elucidate the repertoire of gene products necessary to effect more rapid 

and extensive hydrolysis (and solubilization) of lignocellulosic materials,

systematic advances in our understanding of microbe-mediated plant cellwall deconstruction must be achieved. First, many sequenced microbial 

genomes encode more glycoside hydrolases for plant cell-wall deconstruction than have been identified through historical biochemical and genetic 

studies. Whether or not these multigene families are an evolutionary 

adaptation to provide more “total” activity diversity, to compensate for 

subtleties in substrate conformation and composition, or a combination 

of both is still unknown. In addition, degradative synergism in microbial 

communities often resides in the concerted actions of enzymes from multiple organisms, but mechanistic details and understanding of this concept 

are undeveloped. 

Second, aerobic and anaerobic bacteria with superior degradation capabilities recently have been found to lack canonical processive cellulases 

required in all other well-described systems for substrate solubilization.

The genetics and biochemistry underpinning their lignocellulose degradation, which could represent a new mechanism, must be explored. 

Techncal Mlestones 

GTL will accelerate the development of optimal cellulase systems by providing resources for screening thousands of natural and modified enzyme 

variants, enabling the HTP production and functional analysis of these 

enzymes, elucidating regulatory controls and essential molecular interactions, and generating models for analyzing the structure and activity of 

natural and engineered enzyme systems. 

Wthn 5 years 

•	 Undertake microbiome projects for functional and comparative purposes. The JGI Community Sequencing Program, supported by DOE, is 

now characterizing the microbiome of the termite hindgut. 

•	 Initiate mechanistic characterization (at DNA, protein, and organismal 

levels) of no fewer than three natural microbial communities displaying 

rapid and extensive lignocellulose degradation. Each should be different 

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in terms of prevailing physicochemical conditions (e.g., temperature, pH,

and salt concentration) or dominant feedstock. For example: 

–	 Soils representative of dominant residues in the DOE-USDA Billion-Ton study. Corn stover, rice, switchgrass, and forest residues 


would be appropriate.
 

–	 Decaying biomass (see the sidebar, Examples of Metagenomic 


Analysis: Understanding the Dynamics of Microbial Colonization of 


Decaying Biomass, p. 108).
 

•	 Determine the mechanisms of lignocellulose solubilization by such 

known organisms as F. succinogenes and C. hutchinsonii to help guide

metagenomic analyses, using a combination of biochemical and 

genomic approaches (see sidebar, Sequencing a Soil-Cellulose Degrader,

this page). 

•	 Develop heterologous expression systems for candidate enzymes and 

regulatory proteins, including necessary cofactors and post-translational 

modifications. 

•	 Begin HTP production, characterization, and intercomparison of enzymatic systems discovered in metagenomic analyses. Produce appropriate 

molecular tags to support experimentation in natural and controlled 

systems—including imaging, protein isolation, and detection of interactions. 

Wthn 0 years 

•	 Complete comprehensive analyses of enzymatic systems to isolate different families and high performers. 

•	 Rationally modify, express, and characterize 

native enzymes to understand design principles 

T

and optimize properties. Sequencng a Sol-Cellulose Degrader
 

he DOE Joint Genome Institute is sequenc­ •	 Build computational models of biomass-decay 

microbial communities, and test against experi- ing Cytophaga hutchinsonii, an aerobic Grammental data. negative bacterium commonly found in soil 

that rapidly digests crystalline cellulose. Molecular 

Wthn 5 years analysis of cellulose degradation by C. hutchinsonii is 

•	 Deploy innovative processes based on these dis- now feasible, since techniques for genetically manipucoveries and bioresources to reduce enzyme costs lating the organism recently have been developed.

and loading by tenfold. Provide new options that This microbe exhibits the ability to move rapidly over 

reduce or eliminate the need for nonbiological surfaces by a process known as gliding motility, which 

feedstock pretreatments. is thought to be important in allowing C. hutchinsonii 

to colonize its insoluble growth substrate. The mechaThe Role of GTL and OBP Facltes and 	 nism of gliding motility is not known, but flagella are 

not involved. Analysis of the C. hutchinsonii genome Capabltes sequence will facilitate studies of cellulose degrada- In brief, the facilities will support and enable tion and also will reveal more about bacterial gliding the attainment of these objectives. For instance, motility, which has remained an unsolved biological JGI already has contributed a foundational set mystery for over 100 years (http://genome.jgi-psf. of genome sequences for specialist cellulolytic org/finished_microbes/cythu/cythu.home.html). microbes; this data set is augmented by genomes 

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DECONSTRUCTING FEEDSTOCKS 

Example of Metagenomc Analyss: 

Understandng the Dynamcs of Mcrobal 

Colonzaton of Decayng Bomass 

As primary decomposers, microbial communities 

have evolved both as competitors and collaborators in biomass deconstruction. The ultimate aim 

of this research is to achieve bioprocesses for all steps in 

converting biomass to ethanol. A critical need is to replace 

plant biomass thermochemical pretreatment, which is now 

necessary to convert recalcitrant structural lignoellulose to 

a form in which cellulose is more accessible and amenable 

to hydrolytic enzyme action. By analyzing natural communities that colonize decaying biomass, we can ascertain 

natural mechanisms that can be used to supplant thermochemical treatments. The goal is to better understand the 

complex microbial communities responsible for lignocellulosic biomass deconstruction, harvest key biochemical 

decay mechanisms, and develop predictive modeling and 

control of these complex natural processes. 

Metagenomics will be used to determine microbial-community composition and genetic diversity. Comparative 

genomic tools will initially determine community functionality and identify genes of unknown function. Coupled 

spatial and temporal measurements will reveal principles 

of community formation and dynamics, processes such 

as signaling, and other microbial interactions. Functional 

annotation of unknown proteins through protein production and characterization and other experimentation will 

augment initial gene-function assignment. 

HTP methods from facility and other capabilities will be 

used for measuring enzyme activities: 

•	 Determine cellular and biochemical functions of genes 

discovered in uncultured community members. 

•	 Characterize the temporal composition and functional 

capability of microbial communities—Who is there,

and what metabolic processes are being carried out? 

•	 Characterize expression patterns of cellulolytic enzymes 

and related processes and pathways using transcriptomics, proteomics, and metabolomics. 

Computational tools will predict the metabolic, physiologic, and behavioral characteristics of microbial communities from community DNA sequence data, and 

supporting measurements will be developed. The tools 

will allow design and engineering of microbial systems 

that ultimately can perform all steps in biomass processing to ethanol. 

sequenced at The Institute for Genomic Research 

(called TIGR). Other resources can readily support hypothesis-driven research at the RNA,

protein, organismal, and community levels (see 

sidebar, Example of Metagenomic Analysis, this 

page). This research will validate and produce the 

desired combination of proteins and other biologics necessary to make lignocellulose conversion 

comparable to starch as a feedstock in economic 

and process criteria. 

Characterzng Cell Walls Usng 

Hgh-Throughput Methods 

Understanding the chemical nature and architecture of cell walls at the nano-, micro-, macrochemical, and physical scales, as well as their 

behavior in pretreatment and fermentation, is 

essential in taking a systems-level approach to 

modifying the plant genome or increasing the 

biofuel system’s efficiency. Knowledge and insight 

gained from HTP cell-wall characterization 

will drive several areas of biomass-to-biofuels 

research. These areas include selecting and modifying plants, matching the microbial conversion 

process to the substrates present, and minimizing inhibitors during biological conversion to 

valuable products. Many new instruments and 

analytical methods will be needed to characterize 

a range of biomass feedstocks (e.g., grasses, crop 

residues, short-rotation woods, and early energy 

crops) needed to achieve the Billion-Ton vision.

Ultimately, many different types of feedstocks 

will be at different stages of harvest and hence 

will need to be analyzed rapidly. The robustness 

of these methods and their versatility in addressing the composition of many different materials requires significant further research. Data 

generated by these methods are used to value 

feedstocks, measure conversion efficiency, identify 

regulatory issues, and ultimately establish product 

pricing and investment risk. 

Research must address the disconnect between 

the throughput of genomic and proteomic analysis and that of biomass chemical and structural 

characterization. DNA sequencing and metabolic 

profiling are routinely done at the microliter scale,

where 1000 to 2000 samples can be processed and 

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analyzed in one day. Most biomass analysis requires hundreds of milligrams 

per sample and very few have been automated, so throughput is around 20 

samples per week per person. Recent advancements in biomass analysis have 

demonstrated high throughput using multivariate analysis (MVA) coupled 

with analytical pyrolysis, FTIR, or near infrared spectroscopy to allow characterization of 200 to 500 samples per day per person. These spectroscopic 

methods, however, require calibration and validation using slow wet chemical procedures. MVA often is specific to feedstock and process; for example,

a method developed for hardwood analysis won’t accurately characterize a 

softwood or grass sample. To support the broad goals of this roadmap, many 

new analytical methods will need to be developed. 

Research will focus on two areas: Improving throughput of traditional 

calibration methods to around 500 samples per week and using thse 

data to calibrate “next-generation” capabilities of analyzing 1000 or more 

samples per day. Strategies for improving traditional methods will require 

the adaptation of analytical instruments for biomass applications and 

development of new instruments specifically for this work. For example,

multiplexing robotic sample preparation and multichannel capillary electrophoresis in carbohydrate analysis potentially could offer a tenfold increase 

in sample throughput, but such instruments and methods are not validated 

for biomass samples. New methods and instruments will generate data on 

molecular- and genetic-level relationships needed to (1) improve feedstocks 

by increasing biomass production and making feedstocks more amenable 

to the next generation of biomass-conversion technologies, and (2) develop 

data sets that enable the design of microbial cell conversion strategies that 

will produce desired products in high yield and purity. A special category 

of analytical methods with high sensitivity also will be needed to interpret 

high-resolution images of feedstocks and process substrates. 

The complexity of the biomass matrix presents significant analytical challenges not faced in conventional genomics work. Chemical characterization methods will have to assess the relative weight percent of about 14 

constituents in each feedstock. Structural constituents of potential interest 

are protein, lignin, ash, glucan (cellulose), xylan, mannan, arabinan, galactan, glucuronic acid, ferulic acid, acetyl groups, starch, and galacturonic 

acids (pectin). Some nonstructural materials, which will be specific to each 

feedstock, also may be of interest in saccharification and fermentation studies. These constituents include inorganic salts, sucrose, tars, waxes, gums,

lignans, and others. Analytical methods also will be needed to track the 

constituents and their reaction products through pretreatment, saccharification, and fermentation. Structural characterization methods must quantify 

functional groups of interest (e.g., carboxylic acids, ketones, aldehydes,

esters, and methoxyl and free-phenolic hydroxyl groups). With the requirement for so many measurements, a portfolio of complementary methods 

will be needed to close mass balance of feedstock and process components 

across multiple processing steps. 

This research program addresses a critical component in the feedstocksugars interface by providing analytical instruments and methods with the 

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DECONSTRUCTING FEEDSTOCKS 

precision, accuracy, and throughput required to optimize biomass selection 

and development, biomass pretreatment, and conversion processes leading to economical ethanol production in biorefineries. Biomass-relevant 

analytical tools of this type do not now exist for these applications. 

Tracking individual biomass components from feedstock to products 

requires high-accuracy data obtained in an integrated and consistent 

fashion. A research program focused on analytical methods that coordinate 

data through all stages will improve process integration through consistent 

data on feedstocks, substrates, process inhibitors, and products. Integrated 

data will furnish a systems-level understanding of process streams and 

enable the desired correlations between reactivity and genome expression. 

To enable future development of high-energy biomass feedstocks, method 

portfolios need to be standardized; validated in a consensus environment;

and published in a forum available to plant breeders, field scientists, process engineers, enzyme scientists, and fermentation scientists. Technologies 

will provide data for identifying relationships among plant cell-wall components and interactions among biomass-derived substrates, microbes,

and enzymes. Although progress has been made in chemical procedures 

for biomass analyses, existing portfolios of methods are labor intensive,

time consuming, expensive, and generally not amenable to the HTP needs 

of breeders, agronomists, feedstock processors, and systems approaches to 

fermentation. To bypass this limitation, HTP techniques need to measure 

the amounts and structure of cell-wall components in processes. Correlations among process data and genomic and proteomic data will enable 

identification of genes most important for improved biomass conversion. 

For example, more than 225,000 independent T-DNA insertion lines of 

Arabidopsis have been created that represent almost the entire genome 

space. HTP analysis must be able to screen these large sample sets, as 

well as the much larger number of samples produced for other species 

with more complicated genomes, to determine the role each gene plays in 

different cell-wall chemistry phenotypes. HTP methodologies also will 

enable screening of plant, enzyme, and microbe consortia to allow for 

deeper understanding of different plant and microbe genome interactions.

High-sensitivity methods will integrate with imaging tools to enable 

spatial compositional and functional group determinations in individual 

plant cells. These studies require the analysis of hundreds if not thousands 

of samples and would not be possible without the cost and time saving 

provided by rapid biomass-analysis methods. 

Techncal Mlestones 

Wthn 5 years 

For near-term deployment, portfolios of precise and accurate analytical 

methods for a wide range of structural and nonstructural constituents and 

biomass function groups will be developed and validated in a consensus 

mode and published for general use. Specific techniques and methods will 

be tailored to provide comprehensive characterization of biomass model 

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substrates (feedstocks). Working closely with commercial suppliers, investigators will create instruments and techniques specific to the needs of 

biomass-conversion genomics. Data obtained through customized analytical 

procedures will be used to calibrate and validate rapid MVA methodologies 

for HTP screening of feedstocks and biomass-derived research samples.

This screening will help to select samples of interest from large populations 

representing species whose genome sequence has been determined. Selection will be based on identifiable differences in phenotypes of major plant 

cell-wall biopolymers (lignin, cellulose, hemicellulose, and others) important 

for conversion to fuels and valuable chemicals. Data generated will be used 

to support the breeding and development of new plant lines. In conjunction 

with HTP methods, information-technology strategies will capture, analyze, manage, and disseminate resulting data, enabling creation of a national 

resource for the biofuel and plant biology research community.To the extent 

possible, samples and data used in calibrating new methods will be archived 

safely for future applications. Archived samples may be required for benchmarking, new method development, and crossplatform comparisons. 

Wthn 0 years 

HTP methodologies will be integrated with other studies to provide a 

systems-level understanding of how to alter and improve plant cell-wall 

composition and structure for efficient and economic biofuel production.

High-resolution, molecular-specific images enabled by the availability of 

molecular tags will allow spatial determinations of chemical and structural 

features across individual cell types and structures. 

Wthn 5 years 

Deployment of HTP and high-sensitivity analytical methods and appropriate data-reduction (informatic) techniques will be completed to integrate genomic and proteomic data with cell-wall chemistry information.

A systems-level understanding of interactions between plant cell-wall 

structure and the microbes and enzymes is needed to convert biomass 

effectively to fuels and chemicals. 

The Role of GTL and OBP Facltes and Capabltes 

Many underlying principles and HTP techniques (e.g., robotics, informatics,

and pattern recognition) for HTP chemical analysis of plants have been or 

are being developed by the genomic and metabolomic communities. Collaborations with researchers using the resource will require the adaptation 

and validation of these techniques for biomass-conversion applications. We 

expect technologies developed for plants (e.g., high-sensitivity spatial detection of biopolymers) to have applications for studying other organisms. 

Information obtained from these analytical methodologies will facilitate the 

determination of plant-gene function in synthesis of plant cell walls and 

biopolymers, especially genes related to carbohydrate metabolism, polymerization, and modification. Analytical methods tailored to biomass applications 

will be used to identify protein function related to cell-wall construction. As 

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DECONSTRUCTING FEEDSTOCKS 

additional genomes are sequenced, these tools can be used to characterize gene 

function in other plant lines of interest for biofuel production. 

Crosscuttng Tools, Technologes, and Scence 

Many analytical methods that support a systems approach to biomass 

conversion will require biomass-relevant standards and tags not readily 

available from commercial sources. Obtaining the needed array of smallmolecule standards will require prep-scale isolation of molecules of interest and advanced techniques of carbohydrate and natural product organic 

synthesis. As we learn more about the plant genome, important intermediate molecules and biomarkers will be revealed. Techniques for isolating and 

preparing relevant biomass standards will be transferred to the private sector and made available for biomass research and commercial biorefineries. 

While much existing robotic instrumentation and data-reduction technologies are available for constructing HTP methodologies, several areas must be 

addressed to achieve project goals. New techniques for homogenizing and 

reducing the particle size of biomass samples without degradation will be 

needed to prepare biomass samples for automated and robotic systems. New 

modules, equipment, and accessories for existing analytical techniques such 

as columns for liquid chromatography will have to be adapted specifically 

for biomass samples. Rapid biomass-analysis methods with large dynamic 

ranges will detect multiple compounds with high precision, acceptable accuracy, and short acquisition times. Because these methods are often specific to 

feedstocks and processes, many new ones may be required. Integrated data 

from different analytical methods into a single comprehensive MVA calibration set will need testing and validation. 

Data-reduction and storage methods with predictive platforms for production and processing pipelines are necessary for retrieving and integrating 

data from breeding or field studies. New “hands-off ” bioinformatic methods will combine, analyze, and correlate data (including metabolomic and 

genomic information) from multiple systems. Multivariate analysis tools will 

be applied where pertinent to information extraction from complex systems. 

Appropriate biomass analysis is a necessary first step in applying systems 

biology techniques to bioenergy production. In many cases, analytical 

methods for biomass substrates that demonstrate the required precision,

accuracy, and speed are not available. Analysis using existing methods is too 

slow and expensive for large-scale screening outlined in many basic technology research programs. Before yields of a desired product can be maximized 

or undesirable side reactions minimized, analytical methods must accurately monitor changes in the chemical constituents. These methods also 

must be validated in the complex matrix of biomass-conversion streams. In 

many cases, biomass methods direct the course of research by revealing the 

presence of important products or the nature of yield-reducing side reactions. Biomass-conversion methods will lead us one step closer to those for 

accurately monitoring complex degradation reactions in natural systems. 

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Breakthrough, Hgh-Payoff Opportunty: Smplfyng 

the Boconverson Process by Understandng Cell-Wall 

Deconstructon Enzymes Expressed n Plants 

Scentfic Challenges and Opportuntes 

Complexity is one of the major challenges to economical biomass conversion for large-scale production of liquid transportation biofuels. The 

process currently requires many steps, including pretreatment, detoxification, solid and liquid separation, cellulase production, cellulose hydrolysis,

biomass sugar fermentation, and product recovery supported by such 

ancillary systems as wastewater treatment and utilities. This complexity 

results in added operating and capital costs as well as issues of robustness,

and simpler procedures would be highly desirable. Although engineering potentially could improve the process incrementally, systems biology approaches have the potential to revolutionize it. Various strategies 

envisioned to consolidate processing steps will genetically incorporate 

capabilities within organisms. One potential strategy would include 

incorporating genes that encode lignocellulose-degradative enzymes into 

feedstock plants. 

•	 Enzymes could target wall polysaccharides (backbones, side chains,

and small substituents that can have big effects on activity of other 

enzymes), simple phenolic cross-links between polysaccharides such as 

those that tie together xylans in cereal walls. 

•	 Promoters would be required that are activated from senescence-related 

or harvest insult–triggered genes or by a signal provided exogenously at 

a time deemed appropriate. 

•	 Genes encoding enzymes or other disassembly proteins would be 

required, perhaps those of microbial origin whose discovery would be 

a major “engineering” goal of the larger project and whose expression 

would be driven by the promoters above. 

Research Goals 

While targeting specific objectives using the existing state of technology as 

a benchmark is important, research must be open to new ideas and concepts. Specific areas that need to be addressed in fundamental and applied 

areas include the following. 

Fundamental Scence 

•	 What new learning would enable expression of structural polysaccharide-degrading enzymes in plant tissues? We need new promoters,

genes, transformation tools, and knowledge about the sequestration and 

action of such enzymes when expressed in living plants. 

•	 What is the relative effectiveness of cellulase-enzyme systems acting in 

cellulose-enzyme-microbe complexes as compared to cellulose-enzyme complexes, and what is the mechanistic basis for such enzyme-microbe synergy? 

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DECONSTRUCTING FEEDSTOCKS 

Appled Scence 

•	 Can hemicellulose and cellulose hydrolysis be accomplished without the 

acidic or alkaline high-temperature and -pressure pretreatment step? 

This is particularly important for advantageous expression of hemicellulases and cellulase enzymes in plants. 

•	 Can biocatalysts be developed whose performance is not impacted 

significantly by inhibitory compounds formed as by-products of pretreatment, hydrolysis, or fermentation? If so, they have the potential to 

eliminate the costly and waste-intensive detoxification step. 

•	 Can such upstream processes as storage (ensilage) or biopretreatment 

be developed that make cellulose microfibrils more amenable to enzymatic cellulose hydrolysis without the need for relatively harsh thermochemical pretreatment? 

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References 

Aden, A., et al. 2002. Lignocellulosic Biomass to Ethanol Process Design and 

Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic 

Hydrolysis for Corn Stover, NREL Report No. TP-510-32438. 

Agblevor, F., H. L. Chum, and D. K. Johnson. 1993. “Compositional Analysis of NIST Biomass Standards from the IEA Whole Feedstock Round 

Robin,” pp. 395–421 in Energy from Biomass and Wastes XVI: Proceedings of

the 7th Institute of Gas Technology Clean Fuels and Energy from Biomass and 

Wastes Conference, ed. D. L. Klass, Institute of Gas Technology, Chicago. 

Casler, M. D., and H.-J. Jung. 1999. “Selection and Evaluation of Smooth 

Bromegrass Clones with Divergent Lignin or Etherified Ferulic Acid 

Concentration,” Crop Sci. 39, 1866–73. 

Chum, H. L., et al. 1994. “Status of the IEA Voluntary Standards Activity–Round Robins on Whole Wood and Lignins,” pp. 1701–16 in Advances 

in Thermochemical Biomass Conversion: Papers from the International Conference on Advances in Thermochemical Biomass Conversion Vol. 2, ed. A. V. 

Bridgwater, Blackie Academic & Professional, Glasgow, U. K. 

de la Rosa, L. B., et al. 1994. “Integrated Production of Ethanol Fuel and 

Protein from Coastal Bermudagrass,” Appl. Biochem. Biotechnol. 45–46,

483–97. 

Durot, N., F. Gaudard, and B. Kurek. 2003. “The Unmasking of Lignin 

Structures in Wheat Straw by Alkali,” Phytochem. 63, 617–23. 

Gollapalli, L. E., B. E. Dale, and D. M. Rivers. 2002. “Predicting Digestibility of Ammonia Fiber Explosion (AFEX)- Treated Rice Straw,” Appl. 

Biochem. Biotechnol. 98–100, 23–35. 

Greene, N., et al. 2004. Growing Energy: How Biofuels Can Help End 

America’s Oil Dependence, Natural Resources Defense Council, New York 

(www.nrdc.org/air/energy/biofuels/biofuels.pdf ). 

Hames, B. R., et al. 2003. “Rapid Biomass Analysis: New Tools for Compositional Analysis of Corn Stover Feedstocks and Process Intermediates 

from Ethanol Production,” Proceedings of the Twenty-Fourth Symposium on 

Biotechnology for Fuels and Chemicals, 28 April–1 May, 2002, Gatlinburg,

Tennessee, ed. B. H. Davis et al., Appl. Biochem. Biotechnol. 105-108, 5–16. 

Himmel, M. E., M. F. Ruth, and C. E. Wyman. 1999. “Cellulase for 

Commodity Products from Cellulosic Biomass,” Curr. Opin. Biotechnol. 

10(4), 358–64. 

Kelley, S. S., et al. 2004. “Rapid Analysis of the Chemical Composition 

of Agricultural Fibers Using Near Infrared Spectroscopy and Pyrolysis 

Molecular Beam Mass Spectrometry,” Biomass Bioenerg. 27(1), 77–88. 

Kim, Y., et al. 2005. “Plug Flow Reactor for Continuous Hydrolysis of 

Glucans and Xylans from Pretreated Corn Fiber,” Energy Fuels 19(5), 

2189–2200. 

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DECONSTRUCTING FEEDSTOCKS 

Lam, T.-B. T., et al. 1993. “The Relationship Between in Vitro Enzymatic 

Digestibility of Cell Walls of Wheat Internodes and Compositional 

Changes During Maturation,” Acta. Bot. Neerl. 42, 175–85. 

Lapierre, C., D. Jouin, and B. Monties. 1989. “On the Molecular Origins 

of the Alkali Solubility of Graminae Lignins,” Phytochem. 28, 1401–3. 

Laureano-Perez, L., et al. 2005.“Understanding Factors that Limit Enzymatic 

Hydrolysis of Biomass,” Appl. Biochem. Biotechnol. 121, 1081–1100. 

Lawoko, M., G. Henriksson, and G. Gellerstedt. 2005. “Structural Differences Between the Lignin-Carbohydrate Complexes Present in Wood 

and in Chemical Pulps,” Biomacromolecules 6, 3467–73. 

Lynd, L. R. 1996. “Overview and Evaluation of Fuel Ethanol from Cellulosic BIomass: Technology, Economics, the Environment, and Policy,”

Annu. Rev. Energy Environ. 21, 403–65. 

Lynd, L. R., R. T. Elander, and C. E. Wyman. 1996. “Likely Features and 

Costs of Mature Biomass Ethanol Technology,” Appl. Biochem. Biotechnol. 

57–58, 741–61. 

Moniruzzaman, M., et al. 1997. “Fermentation of Corn Fibre Sugars by an 

Engineered Xylose-Utilizing Saccharomyces Yeast Strain,” World J. Microbiol. 

Biotechnol. 13, 341–46. 

Mosier, N., C. M. Ladisch, and M. R. Ladisch. 2002. “Characterization of 

Acid Catalytic Domains for Cellulose Hydrolysis and Glucose Degradation,” Biotechnol. Bioeng. 79(6), 610–18. 

Mosier, N., et al. 2005. “Features of Promising Technologies for Pretreatment of Lignocellulosic Biomass,” Bioresour. Technol. 96(6), 673–86. 

Sarikaya, A., and M. R. Ladisch. 1997. “An Unstructured Mathematical 

Model for Growth of Pleurotus ostreatus on Lignocellulosic Material in 

Solid-State Fermentation Systems,” Appl. Biochem. Biotechnol. 62, 71–85. 

Scobbie, L., et al. 1993. “The Newly Extended Maize Internode: A Model 

for the Study of Secondary Cell Wall Formation and Consequences for 

Digestibility,” J. Sci. Fd. Agric. 61, 217–25. 

Sedlak, M., and N. W. Y. Ho. 2004. “Production of Ethanol from Cellulosic Biomass Hydrolysates Using Genetically Engineered Saccharomyces Yeast Capable of Cofermenting Glucose and Xylose,” Appl. Biochem. 

Biotechnol. 113–16, 403–5. 

Singh, R., et al. 2005. “Lignin-Carbohydrate Complexes from Sugarcane 

Bagasse: Preparation, Purification, and Characterization,” Carbohydr. 

Polym. 62, 57–66. 

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Wilson, J. R., and Hatfield, R. D. 1997. “Structural and Chemical Changes 

of Cell Wall Types During Stem Development: Consequences for Fibre 

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Cellulosome References 

Cellulosome Revews 

Bayer, E. A., et al. 2004. “The Cellulosomes: Multi-Enzyme Machines for 

Degradation of Plant Cell Wall Polysaccharides,”Annu. Rev. Microbiol. 58,

521–54. 

Bayer, E. A., et al. 1998. “Cellulose, Cellulases, and Cellulosomes,” Curr. 

Opin. Struct. Biol. 8, 548–57. 

Demain, A. L., M. Newcomb, and J. H. Wu. 2005. “Cellulase, Clostridia,

and Ethanol,” Microbiol. Mol. Biol. Rev. 69, 124–54. 

Doi, R. H., and A. Kosugi. 2004. “Cellulosomes: Plant-Cell-Wall-Degrading Enzyme Complexes.” Nat. Rev. Microbiol. 2, 541–51. 

Shoham, Y., R. Lamed, and E. A. Bayer. 1999. “The Cellulosome Concept 

as an Efficient Microbial Strategy for the Degradation of Insoluble Polysaccharides,” Trends Microbiol. 7, 275–81. 

Desgner Cellulosomes 

Bayer, E. A., E. Morag, and R. Lamed. 1994. “The Cellulosome—A Treasure Trove for Biotechnology,” Trends Biotechnol. 12, 378–86. 

Fierobe, H.-P., et al. 2005. “Action of Designer Cellulosomes on Homogeneous Versus Complex Substrates: Controlled Incorporation of Three 

Distinct Enzymes into a Defined Tri-Functional Scaffoldin,” J. Biol. Chem. 

280, 16325–34. 

Fierobe, H.-P., et al. 2002. “Degradation of Cellulose Substrates by Cellulosome Chimeras: Substrate Targeting Versus Proximity of Enzyme 

Components,” J. Biol. Chem. 277, 49621–30. 

Fierobe, H.-P., et al. 2002. “Designer Nanosomes: Selective Engineering 

of Dockerin-Containing Enzymes into Chimeric Scaffoldins to Form 

Defined Nanoreactors,” pp. 113–23 in Carbohydrate Bioengineering: Interdisciplinary Approaches, ed. T. T. Teeri et al., The Royal Society of Chemistry, Cambridge. 

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DECONSTRUCTING FEEDSTOCKS 

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Sugar Fermentaton to Ethanol
 

Fermentation of sugars by microbes is the most common 

method for converting sugars inherent within biomass 

feedstocks into liquid fuels such as ethanol. Bioconversion 

or biocatalysis is the use of microbes or enzymes to transform one 

material into another. The process is well established for some 

sugars, such as glucose from cornstarch, now a mature industry.

Production of fuel ethanol from the mixture of sugars present in 

lignocellulosic biomass, however, remains challenging with many 

opportunities for improvement. More robust microorganisms are 

needed with higher rates of conversion and yield to allow process 

simplification through consolidating process steps. This development would reduce both capital and operating costs, which remain 

high by comparison with those of corn. 

The growing U.S. industry that produces fuel ethanol from cornstarch has opportunities for incremental improvement and expansion. Processes for the bioconversion of lignocellulosic biomass 

must be developed to match the success in starch conversion (see 

sidebar, Starch: A Recent History of Bioconversion Success, this 

page). Technologies for converting cellulosic biomass into fuel 

ethanol already have been demonstrated at small scale and can 

be deployed immediately in pilot and demonstration plants. The 

challenge, with limiting factors of process complexity, nature of 

the feedstock, and limitations of current biocatalysts, remains the 

higher cost (see Fig. 1. The Goal of Biomass Conversion, p. 120).

The discussion in this chapter will focus on process improvements 

that will reduce risk, capital investment, and operating costs. This 

emphasis is driven by the goal to integrate and mutually enhance 

the programs in DOE’s Office of the Biomass Program (OBP) 

and Office of Biological and Environmental Research (OBER) 

related to achieving the president’s goal of a viable cellulosic 

ethanol industry. 

Bioconversion must build on its historic potential strengths of high yield 

and specificity while carrying out multistep reactions at scales comparable to 

those of chemical conversions. Biology can be manipulated to produce many 

possible stoichiometric and thermodynamically favorable products (see Fig. 2.

Examples of Possible Pathways to Convert Biomass to Biofuels, p. 121),

but bioconversion must overcome the limitations of dilute products, slow 

reactions, and often-limited reaction conditions. For commodity products 

such as fuels, biologically mediated conversion represents a large fraction 

Starch: A Recent Hstory of 

Boconverson Success 

Biotechnology has a track record 

of displacing thermochemical 

processing in the biomass starch 

industry. In the 1960s, virtually all starch 

(a sugar polymer in granules) was processed by acid and high temperatures.

Inhibitory by-products and lower conversion rates resulted in a soluble starch 

solution that was lower in quality and 

yield when further fermented to ethanol.

Development of specific thermostable 

high-productivity enzymes (e.g., alphaamylase and glucoamylase) produced a 

higher-quality soluble starch, completely 

displacing the acid process by 1980. This 

new process has allowed technologies for 

producing ethanol from starch to continuously improve to the high yield and 

rate levels seen today in wet and dry corn 

mills. Other starch-conversion enzymes 

(e.g., glucose isomerase) have made 

possible another commodity product,

high-fructose corn syrup, which is used 

in virtually all domestic sweetened beverages and many other products (www.

genencor.com/wt/gcor/grain). 

References: p. 154 

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SUGAR FERMENTATION 

of costs and selling prices (unlike the pharmaceutical industry, where bioconversion costs are small) 

(Lynd, Wyman, and Gerngross 1999). Ultimately,

goals in this roadmap seek to define and overcome 

the biological limitations for key conversion parameters of metabolic flux and product, thermal, and pH 

tolerances to develop a robust bioconversion process.

Several chapters articulate practical advantages and 

some challenges of biocatalysis and biomass conversion. While most biological research has focused 

on systems relevant to basic knowledge or medical 

applications, it has provided a wide base of tools and 

knowledge for application to the bioconversion of 

biobased feedstocks. 

This discussion focuses on defining and prioritizing 

requirements for science and technology pathways 

that reach the maximal potential of biomass bioconversion. Results build on approaches developed in 

prior workshops (Scouten and Petersen 1999; Roadmap for Biomass 2002). This chapter expands that 

focus in light of new biological research tools and 

understanding.The new biology will use such emerging technologies as proteomics, genomics, metabolomics, protein-complex characterization, imaging,

modeling and simulation, and bioinformatics. This 

joint effort will further guide the development of 

new high-throughput (HTP) biological tools (e.g.,

screening, functional assays, and resequencing). 

Some common themes arose during the workshop.

(1) At present, we reaffirm recalcitrance of lignocellulosic biomass as a core 

issue, but portions of both the science and the conversion solution clearly 

are within the microbial world. (2) Understanding microorganisms will 

enable us to manipulate them so they can reach their maximal potential in 

human-designed processes. (3) The first thrust is to develop biocatalysts 

that will allow design and deployment of conversion processes that are 

less costly in operation and capital than current lignocellulose-to-ethanol 

conversion processes. (4) Another major thrust is to eliminate or combine 

separate processing steps by developing a “multitalented” robust microorganism. Research and development are addressing both strategies. (5) Even 

with molecular biology approaches, scientists create alterations (usually 

a single change) and observe the result. While experimental validation 

always is needed, new global genomics methods offer the potential for 

intelligently predicting the impact of multiple simultaneous changes. 

The new omic tools enable a deeper and more complete understanding of 

the microbial “state” and its physiology in its environment—enabling the 

probing of dynamics, regulation, flux, and function. Combining this understanding with the goal of improving microbial traits by manipulation will 

Fig. 1. The Goal of Biomass Conversion. Securing costeffective biofuels from biomass feedstocks requires moving 

biological technology from the laboratory (a. Microbial 

Cultures at Oak Ridge National Laboratory) through the 

pilot plant (b. National Renewable Energy Laboratory’s 

Process Development Unit) to the full industrial biorefinery 

[c. Industrial Biorefinery in York County, Nebraska (Abengoa Bioenergy Corporation)]. 

a 

b c 

20 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 2. Examples of Possible Pathways to Convert Biomass to Biofuels. The dotted lines show examples of factors this 

roadmap can accelerate; solid lines indicate existing paths. [Source: B. Davison, Oak Ridge National Laboratory] 

allow regulation of the microbe to achieve desired outcomes. Many traits 

or phenotypes, such as overall glycolysis rate or ethanol tolerance, will 

be multigenic. To identify further potential improvements, we especially 

need rapid methods to assess the state of microorganisms that have been 

engineered with new properties—either new process traits or industrial 

robustness. A first step is to analyze of how current industrial microbes 

have evolved through human selection from their progenitors to be better 

adapted to their process environments. 

As stated, we need to achieve rapid analysis, modification, and understanding of the biocatalytic system to accelerate implementation of organisms for 

efficient bioconversion of sugars into ethanol. An array of basic microbial 

requirements includes full microbial system regulation and control, tools for 

rapid manipulation of novel microbes, and new microbial platforms. More 

practical requirements for biocatalysts include utilization of all sugars and a 

robust microorganism. The first may require deeper metabolic and regulatory understanding. The second requires an understanding of stress response 

and inhibition. It can be implemented by inserting all capabilities into one 

host or by using multiple microbial species with unique, complementary 

capabilities in a controlled, stable mixed culture. To enable this research 

and development, certain microbial-specific enabling tools are discussed.

Through a deeper understanding of the microbial system, new biocatalysts 

can be developed to reduce process cost and risk in developing a truly sustainable industry. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 2 
 

 

 

 

 

 

 

 

 

 

SUGAR FERMENTATION 

Three core biological barriers have been identified as high-priority research 

areas for improving current bioconversion processes: Optimizing microbial 

strains for ethanol production, developing advanced microorganisms for 

process simplification, and creating tools and technologies to enhance the 

analysis, understanding, and use of microbial systems. We also consider 

several speculative, breakthrough opportunities offering novel approaches 

to biofuel production that could further reduce cost and risk in the moredistant future. These breakthrough, high-payoff opportunities include 

use of microbial communities rather than pure cultures for robust energy 

production, model-driven design of cellular biocatalytic systems, direct 

production of more energy rich fuels such as alkanes or long-chain alcohols, microbial production of up to 40% ethanol from biomass, and microbial conversion of biomass-derived syngas to ethanol and other products.

Although such ideas as a pure in vitro multienzymatic system were considered, they seemed unlikely to compete with advantages microbes offer 

in producing, regulating, and using complex multistep carbon and energy 

metabolic pathways as commodities in the next 20 years. 

Optmzng Mcrobal Strans for Ethanol Producton: 

Pushng the Lmts of Bology 

A major barrier in the efficient use of biomass-derived sugars is the lack of 

microbial biocatalysts that can grow and function optimally in challenging 

environments created by both biomass hydrolysis and cellular metabolism.

The new tools of biology will facilitate the development of these advanced 

biocatalysts. Problems include inhibition by deleterious products formed 

during biomass hydrolysis, yields limited by accumulation of alternative 

products, unnecessary microbial growth, and suboptimal specific productivity resulting from various limitations in the ethanol biosynthetic pathway 

and a mismatch in conditions with the hydrolysis enzymes. Another challenge is that inhibition by the main fermentation product (ethanol) results 

in low alcohol concentration (titer). These problems contribute to the cost 

of lignocellulosic ethanol by increasing capital expenditure, reducing product yields, and increasing water volumes that must be handled as part of 

relatively dilute product streams. The research objective is to mitigate these 

limitations through concerted application of emerging tools for systems 

biology, working with principles from metabolic engineering and synthetic 

biology, and using evolutionary approaches combined with quantitative 

evaluation of candidate high-producing strains. 

To foster an industry based on biomass sugars, process parameters must 

be comparable to those of the cornstarch ethanol industry. Ultimately,

the overall cellulosic process can compete with petroleum, whereas 

cornstarch processes alone cannot achieve the needed quantities. Current technology is based on cornstarch conversion to ethanol utilizing 

yeast. This process uses glucose as the carbon source and converts it at 

high yields (90%), high titers (10 to 14 wt %), and reasonable rates (1.5 

to 2.5 g/L/h). Recombinant ethanologenic organisms (i.e., yeast, E. coli,

and Z. mobilis) have been created to ferment both glucose and xylose, 

22 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 3. Changes in Metabolism Brought About by Genetic Engineering. The E. coli B strain, an organic acid producer, was altered to the E. coli strain KO11, an ethanol producer (ethanologen). The altered KO11 yielded 0.50 g ethanol per g xylose (10% xylose, pH 6.5, 35°C). In the graphs, biomass refers to the cell mass of E. coli. [Source: L. Ingram,

University of Florida. Based on data reported in H. Tao et al., “Engineering a Homo-Ethanol Pathway in Escherichia 

coli: Increased Glycolytic Flux and Levels of Expression of Glycolytic Genes During Xylose Fermentation,” J. Bacteriol. 

183, 2979−88 (2001) .] 

but they currently produce lower ethanol titers (5 to 6 wt % ethanol).

Improvements in ethanol yields and tolerance are needed to increase 

rates of production (>1.0 g/L/h) from all sugar constituents of lignocellulosic biomass. One successful strategy for utilization of both hexose and 

pentose sugars takes known ethanologens like yeast and adds abilities to 

utilize pentose sugars. Another strategy takes mixed-sugar consumers like 

E. coli and replaces native fermentation pathways with those for ethanol 

production. Figure 3. Changes in Metabolism Brought about by Genetic 

Engineering, this page, shows an example of how the output of a microbe 

can be changed. As titers are increased, rates slow down and eventually 

cease at ~6 wt % ethanol, the upper limit for wild-type E. coli. By comparison, wild-type yeast and Z. mobilis can reach titers of >15% ethanol 

from cornstarch glucose but have failed to achieve these levels on pentose 

sugars (see section, Optimal Strains: Fermentative Production of 40% 

Ethanol from Biomass Sugars, p. 149). 

Most methods of biomass pretreatment to produce hydrolysates also produce side products (e.g., acetate, furfural, and lignin) that are inhibitory to 

microorganisms. These inhibitory side products often significantly reduce 

the growth of biocatalysts, rates of sugar metabolism, and final ethanol 

titers. In all cases, the impact of hydrolysates on xylose metabolism is much 

greater than that of glucose. Research described here offers the potential to 

increase the robustness of ethanologenic biocatalysts that utilize all sugars 

(hexoses and pentoses) produced from biomass saccharification at rates and 

titers that match or exceed current glucose fermentations with yeast. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 2 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

SUGAR FERMENTATION 

From the above analysis of present and target states regarding use of biomass hydrolysates for biofuel production, critical parameters needed for a 

cost-competitive process are clearly evident: 

1. High yield with complete sugar utilization, minimal by-product formation, and minimal loss of carbon into cell mass. 

2. Higher final ethanol titer. 

3. Higher overall volumetric productivity, especially under high-solid 

conditions. 

4. Tolerance to inhibitors present in hydrolysates. 

Specifically, the following figures of merit are suggested for a biomass-toethanol process that will be cost-competitive relative to current cornstarch 

ethanol operations: 

•	 Use of both hexoses and pentoses to produce ethanol at a yield greater 

than 95% of theoretical yield. 

•	 Final ethanol titers in the range of 10 to 15 wt %. 

•	 Overall volumetric productivity of 2 to 5 grams of ethanol per liter per 

hour. 

•	 Ability to grow and metabolize effectively in minimal media or on 

actual hydrolysates (with only minerals as added nutrients). 

To achieve the above targets, we must improve our ability to grow organisms in an inhibitory environment of high concentrations of sugars and 

other compounds, including ethanol. In addition, significant increases in 

flux through the sugar-to-ethanol metabolic pathway are needed. We present a roadmap below for meeting these objectives. 

Scence Challenges and Strategy 

Key questions include: 

•	 What are the implications of simultaneous vs sequential consumption 

of 5-carbon and 6-carbon sugars on cellular metabolism, flux, and 

regulation, especially when xylose metabolism has been engineered 

into ethanologens? 

•	 What can allow more rapid and controlled alteration of microbes,

especially regulatory controls and “adaptation” to novel inserted genes 

or deleted genes? This consideration applies also to known industrial 

microbes. 

•	 What mechanisms control glycolytic flux, and what are their implications for cellular metabolism? For example, could the glycolytic 

pathway efficiently handle an excess of carbon flux in an organism 

engineered to rapidly consume a mixture of 5- and 6-C sugars (see 

Fig. 4. Recombinant Yeast, S. cerevisiae, with Xylose Metabolism Genes 

Added, p. 125)? A systems biology approach will allow insights into 

the molecular basis for these processes and development of predictive 

models to refine their design. 

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•	 What molecular mechanisms are used 

by cells to cope with such environmental 

challenges as high concentrations of sugars 

and ethanol and the presence of inhibitors 

from biomass hydrolysis? 

•	 What genetic and physiological characteristics mediate evolution of wild-type 

organisms into robust laboratory or industrial strains, and which ones control their 

functional state in the process environment (see sidebar, Proteomic and Genomic 

Studies of Industrial Yeast Strains and Their 

Ethanol-Process Traits, p. 126)? 

Utilizing a combination of metabolic engineering and systems biology techniques, two 

broad methods for developing more capable 

and more tolerant microbes and microbial 

communities are the recombinant industrial 

and native approaches. 

•	 Recombinant industrial host approach:

Insert key novel genes into known robust 

industrial hosts with established recombinant tools. 

•	 Native host approach: Manipulate new 

microbes with some complex desirable 

capabilities to develop traits needed for a 

robust industrial organism and to eliminate 

unneeded pathways. 

These methods require genetic understanding 

of the trait we wish to be added or preserved 

and robust tools for genetic manipulation. The 

subset of biochemical pathways potentially 

involved in glycolysis is complex (Fig. 5. Some 

Metabolic Pathways that Impact Glucose 

Fermentation to Ethanol, p. 128). Our goal is 

to pare this down to just what is essential for 

xylose and glucose use (Fig. 6. Desired Metabolic Pathways for a Glucose-Xylose Fermenting Ethanologen, p. 129). Both methods 

can have value; for example, either eliminate 

uneccessary pathways in E. coli, which has yielded strains that efficiently 

metabolize both xylose and glucose (and all other sugar constituents of 

biomass) to ethanol, or add xylose-fermenting pathways (and others) to 

ethanol-producing yeast. A number of methods and approaches support 

the two broad strategies. These and other goals will require certain enabling 

microbiological tools (see section, Enabling Microbiological Tools and 

Technologies That Must be Developed, p. 138). 

Fig. 4. Recombinant Yeast, S. cerevisiae, with Xylose Metabolism 

Genes Added. Following rapid consumption of glucose (within 

10 h), xylose is metabolized more slowly and less completely. Ideally, xylose should be used simultaneously with glucose and at the 

same rate, but the xylose is not totally consumed even after 30 h.

Also note that yield is not optimal. Although ethanol is the most 

abundant product from glucose and xylose metabolism, small 

amounts of the metabolic by-products glycerol and xylitol also are 

produced. [Source: M. Sedlak, H. J. Edenberg, and N. Ho, “DNA 

Microarray Analysis of the Expression of the Genes Encoding the 

Major Enzymes in Ethanol Production During Glucose and Xylose 

Cofermentation by Metabolically Engineered Saccharomyces Yeast,” 

Enzyme Microb. Technol. 33, 19−28 (2003). Reprinted with permission from Elsevier.] 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 25 
 

 

 

 

 

 

 

 

 

 

 

 

SUGAR FERMENTATION 

Proteomc and Genomc Studes of Industral Yeast Strans and Ther EthanolProcess Trats: Rapdly Fndng the Genetc and Functonal Bases 

Current industrial yeast strains have been isolated from wild yeast populations for many decades, selected 

for their capacity to produce ethanol under industrial settings. Understanding how these selected 

genotypes and phenotypes differ from undomesticated strains of yeast would help us to understand the 

type of changes needed to develop a robust ethanol producer. Understanding how cells cope with high-ethanol 

media concentrations is essential to improve fermentation yield and titer. Similar studies would be beneficial for 

other industrial organisms, such as Escherichia coli. Gaining insight about an organism’s process of adaptation to 

an industrial biorefinery environment can help us intentionally replicate these changes (see Fig. A. Importance 

of Adaptation for Robust Initial Strains, below). The strategy for studying industrial strains follows. 

• Compare proteomic and genomic sequences of the most common yeast strains manufactured and sold for 

ethanol production with those of their ancestral parent strains. 

• Compare proteomic and genomic sequences of evolved strains produced through metabolic engineering 

and metabolic evolution with those of their parental strains. Proteomic studies will be performed on samples taken from industrial fermentations. Genomic studies of strains from the same processes should reveal 

differences between industrial and laboratory strains that will provide fundamental information regarding 

multigenic traits essential for high metabolic activity, product tolerance, and adjustments to engineered 

changes in metabolism. 

Studying genomes of industrial yeast strains will help us understand common traits of effective ethanol-producing strains. Proteomic studies will reveal proteins generated under actual industrial production conditions.

Complete mapping and reconstruction of the strain’s networks will be needed for proper comparisons. Available modeling tools are being improved continuously. Proteomic analysis of membrane proteins is still a challenge and needs to be developed further to guarantee a more complete and meaningful analysis of samples. 

Data generated through this effort will require full use of all tools available for systems biology and will stimulate hypothesis generation and testing by the academic community. The effect will be similar to those from 

metagenomic studies and community proteomics, in which huge amounts of data were made available and are 

being analyzed by many different groups around the world. 

Fig. A. Importance of Adaptation for Robust Industrial Strains. [Source: H. J. Strobel and B. Lynn. 2004. “Proteomic 

Analysis of Ethanol Sensitivity in Clostridium thermocellum,” presented in general meeting, American Society for Microbiology, New Orleans, La., May 23–27, 2004.] 

26 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Metabolc Engneerng 

Yield and productivity enhancement will be accomplished by applying 

metabolic engineering concepts and methods. Yield maximization is tantamount to by-product minimization, which is achieved by eliminating 

branches of competing pathways that lead to unwanted products. This 

usually is done by deleting genes encoding enzymes that catalyze competing reaction pathways. If such pathways are responsible for synthesis of 

metabolites essential for cell growth and function, downregulation of these 

genes may be preferable to complete gene knockout. In all cases, optimal 

balancing of enzymatic activities is critical for satisfactory function of the 

resulting engineered strain. Current molecular biological methods can be 

deployed successfully to this end, including specific gene knockout, gene 

amplification through promoter libraries or regulated (induced) promoters,

and other methods combining gene knockout or downregulation and gene 

amplification. The ability to measure detailed cell behaviors and develop 

predictive models to refine their design will be critical to speed up and 

enhance these engineering efforts. 

A related part of this work is analysis and regulation of cellular energetics.

Careful alteration of growth, energy, and redox often is needed. Frequently,

decoupling growth from production will increase yield. 

Productivity maximization has been demonstrated in many applications of 

metabolic engineering with E. coli and yeast strains. Examples include 1,3 

propanediol, amino acids such as lysine and threonine, biopolymer biosynthesis, precursors of pharmaceutical compounds, ethanol, and many others 

(see Fig. 7. A 3G Titer from Glucose, p. 130). These examples illustrate 

the feasibility of significant specific productivity enhancements by applying genetic controls, sometimes in combination with bioreactor controls.

Improvements suggest that projected enhancements in specific cell productivity are entirely feasible and that the new technologies of systems biology 

can dramatically increase and accelerate results. 

The first generation of specific productivity improvement will target enzymes 

important for the sugar-to-ethanol pathway. Stable isotopes will be used 

as tracers to map the metabolic fluxes of ethanol, including related pathways producing or consuming energy or redox metabolites (e.g., ATP or 

NADPH), and other key precursors for ethanol biosynthesis. Flux maps,

together with transcriptional profiles, will be generated for control and 

mutant strains to identify enzymes controlling overall pathway flux. Gene 

amplification of rate-limiting steps will be used to overcome flux limitations.This is anticipated to be an iterative process, as new limitations are 

likely to arise as soon as one is removed by gene modulation.The goal will 

be to amplify flux of the entire pathway without adverse regulatory effects on 

the organism’s growth or physiology. Again, balancing enzymatic activities,

removing limiting steps, and pruning unwanted reactions—all supported by 

comprehensive analysis and modeling—will be deployed for this purpose. 

In addition to specific pathway steps, remote genes with regulatory and 

other (often-unknown) functions impact pathway flux. Modulation of 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 27 
 

SUGAR FERMENTATION 

such genes has been found to influence significantly the biosynthesis rate 

of many products. Such genes will be found through inverse metabolic 

engineering, whereby libraries of endogenous and exogenous genes are 

expressed in the host strain and recombinants are selected on the basis of 

drastic improvements in the desirable phenotype (e.g., ethanol production 

and tolerance). Genes conferring these phenotypes can be sequenced and 

identified for expression in clean genetic backgrounds. 

Recombnant Approach 

Tolerance to inhibitors is a multigenic property. In the example systems 

given above, this trait is founded primarily on membrane fluidity and 

other membrane properties and functions. In general, efforts to improve 

microorganism tolerance by recombinant gene manipulation have been 

confounded by the limited ability to introduce multiple gene changes 

simultaneously in an organism. Development and use of a systems approach 

that allows multiple-gene or whole-pathway cell transformation are important milestones. 

Evolutonary Engneerng 

A strategy for increasing ethanol tolerance or other traits could use evolutionary engineering concepts and methods. This strategy would allow the 

microbial process to evolve under the proper selective pressure (in this case,

higher ethanol concentrations) to increasingly higher ethanol tolerances. 

Fig. 5. Some Metabolic Pathways that Impact Glucose Fermentation to Ethanol.

This pathway map demonstrates the complexity of even a simple, widely utilized, and 

relatively well-understood process such as glucose fermentation to ethanol. Glucose 

2 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

Evolutionary engineering can be applied to 


the ethanol-producing 


organism as a whole or


to specific proteins, in 


particular those with 


regulatory functions. In 


the latter case, evolutionary engineering 


emulates the methods of 


directed evolution, which 


has proven very successful in engineering 


protein mutants with 


specific desirable pharmaceutical, regulatory, or 


kinetic properties.
 

Accurate characterization of cell and protein 

mutants will be needed 

to allow an understanding of principles for improving and rationally carrying out the designs.

This task will require sequencing, large-scale binding experiments, 

and ethanol are identified. [E. Gasteiger et al., “Expasy: The Proteomics Server for 

In-Depth Protein Knowledge and Analysis,” Nucleic Acids Res. 31, 3784–88 (2003).

Screenshot source: http://ca.expasy.org/cgi-bin/show_thumbnails.pl.] 

Fig. 6. Desired Metabolic 

Pathways for a Glucose-Xylose 

Fermenting Ethanologen. The 

goal is to genetically engineer 

an industrial organism that can 

metabolize both sugars. Pathways 

indicate the many involved genes 

that would have to be functional in 

such an organism. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 29 
 

 

 

 

 

 

 

 

 

 

 

 

 

SUGAR FERMENTATION 

Fig. 7. A 3G Titer from Glucose. The graph shows 

DuPont-Genencor success in altering E. coli to 

maximize yield and titer of 3G (1,3, propanediol).

Projects such as this could greatly benefit from the 

deeper systems biology understanding that GTL 

seeks. [Source: Adapted from C. E. Nakamura and 

P. Soucaille, “Engineering E. coli for the Production 

of 1,3-Propanediol,” presented at Metabolic Engineering IV, Tuscany, Italy, October 2002.] 

transcriptional studies, proteomics, metabolomics, and 

physiological functional evaluation, all well suited for GTL 

capabilities described in more detail below. 

Evidence is growing that methods of evolutionary engineering and directed evolution of regulatory proteins have the 

potential to achieve the targets of tolerance to ethanol and 

other inhibitory compounds. Recent studies with E. coli have 

increased that organism’s ethanol tolerance by more than 

50% while comparable increases also have been obtained 

for yeast at high ethanol (6%) and glucose (100g/L) concentrations. Applying systems biology methods and HTP 

technologies and computing will accelerate the process by 

revealing the genetic, molecular, and mechanistic impacts of 

evolutionary methods. 

These methods also will be used to isolate fast-growing 

organisms. High growth rates and final biomass concentrations are imperative for achieving high-volumetric productivities, since the latter depend on fermentor biomass 

concentrations. More detailed investigation is needed on 

the effects of various biomass-hydrolysate compounds on cell growth, especially factors responsible for gradual reduction in specific ethanol productivity during fermentation as sugars are depleted and products, particularly 

ethanol and other inhibitors, accumulate. 

Techncal Mlestones 

Wthn 5 years 

•	 Mesophilic microbes demonstrated at scale that are capable of full utilization of all lignocellulosic sugars for reduced commercialization risk.

This requires optimization of developed and partially developed strains. 

•	 Increased strain tolerance to inhibitory hydrolysates and ethanol, with 

the ability to use all sugars, including mesophile and thermophile strains. 

• Understanding of multigenic causes of industrial robustness. 

•	 Candidate microbes such as thermophilic ethanologens compatible with 

desired cellulase enzyme optima. This allows process simplification to 

single-vessel fermentation with efficient use of all biomass-derived 

sugars (see section, Advanced Microorganisms for Process Simplification, p. 132). 

• Development of coproducts. 

Wthn 0 years 

•	 Rapid tool adaptation and regulation of genetically engineered strains,

including use of minimal media. 

•	 Ability to engineer ethanol tolerance and robustness into new strains 

such as thermophiles. 

• Higher-yield microbes via control of growth and energetics. 

0 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

•	 Increased product titer to simplify product recovery and reduce water use. 

•	 Full predictive metabolic pathway systems model for common industrial 

microbes, including regulation and identification of unknown genes (see 

section, Model-Driven Design of Cellular Biocatalytic Systems Using 

System Biology, p. 142). 

Wthn 5 years 

•	 Thermophillic microbes demonstrated at scale to enable simultaneous 

saccharification and fermentation. 

•	 Further refinement of biofuel process and operation. 

The Role of GTL Capabltes 

As discussed, achieving these objectives will require the use of rationalcombinatorial and evolutionary approaches to improve the properties 

of individual enzymes and organisms. To inform, enhance, and accelerate manipulation of new microbes, systems biology analyses (e.g., omic 

measurements, knockouts, tagging of proteins and complexes, visualization, and a bioinformatic core structure for data) will be applied. Once 

the novelties (e.g., pathways, proteins, products, traits, and complexes) 

are identified, additional genetic tools will move desired genes and traits 

into a known industrial host or further manipulate novel microbes into 

an industrial organism by adding gene traits. There are no consistent and 

rapid tools for these manipulations at present. 

The capabilities listed below will play an important role in both cases. 

Proten Producton 

A wide range of proteins (regulatory, catalytic, and structural) will be 

produced and characterized, and appropriate affinity reagents will be 

generated. Modified proteins also will be used to understand functional 

principles and for redesign. Examples include glycolytic proteins and 

alcohol dehydrogenases from other organisms or those evolved in the lab,

structural proteins from high-tolerance organisms, or regulatory proteins 

with altered properties. 

Molecular Machnes 

HTP methods to identify binding sites of global regulatory proteins and 

other aspects of membranes and membrane formation will be required.

Specific protein complexes of interest are sugar transporters, solvent 

pumps, or other porins. These measurements will inform our understanding of, for example, the interaction or association of enzymes along the 

glycolytic pathway. The membrane could be studied as a machine to control inhibitory stress. 

Proteomcs 

Although rational and evolutionary approaches are envisioned, a common component of both is the use of tools that allow quantitative cellular 

characterization at the systems level, including existing tools for global 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy  
 

SUGAR FERMENTATION 

transcript, protein, and metabolite profiling. Additional HTP tools not 

currently available will be required to monitor key players that define the 

redox and cell energy state [e.g., ATP, GTP, NAD(P)H, NAD(P)]. Capabilities could include metabolic flux mapping, a major activity in understanding and manipulating cellular metabolism. The most efficient way to 

estimate in vivo metabolic fluxes is through labeling experiments. Specific 

needs include appropriate nuclear magnetic resonance (NMR) and mass 

spectroscopy (MS) instrumentation and stable isotopes for visualizing pentoses, hexoses, and cellulose. Intensive mathemathical and computational 

power is required to achieve the final goal of flux estimation. HTP technologies for global identification of genes that impact ethanol biosynthetic 

pathways are required to select cells capable of high ethanol production 

and other desired functions. 

Cellular Systems 

The ability to track key molecular species as they carry out their functions 

and create predictive models for systems processes will be critical for developing or enhancing cell properties. 

DOE Jont Genome Insttute 

Sequencing and screening of metagenomic libraries for novel genes and 

processes and analyzing novel organisms will be carried out at DOE JGI.

Exploiting microbial diversity by mining for novel pathways or organisms 

that make a step change in ethanol production could spur the production 

of other chemicals through fermentation. 

Advanced Mcroorgansms for Process Smplficaton 

Methods and technologies discussed above will be applied to consolidating 

process steps, which is widely recognized as a signature feature of mature 

technologies and has well-documented potential to provide leap-forward 

advances in low-cost processing technology. In light of the complexity 

of underlying cellular processes upon which such consolidation depends,

fundamentally oriented work will be a highly valuable complement to 

mission-focused studies and can be expected to accelerate substantially the 

achievement of applied objectives. 

Realizing the benefits of targeted consolidation opportunities requires 

understanding and manipulating many cellular traits, an approach much 

more fruitful at a systems level than at the individual gene level. As 

discussed previously, examples of such traits include transporters, control 

mechanisms, and pathways relevant to use of non-native substrates (e.g., 5­

C sugars and cellulose), microbial inhibition (e.g., by pretreatment-generated inhibitors or ethanol), and the ability to function well in simple and 

inexpensive growth media. Investigation of these traits provides an important way to apply and extend new systems biology tools to nonconventional host organisms such as thermophiles. 

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The current process has undergone many improvements in the last decade.

In Fig. 4 of the Introduction, p. 14, the process cartoon illustrates pretreatment (probably dilute acid hydrolysis), followed by a detoxification 

and neutralization step, then separate fermentation of the soluble pentose 

sugars. Some biomass solids are used to make the cellulases, which then 

are added to the biomass solids to convert cellulose to glucose, followed by 

a separate glucose fermentation. This section discusses recent and ongoing 

developments to make a single microbe for cofermentation of hexose and 

pentose sugars (e.g., glucose and xylose). 

Eliminating process steps may reduce capital and operating costs and allow 

other synergistic benefits. Some of these simplification steps are under 

limited active research. We focus here on three immediate consolidation 

opportunities: 

1. Elimination of a dedicated step to detoxify pretreatment hydrolysates 

before fermentation. These inhibitors can be by-products of the hydrolysis process and include acetate, furfurals, and other undetermined 

substances. Figure 8. Recombinant Yeast Cofermentation of Glucose 

and Xylose from Corn Stover Hydrolysate Without Detoxification (this 

page) shows the impact of these inhibitors. In process configurations 

under consideration (e.g., acid hydrolysis), such detoxification requires 

equipment (e.g., solid-liquid separation and tanks), added materials 

(e.g., base for overliming followed by acid for neutralization before 

fermentation), and added complexity. Obvious savings can be realized 

by developing improved biocatalysts not requiring the detoxification 

step. For detoxification elimination, research will support development 

of organisms having a high tolerance to pretreatment-generated inhibitors or those that 

detoxify these Fig. 8. Recombinant Yeast Cofermenta­ inhibitors (e.g., by 

tion of Glucose and Xylose from Corn consuming them) 


while preserv-
 Stover Hydrolysate Without Detoxification. Note slower xylose use and lower ing other desired 

fermentation ethanol titer and yield than in Fig. 4.

properties. Some Corn stover hydrolysate was prepared by 

inhibitors have aqueous pretreatment followed by enzyme 

been identified, hydrolysis. [Source: M. Sedlak and N. Ho,

such as furfurals “Production of Ethanol from Cellulosic 

and acetate, but not Biomass Hydrolysates Using Genetically 

all are known. Engineered Saccharomyces Yeast Capable 

2. Simultaneous of Co-Fermenting Glucose and Xylose,”

saccharification Appl. Biochem. and Biotechnol. 114, 403–16 

and cofermenta­ (2004) (also see Mosier et al. (2005)]. 

tion (SSCF), in 


which hydrolysis is 


integrated with fermentation of both 


hexose and pentose 


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SUGAR FERMENTATION 

sugars but with cellulase produced in a separate step. For example, development of thermophilic ethanol-producing organisms for use in SSCF 

could allow the consolidated process to run at higher temperatures, thus 

realizing significant savings by reducing cellulase requirements. Previous 

analyses (Svenson et al. 2001) have shown that a midterm strategy to 

produce ethanol from biomass would be to develop new strains capable 

of yielding ethanol at 50°C pH 6.0, the optimal conditions for saccharification enzymes generated today by the industry. 

3. Combining cellulase production, cellulose hydrolysis, and cofermentation of C-5 and C-6 sugars in a single step termed “consolidated 

bioprocessing” (CBP). Widely considered the ultimate low-cost configuration for cellulose hydrolysis and fermentation, CBP has been 

shown to offer large cost benefits relative to other process configurations 

in both near-term (Lynd, Elander, and Wyman 1996) and futuristic 

contexts (Lynd et al. 2005). 

The goal is a process more like Fig. 5 of the Introduction, p. 15. Further 

simplifications can be envisioned beyond these examples. Unique challenges 

are the expression of multiple enzymes for cellulose and hemicellulose 

hydrolysis or the engineering of native cellulose-hydrolyzing organisms 

to produce ethanol. Selecting optimal enzyme targets for expression will 

require extensive screening and characterization of heterologous genes.

Developing a unique enzyme suite capable of complete cellulose and hemicellulose hydrolysis will require insights into the plant cell-wall assembly 

and structure as well as new tools for cell-wall investigations. 

Research must determine which aromatic hydrocarbon degradative pathways can solubilize lignin and how they can be integrated into a productive 

host for additional ethanol production. Fortunately, aromatic hydrocarbon-biodegradation pathways have been studied extensively over the past 

two decades, and many are known. Integrating necessary components into 

single hosts and channeling carbon to ethanol will be major challenges. 

It will be a challenging goal to optimally achieve all the traits at one 

time. Expression, regulation, tolerance, growth, and metabolism must be 

designed and synchronized to function in the process. At present for 

this approach, we appear to be limited to anaerobic bacteria and not the 

aerobic fungi used to make current cellulases. We have limited knowledge and less ability to manipulate most of these bacteria. The cellulolytic 

bacteria also have some interesting differences, such as the cellulosome 

discussed in the biomass deconstruction chapter. 

For all these consolidation opportunities, native or recombinant industrial 

strategies will be employed: 

1. The recombinant industrial strategy, engineering industrial organisms 

with high product yield and titer so cellulose or pentose sugars are used 

by virtue of heterologous enzyme expression. 

2. The native host strategy, engineering organisms with the native ability 

to use cellulose or pentose sugars to improve product-related properties 

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(e.g., yield, titer) and process-related properties (e.g., resistance to toxic 

compounds). 

However, a third combined strategy is possible. 

3. Mixed culture conversion strategy to separately modify microbes to work 

on different parts of the substrates or pathways.This has been suggested 

but not well tested for cofermentation of pentoses and hexoses (see section, Microbial Communities for Robust Energy Production, p. 140). 

As described before, the key difference is how challenging the complex 

trait is. Some cases like the elimination of lactic acid as a by-product 

might involve the deletion of a single gene; in others, the production of 

a complex extracellular cellulase or cellulosome may appear impossible at 

present. Delineating the genetic changes needed to confer resistance to 

toxic compounds generated in biomass processes is even more challenging. Researchers must balance the ease of manipulation against the trait’s 

complexity in pursuing improvements of biocatalysts for industrial ethanol 

from lignocellulose. 

For both SSCF and CBP, causes of cellulosic-biomass recalcitrance need 

to be understood not only with respect to enzymatic hydrolysis, in which 

enzymes act independently of cells (cellulose-enzyme complexes), but 

also to microbial hydrolysis, in which hydrolysis is mediated by celluloseenzyme-microbe complexes. Growing evidence shows that free-enzymatic 

and microbial hydrolyzes differ in substantial ways. Studies of recalcitrance 

in microbial cellulose hydrolysis will build on and complement, but not 

duplicate, investigation of enzymatic hydrolysis. 

Further process simplifications can be considered. For example, development of robust, intrinsically stable pure or mixed microbial cultures could 

eliminate the need for costly sterilization. Alternative routes for process 

simplification also should be considered—such as gasifaction of the entire 

biomass followed by catalytic or biological conversion into fuels like ethanol (see section, An Alternative Route for Biomass to Ethanol, p. 152). 

Scence Challenges and Strateges for Process Smplficaton 

The physiology or microbial state of modified organisms within the conversion process needs to be understood to help determine when simplification is helpful and what conditions must be achieved to make it effective.

Part of this is regulation of native and modified pathways and traits, many 

of which appear to be multigenic, complex, poorly understood, and difficult 

to control. For all three consolidation opportunities, understanding the 

sensitivity of organism performance to growth-medium formulation would 

benefit from use of systems biology tools. Although separate processes are 

more cost-effective at times, simplification tends to win historically. 

A. Elmnaton of Detoxficaton 

•	 Fundamental mechanisms of toxicity and resistance. 

•	 Evaluation of tolerance among a diversity of species and strains with 

and without opportunity for adaptation and evolution. 

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SUGAR FERMENTATION 

•	 Characterization and evaluation of detoxification mechanisms. 

B. Smultaneous Saccharficaton and Cofermentaton 

•	 Fundamentals of fermentation in the presence of high solid concentration. The microbe-enzyme-solid interface should be analyzed. 

•	 Understanding and reconciling factors responsible for differences in 

optimum conditions for cellulase function and fermentation of sugars 

to ethanol. 

C. Consoldated Boprocessng 

•	 A key question is, How do microorganisms break down cellulose? How 

is breakdown in microbially attached cellulosome complexes different 

from enzymatic hydrolysis with added fungal enzymes? A significant 

number of fundamental issues needing to be addressed are over and 

above questions implicit in seeking to understand how enzymes hydrolyze cellulose. They include: 

–	 Bioenergetics, substrate uptake, and metabolic control (including 

regulatory circuits) related to cellulose hydrolysis. 

–	 Relative effectiveness of cellulose-enzyme-microbe complexes as 

compared to cellulose-enzyme complexes and the mechanistic basis 

for such differences and possible synergies. 

–	 Extent to which products of microbial cellulose hydrolysis equilibrate or do not equilibrate with the bulk solution and the fraction of 

hydrolysis products that proceed from the cellulose surface directly 

to adherent cells. 

–	 Features of cellulolytic microorganisms favored by natural selection 

and how selection can be harnessed for biotechnology (especially for 

the recombinant strategy). 

–	 Documentation and understanding of the diversity of cellulose-utilizing organisms and strategies present in nature. 

•	 How do microorganisms respond to cellular manipulations undertaken 

in the course of developing CBP-enabling microorganisms? Specific 

issues include: 

–	 For the native strategy, how cells respond to changes in end-product 

profiles in terms of the cell’s state (transcriptome, proteome, and 

metabolite profiles) as well as key properties of industrial interest 

(product tolerance and growth rate). 

–	 For the recombinant strategy, understanding gained from recombinant cellulolytic microorganisms developed one feature at a time,

including those in addition to hydrolytic enzymes (e.g., for substrate 

adhesion, substrate uptake, and metabolism). Such step-wise organism development provides an outstanding opportunity to advance 

applied goals and gain fundamental insights simultaneously. 

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D. Other Smplficaton Opportuntes 

•	 The development of intrinsically stable cultures could involve contamination-resistant thermophiles or acidophiles or techniques to control 

mixed microbial cultures (see section, Microbial Communities for 

Robust Energy Production, p. 140). 

•	 The development of microbial growth-independent processes will 

reduce waste-treatment volumes of biosolids and allow better return of 

nutrients to the land as sustainable fertilizers. 

Techncal Mlestones 

Of the targeted consolidation opportunities, CBP is the most ambitious and 

probably will require the largest effort to achieve.Thus, we may well see substantial progress toward SSCF and detoxification elimination before CBP. 

Key milestones associated with targeted consolidation opportunities can be 

pursued beneficially by complementary mission-oriented and fundamentalsfocused research activities.These milestones include: 

Wthn 5 years 

•	 Improve hydrolysate-tolerant microbes. 

•	 Achieve SSCF under desirable conditions (high rates, yield, and titer;

solids concentration and industrial media). 

•	 Functionally express heterologous cellulases in industrial hosts, including secretion at high levels and investigation of cell-surface expression. 

•	 Conduct lab tests of modified initial CBP microbes. 

Wthn 0 years 

•	 Eliminate the detoxification step by developing organisms highly tolerant to inhibitors. 

•	 Have the same response with undefined hydrolysates as with defined 

hydrolysates. 

•	 Move to pilot demonstration of CBP. 

Wthn 5 years 

•	 Develop intrinsically stable cultures that do not require sterilization. 

•	 Achieve CBP under desirable conditions (high rates, yield, and titer;

solids concentration and industrial media), first on easily hydrolyzed 

model cellulosic substrates, then on pretreated cellulose. 

•	 Develop methods to use or recycle all process streams such as inorganic 

nutrients, protein, biosolids, or coproduct carbon dioxide (see sidebar,

Utilization of the Fermentation By-Product CO , p. 138). 2 

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SUGAR FERMENTATION 

The Role of GTL Capabltes 

Proten Producton 

Protein production resources could be very useful in synthesizing enzymes 

and mixtures of enzymes as controls in experiments comparing enzymatic 

and microbial hydrolysis. These controls have the potential to be quite 

complex and thus demanding in terms of protein synthesis capability. 

Molecular Machne Analyss 

These resources can provide advanced analytical and computational science 

to study cell and cellulose interaction and particularly to gain insights into 

what is going on in the gap between an adhered cell and cellulose surface. 

Proteomcs 

Proteomic capabilities can assist researchers seeking to understand systemlevel responses to metabolic manipulation in the course of developing 

microorganisms to achieve all three targeted consolidation opportunities as 

well as diagnosis and alleviation of metabolic bottlenecks and flux analysis. 

Cellular Systems 

These capabilities also can assist researchers in understanding system-level 

responses, removing bottlenecks, and conducting flux analysis (e.g., via 

metabolite analysis). 

DOE Jont Genome Insttute 

DOE JGI can play a key role in sequencing genomes of new microorganisms with relevant features (e.g., ability to use C-5 sugars, resistance to 

pretreatment-generated inhibitors, and cellulose utilization), thus enabling virtually all lines of inquiry described 

Utlzaton of the Fermentaton 	 in this chapter and in Crosscutting 21st Century Science,

Technology and Infrastructure for a New Generation of By-Product CO

C

2 Biofuel Research, p. 155. 

arbon dioxide is a major by-product 

of alcoholic fermentation by both Enablng Mcrobologcal Tools and yeast and bacteria. This relatively pure 

gaseous stream requires no primary separation Technologes that Must be Developed 

or enrichment step to concentrate the CO2, Cellulosic biofuel research will use a broad range of powwhich can be sequestered as part of the national erful omic tools targeted for fuller development of the 

climate-protection program or processed by plant, enzyme, and microbial arena. However, some specific 

biological or other means to useful coproducts. microbiological tools will need to be created to further 

Technologies could be developed to produce understand and exploit microorganisms. These tools include 

value-added compounds that might provide analytical technologies and computational approaches and 

income for financing ethanol biorefineries. technologies for revealing the state of a microbial system,

The fundamental challenge is how to supply permitting assessment of perturbation effects on the system,

and providing the information needed to construct useful chemical energy to use and reduce CO2

, pro- models to guide engineering efforts. duce useful compounds, and elucidate factors 

governing efficient use of CO2

. 

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The chapter, Crosscutting 21st Century Science, Technology and Infrastructure for a New Generation of Biofuel Research, p. 155, discusses in detail 

barriers in (1) gene-transfer methods and expression of genes in nonconventional host organisms; (2) tools for rapid analysis and modeling of cellular composition and physiological state; and (3) HTP screening methods 

for novel and evolved genes, enzymes, cells, and communities. Additional 

required tools include the following: 

•	 Devices and requirements for preparing well-controlled microbial 

samples for omic analysis. 

–	 Integrating biological studies into a whole-systems understanding is 

being made possible by new analytical techniques. Systems biology 

needs to be driven by an organism’s biological context and its physiological state, which is linked tightly to its complete history, including that of culture. For experimentation in all aspects of work with 

omic tools, high-quality reproducible samples are paramount for 

subsequent analysis or purification. 

–	 Controlled cultivation is the method to provide these samples.

Cultivation also is that part of the experiment where knowledge of 

the biology is critical, and the quality of subsequent understanding 

is driven by the design of microorganism cultivation. The emphatic 

consensus of workshop attendees was that chemostat or continuous, stirred-tank reactors will provide the highest-quality biological 

samples for measuring multiple properties (omics) because they 

maintain environmental conditions at a steady state. For some omic 

techniques, batch operation will be chosen because of limitations in 

current cultivation technology and because of sample-number and 

amount requirements. Investigators must realize, however, that the 

increased amount or number comes at some cost to quality. 

–	 Apparatus is needed to characterize mixed microbial populations.

Cellulose is degraded in nature by mixed populations needing characterization beyond identification of its members. New tools and 

approaches are required to understand each population’s contribution 

to cellulose degradation. 

•	 Development of novel techniques and approaches is needed to carry out 

evolutionary biotechnology, especially for multigenic traits. New and 

more efficient methods to generate genetic and phenotypic variation in 

microbes are needed to increase capabilities for obtaining new phenotypes that require multiple simultaneous changes. 

•	 Techniques and approaches are required for studying interactions 

between cellulolytic microbes and their substrates. A key step in 

cellulose degradation in nature is adhesion of microbes to the substrate. This dynamic process needs to be characterized with new and 

more quantitative and spatially, temporally, and chemically sensitive 

approaches and techniques. 

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SUGAR FERMENTATION 

Realization of targeted consolidation opportunities and advancement of 

relevant fundamentals will be served by a variety of crosscutting technologies and capabilities, including the following. 

• 

• 

• 

• 

• 

Bioreactors—novel configurations, in some cases—to evaluate performance for consolidation opportunities and to test several key hypotheses. 

Evolutionary biotechnology to develop needed strains, including those 

with new capabilities. Application of these techniques will be advanced 

by miniature reactors and automated or controlled systems (continuous,

semicontinuous, or serial culture) to maximize evolution rates. Some 

special consideration probably will be required to adapt evolutionary 

biotechnology to insoluble substrates. 

Improved gene-transfer and -expression technologies for unconventional host organisms and particularly for Gram-positive organisms,

which have potential to be profoundly enabling with respect to all three 

consolidation targets. 

HTP screening for functional abilities and traits. This is needed for selection of the most improved strains, especially for nongrowth-associated 

functions. It also is needed to identify the function of unknown or hypothetical genes to allow better models and metabolic engineering. 

Tools to understand microbial mixed cultures in an industrial context. 

• Scanning and other microscopic techniques, as well as experimental and 

computational approaches drawn from biofilm research, to characterize 

adhered cells. 

• 

• 

• 

• 

Systems biology tools (e.g., transcriptome, proteome, metabolome) to 

characterize intracellular events associated with targeted consolidation 

opportunities in both naturally occurring and engineered cells. This 

includes omic analysis for characterization of existing industrial microbes 

under production conditions to inform development of new biocatalysts. 

Quantitative modeling at the cellular level to test fundamental understanding and provide guidance for experimental work relevant to all 

three consolidation opportunities. 

Mesoscale molecular modeling to understand critical events occurring 

in the gap between cellulose and an adhered cell and its accompanying 

enzymes (see sidebar, The Cellulosome, p. 102). 

Models to confirm that consolidation and process simplification will be 

more cost-effective than separate optimized steps (see chapter, Bioprocess Systems Engineering and Economic Analysis, p. 181). 

Breakthrough, Hgh-Payoff Opportuntes 

Mcrobal Communtes for Robust Energy Producton 

Most industrial bioconversions rely on pure cultures. All environmental 

bioconversions are based on mixed cultures or communities, with specialists “working” together in an apparently stable fashion. Examples of mixed 

0 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

communities capable of cellulolytic conversion are ruminant cultures and 

termite-gut cultures. Are there intrinsic biological reasons why communities 

could not be used for biofuel production? The fundamental question is, Are 

there stable self-regulating multiplex solutions for biofuels? 

Microbial communities offer flexibility not present in monocultures,

because the collective multiple metabolic pathways of microorganisms 

are activated as conditions demand. For example, microbial communities potentially could produce multiple forms of cellulase enzymes for use 

in industrial production of ethanol. In fact, multiple cellulases have been 

shown to be more effective than a single cellulase at processing complex 

and variable feedstocks. Mixed cultures tend also to be more robust, a characteristic needed for industrial-scale use. 

Research Drectons 

This goal would require the ability to manipulate and use microbial communities to achieve industrial goals—not just the natural microbial goals 

of reproduction, survival, and net energy utilization. Current applications 

of mixed microbial cultures primarily are for waste treatment (i.e., anaerobic digestion or biofiltration). However, these technologies are poorly 

understood and exploit natural selection for survival. There are limited 

examples of products from mixed cultures in the food industry, but modern biotechnology has used only pure cultures for pharmaceuticals or for 

bioproducts such as ethanol. The first steps in applying microbial communities to biofuels are (1) characterize and understand existing cellulolytic microbial communities of microbes (e.g., ruminant, termite, and soil) 

and (2) develop techniques to understand and stabilize intentional mixed 

cultures. Research can elucidate detailed population interactions (e.g., both 

trophic and signaling) that stabilize the community. Support also is needed 

to evaluate robustness and population drift over time, since many mixedculture operations will be continuous. Gaining a deeper understanding 

about community evolution will allow the use of selective pressure methods to evolve consortia with increased cellulose-processing efficiency. As 

an additional benefit, knowledge of mixed-culture dynamics may allow 

development of new methods to make pure cultures resistant to biological 

contamination. 

Scentfic Challenges and Opportuntes 

The most basic requirements are for mixed-culture identification and 

enumeration. Major science challenges are analysis and measurement of 

the mixed-culture “state.” Most current omic analyses are predicated on 

knowledge of the gene sequence. Mixed cultures increase the challenge—

for sequence, transcriptomics, and proteomics. The challenge increases 

geometrically for lower-number (<1%) representatives of the community. Single species existing as a population of clonal variants also adds 

complexity. Therefore, new techniques need to be developed and tested 

for sequence-based “metaomic” analysis. Also possible are nonsequencebased, metaomic techniques (e.g., transcript function–based microarrays) 

(Zhou et al. 2004). The metabolome is the only analysis that will not be 

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SUGAR FERMENTATION 

significantly more difficult compared with that analysis in pure culture.

One subset of mixed cultures is the many industrial strains that live as 

a group of clonal variants (a single-species population). Analysis of how 

these actual strains have adapted to their working environment could be 

helpful. 

After basic analysis, enumeration, and quantitative meta-omics, the goal 

is to understand community structure. Signaling molecules are known to 

be important in many communities, so we need to identify and confirm 

these molecules and determine their importance. Then we can consider 

how to modify these signals to control the community. Regulatory and 

metabolic modeling of individual members, as well as the community, will 

be essential to deciphering the regulatory structure. Understanding the 

physical structure will require imaging technologies to acquire detailed 

visualization and specific labeling of individual species. This might require 

individual-species modification to express tagged marker proteins and 

then to reassemble the mixed culture. Computational models combining 

omics and biochemical and spatial variables will be critical to accomplishing this goal. 

Reproducible samples and improved cultivation techniques in highly 

instrumented chemostats, for example, also will be required, especially 

when lignocellulosic solids are introduced. In this case, reproducible 

samples are especially needed. Mixed cultures may not be deterministic.

Some evidence shows that the final state is highly variable. Previous work 

has shown that parallel enrichments from the same natural source each led 

to different populations after multiple serial transfers. 

GTL Facltes and Capabltes 

A major priority, as described in the GTL Roadmap, is to understand 

microbial communities. Capabilities being developed in the GTL program 

are ideally suited for developing industrial use of microbial communities. GTL will rely heavily on the DOE Joint Genome Institute ( JGI) for 

sequencing and resulting annotation. Integrated proteomic capabilities 

will be useful for a wide range of omic analyses. Signaling molecules and 

tagged proteins and clones would be provided from protein production 

resources. For advanced community metabolic models, cellular systems 

capabilities are needed. When studying community interactions with a 

lignocellulosic medium, the National Renewable Energy Laboratory’s 

Biomass Surface Characterization Laboratory will be valuable. 

Model-Drven Desgn of Cellular Bocatalytc Systems 

Usng Systems Bology 

Systems-level modeling and simulation is the modern complement to the 

classical metabolic engineering approach utilizing all the GTL technologies and computing. Microbial organisms contain thousands of genes 

within their genomes. These genes code for all protein and enzyme components that operate and interact within the cell, but not all proteins are used 

under all conditions; rather, an estimated 25% are active under any given 

2 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

condition in a living cell. We currently understand very little about how 

all these expressed proteins interact and respond to each other to create 

cellular phenotypes that we can measure or try to establish. With improved 

understanding of such complex cellular systems, we should gain the ability to design them in an intelligent manner. Figure 9. Microbial Models 

for Providing New Insights, this page, indicates some of this power and 

complexity. Two general and complementary methods are taken into consideration: The synthetic route, which embraces de novo creation of genes,

proteins, and pathways and the nature-based route, which uses existing 

suites of microorganisms and seeks to improve their properties via rational 

design. This is a qualitative step beyond recombinant and native strategies 

discussed in the Metabolic Engineering section, p. 127. 

The challenge starts with the ability to characterize cellular networks 

and then moves toward establishing computer models that can be used 

to design them. These models would capture all aspects of a microbe’s 

metabolic machinery—from primary pathways to their regulation and 

use— to achieve a cell’s growth. Figure 5 illustrates part of this pathway 

complexity. Through these computational models, we could design and 

optimize existing organisms or, ultimately, create novel synthetic organisms. Specific to biomass-to-biofuels objectives, organisms engineered 

using these technologies potentially will consolidate the overall process 

and reduce unit operations (see Fig. 9. Microbial Models for Providing 

New Insights, this page). 

Fig. 9. Microbial Models for 

Providing New Insights. Information gained in GTL systems 

biology research will enable 

metabolic engineering and 

modeling to enhance microbial 

characteristics. Using defined 

experimental parameters, the 

biology can be changed to perform desired new tasks. This will 

allow new biological system outputs, increase knowledge, and,

ultimately, improve predictive 

models. [Source: M. Himmel,

National Renewable Energy 

Laboratory] 

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SUGAR FERMENTATION 

Research Drectons 

This research would provide the ability to control and optimize a microbial 

transformation from carbohydrates to ethanol and related value-added 

coproducts in a selected microbe. Gaining complete control over cellular 

networks implies a capability for engineering and consistently performing 

transformation with the best available yields, rate, and titers. It also includes 

the ability to design the microbe intelligently by using computational technologies for determining consequences and optimal approaches to intervene 

and engineer within cellular networks. Furthermore, this model would 

enable us to assess the limits of a bioprocess’s microbial biotransformation 

(e.g. the maximal productivity and rates achievable) and potentially to engineer entirely novel biotransformation pathways and systems. 

Specific research directions would include the following. 

1. Enumeration of Cellular Components, Interactions, and Related 

Phenotypes. Before any predictive computational models can be built, we 

need to generate the underlying data sets for relevant process conditions: 

•	 Identification of all proteins and enzymes participating in the metabolic 

pathways relevant to carbohydrate metabolism for cell growth, ethanol 

synthesis, and related by-products. This also would include characterization of key enzyme complexes relevant to cellulose degradation,

carbohydrate transport, and respiratory mechanisms. Experimental 

technologies that may be useful include protein tagging, proteomics, and 

in vivo activity measurements. 

•	 Characterization of novel protein and gene function. About 30% of 

genes have no understood function, yet some of these unknown genes 

are thought to be involved in the microbial metabolic systems and stress 

responses under process conditions. 

•	 Identification and quantification of all metabolites present within the 

cell. Experimental approaches could involve NMR or MS. 

•	 Characterization of the cell’s energetics under various relevant conditions.

These would include measurements to characterize the stoichiometry of 

energy-transducing complexes, parameters such as the P/O and P/H+ 

ratios, and a cell’s maintenance energy associated with cellular functions. 

•	 Characterization of transport mechanisms. In particular, this would 

focus on determining the components associated with transport of 

nutrients into and out of the cell as well as those of the mitochondria 

in eukaryotic organisms. Determining the stoichiometry of transporters 

and transport processes, as well as their kinetics and differential regulation, is envisioned. 

•	 Characterization of membrane composition for process tolerance 

(i.e., alcohol and toxin tolerance) and environmental and community 

interactions. 

•	 Elucidation of regulatory networks enabled by development of experimental approaches to identify protein-protein and protein-DNA 

interactions. 

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•	 Localization data indicating where proteins are operating within the 

cellular and community space. 

2. Knowledgebase to Develop Dynamics and Kinetics Modeling Techniques. One of the most attractive features of a model-driven, rational 

approach is its predictive capacity, requiring the inclusion of regulatory 

events at the genetic and metabolic levels. Creation of such models requires 

data sets, as well as development of HTP tools with capabilities beyond 

those currently available. 

•	 Acquisition of high-quality and dynamic omic data. 

•	 Development of HTP methods to identify binding sites of global regulatory proteins and other interactions. 

•	 Development of HTP tools to monitor key players that define the cell’s 

redox and energy states [e.g., ATP, GTP, NAD(P)H, and NAD(P)]. 

•	 HTP quantification of in vivo enzyme-activity metabolic fluxes. 

3. Network Reconstruction. From data sets generated, we can develop the 

complete mapping and reconstruction of microbial networks and physiology related to the conversion of sugars to ethanol. 

•	 Automated techniques to integrate data sets and rapidly create reconstructed networks. 

•	 Integrated representation of metabolism, regulation, and energetics. 

•	 Approaches to account for the impact of spatial localization of proteins 

and enzyme complexes within integrated models. 

4. Development of In Silico Analysis Tools. Methods are needed to 

interrogate and simulate the functioning of constructed networks to 

address key questions about microbial physiology. Any method should 

develop testable hypotheses that can be integrated with experimental 

studies. Methods should do the following: 

•	 Assist in network reconstruction, particularly in metabolic pathways and 

regulatory networks. These methods may involve new approaches that 

use artificial intelligence. 

•	 Interrogate mechanisms associated with toxic responses and tolerance to 

product and intermediate levels. 

•	 Assess physicochemical limitations of cellular systems and enzyme 

components to determine maximum achievable rates (e.g., identify ratelimiting steps, kinetic as well as diffusion limited). 

•	 Generate prospective designs of cellular networks by modifying and 

testing existing cellular systems. 

•	 Design systems de novo from cellular components. 

5. Design of Cellular Systems. Designing engineered and synthetic 

organisms to convert carbohydrates to ethanol through the use of computational models and methods would include the following: 

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SUGAR FERMENTATION 

•	 Dedicated transforming microbes with focused abilities to perform biotransformation and necessary supporting operations for the conversion 

of carbohydrates to ethanol. 

•	 Self-replicating synthetic microbes to support biofuel production under 

optimal conditions. 

•	 Novel pathways for producing biofuels and value-added coproducts 

from biomass could involve the generation of new enzymes and organisms through the use of evolutionary design concepts (e.g., directed 

evolution and adaptive evolution). 

Scentfic Challenges and Opportuntes 

To address this model-driven design goal, a number of broad scientific and 

conceptual challenges will need to be overcome, including the ability to 

make high-quality measurements of cellular components and states to simulate physiology and design networks with models generated from these data. 

The Role of GTL Capabltes 

Many GTL capabilities, either centralized or distributed, can be leveraged 

to aid in accomplishing these goals. Particular ones are noted below. 

Protein Production 

GTL capabilities will be used to characterize proteins by rapid isolation,

production, and biochemical characterization in an HTP manner. 

Molecular Machines 

Molecular machine analysis will enable characterization of large complexes 

containing many active components of biotransformation networks. 

Proteomics 

HTP analysis of all proteins present in the cell, their relative abundance, spatial distribution, and interactions will be important to model development. 

Cellular Systems 

Ultimately, cellular systems analysis is about developing computational 

models of systems that can be used reliably to engineer microbes. Resources 

dedicated to the analysis and modeling of cellular systems can be used 

reliably by technologists to engineer microbes for biofuel production on an 

industrial scale. 

Outcomes and Impacts 

The GTL Roadmap describes scientific goals and milestones and the 

technology and computing needed to meet these research directions. These 

resources can be focused on the problem of engineering existing or synthetic organisms for biofuel production from biomass. This type of “rational 

design” and organism engineering has the potential to transform various 

stages of biomass conversion to biofuels consistent with goals of consolidating the overall process and reducing unit operations. The practical impact 

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is in reducing the time required to modify a microorganism to perform as 

desired in an industrial setting. 

Although the direct applied benefit will be in biomass-to-biofuel processes,

technologies and methods derived from the ability to reliably engineer 

biological systems will have far-reaching impacts on basic and applied 

research across many sectors of biotechnology. 

Drect Boproducton of Energy-Rch Fuels 

This breakthrough, high-payoff opportunity focuses on microbes for direct 

production of hydrophobic alternative fuels (i.e., alkanes, longer-chain 

alcohols, and fatty acids). This would overcome one limitation of nearly all 

bioconversions—they result in dilute aqueous mixtures. Typical industrial 

product concentrations are 100 to 150 g/L for ethanol and other such 

products as organic acids. This limitation imposes separation requirements 

that increase process and energy costs. New fermentation systems would 

be highly desirable to allow significant increases in product concentration,

new types of products, and new processes for product recovery. Strong 

increases in efficiency also could be achieved by developing continuous 

processes. 

Research Drectons 

Microorganisms produce a wide variety of potentially useful compounds 

but in relatively low amounts. Recently, because of expanded knowledge 

about the identity of genes for important pathways and mechanisms of 

pathway regulation, increasing the flux of microbially produced chemicals 

by up to six orders of magnitude (Martin et al. 2003) has been possible 

(from trace levels of primary products). A new opportunity is now offered 

to explore whether or not similar methods can be applied to developing 

modified microorganisms that secrete nontoxic molecules possibly useful 

for fuels. Examples may include alkanes, longer-chain alcohols, fatty acids 

(Voelker and Davies 1994), esters, and other types of molecules with low 

aqueous solubility that facilitate continuous product removal during 

fermentation. Advances in understanding how hydrophobic molecules are 

secreted by specialized cell types (Zaslavskaia et al. 2001) may facilitate 

the development of radically new production systems. The challenges 

described here for fermentation into hydrophobic fuels also would apply 

to potential photosynthetic systems. 

Additionally, advances in systems biology and protein engineering may 

facilitate new approaches to the overall process of fermentation. For 

instance, developing chemical regulators of cellular processes such as cell 

division may be possible to allow cultures to be held in highly efficient 

steady states for prolonged periods. Such process controls may be synergistic with the development of novel product types not normally produced in high concentrations by microorganisms. For example, cocultures 

may possibly be used for directly combining alcohols and organics into 

ether or ester production. This would be an advantageous use of acetate 

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SUGAR FERMENTATION 

released from biomass hydrolysis–—taking it from a harmful by-product 

to a fuel cosubstrate. 

Scentfic Challenges and Opportuntes 

The explosion of sequence information resulting from GTL and other 

genome sequencing programs has greatly facilitated identification of genes 

for a wide variety of processes. This information expansion also has allowed 

the development of systems tools such as whole-genome DNA chips for 

measuring gene expression. A next-phase challenge is to bring that information and associated tools to bear on identifying entire pathways and 

cellular processes of relevance to biofuel production. Additionally, understanding how such pathways and processes are regulated is essential. New 

protein-production and proteomic tools envisioned for GTL will greatly 

facilitate the elucidation of pathways and their regulation. Important challenges are to understand how the permeability properties of membranes 

are controlled by composition and how the structure of membrane proteins 

such as transporters relates to function. Progress has been slow in elucidating membrane protein structure by conventional methods, requiring new 

approaches that may be addressed by GTL. Identification of microorganisms with high levels of resistance to biofuel compounds (but not necessarily to any production capabilities) could provide useful insights into 

strategies for improving fermentation efficiency. 

The Role of GTL Capabltes 

The full suite of GTL resources for genomics and systems tools will be 

essential in clarifying the underlying mechanisms associated with these 

and related problems. Examples of the types of contributions envisioned 

are listed below. 

Protein Production 

Protein production capabilities will enable elucidation of enzyme function in 

novel pathways for biofuel production; optimization of enzymes and transporters by protein engineering and evolution; and revelation of components 

for in vitro pathways.This could lead to development of novel chemical 

regulators of microbial cellular processes for use in industrial fermentation. 

Molecular Machines 

These resources will allow nanoscale interrogation of membrane interactions with biofuel compounds (e.g., using patterned membranes);

identification of protein complexes; and mechanistic understanding of 

transporters involved in biofuel secretion. Development of nanoscale materials will facilitate product separations. 

Proteomics 

The proteomic approach involves biological-state omics for microbes under 

inhibitory stress; characterization of post-translational modifications of 

proteins that regulate enzymes or pathways for biofuel production; and 

analysis of biofuel exposure effects on microbial gene expression. 

 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Cellular Systems 

Cellular system capabilities include modeling of cellular carbon flux from 

uptake of biomass-derived sugars to secretion of finished biofuel compounds, systems engineering of batch and continuous fermentation for 

biofuel production, and modeling of protein structures in aqueous and 

nonaqueous environments. 

DOE Joint Genome Institute 

DOE JGI will characterize organisms with such useful properties as high 

productivity of or resistance to prospective biofuel compounds and will 

develop gene-expression interrogation systems. 

Other Needs 

Other needs (e.g., screening for new pathways and functions) include 

assessment of maximal redox balances (reduced fuel products yield more 

CO in fermentation). 2

Outcomes and Impacts 

If alternate fuels were made with higher fuel value (i.e., diesel, alkanes,

lipids), both separations and life-cycle costs would be altered because these 

hydrophobic fuels would separate spontaneously from water. Fuel-density 

issues of ethanol also would be reduced. Additionally, transportation costs 

might be lowered because compounds such as alkanes would be significantly less corrosive than ethanol. These biofuels could be used more easily 

in the nation’s current transportation infrastructure. If continuous fermentation with product removal were implemented, higher throughput would 

result in lower capital expenditures as well as costs associated with product 

dehydration, as in ethanol production. 

Translaton to Applcatons 

DOE EERE would lead in pilot-scale tests of strains that produce novel 

biofuels and in developing fermentation processes based on new strains,

products, and product-recovery processes. EERE would analyze the potential market and cost impacts for new and existing biofuels and then take 

the lead in separation technologies and in integrative separations. Additionally, EERE would carry out testing and possible engine-design modifications for new types of biofuels. 

Optmal Strans: Fermentatve Producton of 0% Ethanol from 

Bomass Sugars 

Current corn-to-ethanol processing plants typically produce titers of 10 to 

14% w/w. Because of limits on biomass sugar concentrations, these ethanol 

levels are at least threefold higher than those produced from lignocellulose 

using current technology and biocatalysts. Tthe dilute product stream for 

lignocellulosic ethanol imposes a two- to threefold increase in fermentation volume per annual gallon of ethanol, with corresponding increases 

in pumps, nutrients, and management. Use of modern molecular tools to 

harvest the catalytic diversity of nature should facilitate construction of 

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SUGAR FERMENTATION 

revolutionary biocatalysts that could increase ethanol titers up to 40% and 

lower needed investments in capital and operating costs for biomass-toethanol plants. This challenge is presented as an example of how systems 

biology may allow applications to exceed current biological limits. Strategies for obtaining organisms that produce and tolerate high-ethanol 

concentrations include: 

•	 Engineering current ethanol producers to retain high metabolic activity. 

•	 Engineering naturally ethanol-tolerant organisms to produce ethanol. 

•	 Exploring native diversity of microorganisms to identify those that 

retain glycolytic and fermentative activity in the presence of highethanol titers. 

Scentfic Challenges and Opportuntes 

If achieved, production of fermentation broth containing 40% ethanol from 

biomass or starch sugars would revolutionize process designs. Incremental 

progress toward this goal would reduce dramatically the size of fermentation 

plants by decreasing fermentor capacity and associated pumps, nutrient cost,

water usage, and waste-water treatment and recovery. For instance, doubling 

ethanol titers from 5 to 10% would reduce process water volume by 63% for 

equivalent ethanol production. Further doubling the titer from 10 to 20% 

would reduce the water needed by an additional 55%. For fermentation 

broth with a 40% titer, water usage would be only one-tenth the amount currently needed for the biomass-to-ethanol technology that produces 5% titer.

At ethanol titers above 40%, viable alternatives to distillation could reduce 

energy costs associated with purification. However, distillation is a mature 

technology and its energy costs do not substantially limit the current process 

because waste heat is reused. 

Improving the distillation process also would require developing better 

upstream processes or conversion technologies to provide highly effective 

sugar concentrations. A 30% biomass slurry can yield only a 15 to 20% 

sugar stream that results in just 7 to 10% ethanol. However, reflecting on 

this challenge illustrates how to further push biology beyond current limits. 

Grain ethanol plants produce from 10 to 15% ethanol (Lynd, Wyman,

and Gerngross 1999), reaching up to 20% ethanol when provided with 

very high substrate levels (Scouten and Petersen 1999). Even higher levels 

of ethanol (25%) are produced very slowly by sake yeasts at a low temperature. The most ethanol-tolerant microorganisms known, Lactobacillus 

homohiochii and L. heterohiochii, were isolated as spoilage bacteria in sake 

(Roadmap for Biomass 2002; Svenson et al. 2001; Lynd et al. 2005). The 

membrane lipids in these bacteria contain unusually long fatty chains 

that supposedly are an adaptation to growth in high ethanol. However, no 

systematic search for a class of extremophiles with resistance to ethanol has 

been reported. 

Cells need not be viable to metabolize sugars to ethanol. At a biochemical level, ethanol production from sugars is a strongly exergonic reaction 

when coupled to ATP hydrolysis. Individual glycolytic enzymes in yeast 

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and Zymomonas mobilis have been shown to function well in the presence 

of 20% ethanol (Zhou et al. 2004) and are progressively more inhibited at 

increasingly higher ethanol concentrations. In vitro disrupted cell preparations of both organisms have been shown to remain active and continue to 

produce ethanol even in the presence of 20% ethanol. Although organisms 

continuing to grow at ethanol concentrations above 30% are unlikely to 

be found, developing microorganisms that remain catalytically active and 

metabolize sugars to achieve very high levels of ethanol is quite plausible.

For instance, over half the ethanol in commercial yeast fermentations is 

produced after growth has been inhibited by accumulated by-products.

Formulating new biocatalysts for biomass presents a challenge and opportunity for engineering improvements to provide concentrated sugar feedstocks 

(Zhou et al. 2004). 

Described below are several approaches for moving toward the goal of 

producing fermentation broth containing 40% ethanol, including the isolation of native novel microorganisms capable of growing or surviving in the 

presence of high levels of ethanol. Such organisms can be used as a platform 

from which to engineer ethanol production, if needed, and as a source of 

genes and enzymes to improve alcohol production in current ethanologenic biocatalysts.This work is expected to rely heavily on GTL resources 

for sequencing and transcriptome and proteome investigations, which will 

identify molecular requirements for ethanol tolerance during growth and for 

maintenance of active metabolism in the presence of high levels of ethanol. 

Retaining the traits of current biocatalysts that do not disturb the metabolisms of all carbohydrate constituents of lignocellulose (hexoses, pentoses,

and uronic acids) will be important. Additional genes for using various 

biomass carbohydrates and other components may be needed to provide 

high-substrate levels for high ethanol titers. These genes perhaps include 

hydrolases for cellulose and hemicellulose as well as uptake systems for 

solubilized products. 

Other biomass components offer further opportunities to increase yield.

Acetate levels equivalent to 10% of hemicellulose weight represent a potential source of oxidized substrate. Equivalent levels of solubilized lignins 

represent a source of reduced substrate, a potential electron donor to convert 

portions of both substrates into additional ethanol. Cometabolizing these 

substrates by engineering known genes and pathways from soil organisms 

could increase ethanol yield up to 5%. Acid-stable products represented 

by 4-O-methyl-glucuronoxylose and 4-O-methyl-glucuronoxylobiose in 

acid hydrolysates of hemicellulose currently are not metabolized by any 

ethanologenic biocatalysts. These recalcitrant products typically are not 

measured by high-performance liquid chromatography analysis and can 

represent up to 10% of total carbohydrate. Discovery of new genes and 

organisms to metabolize these saccharides and incorporate them into biocatalysts could provide a further incremental increase in ethanol yield with 

no increase in capital or operating costs. Together, the more complete use of 

all solubilized components from lignocellulose and the increase in ethanol 

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SUGAR FERMENTATION 

titers would reduce dramatically the size of future ethanol plants and the 

amount of water use. 

Research Drectons 

The burgeoning amount of sequence information and GTL’s current ability 

to rapidly determine genome sequences for new alcohol-resistant organisms from nature provide an excellent opportunity. We can identify the 

genes and functions required for growth, survival, and continued metabolism in the presence of high levels of ethanol. Transcriptome and proteomic analyses resources will greatly assist these studies. 

Characterizing microbial membrane and wall structures, including lipids,

proteins, and carbohydrates, represents a difficult analytical problem and a 

necessary challenge for future GTL capabilities. Envelope structure is presumed to represent a major determinant for continued metabolic cell activity 

in high-ethanol and other extreme environments. New tools are needed to 

facilitate design and modification of biocatalysts for many future processes. 

Other yet-to-be-discovered opportunities include potential metabolic 

pumps for solvents and products, possibly evolved by cells to maintain low 

intracellular product concentrations. New analytical tools will be needed to 

investigate properties and functions of these biomachines. 

Process improvements also will be needed to make available the high 

amounts of sugars needed to achieve these ethanol titers, leading to solidstate fermentation in the future. 

An Alternatve Route for Bomass to Ethanol: Mcrobal Converson 

of Syngas 

Biomass can be gasified to produce syngas (mostly a mixture of CO and 

H ). Perhaps surprisingly, syngas has been shown to be converted by cer­ 2

tain microbes into products including ethanol (Klasson et al. 1990; Gaddy 

2000). These microbes are not well understood, but the process has been 

taken to small pilot scale. The attraction of this alternative approach to 

bioethanol is that the theoretical yield is quite high since all the biomass 

potentially is available as syngas for anaerobic fermentation. This gives 

theoretical yields greater than 130 gal per dry ton of biomass. 

Background 

Gasification is a combination of pyrolysis and combustion reactions for 

converting a solid material, such as biomass, to a gasified product (syngas).

Gasification is a robust and traditional technology, yet not extensively 

implemented. 

Biopower can use this syngas as a fuel for power production. Once sulfur 

compounds have been removed, this gas can be converted to other products 

through catalytic Fisher-Tropsch reactions at high temperatures and pressures. However, these precious-metal catalysts for gas-to-liquid conversion 

have been explored for over 50 years with incremental improvements. Biocatalysts for some conversion methods are relatively unstudied, operating in 

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aqueous media with the syngas bubbled past at ambient temperature and 

pressures and representing a strong alternative to traditional catalysis. 

Challenges 

How do these biocatalysts carry out transformations that otherwise work 

only with precious metals at high temperature and pressure? Which 

enzymes and molecular machines allow these transformations? Can 

increased understanding of these protein structure-function relationships 

aid development of either better biocatalysts or insights to improved inorganic metal catalysts? 

Trial and error experimentation has shown that process conditions and 

reactor design will shift the microorganisms to higher product yields. This 

is the fundamental and unexplored biological question: How does the 

regulation of the fermentation pathway allow these environmental shifts 

(e.g., pH, and medium composition) to induce higher yields? 

Syngas Status n Industry 

Bioengineering Resources, Inc. is a small company developing and soon 

to be demonstrating its pilot syngas bioethanol process (EERE 2005; BRI 

Energy 2006). The University of Oklahoma has assembled an integrated 

gasification and biology program; however, rates remain slow and are limited by the fundamental biology and mass transfer (Klasson et al. 1990). 

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SUGAR FERMENTATION 

Cted References 

BRI Energy. 2006. “Technology Summary” (www.brienergy.com/pages/

process01.html). 

EERE. 2005. “Synthesis Gas Fermentation” (www.eere.energy.gov/biomass/

synthesis_gas_fermentation.html). 

Gaddy, J. L. 2000. “Biological Production of Ethanol from Waste Gases 

with Clostridium ljungdahlii,” United States Patent 6,136,577. 

Klasson, K. T., et al. 1990. “Biological Production of Liquid and Gaseous 

Fuels from Synthesis Gas,” Appl. Biotech. Biochem. 24–25, 857–73. 

Lynd, L. R., et al. 2005. “Consolidated Bioprocessing of Cellulosic Biomass: An Update,” Curr. Opin. Biotechnol. 16, 577–83. 

Lynd, L. R., C. E. Wyman, and T. U. Gerngross. 1999. “Biocommodity 

Engineering,” Biotechnol. Prog. 15, 777–93. 

Lynd, L. R., R. T. Elander, and C. E. Wyman. 1996. “Likely Features of 

Mature Biomass Ethanol Technology,” Appl. Biochem. Biotechnol. 57–58,

741–61. 

Martin, V. J. J., et al. 2003. “Engineering a Mevalonate Pathway in Escherichia coli for Production of Terpenoids,” Nat. Biotechnol. 21, 796–802. 

Mosier, N., et al. 2005. “Optimization of pH-Controlled Liquid Hot Water 

Pretreatment of Corn Stover,” Bioresour. Technol. 96, 1986–93. 

Roadmap for Biomass Technologies in the United States. 2002. Biomass 

Research and Development Technical Advisory Committee (www.biomass.

govtools.us/pdfs/FinalBiomassRoadmap.pdf). 

Scouten, W., and G. Petersen. 1999. New Biocatalysts: Essential Tools for a 

Sustainable 21st Century Chemical Industry, Council for Chemical Research 

(www.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf ). 

Svenson, C. J., et al. 2001. “Strategies for Development of Thermophiles 

for Ethanol Production from Lignocellulosics,” presented at 23rd Symposium on Biotechnology for Fuels and Chemicals, Breckenridge, Colo., May 

2001. 

Voelker, T. A., and H. M. Davies. 1994. “Alteration of the Specificity and 

Regulation of Fatty Acid Synthesis of Escherichia coli by Expression of a 

Plant Medium-Chain Acyl-Acyl Carrier Protein Thioesterase,” J. Bacteriol. 

176, 7320–27. 

Zaslavskaia, L. A., et al. 2001. “Trophic Obligate Conversion of a Photoautotrophic Organism Through Metabolic Engineering,” Science 292,

2073–75. 

Zhou, J., et al. 2004. Microbial Functional Genomics, New York, John Wiley 

& Sons, Inc. 

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Crosscuttng 2st Century Scence, 

Technology, and Infrastructure for 

a New Generaton of Bofuel Research 

Opportuntes and Challenges 

Efficiently and inexpensively producing ethanol or alternative products such as alkanes, fatty acids, and longer-chain alcohols from 

biomass will require significant advances in our understanding and 

capabilities in three major areas explored at this workshop: Feedstocks for 

Biofuels, Deconstructing Feedstocks to Sugars, and Sugar Fermentation 

to Ethanol. A systems-level approach to understanding and manipulating 

plants and microorganisms central to processing biomass into liquid fuels 

depends on obtaining and using detailed chemical and biochemical information on organism states and structures to build functional models that 

guide rational design and engineering. A systems-level understanding of 

model plants will facilitate rational improvement of plant cell-wall composition in crops dedicated to conversion into biofuels. New approaches 

and tools will be necessary to characterize definitively the detailed organizational structures of principal types of plant cellulose and their relative 

energies and interrelationships with such other structural components as 

lignins and noncellulosic polysaccharides. 

Similarly, systems-level explorations are needed to determine genetic 

makeup and functional capabilities of such microbial communities as 

those involved in biomass decomposition and sugar fermentation. Emerging tools of systems biology—together with principles and approaches 

from metabolic engineering, synthetic biology, directed evolution, and 

evolutionary engineering—will help to overcome current obstacles to 

bioprocessing cellulosic feedstocks to ethanol. Increased emphasis must 

be placed on achieving a predictive understanding of plant and microbial biology including dynamics, regulation, flux, and function, with the 

ultimate goal of rational design to improve traits of bioprocessing microorganisms and plant feedstocks. For example, a major barrier in efficient 

use of biomass-derived sugars is the lack of microorganisms that can grow 

and function optimally in the challenging environment created through 

biomass pretreatment, hydrolysis, and cellular metabolism. A milestone in 

surmounting this barrier is to identify and understand molecular mechanisms used by cells to cope with such environmental challenges as high 

sugar and ethanol concentrations and the presence of inhibitors from biomass pretreatment and hydrolysis. Because resistance mechanisms typically involve complex subsystems and multiple genes, systems biology is 

needed for rationally engineering microbes to overcome these limitations.

Ultimately, the systems biology focus of the Genomics:GTL program References: p. 180 

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CROSSCUTTING RESEARCH 

(GTL) has the potential to enable consolidation of the overall bioconversion process and reduce the number of biorefinery operations. 

Powerful new tools and methods, including high-throughput analytical 

and imaging technologies and data-handling infrastructure, also will be 

needed to obtain, manage, and integrate information into models. Related 

tasks involve sequencing model crop-plant genomes, understanding the 

structure and function of biomass deconstruction enzymes, and modeling 

metabolic and regulatory networks of microbial systems involved in bioconversion. Some of these technologies, which either exist now or are envisioned in the GTL Roadmap, are planned components of the proposed 

GTL capability investments; others will need to be enhanced or developed 

for specific research outlined in this report. 

Analytcal Tools to Meet the Challenges 

of Bofuel Research 

Systems for effectively converting biomass to liquid fuels will require a 

wide array of innovative analytical capabilities to facilitate fundamental and 

applied science. These capabilities encompass new methods for rapid and 

sensitive analysis of biomass polymers and subunits and high-throughput 

characterization of plant cell walls and microbial populations catalyzing 

bioconversion reactions. Analytical methods should provide detailed information on chemical moieties, chemical bonds, and conformation of plant 

cell-wall polymers; they should rapidly assess the state of microbes having 

new properties or growing under various defined environmental conditions.

As discussed in preceding sections, a capability requirement is analysis of 

membrane components including lipids, proteins, and carbohydrates. These 

structures possibly represent major determinants for continued microbial activity in high-ethanol and other extreme environments. Membrane 

analytics are a challenging problem but an essential capability. New tools 

are needed to facilitate biocatalyst design and modification for many future 

processes, and technologies, methods, and computational and informational 

tools will be established to support core systems biology. Such tools include 

genomics, transcriptomics, proteomics, glycomics and lignomics, and 

fluxomics as described below; imaging technologies at various lengths and 

time scales; structure-characterization techniques; biomass characterization;

cultivation; and accompanying modeling and simulation capabilities. 

Genomcs 

Capitalizing on potential biofuel production from cellulosic biomass 

requires the continued commitment of DOE’s Joint Genome Institute to 

sequence various organisms that contribute to the process. These organisms 

include crop plants, industrial yeast strains, and microbial communities 

involved in biomass decomposition and soil productivity. Genetic blueprints provided by DNA sequences will allow the use of systems biology 

for rational design and process consolidation to increase production of 

biomass crops and optimize biofuel conversion processes. Support will be 

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required for sequence assembly, optical mapping, and other techniques to 

place assembled contigs from a plant, microbe, or community on a physical 

map. Comparative genome analyses will infer similarities or differences in 

regard to other organisms, and expressed sequence tag and other libraries 

will facilitate genetic and molecular analysis of feedstock plants. 

Genomic information will enable identification and molecular characterization of the diversity and activity of relevant biomass deconstruction enzymes from such new sources as fungi, bacteria, and uncultured 

microbes. Genomic analysis will open the door to use of genetic resources 

present in microbial communities specializing in lignocellulose degradation. Genomic projects are envisioned in the following general areas. 

Feedstock Plants 

This report outlines the importance of new and improved sources of feedstock biomass for conversion to liquid fuels. Considering project scale, soil 

and climate differences across the United States, and the desire to maintain 

biological diversity on agricultural lands, investigators recognize that a variety of biomass crops must be used. Grasses potentially are a prime source 

of biomass, so determining the DNA sequence of one or more grasses is a 

high priority. The genetic blueprint will assist in short-term development 

of grass-breeding strategies and the use of longer-term systems biology 

methods to realize the plant’s potential as a source of biomass. Some plant 

systems to be studied include Brachypodium (slated for sequencing at DOE 

JGI) and Populus (already sequenced). 

Sol Mcrobal Communtes 

Conversion into a high-value commodity is likely to alter the amount 

and composition of postharvest plant material and residues returned to 

the soil. Sustainability of biofuel technologies requires a scientific assessment of production practices and their influence on soil quality, including 

impacts on microbial communities and the processes they catalyze. Given 

that the technology involves crops tailored to thrive in individual regions,

a number of sites representing various ecosystems must be chosen for soil 

metagenomic analysis. In the short term, such analyses will contribute to 

an understanding of effects on soil sustainability. The data also will be critical in preventing unwanted long-term effects on soil sustainability and for 

assessing more-direct effects of soil microbes on plant growth. 

Fermentaton and Bomass Decay Communtes 

Biomass conversion to sugars and biofuels requires optimizing microbial 

breakdown of structural sugars and fermentation of complex sugar mixtures.

A number of microbial communities have evolved over millions of years to 

maximize and coordinate these capabilities, with several of the better-studied 

ones associated with ruminants and the hindgut of termites.Thus, a reasonable number of model fermentative communities that can degrade lignocellulose are critical targets for metagenomic analysis. In the short term, new 

insights into activities present or coordinated among members of these wellestablished communities will result.This information potentially can be used 

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CROSSCUTTING RESEARCH 

to design second-generation biofuel systems composed of microbial communities or consortia considered more robust and diverse in regard to environmental conditions they can withstand and types of substrates they can use. 

Increased Producton Systems 

Adaptive evolution of microorganisms using selective pressure in fermentors or chemostats has enormous potential to select for traits that benefit 

biofuel production. To understand mutations that give rise to desired traits,

determining the molecular basis of such changes is critical; we need to 

sequence industrialized strains and compare them to their progenitors. In 

the long term, this information is vital in designing new microbial systems 

that increase bioconversion efficiency and lower biofuel cost (see sidebar,

Proteomic and Genomic Studies of Industrial Yeast Strains and Their 

Ethanol-Process Traits, p. 126). 

Transcrptomcs: Hgh-Throughput Expresson Analyses 

Measuring RNA expression (transcriptomics) provides insight into genes 

expressed under specific conditions and helps to define the full set of cell 

processes initiated for coordinated molecular response. This information can 

be used to elucidate gene regulatory networks and evaluate models of cellular metabolism. Current technologies include microarray-based approaches 

applicable to the study of plants and homogeneous microbial populations 

in well-mixed systems. Custom microarrays targeted to new plant varieties 

or microorganisms are critically needed. In heterogeneous systems such as 

those in mixed microbial populations, current microarray-based approaches 

are less useful because they provide only an average gene-expression profile 

across an entire population. Therefore, single-cell gene-expression methods 

applicable to diverse cell types are required. 

Proteomcs 

Although carbohydrates are biomass conversion’s primary substrate, proteins are workhorse catalysts responsible for constructing biomass-forming 

polymers, depolymerizing biomass before fermentation, and converting 

sugars to desired end products such as ethanol. To interrogate these various 

processes, identifying and quantifying proteins and protein complexes for 

various plant and microbial systems and subsystems are vital. The genome’s 

information content is relatively static, but proteins produced and molecular machines assembled for specific purposes are dynamic, intricate, and 

adaptive. All proteins encoded in the genome are collectively termed the 

“proteome.”The cell, however, does not generate all these proteins at once;

rather, the particular set produced in response to a specific condition is 

precisely regulated, both spatially and temporally, to carry out a process or 

phase of cellular development. 

Proteomics can be used to explore a microbe’s protein-expression profile 

under various environmental conditions as the basis for identifying protein 

function and understanding the complex network of processes facilitated 

by multiprotein molecular machines. Identifying the suite of proteins 

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involved in construction and breakdown of cell-wall polymers is important 

in understanding biomass conversion to biofuels. In addition to analysis of intracellular proteins, all degradation enzymes and other proteins 

secreted by biomass-degrading microbes need to be characterized because 

they probably are key to the depolymerization process. Membrane protein 

systems are particularly important and need to be improved because they 

control cellulose production, cellulolytic enzyme excretion, and fermentable sugar transport. 

To facilitate mass spectroscopy (MS) detection and quantitation of proteins in complex mixtures, improvements in instrumentation and chemistry are needed to enhance protein and peptide ionization, increase 

sensitivity and mass resolution, quantify protein levels in complex mixtures,

and widen the dynamic range of their detection. New information and bioinformatic tools are required to analyze proteomic data. 

Metabolomcs 

Analysis of the cell’s metabolite content has lagged behind transcript and 

protein profiling but is an equally important indicator of cellular physiology. The metabolite profile may be a better indicator of cell physiology 

because metabolite concentrations (and fluxes) occur sooner in response 

to changes in the extracellular environment than do gene expression and 

protein production. Also, metabolites are precursors to the cell’s transcripts 

and proteins and all other cell macromolecules, and regulation of cellular 

processes may not always be reflected in transcript or protein profiles. 

The difficulty in profiling metabolites derives from their structural heterogeneity and short lifetimes inside the cell. Proteins and RNA are each 

composed of a constrained or limited set of precursors (amino acids in 

proteins, nucleic acids in RNA). Differences lie in amino acid or nucleic 

acid sequences, enabling a single separation method to analyze the entire 

transcript or protein profile at once. Unlike RNAs and proteins, metabolite heterogeneity makes analysis nearly impossible with a single separation technique. Although such techniques (e.g., high-performance liquid 

chromatography, thin layer chromatography, gas chromatography, and 

capillary electrophoresis) can separate metabolite groups having common 

structures, very few reports on separation and analysis of a comprehensive 

set of cellular metabolites have been issued. Furthermore, metabolites tend 

to have much shorter (seconds or less) half-lives than do proteins (hours) 

or RNAs (minutes). Shorter half-lives make rapid metabolism sampling 

and quenching even more important than analysis of RNAs and proteins.

Methods such as nuclear magnetic resonance (NMR) that can effectively 

integrate and store information about flux through particular pathways and 

potentially can be applied to living systems. Localized metabolite concentrations such as those within an organelle also need to be measured. 

Another complication is that a metabolite’s intracellular concentration 

says little about its importance in cellular physiology. Indeed, some of the 

most potent cellular-signaling molecules found recently exist at relatively 

low concentrations inside the cell, and some metabolites produced and 

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CROSSCUTTING RESEARCH 

consumed at the highest rates have relatively low intracellular concentrations. In the former case, extremely sensitive methods are necessary to 

measure these metabolites. In the latter case, the metabolic flux profile 

is more important than absolute metabolite concentrations (see section,

Fluxomics, p. 161). 

Metabolomic research will address fuel production, including methods to 

isolate, extract, and analyze labile metabolites such as those involved in cellular energy metabolism (e.g., ATP, GTP, NADP, and NADPH). Multidimensional separation methods are vital to analyzing plant or microbial-cell 

metabolites. New analytical techniques are required for continuous metabolite measurements, monitoring processes, and in vivo metabolite analyses. 

Glycomcs and Lgnomcs 

As parallel concepts to transcriptomics, metabolomics, and proteomics 

described in the GTL Roadmap, biomass-to-biofuels research will use 

similar high-throughput and high-content analysis with compounds having low molecular weight and involvement in synthesis and degradation of 

plant cell-wall polymers. Glycomics (profiling materials related to structural polysaccharides) and lignomics (profiling materials related to lignin) 

are essential capabilities. New powerful analytical tools will provide information for systems-level understanding of biomass structure and chemistry 

and its role in biomass conversion to fuels and valuable chemicals. 

Information from these analyses will help us understand native and 

modified pathways for synthesis of cell-wall polymers by tracking precursor consumption, generating and utilizing intermediate structures, and 

exploring their connection to plant cell-wall chemical composition and 

physical-chemical structure. A “toolbox” of options for plant breeders and 

crop scientists eventually will be available to improve feedstock substrates 

specifically for fuel production. New glycomic and lignomic tools also will 

be applicable directly to other plant-development needs. 

Information obtained from lignomics and glycomics will elucidate substrate modification by tracking structural changes and concentration fluxes 

in saccharification products that may be linked to harsh pretreatments.

Saccharification-product profiling also will guide selection of appropriate 

enzymatic cocktails by identifying specific chemical bonds and functional 

groups in solids and larger oligomeric products. The addition of enzymes 

tailored to known recalcitrant structures could significantly enhance 

product yields. Information on the nature of recalcitrant structures also 

can be used to guide plant breeding and pretreatment conditions. These 

approaches will provide insight into rate-limiting steps that arise in substrate- and enzyme-limited systems. 

Accurate and robust glycomic and lignomic analytical tools will play an 

important role in fermentation research. Many lignin- and carbohydratederived compounds are fermentation inhibitors, and other materials may 

be converted to undesirable side-products. Tracking these materials from 

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feedstock through products will provide valuable insights into possible 

improvements in all stages of biomass-to-biofuels conversion. 

Several specific challenges persist in the application of high-sensitivity,

high-throughput tools for lignin and carbohydrate analysis. Many robotic 

and automated tools used in conventional metabolomics research cannot 

accommodate the larger sample sizes required for representative sampling 

of biomass substrates. Multivariate analysis tools will need to be developed 

and validated for accurate quantification of complex mixtures of substrates,

pretreatment catalysts, conversion enzymes, and microbes. Many analytical methods for supporting a systems approach to biomass conversion will 

require biomass standards not readily available from commercial sources.

Obtaining small-molecule standards will require prep-scale synthesis and 

isolation of molecules of interest and the ability to modify (e.g., by isotopically labeling) isolated materials using advanced techniques of carbohydrate and natural-product organic synthesis. 

Fluxomcs 

Use of microorganisms for liquid-fuel production from lignocellulosic 

biomass depends on fluxes of diverse substrates through complex networks 

of metabolic pathways. Quantifying metabolic fluxes in microorganisms 

allows identification of rate-limiting steps in a biosynthetic pathway that 

could be improved by genetic manipulation or by alterations in cultivation conditions. Substrate turnover by an enzyme in a metabolic pathway 

can be determined by measuring changes in isotopically labeled substrate 

levels over a specific period of time. Using isotopomer analysis, quantities 

of labeled substrates and metabolic products derived from them can be 

measured and compared. Similar approaches can be used to map metabolic 

sugar fluxes to ethanol and related pathways. 

Simultaneously measuring the turnover of all intracellular metabolites 

often is difficult, if not impossible. Once flux for certain enzymatic reactions is measured in the laboratory, flux through other cellular pathways 

can be calculated using mathematical models of the entire metabolic network. By modeling a given organism’s metabolism, scientists can quantify 

the effects of genetic manipulations or changes in growth conditions on 

the cell’s entire metabolic network. Inputs to flux-based models are the set 

of potentially active metabolic reactions and measurements of the steadystate production rates of such metabolites as DNA, RNA, carbohydrates,

fatty acids, and proteins. Improved estimates of cell fluxes can be obtained 

by feeding a labeled carbon source and using measured transformed fluxes 

as input to the model. 

A range of techniques and methods will be needed to determine fluxes in 

metabolic pathways of microbial cells employed for biomass fermentations.

These techniques include stable and radioactive isotope labeling and associated methods for estimating unmeasured fluxes from isotope distribution 

in metabolites and macromolecules. MS- and NMR-based methods will 

determine isotope distributions in cellular metabolites and macromolecules. 

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CROSSCUTTING RESEARCH 

The Super Imager 

The potential is to create compound,

multifunctional instruments that 

individually include many of the 

following capabilities: 

•	 Mapping of molecular species such as 

RNA, proteins, machines, and metabolites through the use of fluorescent tags of 

various kinds 

•	 Multiple excitation and detection wavelengths including both fluorescent and 

infrared absorption methods 

•	 High-speed 3D imaging 

•	 Nonlinear contrast imaging including 

second- and third-harmonic generation 

and coherent Raman scattering 

•	 Lifetime mapping as sensitive probes of 

local environments 

•	 Rotational correlation mapping for in 

situ analysis of protein structure and 

function 

•	 Magnetic resonance imaging with 

10-micron–scale analyses of metabolite 

concentrations and providing data on diffusion properties and local temperatures 

•	 Acoustical imaging at micron-scale resolution of the system’s physical parameters 

•	 Atomic force microscopy (AFM) 

mapping of structures with added 

information provided by the controlled 

interaction of light and sharp metallic 

AFM tips to obtain optical resolutions of 

~20 nm, one-tenth the diffraction limit 

•	 High spatial resolution (nanometer scale) 

using X-ray and electron microscopies,

including the use of special DOE facilities or perhaps the development of laboratory-based X-ray sources for imaging 

[Sources: Report on the Imaging Workshop 

for the Genomes to Life Program, Office of 

Science, U.S. Department of Energy, 2002 

(www.doegenomestolife.org/technology/

imaging/workshop2002/); GTL Roadmap,

pp. 182–87] 

New analytical techniques are needed to quantify extracellular 

metabolites and quickly and easily determine biomass composition. 

Enzyme Structure and Functon 

Developing and deploying improved enzymes and multienzyme 

complexes for biomass deconstruction and conversion will require 

more understanding and the production of suitable substrates as 

well as enzymes and their appropriate complexes. They will be 

used to achieve a mechanistic understanding of cellulose and cellwall interactions with degrading enzymes. 

Definng and Producng Substrates 

Despite nearly 100 years of pretreatment research, detailed understanding of cellulosic biomass’s fundamental physical and chemical features is still lacking. The plant cell wall—the substrate for 

degradative enzyme systems—is complex, containing various forms 

and quantities of cellulose, hemicellulose, and lignin. Chemical 

and physical pretreatment may alter biomass to make enzymatic 

feedstock digestion more difficult, so merging pretreatment and 

deconstruction steps would simplify processing. Critical to understanding any enzymatic reaction is having both a reliable assay 

and a defined substrate, thus allowing activities to be measured 

accurately. Constructing standardized substrates suitable for highthroughput assays is required to optimize these enzymes. Synthesis 

and characterization of model substrates require multiple technologies, first to define the substrates by employing multiple analytical 

and computational methods and then to synthesize the substrates.

High-throughput methods are needed for compositional analysis 

and characterization of biomass substrates, once a family of basic 

reference structures has been identified. 

Identfyng Enzymes and Degradatve Systems 

Multiple classes of enzymes are required for biomass conversion 

to achieve maximum sugar yields including hemicellulases and 

ligninases. The rate-limiting step is making cellulose accessible for 

subsequent saccharification steps. Surveying and identifying suitable enzymes require many technologies listed in the GTL Roadmap, including metagenomic analyses of model biomass-degrading 

communities, high-throughput protein expression, generation 

of affinity tags, reassembly of complexes, activity measurements,

and biochemical and biophysical characterizations of enzymes 

and complexes. Degradative systems fall into two general classes:

Individual enzymes working synergistically; and the cellulosome,

a large, mega-Da complex normally attached directly to cells and 

found to date only in anaerobic bacteria. The cellulosome is a 

LEGO-like system with many interchangeable structural and degradative protein components deployed when specified for particular 

substrates. This system provides the basis for engineering to create 

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cellulosomes optimized for particular substrates. The cellulosome can be detached from cells and functions, even under 

aerobic conditions. With appropriate modifications, the cellulosome has potential for enhanced stability and activity. Individual 

enzymes and those in cellulosomes might benefit from immobilization technologies designed to increase activity and stability.

How membrane proteins such as sugar transporters interface 

with cellulosomes attached to cell walls is unknown (see sidebar,

The Cellulosome, p. 102). 

Imagng Technologes 

“Thought is impossible without an image.” —Aristotle, 325 B.C. 

Imaging technologies with wide applications will be critical to 

many of the research challenges identified in this report.

Biomass deconstruction is a crucial step in conversion, yet 

relatively little is known about the detailed molecular structure 

of plant cell walls and how they are constructed from various 

components. To address this limitation, new and improved 

methods are needed to analyze plant cell-wall composition and 

structure at the nanometer scale. Organization of polymer 

components in biomass structures should be analyzed in three 

dimensions using noninvasive tools. Such new capabilities are 

anticipated to reveal key molecular processes occurring in real 

time during the full life cycle of cell-wall formation and 

maturation and during experiments to transform crop species 

into feedstocks suitable for bioconversion by optimizing cellwall makeup. Many requirements generally have been anticipated by GTL and by capability development and planning as 

documented on pp. 182–87 in the GTL Roadmap (U.S. DOE 

2005) and Imaging Report (U.S. DOE 2002). 

Imaging needs for each specific research area are summarized 

below. They include advances in a wide range of imaging technologies using NMR, optical, X-ray, and electron-based methods as well as atomic force and scanning tunneling microscopies.

Given differences in resolution, sensitivity, and selectivity, the 

full impact of these methods will be realized best when used 

in combinations, either within a single instrument (see sidebar,

The Super Imager, p. 162) or parallel applications assembled and 

correlated through advanced image-management software. 

Imagng Needs for Feedstock Research 

Within 5 years, methods will be developed and deployed for 

chemical-specific imaging over a wide range of spatial scales 

(0.5 nm to 50 μm), with contrast methods and tags enabling 

many molecular components to be distinguished easily. Imaging of living (or never-dried) materials is perhaps just as 

Some Imagng Technologes 

Relevant to Feedstock 

Characterzaton 

Atomc Force Mcroscopy (AFM) 

In AFM, a scanning-probe technique 

allows the direct study of surface using 

tapping probes, the tips of which project 

less than a micron. The dynamic behavior 

of surfaces and molecules often can be 

observed. A great advantage of scanning 

force microscopy over most other highresolution techniques is its ability to operate 

in a liquid environment. High-resolution 

AFM images of a cellulose surface recently 

have been reported. Using AFM to support 

interpretation of pretreatment and enzyme 

action on biomass surfaces represents a 

tremendous opportunity. 

Scannng Electron Mcroscopy (SEM) 

SEM really is the backbone of traditional 

biological surface analysis. New developments in the design of SEM sample 

chambers and “optics” permit the analysis of 

samples containing some natural moisture,

which is critical for biomass fractions. 

Also, new strategies for creating replicas 

of biological samples provide more versatility in analyzing proteins and microbialcell surfaces. 

Transmsson Electron Mcroscopy (TEM) 

Cryoelectron microscopy is the leading 

technique for high-resolution molecular 

machine structural-biology research, and 

TEM is the most common platform for 

this method. The technique makes the 

following special demands on electron 

microscopy: Ultrahigh and clean vacuum 

for contamination-free observation,

stable low-drift cryotemperature holders, special functions to provide low-dose 

imaging conditions, and cooled slowscan charge coupled display cameras for 

low-dose digital image recording. 

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CROSSCUTTING RESEARCH 

critical as imaging harvested materials that have been dried, either for 

storage in a processing facility or for structure examination. These imaging capabilities should enable observation of cell-wall deconstruction and 

construction. New optical technologies and advances at synchrotron light 

sources will allow some imaging methods to function as high-throughput 

devices, producing hundreds to thousands of images per minute with a 

single instrument. Important possible consequences are dramatic reduction of cost and processing time per sample and greater community 

access to these technologies as part of a user facility. Second, enhanced 

imaging speed enables real-time observation of biological processes.

Availability of real-time data is vital to building and validating models 

that allow a systems biology approach. 

Temporal imaging of dynamic subcellular small molecules, including 

metabolites, is among advances expected to take longer development 

times. Improved molecule-specific imaging tags and a means for introducing them into cells without disrupting function are critical needs.

The scale of various biomass conversions will require enhanced imaging 

for obtaining high-resolution, large-volume tomographic images as well 

as real-time imaging of living systems at high spatial resolution and for 

use in the field at lower spatial resolution. The critical aspect of enzyme 

engagement with biomass components, such as cellulase interfacing with 

cellulose, requires new capabilities for characterizing enzyme binding sites 

by atomic force microscopy, scanning electron microscopy, transmission 

electron microscopy, and electron spectroscopy for chemical analysis [see 

sidebar, Some Imaging Technologies Relevant to Feedstock Characterization, p. 163, and Image Analysis of Bioenergy Plant Cell Surfaces at the 

OBP Biomass Surface Characterization Lab (BSCL), p. 40]. Similarly,

capabilities are needed to characterize interactions between microbial cells 

and their solid-phase substrates. 

Imagng Needs for Mcrobal Communtes n Deconstructon and 

Converson of Bomass to Ethanol 

Meeting imaging requirements for processing biomass to ethanol will draw 

on the above capabilities but will focus more on microbial-cell and -community imaging to aid in delineating sub- and extracellular organization of 

proteins, protein complexes, transporters, and metabolites. Appropriate tags 

and methodologies will be required for investigating multicellular interactions in mixed microbial populations (e.g., those observed in fermentation) 

and communities (e.g., those observed in colonization of decaying biomass) 

with solid substrates such as plant cell walls. Understanding these interactions will require information on amounts, types, and locations of secretion 

products, reactions they catalyze, and how these reactions and products 

impact cell viability. Imaging-based approaches are essential to achieve 

temporal and spatial resolution sufficient for understanding these processes 

(see Fig. 1. Probing Microbial Communities, p. 165). 

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Fig. 1. Probing Microbial Communities. Microbial communities and ecosystems must be probed at the 

environmental, community, cellular, subcellular, and molecular levels. The environmental structure of a 

community will be examined to define members and their locations, community dynamics, and structurefunction links. Cells will be explored to detect and track both extra- and intercellular states and to 

determine the dynamics of molecules involved in intercellular communications. Probing must be done at 

the subcellular level to detect, localize, and track individual molecules. Preferably, measurements will be 

made in living systems over extended time scales and at the highest resolution. A number of techniques 

are emerging to address these demanding requirements; a brief listing is on the right side of the figure.

[Figure source: GTL Roadmap, p. 176 (http://doegenomestolife.org/roadmap/).] 

Mcrobal Cultvaton 

The biorefinery environment is complex, and cultivation technologies must 

be capable of reproducing critical aspects of industrial systems. Efficient 

conversion of biomass to liquid fuels will require new and innovative 

approaches for controlling cultivation and simultaneously monitoring cell 

physiological states and metabolic processes under a range of conditions.

Such capabilities are needed to identify gene regulatory and metabolic 

networks and to develop and evaluate modes of cell metabolism. Obtaining a systems-level understanding of fermentation organisms will require,

in some cases, thousands of samples from single- and multiple-species 

cultures; and technologies for continuously monitoring and controlling 

culture conditions and interrogating the physiological state of microbial 

cells. Relatively homogeneous and complex microbial populations will 

be tested in physically and chemically heterogeneous environments such 

as those associated with solids (plant biomass). Necessary infrastructure 

will support cultivation at scales sufficient to obtain adequate amounts of 

sample for analysis and to grow microbial cells in monocultures and in 

nonstandard conditions (e.g., in association with solids or biofilms). These 

cultivation systems will be enhanced by advanced computational capabilities 

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CROSSCUTTING RESEARCH 

Laboratory Cultvaton Technques to Smulate Natural Communty Structure 

To identify the function of genes preferentially expressed by specific populations in a structured microbial community, such as those deconstructing biomass in soils or in a bioreactor, new cultivation 

techniques are being devised. During the past decade, researchers have developed reactors in which 

biofilms can be imaged using confocal scanning laser microscopy (CSLM) and 

other light-microscopic techniques (Wolfaardt et al. 1994). When combined with 

fluorescent in situ hybridization to distinguish populations of cells in multipopulation biofilms and fluorescent reporters (green fluorescent protein) of functional 

gene expression, CSLM has been used to demonstrate how gene expression by 

one population affects gene expression in another proximally located population 

(Moller et al. 1998). 

The mobile pilot-plant fermentor shown here has a 90-L capacity and currently is 

used to generate large volumes of cells and cell products such as outer-membrane 

vesicles under highly controlled conditions. Future generations of fermentors will 

be more highly instrumented with sophisticated imaging and other analytical 

devices to analyze interactions among cells in microbial communities under an 

array of conditions. 

Pacifi c Northwest National Laboratory 

that allow simulation of cultivation scenarios and identification of critical 

experimental parameters. 

Most industrial bioconversions rely on pure cultures while, in contrast,

environmental bioconversions are more commonly catalyzed by mixed 

populations or communities with “specialists” working together in an 

apparently integrated and stable fashion. A fundamental question is, Are 

there stable, self-regulating multiplex solutions for biofuels? In this regard,

new techniques are needed to understand and reproduce composition and 

function in stabilized mixed cultures (see sidebar, Laboratory Cultivation 

Techniques to Simulate Natural Community Structure, this page). 

Biological systems are inherently inhomogeneous; measurements of the 

organism’s average molecular-expression profile for a cell population cannot be related with certainty to the expression profile of any particular cell.

For example, molecules found in small amounts in samples with mixed 

microbial species may be expressed either at low levels in most cells or at 

higher levels in only a small fraction of cells. Consequently, as a refinement, new analytical and imaging techniques will be necessary to interrogate individual cell physiological states in heterogeneous culture systems.

For experimentation in all aspects of work with omic tools, production of 

high-quality reproducible samples is paramount. 

Data Infrastructure 

Progress toward efficient and economic processes for converting biomass 

to biofuels will benefit from a research-data network able to manage,

preserve, query, and efficiently disseminate large amounts of experimental and analytical data, as outlined in the GTL Roadmap. Because many 

research activities are data intensive and will engage a large number of 

investigators from national laboratories, academia, and the private sector, these activities will benefit from a distributed data environment. 

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Fig. 2. GTL Integrated Computational Environment for Biology: Using and Experimentally Annotating 

GTL’s Dynamic Knowledgebase. At the heart of this infrastructure is a dynamic, comprehensive knowledgebase with DNA sequence code as its foundation. Offering scientists access to an array of resources, the 

knowledgebase will assimilate a vast range of microbial and plant data and knowledge as they are produced.

[Figure source: GTL Roadmap, p. 83 (http://doegenomestolife.org/roadmap/).] 

Consisting of federated databases and repositories, the data environment 

will comprise descriptive, quantitative, and visual information, method 

libraries, query and data-mining tools, and a communication network.

This environment provides controlled sharing and management and communication of biochemical, genetic, and other types of biological data 

and information. When available through a single portal, such a cyberinfrastucture can have a dramatic impact in efficient use of new knowledge.

Additional benefits from efficient data sharing include fostering collaboration among projects, facilitating standards development, and accelerating implementation of new enabling technologies. GTL also will need a 

centralized research data network for model plants and specific biomass 

crop plants. 

Data-infrastructure requirements of biomass-to-biofuels research align 

extensively with the Integrated Computational Environment for Biology 

described in the GTL Roadmap (see Fig. 2. GTL Integrated Computational Environment for Biology, this page), reproduced as a programmatic subset of the GTL information infrastructure. Biomass-to-biofuels 

research will require multiple databases, imaging archives, and LIMS in 

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CROSSCUTTING RESEARCH 

each of its major research areas: Feedstock for Biofuels, Deconstructing 

Feedstocks to Sugars, and Sugar Fermentation to Ethanol. 

For example, research on deconstructing feedstocks will necessitate (1) 

LIMS for tracking and documentation of samples for enzymatic pretreatment experiments and high-throughput compositional analysis of cell-wall 

material; enzyme databases for characterizing such key enzymes as glycohydrolases, esterases, and ligninases; metagenomic sequence databases for 

characterizing lignocellulolytic and other microbial communities; imaging 

repositories for a vast array of microscopies including optical, X ray, electron, and atomic force; and archives for molecular dynamic simulations of 

the molecular machinery that breaks down plant cell-wall components. 

Feedstock and fermentation research goals have comparable data-infrastructure needs, with descriptive and quantitative data types differing mainly in 

experimental and analytical methods employed. Proteomic, metabolomic,

transcriptomic, and plant and microbial genomic data will require corresponding databases and integration within the GTL Knowledgebase. 

A variety of computational tools will be needed to support biomassto-biofuels research and such enabling technologies as proteomics and 

imaging. For example, computational tools are necessary for streamlining 

the conversion of isotopomer flux-tracing data into flux distributions for 

subsequent analysis; the process currently is limited by the computational 

sophistication of required methods rather than by the ability to generate raw data. High-throughput, automated image acquisition, storage,

processing, and analysis will be required for examining data on molecular 

machines in vivo. These tools also will aid in localizing and validating 

complexes, dynamics, docking, intercellular communication, extracellular 

matrix, and metabolite distribution. 

Computatonal Modelng 

Computational technologies will be critical to the overall platform that will 

enable success in developing effective technologies for converting biomass 

to biofuels. Significant amounts of data generated at a number of investigative levels must be made accessible to investigators at different institutions. Data sets will need to be analyzed and used to develop and evaluate 

computer models that will contribute to guiding overall decision making 

and program design. These decisions could involve the best lignocellulosic 

composition of biomass for a given process and how to create it as well as 

designing enzymes or engineering microbes for optimal ethanol production. 

Computational modeling technologies and tools will be needed to address 

these challenges (see Fig. 3. From Genome Data to Full Cell Simulation,

p. 169). Some key needs and examples are discussed below. 

Modelng: Genome Sequence Analyss 

A wide range of organisms across many different kingdoms of life—

including plants, fungi, and bacteria—are relevant to biomass conversion to 

biofuels. With the power of modern genome sequencing and capabilities at 

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Fig. 3. From Genome Data to Full Cell Simulation. This concept diagram schematically illustrates a path from 

basic genome data to a more detailed understanding of complex molecular and cellular systems. New computational 

analysis, modeling, and simulation capabilities are needed to meet this goal. The points on the plot are very approximate, depending on the specifics of problem abstraction and computational representation. Research is under way 

to create mathematics, algorithms, and computer architectures for understanding each level of biological complexity.

[Figure source: GTL Roadmap, p. 89 (www.doegenomestolife.org/roadmap/).] 

DOE’s Joint Genome Institute, obtaining genome sequences for essentially 

any organism is possible. Research will use sequence-analysis tools surpassing traditional genome annotation. The complexity of biomass conversion 

to biofuels will necessitate the evaluation and integration of sequence data 

from multiple organisms. The need to establish functional roles for a multitude of proteins involved in mediating plant cell-wall biology and defining metabolic and regulatory pathways will require robust bioinformatic 

capabilities. Within each genome sequence is a wealth of information that 

can be mined and accessed to improve our knowledge about these organisms. In some cases, traditional bioinformatic approaches may be sufficient.

Many areas, however, still need research advances. 

Sequence-based analysis methods and tools are vital in many areas. Creation of an improved-quality standardized pipeline for genome-sequence 

annotation, including definition of open reading frames and functional 

assignment of genes, is a critical need for essentially all sequenced organisms that are subjects of GTL mission-directed research. These annotations 

need to be updated continuously because advancements can be rapid in 

many areas and should be applied to single organisms, including plants,

and metagenomes of soil ecosystems and fermentative consortia. Specific 

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CROSSCUTTING RESEARCH 

to biomass-to-biofuels research are computational methods for improving 

analysis of metagenomic data to identify new enzymes related to lignocellulose degradation and fermentation. Directed evolution has the potential 

to rapidly evolve new and useful microbial strains for fermentation reactions. Comparative genomics of evolved microbes following resequencing will identify specific changes in the genome. Comparative analyses of 

multiple plant genomes will generate family clusters of gene components 

mediating cell-wall biosynthesis (i.e., enzymes, regulatory factors, and 

processing proteins). A more generic requirement is enhanced annotation related to recognition of interacting proteins, regulatory signals, and 

structures. 

Modelng: Molecular 

A number of critical biochemical processes, occurring at the molecular 

scale, govern the overall conversion of biomass to biofuels. These processes 

include, for example, mobilization of building blocks for plant cell-wall 

synthesis, machinery for cell-wall formation in plants and their biophysical characteristics, degradation of lignocellulosic materials by enzymes, and 

transport of sugars into microbes for further metabolism and conversion to 

desired end products. In combination with structural information, mechanistic models of plant-derived polymeric substrates and their enzymatic 

conversion are needed to identify “bottlenecks” in bioconversion of lignocellulosic biomass. Gaining a detailed understanding of these mechanisms 

will help in designing superior technologies to address key barriers. As a 

component of this research, new computer programs and codes are needed 

for ultralarge biological models of more than a million atoms to enable the 

analysis of complex molecular machines such as the cellulosome. 

Molecular-scale models are critical to a number of biomass-to-biofuels 

aspects. For example, biophysical models of plant cell-wall composition 

are needed to delineate pathways for synthesis and transport of key cellwall components. Our knowledge of fundamental polysaccharide-water 

interactions within lignin carbohydrate complexes will benefit greatly 

from molecular dynamics simulations, as will our understanding of water 

and chemical transport along or through the plant cell wall. Dynamic 

molecular models will aid in determining cell-wall enzymatic degradation 

and interactions among these enzymes and their substrates. Specifically,

molecular models of such cell-wall–degrading enzymes as hemicellulases 

and ligninases will help to establish structure-function relationships for 

substrate interactions and guide the development of novel enzymes. Similarly, dynamic modeling of large molecular machines and their substrates 

on nano- to millisecond measures will provide insights into the structure 

and function of key protein complexes such as the cellulosome. Molecular modeling at a scale exceeding that for individual enzymes will enable 

understanding of critical events occurring at the interface between cellulose 

and an adhered cell. On the microbial side, models are needed to predict 

membrane composition and changes in response to stress induced by high 

concentrations of fermentation end products. 

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Modelng: Pathways and Networks 

Molecular machines that carry out individual transformations and processes important in biomass-to-biofuels conversions often operate within 

much larger systems of interacting proteins and metabolic pathways in cells.

Understanding, controlling, and manipulating the overall phenotypical state 

of these systems in either plant or microbial cells will be necessary to overcome identified technical barriers. Computational models of these cellular 

systems and pathways will facilitate integrative analysis of experimental omic 

data sets while also providing the basis for predictive simulations that can 

generate testable hypotheses.To increase the efficiency of fermentation reactions, improved metabolic and regulatory models are needed to understand 

mechanisms that control glycolytic flux and its impact on cellular metabolism. Because careful alterations of growth, energy, and redox conditions 

often are required to optimize fermentation reactions, robust models focused 

on analysis and regulation of cellular energetics are necessary. Regulatory 

and metabolic modeling at community and individual levels is essential for 

identifying and understanding gene regulatory networks. 

A number of specific biomass-to-biofuels topics will require systems-level 

models and simulation methods. Genome-scale models of key industrial 

microbes are needed to understand metabolism details and allow rational 

design for improved biofuel production, including ways to identify novel 

biotransformation routes. Dynamic pathway models incorporating key 

enzyme kinetics for ethanol-producing pathways and other relevant cellular subsystems are a specific need. Methods also are required to assess 

physicochemical limitations of cellular systems and enzyme components 

to determine maximum-achievable metabolism rates (e.g., identify ratelimiting steps, both kinetic and diffusion limited). From the plant perspective, detailed pathway models of cell-wall biosynthesis in the context 

of overall plant metabolism are needed to generate testable hypotheses for 

controlling cell-wall composition. These models should incorporate information about catalytic activity, gene expression, mechanisms of control,

and interspecies variations. 

Generic modeling capabilities, many of which are required in the broader 

GTL program, include integrated modeling of metabolic pathways and 

regulatory networks and simulation of their functional capabilities. In support, automated techniques are needed to integrate data sets and rapidly 

reconstruct metabolic pathways and gene regulatory networks. Methods 

also will be required to incorporate the next level of complexity into systems-level cell models by adding information from cellular-component 

imaging. Other methods will account for the impact of protein and enzyme 

spatial localization within integrated models of cellular systems. 

Modelng: Borefinery Process 

The long-term vision for biomass-to-biofuels research involves all cellularand molecular-based procedures within an integrated biorefinery. To 

determine design requirements and alternatives, the overall process should 

be modeled computationally. Modeling is important, as is obtaining 

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CROSSCUTTING RESEARCH 

experimental validation of large-scale designs that will enable implementation of genomic and fundamental science research. In addition,

physical properties and material handling including feed, mass, and fluidic 

transport parameters need to be determined. Without these parameters,

designing bioprocessing facilities—including bioreactors, heat exchange,

filtration, bioseparations, centrifugation, pumps, valves, and other chemicalengineering unit operations—is difficult and not comprehensive. This capability encompasses economic models and those that assess the net carbon 

balance impact of different processing schemes and the advantages and 

disadvantages of various coproduct processes. Models do exist for some 

of these processes, but they are not readily available to the community.

To allow researchers to assess the feasibility of different design concepts,

results and methodologies should be readily accessible and subject to continuous feedback-based improvements. 

Process modeling will be very useful in evaluating the feasibility of 

consolidated bioprocessing. Considering the goal to provide the science 

underpinning one or more commercial processes for producing liquid 

fuels from biomass, a proper evaluation of most-probable future scenarios is needed. For example, one scenario envisioned for more efficiency 

and economy is simultaneous saccharification and fermentation (SSF).

Saccharification enzyme mixes currently are optimal at 50°C and at a 

pH between 4 and 6, so as indicated by other study groups, “none of the 

current ethanol-producing microorganisms is suitable.” Process modeling 

will evaluate the importance and sensitivity of SSF vs a two-step process.

Modeling is needed to demonstrate rational design of integrated biomass 

processing by predicting and then verifying overall hydrolysis yields for 

native and modified biomass species using different pretreatment chemistries, temperatures, and specific enzymes. 

In summary, research is needed to develop process models for assessing 

mass balances and economic models for bioprocess engineering and to 

disseminate them for community benefit (see chapter, Bioprocess Systems 

Engineering and Economic Analysis, p. 181). 

Capablty Sutes for Boenergy Research 

and Faclty Infrastructure 

To address mission science needs in energy and environment, the DOE 

Office of Science has proposed establishing vertically integrated research 

centers that will draw upon the range of advanced, high-throughput technologies and information-management computing described in the GTL 

Roadmap. The first of these centers will focus on bioenergy. This section 

provides an overview of existing and developing GTL capabilities that will 

be required to accelerate biomass-to-biofuels research. The GTL program 

has established pilots of most key technologies required to perform systems biology that might be incorporated into research centers. Biological 

capabilities need to be investigated at many scales (see Fig. 9. Understanding Biological Capabilities at All Scales Needed to Support Systems 

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Biology Investigations of Cellulosic Biomass, p. 21). Over the past decade,

genomic sequencing has been established as a highly efficient production 

component at the DOE Joint Genome Institute. Such capabilities provide 

the genomic foundation for research described in this report. 

DOE Jont Genome Insttute 

DOE JGI’s mission is to provide integrated high-throughput sequencing and computational analyses to enable genomic-scale, systems-based 

scientific approaches to DOE challenges in energy and environment. JGI 

capabilities will have an immediate impact on biomass-to-biofuels science 

by providing high-quality genome sequences of relevant crop plants and 

microorganisms, including naturally occurring and engineered microorganisms used for biomass conversion and soil microbial communities as they 

relate to soil quality and sustainability. 

Identification of genes that control cell-wall composition in biomass species and development of tools such as gene chips depend on the availability 

of genome sequences for biomass crop species. Since micro-RNAs are 

expected to play a role in controlling expression in many relevant genes,

DNA sequencing must be comprehensive enough to identify all microRNAs in these species. 

Implementating effective biomass conversion will require JGI to rapidly 

and cost-effectively determine the sequences of a number of organisms,

consortia, and metagenomes. In addition to biomass crop plants, JGI could 

provide genome sequence for new species of white rot fungi and brown rot 

fungi, actinomycetes, and such other biofuel-relevant organisms as natural consortia and communities involved in biomass decomposition. Full 

metagenomic sequencing will elucidate hemicellulose and lignin breakdown observed in mixed microbial populations and will allow harnessing 

of their collective processes. 

Producton and Characterzaton of Protens and Molecular Tags 

Functonal Capabltes 

These capabilities would enable the high-throughput expression of proteins mediating cell-wall biosynthesis (laccases, peroxidases, and glycosyl 

transferases) in Arabidopsis and other model and feedstock plants. Most 

enzymes of interest in cell-wall biosynthesis, such as glycosyl transferases,

are thought to be membrane associated and, therefore, difficult to purify 

and characterize by conventional methods. Heterologously expressed 

proteins would be characterized biochemically and used as a resource for 

first-pass identification of function and the generation of antibodies and 

tags. To improve enzymes for lignocellulose deconstruction, the protein 

production capability would be a resource for native and recombinant 

forms of enzymes and modified enzymes from directed evolution and 

from rational-design, site-directed mutagenesis approaches. For pilot and 

validation studies, significant quantities of protein would be produced.

Proteins, natural and modified, could be used to rapidly reconstitute 

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CROSSCUTTING RESEARCH 

machines such as cellulases and cellulosomes for improved performance.

Antibody resources would have multiple applications, including pinpointing cellular localization of target proteins; protein complex identification;

quantification of natural functional diversity; and quantitation of molecular 

outcomes from breeding experiments. In addition, this resource would 

generate tags and probes for imaging cell-wall polymers and progress of 

enzymes that degrade these polymers. 

Assays for many cell-wall synthesis and deconstruction enzymes will 

require new approaches to screen for activity. In particular, they will involve 

access to different polysaccharide substrates and capabilities. In combination with proteomic capabilities, the resource would provide valuable 

information about proteins and mechanisms through which biomass is 

converted to biofuels. This knowledge will enable engineered-organism 

combinations to optimize biofuel production. 

These capabilities will be critical for generating and characterizing new 

proteins and biomarkers for imaging. For example, a collection of fluorescently labeled proteins for such specific model plants as Arabidopsis or 

Brachypodium would be particularly valuable. 

Instrumentaton and Methods 

From genome sequences, protein production and characterization methods 

will express proteins and generate reagents for interrogating cell function.

Specifically, the goal will be to create capabilities to produce on demand all 

proteins encoded in any genome; create molecular tags that allow each protein to be identified, located, and manipulated in living cells; gain insights 

into function; and perform biophysical and biochemical protein characterizations. Using high-throughput in vitro and in vivo techniques (i.e., cellular 

and cell free) will lower protein-production costs to levels that will allow 

comprehensive analysis of cellular proteins. These methodologies will be 

applied to the production and characterization of modified and native proteins. Products and analysis capabilities will be made available to scientists 

and technologists. 

“Affinity reagents” will be generated in parallel with protein production,

using many of the same technologies. Tagged proteins or nucleic acids 

will permit detection and tracking of individual proteins in living systems,

including complex molecular assemblages; intracellular position of all proteins and their spatial dynamics; if secreted, extracellular localization and 

interaction with other community members; and techniques for manipulating protein activity in the environment. 

Potential core instrumentation: 

•	 Gene-synthesis and manipulation techniques. 

•	 High-throughput microtechnologies for protein-production screening. 

•	 Robotic systems for protein and affinity-reagent production and characterization. 

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•	 Computing for data capture and management, genomic comparative 

analyses, control of high-throughput systems and robotics, and 

production-strategy determination. 

Characterzaton and Imagng of Molecular Machnes 

Functonal Capabltes 

These capabilities will enable identification and characterization of protein 

complexes responsible for essential functions of energy-relevant phenotypes, including cell-wall biosynthesis. Understanding the function of these 

cell-wall synthesizing complexes and their interactions with metabolic 

pathways that produce sugar nucleotides in the cytosol will be important 

for understanding polysaccharide biosynthesis. Identifying key proteinprotein interactions (interactomics) involving such biomass-deconstruction enzymes as 

polysaccharidases, hemicellulases, ligninases,

and esterases, along with interactions with their 

substrates, also is a priority. Because polysaccharides probably are synthesized by a multienzyme complex in the Golgi, that also will be 

identified and analyzed. 

Advanced capabilities are needed to understand 

the structure and function of cellulase and cellulosome molecular machines and improve designer 

cellulosomes by linking essential enzymes to the 

desired substrate and eliminating superfluous 

proteins and reactions.These advanced capabilities include analysis and computation for probing 

interactions among cells, their associated secreted 

enzyme complexes, and cellulose; and particularly 

for understanding the biology occurring at the 

cell surface–cellulose interface. 

Instrumentaton and Methods 

Capabilities will identify and characterize Fig. 4. Visualizing Interaction Networks. Graphical maps 

molecular assemblies and interaction networks display protein interaction data in an accessible form. These 

(see Fig. 4. Visualizing Interaction Networks, visualizations summarize data from multiple experiments and 

this page). Resources will isolate and analyze also allow quick determinations of proteins that might be core molecular machines from microbial cells and constituents of a particular protein complex and those that might plants; image and localize molecular machines in play roles in bridging interactions among different complexes. cells; and generate dynamic models and simula- The figure above, generated using Cytoscape, summarizes protein tions of the structure, function, assembly, and interactions in complexes isolated by affinity approaches from disassembly of these complexes. High-through- Shewanella oneidensis. Nodes (yellow or red circles) represent put imaging and characterization technologies proteins. [Source: GTL Center for Molecular and Cellular Sys­ will identify molecular machine components, tems at Oak Ridge National Laboratory and Pacific Northwest characterize their interactions, validate their National Laboratory; previously published in GTL Roadmap, occurrence, determine their locations within the 

p. 68 (doegenomestolife.org/roadmap/).] cell, and allow researchers to analyze thousands 

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CROSSCUTTING RESEARCH 

of molecular machines that perform essential functions inside a cell. The 

capability for completely understanding individual molecular machines will 

be key in determining how cellular molecular processes work on a wholesystems basis, how each machine is assembled in 3D, and how it is positioned in the cell with respect to other cellular components. 

Potential core instrumentation: 

•	 Robotic culturing technologies to induce target molecular machines in 

microbial systems and support robotic techniques for molecular-complex 

isolation. 

•	 State-of-the-art mass spectroscopy and other techniques specially configured for identification and characterization of protein complexes. 

•	 Various advanced microscopies for intracomplex imaging and structure 

determination. 

•	 Imaging techniques for both spatial and temporal intra- and intercellular localization of molecular complexes. 

•	 Computing and information systems for modeling and simulation of 

molecular interactions that lead to complex structure and function. 

Analyss of Genome Expresson: The Omcs 

Functonal Capabltes 

An organism selectively produces portions of its proteome in response to 

specific environmental or intracellular cues. Studying its constantly changing protein expression thus leads to 

better understanding of how and why 

an organism turns genome portions 

“on” and “off.” Identifying, quantifying, and measuring changes in 

global collections of proteins, RNA,

metabolites, and other biologically 

significant molecules mediated by 

proteins—including lipids, carbohydrates, and enzyme cofactors—are 

important to this understanding.The 

ability to measure time dependence 

of RNA, protein, and metabolite 

concentrations will reveal the causal 

link between genome sequence and 

cellular function (see Fig. 5. GeneProtein-Metabolite Time Relationships, this page). High-throughput 

omic tools (transcriptomics, proteomics, interactomics, metabolomics,

glycomics, lignomics, and fluxomics) 

will be critical for systems-level 

investigation of plant cell-wall 

Fig. 5. Gene-Protein-Metabolite Time Relationships. To accurately establish causality among measured gene, protein, and metabolite events, sampling 

strategies must cover the full characteristic time scales of all three variables.

Little is known about the time scale of gene, protein, and metabolite responses 

to specific biological stimuli or how response durations vary in genes and species. [Figure adapted from J. Nicholson et al., “Metabonomics: A Platform for 

Studying Drug Toxicity and Gene Function,” Nat. Rev. 1, 153-61 (2002); previously published in GTL Roadmap, p. 158 (doegenomestolife.org/roadmap).] 

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construction and processes whereby the cell wall is deconstructed and 

converted to ethanol. Multiple analytical capabilities will be seamlessly 

integrated with modeling to achieve the needed level of understanding. 

Use of proteomics will allow comprehensive analysis of plant protein complexes, factors controlling their creation and function, and ensuing processes, beginning with the model plant Arabidopsis. The thousand or more 

proteins involved in cell-wall synthesis or modification are highly interacting or located in complexes. Knowing which proteins are in complexes 

before attempting to develop surrogate expression systems for enzyme 

characterization will be essential. Many such enzymes are membrane 

associated, so innovative methods for characterizing membrane complexes 

must be developed. These capabilities are vital to documenting the molecular makeup of living cell types in vascular and other tissues, including ray 

parenchyma and phloem. 

MS and NMR would be used to develop very sensitive, high-throughput 

assays for glycosyl transferase activity. Synthesis, purification, and characterization of glycans needed as acceptors would be important. 

A “glycochip” or related method for assaying enzymes involved in polysaccharide synthesis and modification also would be needed. High-throughput MS capabilities would be well suited for developing the analytical 

aspects of a glycochip. These resources also would benefit analysis of microbial systems used for plant cell-wall deconstruction and sugar fermentation. For example, such capabilities could quantify response of white rot 

fungi to changing culture conditions and provide a systems-level understanding for improving the energetics and carbon-allocation efficiency for 

cellulase and cellulosome production. 

Optimizing microbial cultures for bioconversion will require tools that 

allow quantitative cellular characterization at the systems level, including 

those for global transcript, protein, and metabolite profiling in conjunction with metabolic and regulatory modeling. HTP methods to identify 

binding sites of global regulatory proteins will be required for models of 

global gene regulation. Additional HTP tools will monitor key metabolites 

that define cell redox and energy states [e.g., ATP, GTP, NAD(P)H, and 

NAD(P)] for fermentation optimization. 

Instrumentaton and Methods 

Consolidated omics will be capable of gaining insight into microbial functions by examining samples to identify (1) all proteins and other molecules 

created by a plant or individual or community of microbes under controlled conditions and (2) key pathways and other processes. Integrating 

these diverse global data sets, computational models will predict microbial 

functions and responses and infer the nature and makeup of metabolic and 

regulatory processes and structures. 

Potential core instrumentation: 

•	 Large farms of chemostats to prepare samples from highly monitored 

and controlled microbial systems under a wide variety of conditions. 

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CROSSCUTTING RESEARCH 

•	 Numerous specialized mass and NMR spectrometers and other instruments capable of analyzing the molecular makeup of ensemble samples 

from thousands of diverse molecular species. 

•	 High-performance computing and information capabilities for modeling and simulating plant or microbial-system functions under different 

scenarios. These studies will inform the design of systems-level experiments and help to infer molecular processes from ensuing data. 

Analyss and Modelng of Cellular Systems 

Functonal Capabltes 

Biomass complexity, coupled with intricate processes involved in conversion to ethanol, requires experimental capabilities and models for analyzing 

the biomass-enzyme-microbe system, specifically cell-wall synthesis and 

microbial decomposition of biomass. These capabilities also can contribute 

to determining the response of complex soil microbial communities to 

various cropping regimes. 

On the basis of similarity with genes of known function, advanced computation will be used for comprehensive analysis of genes from model and 

biomass crop plants implicated in polysaccharide synthesis or modification. Acquisition and analysis of HTP gene-expression data from model 

and crop plants would be powerful in assigning probable function to genes 

implicated in cell-wall synthesis. As enzyme function emerges, a systemslevel plant-cell model would incorporate biophysical and structural properties and knowledge about pertinent proteins. Models of this type would 

greatly facilitate the rational development of feedstock species based on 

“design principles,” in which wall chemical composition is optimized as 

feedstock while plant productivity is simultaneously maximized. 

These resources should include capabilities for generating models that 

enable improved cellulase production from near-term hosts such as Trichoderma reesei and proceed to bacterial and yeast systems that will require 

longer times for research and development. Other goals are to improve 

production of specific preferred components and foster a more global 

understanding of hyperproduction at industrial scales in industrial strains. 

An integrated model of fermentative microorganisms and the ability to 

test it experimentally will support new concepts to produce liquid fuels and 

understand the producers’ underlying metabolism and responses to stress in 

producing biofuels such as ethanol. Models and new rounds of modification and experimentation then will be used to alleviate stress responses and 

increase carbohydrate degradation, minimize cell growth and side products 

of synthesis, and maximize fuel production. 

Metabolic flux analysis (as described earlier) is essential in improving 

understanding and manipulation of cellular metabolism. Possible analytical 

needs include appropriate NMR and MS instrumentation; stable isotopic 

labeling for key molecular moieties involved in metabolic processes; and 

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synthesis of process intermediates. Intensive mathematical and computational power is required to achieve the final goal of flux estimation. 

Instrumentaton and Methods 

Technologies and methodologies for analyzing cellular systems will be the 

capstone for ultimate analytical capabilities and knowledge synthesis to 

enable a predictive understanding of cell, organism, tissue, and community function critical for systems biology. Imaging methods will monitor 

proteins, machines, and other molecules spatially and temporally as they 

perform their critical functions in living cells and communities. Within 

their structures, microbial communities contain numerous microniches 

that elicit unique phenotypical and physiological responses from individual 

microbial species. The ability to analyze these niches and the microbial 

inhabitants within is crucial to our ultimate goal. This grand biological 

challenge must be addressed before scientists can predict the behavior of 

microbes and take advantage of their functional capabilities. Modeling 

resources will enable description of biological interactions with the physicochemical environment and predict how the system will evolve structurally and functionally. 

Potential core instrumentation: 

•	 Highly instrumented cultivation technologies to prepare structured 

microbial communities for simulating natural conditions in a highly 

controlled environment. 

•	 Instruments integrating numerous analytical-imaging techniques to 

spatially and temporally determine, in a nondestructive way, relevant 

molecular makeup and dynamics of the community environment, community, and microbes that comprise it. 

•	 Computing and information capabilities to model and simulate complex 

microbial systems, design experiments, and incorporate data. 

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CROSSCUTTING RESEARCH 

Cted References 

Moller, G. M., et al. 1998. “In Situ Gene Expression in Mixed-Culture 

Biofilms: Evidence of Metabolic Interactions Between Community 

Members,” Appl. Environ. Microbiol. 64, 721–32. 

U.S. DOE. 2005. Genomics:GTL Roadmap: Systems Biology for Energy and 

Environment, U.S. Department of Energy Office of Science 

(doegenomestolife.org/roadmap/). 

U.S. DOE. 2002. Report on the Imaging Workshop for the Genomes to Life Program, April 16–18, 2002, Office of Science, U.S. DOE (doegenomestolife.

org/technology/imaging/workshop2002). 

Wolfaardt, G. M., et al. 1994. “Multicellular Organization in a Degradative 

Biofilm Community,” Appl. Environ. Microbiol. 60, 434–46. 

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Boprocess Systems Engneerng 

and Economc Analyss 

Complete process modeling should be initiated to guide the scientific work described in this Roadmap, including systems engineering and economic analyses, to evaluate the most-probable 

scenarios; to coordinate advances and needed research across the feedstock,

deconstruction, and fermentation domains; and to reduce risk as the 

development cycle proceeds. Results and methodologies should be made 

available to the community through the web and should be subject to continuous improvements based on community feedback. 

Biomass-conversion literature has many examples of pretreatment, hydrolysis, and fermentation systems that are technically effective but have no 

real chance to be competitive economically. A common tendency among 

biotechnologists is to attempt processes that will achieve, for example,

the highest yields, rates, and titers. Process modeling, however, very often 

reveals that different combinations of these parameters are needed to produce a commercially viable process. Disciplined systems engineering and 

economic analysis using mass balances and standard analytical methods 

can eliminate ineffective approaches rather quickly, narrowing the focus to 

the most-promising options. Current advanced saccharification enzyme 

systems have demonstrated what a concerted, focused program of enzyme 

development can achieve. These systems, however, were designed for a specific acid-based pretreatment and the biomass raw material of corn stover.

Other combinations of pretreatments and biomass materials will need to 

be analyzed and subjected to process engineering and economic analyses as 

they mature. 

Research Goals 

This work seeks to integrate improvements in the molecular biology of 

plant materials and those in pretreatment, enzyme hydrolysis, and fermentation while conducting rigorous bioprocess systems engineering (incorporating new processes into a model refinery) and economic analysis. The 

approach integrates all elements of enhanced and cost-effective means to 

convert biomass into ethanol and other bioproducts. 

Practical options for lignocellulose conversion to liquid fuels are sharply constrained by the need to produce fermentable sugar costing a few cents per 

pound, preferably with minimal sugar loss and degradation. Furthermore,

in a biorefinery, all downstream processes will be affected by pretreatment 

choice. Process integration, therefore, is crucial. Finally, no evidence suggests 

that biological methods alone (i.e., without a thermochemical pretreatment 

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SYSTEMS ENGINEERING 

step) will be able to obtain the high-yield, low-cost conversions of cellulose and hemicellulose required for economic viability. Research elements 

would include (1) correlation of cell-wall chemistry and structure with the 

efficiency of cellulase polysaccharide depolymerization, with and without 

pretreatment; (2) multidisciplinary studies of specific targeted lignocellulosic materials with promising pretreatments and advanced enzymes, using 

standard analytical methods and detailed mass balances; (3) examination 

of the effects of individual and combined enzymatic activities on pretreated 

substrates, with the primary technical-evaluation criterion of maximum 

sugar yield at minimum enzyme loadings; and (4) process engineering and 

economic modeling of these integrated pretreatment and hydrolysis studies 

to identify promising combinations and eliminate less-attractive options.

This work must be coordinated with all domains of the research portfolio to 

have the desired effect—optimal process development. 

Considering the goal to develop one or more commercial processes for 

ethanol production from biomass, a proper evaluation of most-probable 

future scenarios is needed. For example, several groups envision simultaneous saccharification and fermentation (SSF) for a more viable process. Since 

saccharification enzyme mixes work better at 50o

C and a pH between 4 

and 6, then as indicated by other study groups, “none of the current ethanol 

producers’ microorganisms is suitable.” Under this scenario the parameters 

of saccharification must be included in designing new production microorganisms. Process modeling is needed to evaluate the importance and 

sensitivity of SSF vs a two-step process. Similarly, ethanol fermentations 

will be evaluated by three main parameters: Yield from carbohydrates, rate 

of production, and final titer. 

Modeling will be very useful in evaluating the feasibility of consolidated 

bioprocessing. As an example, current specific-activity values of the three 

enzymes used today to degrade cellulose (i.e., exoglucanases, endoglucanases, and beta-glucosidases) may possibly allow prediction of the protein 

amount that a “consolidated strain” will have to produce per unit of time to 

degrade cellulose and liberate sugars quickly enough to sustain fast rates of 

ethanol production. 

Mlestones 

Wthn 5 years or less 

Initial research should address correlation of changes in plant cell-wall 

structure with ease of hydrolysis. This could be done through chemical 

composition or nanoscale and molecular-scale imaging (such as would be 

available from National Renewable Energy Laboratory). The relationship 

between changes in plant cell-wall structure (e.g., lignin composition) 

would enable better formulation of more-effective and more-productive 

advanced enzymes in their interactions with a solid substrate. Emphasis 

would be on coupling changes in plant cell-wall structure, changes in pretreatment conditions, and decreases in enzyme usage to achieve conversion 

extent and rate currently attained with much-higher enzyme loadings. 

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Wthn 0 years 

Once scientists determine the impact of changes in plant cell-wall structure on hydrolysis extent and the ease with which a plant cell wall can be 

deconstructed, further mechanistic studies for improving the efficiency of 

enzyme hydrolysis will be carried out. Certain types of plant cell walls may 

have lignin content higher than or different from current biomass materials; utilization of enzymes including lignases, hemicellulases, and cellulases 

could be considered. A model would be helpful, for example, in which a 

lignin-rich material is converted efficiently into its fractions of lignin, celloligosaccharides, and xylans. Lignin could be used as a source of energy 

for a cellulose-to-ethanol processing plant. The proper composition balance 

in lignin to polysaccharides could make such a facility self-sufficient with 

respect to energy usage. Systems integration of these various steps will be 

carried out in a longer-term project. Scientists expect initial improvements 

in the efficiency of cellulose-to-ethanol conversion in an existing plant 

(such as a dry mill) to be possible within 10 years. 

Wthn 5 years 

Engineering, molecular evolution of new enzymes, and genetic modification of agronomic traits should be combined to yield a vertically integrated technology for converting biomass materials to ethanol and other 

bioproducts. Combining molecular biology with process engineering may 

yield plants that are robust in the field but easily accessible for conversion 

using specialized enzymes added after harvest. Transportation of biomass 

materials is known to be a key cost factor, so enzymes integrated into plant 

material could make it more compressible after harvest. Lignin-degrading 

enzymes may make possible high-lignin plants (including certain types of 

grasses) that are easily deconstructed once exposed to proper conditions in 

a processing plant, yielding an adequate amount for powering the facility. 

The Role of GTL and OBP Facltes and Capabltes 

The challenge in using advanced analytical and modeling capabilities is to 

integrate research vertically across several domains into a viable, technically and economically sound biorefinery concept. Research results must be 

rapidly assimilated into an overall systems model that will provide insight 

into the impact of new phenomena or processes; concomitant technologies must be incorporated into the system to optimize these effects. Such 

analysis will guide a vertically integrated research portfolio that supports 

all aspects of bioprocess engineering. A summary follows. 

Proten Producton 

Protein production capabilities will be critical in generating and characterizing new proteins and biomarkers for imaging and for identifying key 

protein-protein interactions, including enzymes for lignin, polysaccharide,

and hemicellulose ester degradation. 

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SYSTEMS ENGINEERING 

Proteomcs 

Proteomic measurements can provide valuable information about proteins 

and mechanisms by which monosaccharide conversion to ethanol and 

other products is achieved. Combinations of native and genetically modified organisms can achieve better conversion in existing ethanol facilities. 

Cellular Systems 

Cellular systems analysis and modeling may be employed to generate and 

monitor microbial cells and communities that would enable expression of 

new types of enzymes or proteins so sufficient quantities are available for 

research. 

DOE Jont Genome Insttute 

DOE JGI will be important in discovering new microorganisms and 

enzymes that may be useful in converting more-recalcitrant forms of cellulosic materials to monosaccharides. 

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APPENDICES 

Appendix A. Provisions for Biofuels and Biobased Products in the Energy Policy Act of 2005 ....................... 186
 

Appendix B. Workshop Participants .......................................................................................................... 188
 

Appendix C. Workshop Participant Biosketches ......................................................................................... 192
 

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APPENDICES 

Appendx A. Provsons for Bofuels and Bobased 

Products n the Energy Polcy Act of 2005 

On July 29, 2005, Congress passed the Energy Policy Secton 9. Amendments to the Bomass Research and 

Act of 2005,1 and President George Bush signed it into Development Act of 2000. law on August 8, 2005. The $14 billion national energy This section presents several changes in the wording of plan includes provisions that promote energy efficiency the Biomass Research and Development Act of 2000. and conservation, modernize the domestic energy in- Some important changes include redefining the objec­ frastructure, and provide incentives for both traditional tives of the Biomass Research and Development Initia­ energy sources and renewable alternatives. The follow­ tive to state that the initiative will develop ‘‘(1) tech­ ing sections of the Energy Policy Act relate to biofuels nologies and processes necessary for abundant commer­ and biobased products. cial production of biobased fuels at prices competitive 

with fossil fuels; (2) high-value biobased products... to Ttle IX—Research and Development enhance the economic viability of biobased fuels and 

Secton 92. Boenergy Program. power... and [serve] as substitutes for petroleum-based 

Research, development, demonstration, and com- feedstocks and products; and (3) a diversity of sustainmercial application activities under this program will able domestic sources of biomass for conversion to bioaddress biopower, biofuels, bioproducts, integrated based fuels and biobased products.” Another important 

biorefineries, crosscutting research and development amendment introduces four technical areas that the 

in feedstocks; and economic analysis. Goals for DOE initiative will address in its research and development 

biofuel and bioproduct programs include partnering activities: (1) develop crops and systems that improve 

with industrial and academic institutions to develop: feedstock production and processing, (2) convert recal­

“(1) advanced biochemical and thermochemical con- citrant cellulosic biomass into intermediates that can 

version technologies capable of making fuels from lig- be used to produce biobased fuels and products, (3) denocellulosic feedstocks that are price-competitive with velop technologies that yield a wide range of biobased 

gasoline or diesel in either internal combustion engines products that increase the feasibility of fuel production 

or fuel cell-powered vehicles; (2) advanced biotech- in a biorefinery, and (4) analyze biomass technologies 

nology processes capable of making biofuels and for their impact on sustainability and environmental 

bioproducts, with emphasis on development of bio- quality, security, and rural economic development. 

refinery technologies using enzyme-based processing Secton 92. Producton Incentves for Cellulosc Bofuels. systems; (3) advanced biotechnology processes capable 

of increasing energy production from lignocellulosic This section calls for the establishment of a program 

feedstocks, with emphasis on reducing the dependence that provides production incentives on a per-gallon 

of industry on fossil fuels in manufacturing facilities; basis for cellulosic biofuels. The purpose of the incentive 

and (4) other advanced processes that will enable the program is to ensure that 1 billion gallons in annual celdevelopment of cost-effective bioproducts, including lulosic biofuel production are achieved by 2015; cellulosbiofuels.”Through Integrated Biorefinery Demonstra- ic biofuels are cost-competitive with gasoline and diesel;

tion Projects, DOE will demonstrate the commercial and small feedstock producers and rural small businesses 

application of integrated biorefineries that use a wide are full participants in the cellulosic biofuel industry. 

variety of lignocellulosic feedstocks to produce liquid 

transportation fuels, high-value biobased chemicals, Secton 977. Systems Bology Program. 

electricity, substitutes for petroleum-based feedstocks, This section provides for the establishment of a research 

and useful heat. program in microbial and plant systems biology, protein 

science, and computational biology to support DOE 

energy, national security, and environmental missions.

Funds will be available for projects to plan, construct, 1

U.S. Congress. 2005. Energy Policy Act of 2005, Pub. L. 109- or operate special instrumentation or facilities for 58 (www.epa.gov/OUST/fedlaws/publ_109-058.pdf ). 

6 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Appendix A. Energy Policy Act 

researchers in systems biology and proteomics and associated biological disciplines. Biomedical research on 

human cells or human subjects is prohibited. 

Ttle XV—Ethanol and Motor Fuels 

Secton 50. Renewable Content of Gasolne (Renewable 

Fuels Standard). 

This section amends Section 211 of the Clean Air 

Act. An important amendment is the establishment of 

the renewable fuel program. In this program, gasoline 

sold in the United States is required to be mixed with 

increasing amounts of renewable fuel (usually ethanol) 

on an annual average basis. In 2006, 4 billion gallons of 

renewable fuels are to be mixed with gasoline, and this 

requirement increases annually to 7.5 billion gallons 

of renewable fuel by 2012. For 2013 and beyond, the 

required volume of renewable fuel will include a minimum of 250 million gallons of cellulosic ethanol. 

Secton 505. Publc Health and Envronmental Impacts of 

Fuels and Fuel Addtves. 

This section amends Section 211(b) of the Clean Air 

Act by requiring study of the impacts of increased use 

of fuel additives (e.g., ethanol and other chemicals) on 

public health, air quality, and water resources. 

Secton 506. Analyss of Motor Vehcle Fuel Changes. 

This section amends Section 211 of the Clean Air Act 

by requiring study of the effects of ethanol-gasoline 

mixes on permeation of fuel molecules into plastic and 

rubber components of fuel systems and the evaporative 

emissions resulting from this permeation. 

Secton 5. Renewable Fuel. 

This section amends the Clean Air Act by adding a 

section that allows funds to cover loan guarantees for 

no more than four projects commercially demonstrating the feasibility of producing cellulosic ethanol. Each 

project is expected to produce 30 million gallons of 

cellulosic ethanol annually. Funds also have been authorized for a resource center that further develops bioconversion technologies for ethanol production. 

Secton 52. Converson Assstance for Cellulosc Bomass, 

Waste-Derved Ethanol, Approved Renewable Fuels. 

This section amends Section 211 of the Clean Air Act 

by authorizing DOE to provide grants for building 

production facilities to producers of cellulosic ethanol 

and other approved renewable fuels. 

Secton 5. Advanced Bofuel Technologes Program. 

This section authorizes the EPA Administrator to 

establish a program to demonstrate advanced technologies for the production of alternative transportation 

fuels. Priority is given to projects that increase the geographic distribution of alternative fuel production and 

make use of underutilized biomass feedstocks. 

Secton 56. Sugar Ethanol Loan-Guarantee Program. 

This section authorizes DOE to issue loan guarantees 

to projects that demonstrate the feasibility of producing 

ethanol from sugar cane or sugar cane by-products. 

Ttle II—Renewable Energy
 

Secton 20. Sugar Cane Ethanol Program. 


The Environmental Protection Agency is to establish a 

$36 million program to study sugar cane, production of 

ethanol from sugar cane, and sugar cane by-products.

The project will be limited to sugar and ethanol producers in Florida, Louisiana, Texas, and Hawaii. Information will be gathered on how to scale up production 

once the sugar cane industry is ready to locate sites for 

and construct ethanol-production facilities. 

Secton 20. Grants to Improve the Commercal Value 

of Forest Bomass for Electrc Energy, Useful Heat, 

Transportaton Fuels, and Other Commercal Purposes. 

The Secretary of Agriculture or the Secretary of the 

Interior may provide grants to any person in a preferred 

community (e.g., Indian tribe, town or unit of local 

government with fewer than 50,000 individuals, or one 

that has been determined to pose a fire, disease, or insect-infestation hazard to federal or Indian land). Owners of facilities that use biomass to produce electricity,

useful heat, or transportation fuels may receive grants of 

up to $20 per green ton of biomass delivered to offset 

the cost of raw material. Grants of up to $500,000 may 

be awarded to offset the costs of developing research 

opportunities that improve the use of or add value to 

biomass. For each fiscal year from 2006 through 2016,

$50 million is authorized to be appropriated to carry 

out this section. 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 7 
 

 

 

 

APPENDICES 

Appendx B. Workshop Partcpants
 

Ackerman, Eric

Pacific Northwest National Laboratory

eric.ackerman@pnl.gov 

Adams, Justin

BP PLC 

justin.adams@uk.bp.com 

Armstrong, Katherine

Dow Agrosciences LLC

karmstrong@dow.com 

Atalla, Rajai

33/314 Forest Products Laboratory 

rhatalla@wisc.edu 

Baldwin, Sam

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

sam.baldwin@ee.doe.gov 

Bayer, Ed

Department of Biological Chemistry

Weizmann Institute of Science 

ed.bayer@weizmann.ac.il 

Bownas, Jennifer

Genome Management Information System

Oak Ridge National Laboratory

bownasjl@ornl.gov 

Brady, John

Department of Food Science

Cornell University

jwb7@cornell.edu 

Bull, Stan

National Renewable Energy Laboratory

stan_bull@nrel.gov 

Cameron, Doug

Cargill, Incorporated 

doug_cameron@cargill.com 

Casey, Denise

Genome Management Information System

Oak Ridge National Laboratory

caseydk@ornl.gov 

Cavalieri, Ralph

Washington State University

cavalieri@wsu.edu 

Chapple, Clint

Department of Biochemistry

Purdue University

chapple@purdue.edu 

Chum, Helena

National Renewable Energy Laboratory

helena_chum@nrel.gov 

Cleary, Michael

San Diego Supercomputer Center

University of California, San Diego

mcleary@sdsc.edu 

Collart, Frank

Argonne National Laboratory

fcollart@anl.gov 

Colson, Steven

Fundamental Science Directorate 

Pacific Northwest National Laboratory

steven.colson@pnl.gov 

Cotta, Mike

National Center for Agricultural Utilization Research

Agricultural Research Service

U.S. Department of Agriculture

cottama@ncaur.usda.gov 

Dale, Bruce

Department of Chemical Engineering and Materials Science

Michigan State University

bdale@egr.msu.edu 

Davison, Brian

Life Sciences Division 

Oak Ridge National Laboratory 

davisonbh@ornl.gov 

Dean, William

Danisco Genencor International 

bdean@danisco.com 

Donohue, Tim

Bacteriology Department

University of Wisconsin, Madison

tdonohue@bact.wisc.edu 

Drell, Daniel

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

daniel.drell@science.doe.gov 

Ferrell, John

Biomass Program

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

john.ferrell@hq.doe.gov 

Foust, Tom

National Renewable Energy Laboratory

thomas_foust@nrel.gov 

Fredrickson, Jim

Pacific Northwest National Laboratory

jim.fredrickson@pnl.gov 

Gonzalez, Ramon

Department of Chemical and Biomolecular Engineering

Rice University

ramon.gonzalez@rice.edu 

 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

Appendix B. Workshop Participants 

Greene, Rich

Office of International Research Programs

Agricultural Research Service

U.S. Department of Agriculture

richard.greene@nps.ars.usda.gov 

Hames, Bonnie

National Renewable Energy Laboratory

bonnie_hames@nrel.gov 

Harrison, Maria

Boyce Thompson Institute for Plant Research

mjh78@cornell.edu 

Heineken, Fred

National Science Foundation 

fheineke@nsf.gov 

Hennessey, Susan

DuPont Central Research and Development

susan.m.hennessey@usa.dupont.com 

Himmel, Mike

National Bioenergy Center

National Renewable Energy Laboratory

mike_himmel@nrel.gov 

Hladik, Maurice

Iogen Corporation

mauriceh@iogen.ca 

Houghton, John

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

john.houghton@science.doe.gov 

Ingram, Lonnie

Florida Center for Renewable Chemicals and Fuels 

Department of Microbiology and Cell Science

University of Florida

ingram@ufl.edu 

Jacobs-Young, Chavonda

National Research Initiative 

Cooperative State Research, Education, and Extension Service

U.S. Department of Agriculture

cjacobs@csrees.usda.gov 

Jofuku-Okamura, Diane

Division of Biological Infrastructure

National Science Foundation 

dbipqr@nsf.gov 

Kaempf, Doug

Biomass Program

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

douglas.kaempf@ee.doe.gov 

Kahn, Michael

Basic Energy Sciences

Office of Science 

U.S. Department of Energy

michael.kahn@science.doe.gov 

Kaleikau, Ed

National Research Initiative 

Cooperative State Research, Education, and Extension Service

U.S. Department of Agriculture

ekaleikau@csrees.usda.gov 

Keasling, Jay

Physical Biosciences Division

Lawrence Berkeley National Laboratory

jdkeasling@lbl.gov 

Keegstra, Ken

MSU-DOE Plant Research Laboratory

Michigan State University

keegstra@msu.edu 

Klembara, Melissa

Biomass Program

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

melissa.klembara@hq.doe.gov 

Knotek, Mike

m.knotek@verizon.net 

Ladisch, Mike

Laboratory of Renewable Resources Energy

Purdue University

ladisch@purdue.edu 

Lohman, Kent

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

kenton.lohman@science.doe.gov 

Lynd, Lee

Thayer School of Engineering

Dartmouth College

lee.r.lynd@dartmouth.edu 

Mansfield, Betty

Genome Management Information System

Oak Ridge National Laboratory

mansfieldbk@ornl.gov 

Matteri, Bob

Agricultural Research Service

U.S. Department of Agriculture

rmatteri@pw.ars.usda.gov 

McLean, Gail

National Research Initiative 

Cooperative State Research, Education, and Extension Service

U.S. Department of Agriculture

gmclean@csrees.usda.gov 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

APPENDICES 

Michaels, George

Bioinformatics and Computational Biology

Pacific Northwest National Laboratory

george.michaels@pnl.gov 

Miranda, Amy

Biomass Program

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

amy.miranda@hq.doe.gov 

Mitchinson, Colin

Danisco Genencor Intl. Inc. 

cmitchinson@danisco.com 

Moorer, Richard

Office of Energy Efficiency and Renewable Energy

U.S. Department of Energy

richard.moorer@hq.doe.gov 

Morrison, Mark

Department of Microbiology

Ohio State University

morrison.234@osu.edu 

Palmisano, Anna

Cooperative State Research, Education, and Extension Service

U.S. Department of Agriculture

anna.palmisano@usda.gov 

Patrinos, Ari

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

ari.patrinos@science.doe.gov 

Ragauskas, Art

Institute of Paper Science and Technology

Georgia Institute of Technology

art.ragauskas@ipst.gatech.edu 

Ralph, John

U.S. Dairy Forage Research Center 

Cooperative State Research, Education, and Extension Service

U.S. Department of Agriculture

Department of Forestry

University of Wisconsin, Madison

jralph@wisc.edu 

Remington, Karin

J. Craig Venter Institute 

Sarkanen, Simo

Kaufert Laboratory

Department of Bio-Based Products

University of Minnesota, Twin Cities

sarka001@umn.edu 

Schilling, Christophe

Genomatica, Inc.

cschilling@genomatica.com 

Shanklin, John

Biology Department

Brookhaven National Laboratory

shanklin@bnl.gov 

Shoemaker, Sharon

Department of Food Science and Technology

University of California

spshoemaker@ucdavis.edu 

Shoham, Yuval 

Department of Biotechnology and Food Engineering

Technion-Israel Institute of Technology

yshoham@tx.technion.ac.il 

Smith, Lloyd

Department of Chemistry

University of Wisconsin, Madison 

smith@chem.wisc.edu 

Somerville, Chris

Department of Plant Biology

Department of Biological Sciences

Carnegie Institution of Washington

Stanford University

crs@stanford.edu 

Stephanopoulos, Greg

Department of Chemical Engineering

Massachusetts Institute of Technology

gregstep@mit.edu 

Stevens, Walt

Chemical Sciences, Geosciences, and Biosciences Division

Basic Energy Sciences

Office of Science 

U.S. Department of Energy

walter.stevens@science.doe.gov 

Stone, Bruce

Department of Biochemistry

La Trobe University

b.stone@latrobe.edu.au 

Stults, Ray

National Renewable Energy Laboratory

ray_stults@nrel.gov 

Tabita, Bob

Department of Microbiology

Ohio State University

tabita.1@osu.edu 

Thomas, Steve

Ceres, Inc.

sthomas@ceres-inc.com 

Thomassen, David

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

david.thomassen@science.doe.gov 

90 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

Appendix B. Workshop Participants 

Tuskan, Gerald

Environmental Sciences Division 

Oak Ridge National Laboratory

tuskanga@ornl.gov 

Uberbacher, Ed

Life Sciences Division 

Oak Ridge National Laboratory

ube@ornl.gov 

Valle, Fernando

Danisco Genencor, Inc.

fvalle@Danisco.com 

Vogel, John

Western Regional Research Center

jvogel@pw.usda.gov 

Vogel, Kenneth

Agricultural Research Service

U.S. Department of Agriculture

Department of Agronomy and Horticulture

University of Nebraska, Lincoln

kpv@unlserve.unl.edu 

Weatherwax, Sharlene

Biological and Environmental Research

Office of Science 

U.S. Department of Energy

sharlene.weatherwax@science.doe.gov 

Wheeler, Nick

Molecular Tree Breeding Services, LLC

nickwheeler@scattercreek.com 

Wilson, David

Department of Molecular Biology and Genetics

Cornell University

dbw3@cornell.edu 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

 

 

APPENDICES 

Appendx C. Workshop Partcpant Bosketches
 

Erc Ackerman 

Eric Ackerman is a molecular biologist at Pacific Northwest 

National Laboratory, having earned a doctorate in biophysics from the University of Chicago in 1979. He was a Helen 

Hay Whitney Fellow at the Laboratory of Molecular Biology 

in Cambridge, England, for 3 years and then a staff scientist at 

NIH National Institute of Diabetes and Digestive and Kidney 

Diseases until 1996. He has been involved in developmental 

biology projects with Xenopus laevis, the methods by which 

toxins kill cells, mechanisms of nucleases, biochemistry of 

nucleotide excision DNA repair, and an extremely sensitive and 

quantitative assay to measure radiation effects on multiple kinds 

of DNA repair using as few as 3000 cells and 0.1-ng DNA. 

Most relevant for the Biomass to Biofuels workshop have been 

Ackerman’s studies of novel mechanisms for immobilizing 

enzymes in functionalized nanoporous materials for enhanced 

activity and stability. Recently, he began implementing highthroughput, cell-free production of proteins and their characterizations in hopes that this approach might yield optimized,

stable protein complexes that could contribute to energy generation. He also was involved in strategic planning for Genomics:

GTL production facilities, particularly for the Facility for Production and Characterization of Proteins and Affinity Reagents. 

Justn Adams 

Justin Adams joined BP PLC in 2003 and currently is director 

of long-term technology strategy in BP’s Office of the Chief 

Scientist. In this role he helps build and shape the strategic 

agenda, coordinates all long-term activities across the company 

to ensure alignment and balance, and oversees specific programs 

managed by the central technology function. 

Before joining BP, Adams was founder and CEO of High 

Power Lithium, a Swiss company developing next-generation 

battery materials for hybrid electric vehicles in collaboration 

with Toyota. He also was an advisor to Konarka Technologies, a 

Massachusetts-based startup developing next-generation solar 

cells using conducting polymers and nanostructured materials.

He previously worked as a consultant with Arthur D. Little,

ultimately leading its Advanced Energy Systems practice in 

Europe. Arthur D. Little delivers strategic and technoeconomic 

consulting on emerging energy technologies to many of the 

world’s leading energy majors. 

Adams holds joint honors in management and technology from 

the University of Bath (England) and the University of Richmond (Virginia). 

Katherne Armstrong 

Katherine Armstrong is global leader for trait genetics and 

technologies R&D at Dow AgroSciences in Indianapolis, 

Indiana. She earned her bachelor’s degree from the University of 

Virginia and master’s in molecular and population genetics from 

the University of Georgia. She has been an R&D scientist with 

Dow for 25 years and has studied plant gene expression at the 

molecular and cellular levels. Currently she oversees the development of corn traits through product launch. She holds seven 

U.S. patents in the area of plant gene expression and has written 

numerous publications. Relevant areas of research include optimization of corn genetics for ethanol extraction from both grain 

and cellulosic feedstocks. 

Raja H. Atalla 

Rajai Atalla received his bachelor’s degree from Rensselaer Polytechnic Institute in 1955 and master’s and doctorate in chemical engineering and physics from the University of Delaware 

by 1960; his work focused on spectroscopic studies of flames.

During 8 years at Hercules Research Center, he studied phase 

transitions in semicrystalline polymers and evaluated anomalous spectra of many compounds. He was first to recognize that 

anomalous proton nuclear magnetic resonance (NMR) spectra 

of (alkyl phosphito) hydrides of cobalt and iron indicated the 

occurrence of fluxional molecules. He also developed the first 

theoretical model for photodegradation of inorganic pigment. 

As professor of chemical physics and engineering at the Institute 

of Paper Chemistry, Atalla pioneered the application of Raman 

spectroscopy to studies of celluloses. Finding accepted crystal 

structures inconsistent with Raman spectra, he investigated the 

13C solid-state NMR spectra of native celluloses with David 

VanderHart of the National Institute of Standards and Technology. They determined that all native celluloses are composites 

of two forms—I and I (1984). With Umesh Agarwal, using a α β

Raman microprobe, Atalla developed the first direct evidence of 

lignin orientation in secondary walls (1984). 

In 1989, as head of chemistry and pulping research at the U.S.

Department of Agriculture (USDA) Forest Service and adjunct 

professor in chemical and biological engineering at the University of Wisconsin, Madison, Atalla led development of inorganic 

analogs of lignin peroxidases for use in liquid-effluent–free pulping and bleaching systems. The processes were feasible economically, but the industry’s economic condition led to suspension of 

the program. Freed of administrative responsibilities in 1999 and 

elevated to senior and pioneering research scientist in 2005, he 

returned to studies of molecular architecture in plant cell walls,

with emphasis on secondary walls and native celluloses. 

Atalla has published more than 150 papers, edited a book on 

cellulose structures, and is a fellow of the International Academy 

of Wood Science and of the Technical Association of the Pulp 

and Paper Industry. He received the Anselme Payen Award of 

the American Chemical Society’s Cellulose Division as well as 

92 Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendix C. Participant Biosketches 

multiple USDA awards, including the Forest Service Chief ’s 

Distinguished Scientist Award. 

Ed Bayer 

Ed Bayer is a professor in the Department of Biological Chemistry at the Weizmann Institute of Science, Rehovot, Israel. He 

was awarded a bachelor’s degree in liberal arts from the University of Michigan in 1969, a master’s in biology from Wayne 

State University in 1971, and a doctorate in biophysics from the 

Weizmann Institute of Science in 1976. Since the early 1970s,

he has been involved in developing the avidin-biotin system 

as a general tool in the biological sciences. He was first to use 

biotinylation procedures for antibodies and other proteins and 

carbohydrates as well as avidin-conjugation and complexation 

techniques. The work initially was published in the mid-1970s,

and many of the procedures are still in routine use today. He 

received the Sarstedt Award for his contributions to the avidinbiotin system for biomedical analysis. 

Together with Raphael Lamed, Bayer introduced the cellulosome concept in the early 1980s. In 1999, he was organizer and 

cochair of the first Gordon Research Conference on Cellulases 

and Cellulosomes, and he served as chairman of the same 

conference in 2001. In 1994, he proposed the use of “designer 

cellulosomes” for biomass degradation and waste management 

and as a general tool in the biological sciences. Since then, he 

has worked systematically toward the controlled construction of 

such artificial cellulosomes via self-assembly and has produced 

a growing repertoire of divergent cellulosomal components for 

this purpose. 

During his career, he has collaborated with groups in the United 

States, Canada, Holland, Belgium, Germany, Great Britain,

France, Spain, Finland, Denmark, Guatemala, and the Republic 

of Georgia, and he has authored more than 250 articles and 

reviews in both fields. He coedited Vol. 184 on avidin-biotin 

technology in the Methods in Enzymology series and since 1999 

has served as editor of the review journal Biotechnology Advances. 

In 2002, he was elected a fellow of the American Academy of 

Microbiology. He continues his work in both the avidin-biotin 

and cellulosome fields, and his interests still focus on protein 

engineering, nanobiotechnology, and the structural and functional consequences of protein-protein and protein-ligand 

interactions. 

John Brady 

John Brady is a professor in the Department of Food Science at 

Cornell University. He received a bachelor’s degree in chemistry 

from the University of North Carolina, Chapel Hill, in 1975 

and a doctorate in chemistry from the State University of New 

York at Stony Brook in 1980. During much of his graduate 

study, he was a visiting staff member at Los Alamos National 

Laboratory in New Mexico. He received his postdoctoral training in chemistry at Harvard University, working with Martin 

Karplus. 

His research primarily involves biopolymer dynamics and 

hydration and the relationships among structure, conformation,

and function in biological systems. Specific examples include 

the solution behavior of biopolymers, factors that determine 

secondary and tertiary structure in polymers, enzymatic reaction 

mechanisms, rational drug design, effects of point mutations 

in proteins, and the possibility of engineering desirable modifications in the function of wild-type proteins. His work uses 

techniques of computational theoretical chemistry to model 

properties of biopolymers and solutions numerically. These 

techniques, often called molecular mechanics, include computer 

graphics–based molecular docking, energy minimization and 

conformational energy calculations, and molecular dynamics 

simulations. 

A principal focus of Brady’s research is on carbohydrate structure, dynamics, and hydration. He has contributed to advances in 

carbohydrate modeling, including the first molecular dynamics 

simulations on a sugar, the first relaxed conformational energy 

map for a disaccharide, the first free-energy simulations of sugar 

energy differences in solution, and the first potential of meanforce or conformational free-energy map for a disaccharide. As 

an outgrowth of his primary interests in carbohydrates, he is 

studying carbohydrate interactions with proteins. In a current 

project, Brady is using molecular mechanics simulations to study 

the catalytic mechanism and mode of substrate binding in various cellulases, including E2 from Thermomonospora fusca, in the 

hope of designing a more active enzyme that could be produced 

by site-directed mutagenesis. 

Doug Cameron 

Doug Cameron received a bachelor’s degree in biomedical engineering in 1979 from Duke University and a doctorate in biochemical engineering from MIT in 1986. He serves as director 

of biotechnology for Cargill Research, with an adjunct professorship in the Department of Chemical and Biological Engineering 

at the University of Wisconsin, Madison (UWM). From 1986 to 

1998, Cameron was a professor in the Department of Chemical 

Engineering and an affiliate in the Molecular Biology Program 

at UWM. In 1996 he was a guest professor in the Institute for 

Biotechnology at the Swiss Federal Institute of Technology in 

Zurich. From 1979 to 1981, he held the position of biochemical 

engineer at Advanced Harvesting Systems, a plant biotechnology company funded by International Harvester. 

Cameron is a fellow of the American Institute of Medical 

and Biological Engineering and is on the editorial boards of 

Metabolic Engineering and Biomacromolecules. He serves on the 

Minnesota Governor’s Bioscience Council and the board of 

directors of Minnesota Biotechnology Industry Organization.

He is a member of the MIT Biological Engineering visiting 

Bofuels Jont Roadmap, June 2006 • Office of Scence and Office of Energy Efficency and Renewable Energy • U.S. Department of Energy 9 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

APPENDICES 

committee and on the managing board of the newly formed 

Society for Biological Engineering. Cameron also is a consulting professor in the Department of Chemical Engineering at 

Stanford University. 

Clnt Chapple 

Clint Chapple received his doctorate in chemistry from the University of Guelph in 1989. After doing postdoctoral work with 

Chris Somerville at Michigan State University, he joined the 

faculty of Purdue University in the Biochemistry Department.

Chapple’s research in the area of lignin biosynthesis and plant 

secondary metabolism, using Arabidopsis as a model system, has 

earned him the title of Purdue University Scholar and a fellowship in the American Academy for the Advancement of Science.

Research by the Chapple group helped to change the traditional 

paradigm of the role of ferulic and sinapic acids in building plant 

cell walls. Rather than contributing to the production of lignin,

the group found that these two acids serve as end products in an 

essential biochemical pathway for cell-wall construction. 

The phenylpropanoid pathway gives rise to a wide array of soluble metabolites in plants. These compounds participate in many 

plant defense responses and absorb potentially damaging UVB 

radiation. The pathway also generates the monomers required 

for lignin biosynthesis—ferulic acid and sinapic acid. Lignin is 

integrated into the plant secondary cell wall, where it provides 

structural rigidity to plant tissues and enables tracheary elements 

to withstand the tension generated during transpiration. 

Chapple received the 2001 Agricultural Researcher Award from 

the Purdue School of Agriculture for his patented work in engineering plants to store and stabilize plastic monomer precursors 

in vacuoles. 

Helena L. Chum 

Trained in physical and industrial chemistry, Helena Chum 

has worked in bioenergy and renewable energy since 1979 at 

the Solar Energy Research Institute, now National Renewable Energy Laboratory (NREL), and has led R&D branches,

divisions, and centers at NREL since 1992. Her general research 

involves technology development for conversion of biomass and 

a variety of organic wastes into biofuels, chemicals, electricity,

and high-value materials. She has coauthored a book, 85 peerreviewed publications, and 150 meeting papers. She has presented 

100 invited lectures worldwide and jointly holds 18 patents. 

Her specific research areas are biomass chemical analyses,

standards development, and rapid spectrometric analysis methodologies; biomass and urban and plastic residue conversion to 

chemicals and biofuels; thermochemical conversion to multiple 

products; biomass fractionation; electrochemistry applied to 

biomass and derived compounds; environmental technologies;

thermally regenerative electrochemical systems and fuel cells;

technology development and government-industry-academia 

partnerships in R&D; and analyses of U.S. governmental 

biomass and hydrogen programs and their impact on commercial tools and systems to support recommendations for future 

programs. 

Chum is a fellow of the American Association for the Advancement of Science for integrating industry-academia-government research partnerships in biomass and biobased materials 

and also of the International Academy of Wood Science for 

demonstrated leadership of biomass analysis and standards 

activities worldwide. She received a certificate of appreciation 

from the U.S. DOE Assistant Secretary of the Office of Energy 

Efficiency and Renewable Energy (EERE) for contributions 

and leadership in departmental, presidential, and congressional 

environmental initiatives, including the National Environmental 

Technology Strategy, and for dedication to EERE programs 

(1995). 

Mke Cleary 

Joseph Michael Cleary is Executive Division Director of Sciences R&D at the San Diego Supercomputer Center (SDSC),

University of California, San Diego. He received his bachelor’s 

degree in biology from Stanford University in 1970 and his doctorate in molecular biology from the University of California,

Los Angeles, in 1980. At SDSC, he directs groups that support 

the cyber infrastructure needs of researchers by producing data 

systems and computational tools to facilitate discoveries in the 

natural sciences. His responsibilities include the initiation of 

interdisciplinary research projects for life science programs with 

biomedical and biology researchers at university, government,

and independent research institutions. 

Before joining SDSC in 2003, Cleary held biotechnology 

research and management positions for over 20 years at Merck 

and Monsanto, where he worked in fermentation and microbialstrain development, with emphasis on biosynthetic pathways for 

producing commercially valuable bacterial polysaccharides. He 

is an adjunct professor of biology at San Diego State University 

and serves as a consultant to several biotechnology businesses on 

matters related to industrial microbiology. 

Frank Collart 

Frank Collart is manager of the Robotic Molecular Biology Facility in the Biosciences Division at Argonne National 

Laboratory and Cloning and Expression group leader of the 

Midwest Center for Structural Genomics (MCSG). He received 

his doctorate in medical sciences from the Medical College of 

Ohio and master’s degree in chemistry from Bowling Green 

State University. He has used cultured cell models to delineate 

critical signal transduction events involved in differentiation of 

hematopoietic, melanoma, and breast cell lineages and has over 

50 scientific publications and 4 patents. 

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Appendix C. Participant Biosketches 

Collart manages a research program for DOE that focuses on 

the development of genome-scale methods for cloning and 

expression of proteins from the genomes of Shewanella oneidensis and Geobacter sulfurreducens. These organisms have potential 

for degrading organic pollutants and bioremediating metals. The 

program uses in vivo and cell-free approaches to address protein 

classes that represent a challenge for current cellular expression 

systems but are essential experimental targets for DOE research 

programs. With colleagues at MCSG, he has developed automated protocols for high-throughput generation and analysis of 

bacterial expression clones. 

Steve Colson 

Steve Colson received his bachelor’s in chemistry from Utah 

State University in 1963 and his doctorate from the California 

Institute of Technology in 1968. He became associate laboratory 

director of the Fundamental Science Directorate in 2003 when 

he joined the leadership team at Pacific Northwest National 

Laboratory. Directorate divisions include Atmospheric Sciences 

and Global Change, Chemical Sciences, and Biological Sciences. 

Colson has published more than 130 papers in peer-reviewed 

journals and has one patent. Before moving to PNNL, he spent 

21 years at Yale University as a professor of chemistry. He 

research focused on the combination of optical and mass spectrometric methods to address fundamental problems in physical and analytical chemistry. General research interests include 

high-sensitivity spectroscopy and microscopy, photochemistry,

photophysics, molecular dynamics, electronic structures of molecules, radical and molecular ions, process at the molecule-surface 

interface, and intermolecular interactions in molecular solids. 

Before leaving Yale, he built up a strong collaborative team of 

faculty from the chemistry, physics, and engineering departments and industry. The power and excitement of interdisciplinary, collaborative research led him naturally to join the team 

created to establish the W. R. Wiley Environmental Molecular 

Sciences Laboratory, with a focus on the integration of modern 

physical, biological, and computational sciences. 

Mchael Cotta 

Michael Cotta is research leader for the Fermentation Biotechnology Research Unit (FBT), U.S. Department of Agriculture 

(USDA) Agricultural Research Service (ARS), National Center 

for Agricultural Utilization Research, in Peoria, Illinois. FBT 

conducts a broad-based program of microbial, biochemical,

genetic, and engineering research to develop bioproducts and 

bioprocesses for conversion of agricultural commodities into 

biofuels and chemicals, enzymes, and polymers. 

Cotta obtained his bachelor’s and master’s degrees in animal 

science in 1977 and 1979, respectively, from the University of 

California, Davis, where he worked under the direction of R. L. 

Baldwin. Upon completion of these studies, he continued his 

education at the University of Illinois in the laboratories of R. B.

Hespell and M. P. Bryant. Cotta earned a doctorate in dairy science in 1985 and joined USDA ARS as a research microbiologist 

in October 1984. His research interests include microbial ecology 

of gastrointestinal environments and animal waste–handling 

systems, ecophysiology of ruminal microorganisms, and microorganism interactions in the bioconversion of complex polysaccharides. 

Bruce E. Dale 

Bruce Dale is professor of chemical engineering and former 

chair of the Department of Chemical Engineering and Materials Science at Michigan State University (MSU). He earned his 

bachelor’s degree (summa cum laude) in chemical engineering 

from the University of Arizona, Tucson, in 1974 and master’s 

degree from the same university in 1976. He then studied under 

George T. Tsao at Purdue University, receiving his doctorate 

in 1979. His first academic position was in the Department of 

Agricultural and Chemical Engineering at Colorado State University, where he rose to professor in 1988. In that same year, he 

joined Texas A&M University, where he was professor of chemical engineering and of agricultural engineering. He also directed 

two multimillion-dollar interdisciplinary research centers at 

Texas A&M: Engineering Biosciences Research Center and the 

Food Protein Research and Development Center. 

In 1996 Dale became professor and chair of the Department of 

Chemical Engineering at MSU, where he also holds an appointment in the Michigan Agricultural Experiment Station. In 1996 

he won the Charles D. Scott Award for contributions to the 

use of biotechnology to produce fuels and chemical and other 

industrial products from renewable plant resources. In 2001 he 

stepped down as department chair to return to full-time research 

and teaching. 

His research and professional interests lie at the intersection 

of chemical engineering and the life sciences. Specifically, he is 

interested in the environmentally sustainable conversion of plant 

matter to industrial products—fuels, chemicals, and materials—

while meeting human and animal needs for food and feed. Dale 

expects to devote the rest of his MSU career to teaching and 

research aimed at developing such resources while the Hydrocarbon Age is winding down during the current century. His 

concern with sustainable resources was influenced by growing 

up in the copper-mining town of Ruth in eastern Nevada—a 

vibrant small community that became a ghost town when the 

mine ran out. Dale has distrusted societies that rely on mining 

natural resources (petroleum, for example) ever since. 

He led production of the May 2000 National Research Council 

report, Biobased Industrial Products: Research and Commercialization Priorities. He has authored more than 100 refereed journal 

papers and is an active consultant to industry and an expert 

witness. He also holds 13 U.S. and foreign patents. 

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APPENDICES 

Bran H. Davson 

Brian H. Davison is chief scientist for systems biology and 

biotechnology at Oak Ridge National Laboratory (ORNL),

where for 2 years he was director of the Life Sciences Division.

He previously was a distinguished researcher and leader of the 

Biochemical Engineering Research Group. In his 20 years at 

ORNL, he has conducted biotechnology research in a variety of 

areas, including bioconversion of renewable resources (ethanol,

organic acids, and solvents); nonaqueous biocatalysis; systems 

analysis of microbes (cultivation and proteomics); biofiltration 

of volatile organic compounds; mixed cultures; immobilization 

of microbes and enzymes; metal biosorption; and extractive 

fermentations. His research has resulted in more than 80 publications and 6 patents. 

Davison received his doctorate in chemical engineering from the 

California Institute of Technology and his bachelor’s in chemical 

engineering from the University of Rochester. He chaired the 

15th to 26th Symposia on Biotechnology for Fuels and Chemicals 

and served as proceedings editor in Applied Biochemistry and 

Biotechnology from 1994 to 2005. The symposium grew from 150 

to 400 attendees during his 12 years as chair. Davison received 

an R&D 100 Award in 1977 for “Production of Chemicals 

from Biologically Derived Succinic Acid.” He also is an adjunct 

professor of chemical engineering at the University of Tennessee, Knoxville. 

Bll Dean 

Bill Dean received his doctorate in biochemistry from Syracuse 

University. He is vice president of development and process 

sciences at Danisco Genencor International, Inc.; previously, he 

held the positions of vice president of technology programs and 

vice president of manufacturing development. Before joining 

Genencor, he was in the Research Division of Corning Glass 

Works, where he worked on enzyme immobilization techniques 

and bioreactor design and was responsible for overseeing the 

enzyme subcontract with the National Renewable Energy Laboratory to develop cost-effective biomass cellulases. Currently, he 

is responsible for grain-processing technology at Genencor. 

Tm Donohue 

Tim Donohue is professor of bacteriology at the University of 

Wisconsin, Madison. He earned his doctorate from Pennsylvania State University in 1980. He studies photosynthetic bacteria 

that convert solar energy into alternative fuels (hydrogen) or 

remove greenhouse gases and other environmental pollutants.

He has used molecular genetic, biochemical, and systems biology techniques to study global signal-transduction pathways,

alternative sigma factors, and signals that control expression of 

well-studied components of the respiratory and photosynthetic 

electron-transport chains. 

Donohue’s most recent work includes identification of cellular 

pathways used by photosynthetic microbes to sense the presence of singlet oxygen and defend themselves from this toxic 

substance. This knowledge may lead to the ability to fine tune 

the design of microbial and plant photosynthetic systems to 

minimize the harmful effects of singlet oxygen and to enhance 

energy production. Photosynthesis provides >90% of net energy 

input into the biosphere. Therefore, light-driven processes within 

photosynthetic organisms have enormous capacity for the production of sustainable, carbon-neutral, solar-powered technologies that reduce the global dependency on fossil fuels. 

The long-range goals of Donohue’s projects are to identify 

important metabolic and regulatory activities; obtain a thorough 

understanding of energy-generating pathways of agricultural,

environmental and medical importance; and generate computational models to help design microbial machines with increased 

capacity to use solar energy, generate renewable sources of 

energy, remove toxic compounds, or synthesize biodegradable 

polymers. 

Tom Foust 

Tom Foust joined the National Renewable Energy Laboratory (NREL) in 2004 as director of biomass research. He has 

a doctorate from the University of Idaho, master’s from Johns 

Hopkins University, and bachelor’s from Pennsylvania State 

University, all in mechanical engineering. He also is a licensed 

professional engineer. 

In his current role, he guides and directs NREL’s research efforts 

to develop biomass conversion technology via both bioconversion and thermoconversion. This research is focused on developing the necessary science and technology for converting biomass 

to biofuels in an economical manner and covers the gamut of 

fundamental to applied science. His particular area of expertise 

is in complex flow and chemical-reaction modeling as it relates 

to biomass-conversion processes and in-process separations. 

Before joining NREL, Foust spent 7 years with the Idaho 

National Laboratory where he was the research lead for the 

biomass feedstocks program. His primary area of research was 

in complex multiphase flow analysis as it related to physical 

fractionation of biomass. He has over 20 years of experience 

in research and research management, specializing in biomass 

feedstocks and conversion research. He has written more than 

15 peer-reviewed publications related to biomass fractionation 

and technology-development issues. 

Jm Fredrckson 

Jim Fredrickson obtained a bachelor’s in soil science from the 

University of Wisconsin, Stevens Point, and advanced degrees 

in soil chemistry and soil microbiology from Washington State 

University. He is a chief scientist within the Biological Sciences 

Division at Pacific Northwest National Laboratory, specializing 

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Appendix C. Participant Biosketches 

in microbial ecology and environmental microbiology. With his 

research focused on subsurface microbiology and biogeochemistry, he has been responsible for laboratory and field research 

programs investigating the microbial ecology and biogeochemistry of geologically diverse subsurface environments and is 

recognized nationally and internationally for these studies. 

Fredrickson also has served as subprogram coordinator for 

DOE’s Subsurface Science Program from 1991 to present. In 

this role, he coordinated the technical aspects of DOE’s Deep 

Subsurface Microbiology Subprogram at the national level and 

assisted the program manager in setting programmatic research 

directions. This subprogram involved more than 15 projects at 

universities and national laboratories nationwide and focused on 

multidisciplinary field-scale research. At the request of DOE,

he currently is national coordinator for the Shewanella Microbial Cell Project, part of the Genomics:GTL program. He was 

appointed chief scientist in 2005 to serve as spokesperson to the 

science community for the GTL program and facilities. 

Ramon Gonzalez 

Ramon Gonzalez is the William W. Akers Professor in the 

Department of Chemical and Biomolecular Engineering at 

Rice University. He holds degrees in chemical engineering 

from the University of Chile and the Central University of Las 

Villas, Cuba, and also is a licensed professional engineer. His 

research addresses such challenging issues in microbial catalysis as understanding and manipulating vitamins and cofactor 

biosynthesis, anaerobic fermentation of nontraditional carbon 

sources, simultaneous metabolism of sugars in sugar mixtures,

and understanding and modifying respiratory and fermentative systems for synthesis of reduced products. Specific research 

areas include metabolic engineering and inverse (metabolic) 

engineering, functional genomics and systems biology, microbial 

fermentation, molecular modeling, and high-performance liquid 

chromatography optimization. 

Gonzalez uses a wide spectrum of approaches and state-of-theart techniques typically viewed under such different scientific 

and engineering disciplines as molecular biology, biochemistry,

and chemical engineering. He currently is using transcriptomics 

and proteomic tools in conjunction with fluxomic tools to elucidate biological function of individual genes at cellular levels. He 

advocates the systemic method for its integration of mathematical and computational tools and is using this global and integrative approach to understand complex metabolic and regulatory 

networks in bacterial systems, the basis for understanding 

similar processes in more complex organisms. The ultimate goal 

of his research is the design of specific genotypes based on the 

desired phenotype. 

Bonne Hames 

Bonnie Hames leads the biomass chemical characterization 

teams within the National Bioenergy Center at the National 

Renewable Energy Laboratory (NREL). She earned a bachelor’s 

degree in chemistry from Regis University and a doctorate in 

organic chemistry from the University of Denver. Working in 

the group of Bernard Monties, she also completed a postdoctoral assignment in lignin chemistry at the Centre National de 

la Recherche Scientifique, Institute National de la Recherche 

Agronomique, Laboratoire de Chimie Biologique, ThivervalGrignon-Paris, France. 

Her extensive experience in biomass chemistry includes more 

than 18 years of developing standard wet chemical methods for 

characterizing biomass feedstock and biomass-derived materials, preparing standard reference materials for quality assurance 

and quality control, and applying standard methods to fuel and 

chemical production from biomass. She currently leads the 

NREL Biomass Program to develop new, rapid, and inexpensive 

methods for biomass compositional analysis. These methods 

include advanced tools for chemical characterization of biomass 

feedstocks and biomass-derived materials based on infrared 

spectroscopy and advanced multivariate analysis techniques first 

developed in the Agenda 2020 program sponsored jointly by 

DOE Office of Industrial Technologies and the forest products 

industry. In 2000, her program’s real-time biomass analysis won 

an R&D 100 award and was honored by DOE as a Best of 

Agenda 2020 Project. 

Hames also developed patented techniques for biomass fractionation and lignin isolation. She has extensive experience in 

lignin chemistry including structural characterization, synthesis 

of lignin model compounds, and development of methods for 

the selective oxidation of lignin using organ metallic catalysts 

and biomimetic systems. She holds 3 U.S. patents, has authored 

3 book chapters and more than 25 papers in peer-reviewed 

journals, and has made more than 80 presentations at technical 

meetings. She currently chairs the American Society for Testing 

and Materials committee E48 on standards for biotechnology 

and subcommittee E48.05 on standards of biomass conversion. 

Mara Harrson 

Maria Harrison earned her bachelor’s degree with honors in 

microbiology from the University of Newcastle Upon Tyne,

England, and her doctorate in 1987 from the Institute of Science and Technology, University of Manchester. She conducted 

postdoctoral research at the Samuel Roberts Noble Foundation 

in Ardmore, Oklahoma, under the direction of R. A. Dixon. She 

has served as an adjunct professor at Oklahoma State University 

and Texas A&M University; in 2003, she joined the staff at the 

Boyce Thompson Institute for Plant Research, with an adjunct 

appointment in the Department of Plant Pathology at Cornell 

University. 

Most vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal (AM) fungi. These associations develop in the roots, where the fungus colonizes cortical 

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APPENDICES 

cells to access carbon supplied by the plant. The fungal contribution to symbiosis includes transfer of mineral nutrients, particularly phosphorus, from the soil to the plant. In many soils,

phosphate levels are limiting to plant growth. Consequently,

additional phosphate supplied via AM fungi can have a significant impact on plant development, and this symbiosis influences 

the structure of plant communities in ecosystems worldwide. 

The long-term goals of Harrison’s research are to understand 

the mechanisms underlying development of AM symbiosis 

and phosphate transfer among symbionts. She uses a model 

legume Medicago truncatula and AM fungi Glomus versiforme, G. 

intraradices, and Gigaspora gigantea for these analyses. Currently,

a combination of molecular, cell biology, genetic, and genomic 

approaches is being used to obtain insights into symbiosis development, communication among plant and fungal symbionts, and 

symbiotic phosphate transport. 

Susan Hennessey 

In almost 20 years at DuPont, Susan Hennessey has applied 

her expertise as a chemical engineer to a number of improved 

chemical production processes. Her team of scientists has used 

immobilized bacterial cells and other encapsulation techniques 

to increase enzyme durability and concentration for continuous production. Her work on the introduction of the herbicide 

Milestone for crop protection earned her team the 2002 Industrial Innovation Award from the Mid-Atlantic Region of the 

American Chemical Society. Other work combining the synergy 

of biology with new chemical synthesis technologies continues 

to result in new high-value products using environmentally 

preferred processes. 

Mke Hmmel 

During his 23-year term at DOE’s National Renewable Energy 

Laboratory (NREL, formerly SERI) in Golden, Colorado, Mike 

Himmel has worked to support many technical aspects of DOE’s 

Biomass Program. More recently, he has been responsible for 

establishing the facilities and staff necessary to pursue projects in 

protein engineering, specifically cellulases. Today, he manages the 

award-winning Enzyme Technology Team and the major industrial and academic subcontracts that support this work. The team 

has assembled world-class protein purification and characterization facilities at NREL, with special emphasis on robotic systems 

for screening libraries derived from directed evolution technology. Himmel has contributed 300 peer-reviewed papers and 

meeting abstracts, 4 books, and 16 patents to the literature. He 

also chaired or cochaired 15 international meetings in the field 

of biochemistry and biotechnology, including the 2003 Gordon 

Research Conference on Cellulases and Cellulosomes. 

Maurce Hladk 

Maurice Hladik is director of marketing for Iogen Corporation,

which specializes in developing, manufacturing, and marketing 

enzymes to modify and improve the processing of natural fibers 

within the textile, animal-feed, and pulp and paper industries.

One of Iogen’s major activities is research on cellulosic ethanol.

Hladik has extensive international business experience, particularly in the United States, Germany, and the United Kingdom 

as well as several Asian countries including China (also Hong 

Kong), South Korea, and Thailand. A particular strength is his 

ability to locate business contacts and commercial intelligence in 

a foreign setting. 

Before joining Iogen, from 1978 to 1998 he served in the Canadian Foreign Service. His assignments included senior Canadian 

trade officer posted to Bangkok (3 years), Hong Kong (3 years),

Beijing (1 year), Seoul (2 years), and Munich as consul general 

(3 years); and director general of the Grain Marketing Bureau at 

Agriculture Canada. In this position, Hladik was chief advisor 

to the Canadian government on international grain policy and 

marketing issues. He also served the Canadian International 

Development Agency as director for Asia, Industrial Cooperation Division. 

Lonne Ingram 

Lonnie Ingram is a distinguished professor of microbiology 

and director of the Florida Center for Renewable Chemicals 

and Fuels at the University of Florida. Elected to the National 

Academy of Sciences in 2001, Ingram and his coworkers were 

the first in the world to develop genetically engineered E. coli 

bacteria capable of converting all sugar types found in plant cell 

walls into fuel ethanol. Ingram’s organism produces a high yield 

of ethanol from such biomass as sugarcane residues, rice hulls,

forestry and wood wastes, and other organic materials. 

Ingram’s breakthrough bioconversion technology was selected to 

become Landmark Patent No. 5,000,000 by the U.S. Department 

of Commerce. More than 30 additional patents are pending or 

have been issued for this technology, which is being commercialized with assistance from the U.S. Department of Energy. BC 

International Corp., based in Dedham, Mass., holds exclusive 

rights to use and license the engineered bacteria, dubbed “K011”

by Ingram. In 1993, Ingram received a U.S. Department of 

Agriculture Distinguished Service Award for his breakthrough 

research. The agency’s highest honor, the award recognized his 

outstanding contributions to research and the consumer. 

Jay Keaslng 

Jay Keasling earned a bachelor’s degree in chemistry and biology in 1986 from the University of Nebraska, Lincoln, and 

a doctorate in chemical engineering from the University of 

Michigan in1991. He is professor of chemical engineering and 

bioengineering at the University of California, Berkeley. He also 

serves as director of the Physical Biosciences Division and heads 

the new Synthetic Biology Department at Lawrence Berkeley 

National Laboratory. The idea behind this department is to 

design and construct novel organisms and biologically inspired 

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Appendix C. Participant Biosketches 

systems to solve problems unsolvable by natural biological cultural and Biological Engineering at Purdue University, with 

systems and also provide new information about living cells. a joint appointment in biomedical engineering and a courtesy 

Keasling’s many honors include election as fellow of the Ameri- appointment in food science. In 1973 he earned his bachelor’s 

can Institute of Medical and Biological Engineering and recipi- degree from Drexel University and in 1974 and 1977, respecent of the AIChE Award for Chemical Engineering Excellence tively, his master’s and doctorate from Purdue University, all in 

in Academic Teaching. He also is founder of two companies, chemical engineering. 

Amyris Biotechnologies and Codon Devices, which have grown He has a broad background in bioscience and bioengineer­ from discoveries at his laboratory. ing and has authored a textbook, Bioseparations Engineering: 

Keasling and collaborators were awarded a Gates Foundation Principles, Practice and Economics (Wiley, 2001) and 150 journal 

grant that will seek to create in the laboratory an inexpensive and proceedings papers. He has 14 patents (issued and applied 

antimalarial drug, artemisinin. This drug could be sold for one- for) and has presented over 100 papers. He received the Marvin 

tenth of today’s price and compete with the formerly front-line J. Johnson Award in Biochemical Technology of the American 

antimalarial now confronted by disease-resistant strains around Chemical Society in 2002 and the Food, Pharmaceutical, and 

the world. Keasling and his team at Berkeley already have Bioengineering Division Award of the American Institute of 

worked out methods for extracting the genes responsible for Chemical Engineers in 2001. He was elected to the National 

making artemisinin and have transplanted them into a harmless Academy of Engineering in 1999. 

strain of E. coli. Ladisch served as a member of U.S. delegations and advisory 

Research in the Keasling laboratory focuses on the metabolic panels to Russia, Thailand, China, and Japan to review the status 

engineering of microorganisms for degradation of environmen- of biotechnology programs. In 1991 and 1992, he chaired the 

tal contaminants or for environmentally friendly synthesis. To National Research Council’s Committee on Bioprocess Engithat end, he has developed a number of new genetic and math- neering, which studied research priorities and policy issues 

ematical tools to allow more precise and reproducible control of related to commercialization of biotechnology and published 

metabolism. These tools are being used in such applications as the report Putting Biotechnology to Work: Bioprocess Engineering

synthesis of terpene drugs and biodegradable polymers, accumu- (National Academy Press, 1992). 

lation of phosphate and heavy metals, degradation of chlorinated His research addresses fundamental topics in bioprocess engi­ and aromatic hydrocarbons, biodesulfurization of fossil fuels, neering as they apply to bioenergy, bioproducts, biorecovery, and and complete mineralization of organophosphate nerve agents bionanotechnology. The research addresses transformation of and pesticides. Genomics, proteomics, and metabolomics are renewable resources into bioproducts, properties of proteins and being employed to investigate effects of these changes on cellular living organisms at surfaces, rapid prototyping of microfluidic physiology and to optimize cellular redesign. biosensors, and bioseparations. This work has resulted in new 

Ken Keegstra industrial bioenergy processes and systematic approaches and 

correlations for scaleup of laboratory chromatographic purifi- Ken Keegstra is director of the Plant Research Laboratory and cation techniques to process-scale manufacturing systems; it a University Distinguished Professor in the departments of also has resulted in scaledown of bioseparations and the rapid Biochemistry and Plant Biology at Michigan State University prototyping of microfluidic biochips for quick detection of (MSU). He received his doctorate in chemistry at the Univer­ pathogenic microorganisms. Ladisch teaches bioseparations, sity of Colorado, where he investigated the structure of plant bioprocess engineering, and biotechnology at both the graduate cell-wall components and their interactions within the wall. For and undergraduate levels. more than 20 years he studied other biological problems, mainly 

chloroplast biogenesis and the targeting of nuclear-encoded pro- Lee Lynd tein into chloroplasts. At the time of his move to MSU in 1993, Lee Rybeck Lynd is a professor of engineering and adjunct he reinitiated work on plant cell walls. The major focus of his professor of biological sciences at Dartmouth College and a current research is the biosynthesis of plant cell-wall polysaccha­ professor extraordinary of microbiology at the University of rides produced in the Golgi before delivery to the cell wall. The Stellenbosch in South Africa. He received a bachelor’s degree in greatest amount of effort has centered on xyloglucan biosynthe­ biology from Bates College; master’s in bacteriology from the sis, but his research group also has investigated the biosynthesis University of Wisconsin, Madison; and master’s and doctorate of mannans, glucomannans, and arabinoxylans as well as a few from the Thayer School of Engineering. other wall components. 

Lynd leads a research group in biochemical engineering and Mchael Ladsch applied biology relevant to processing cellulosic biomass. His 

Michael Ladisch is director of the Laboratory of Renewable laboratory’s research topics are chosen to address a primary 

Resources Engineering and Distinguished Professor of Agri- technical impediment to realizing a “carbohydrate economy”: 

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APPENDICES 

Overcoming the recalcitrance of cellulosic materials to biological 

conversion. Specific topics include, in order of increasing scale,

metabolic engineering to improve product yields in thermophilic 

bacteria, microbial physiology of anaerobic cellulolytic microorganisms, kinetics and reactor design for enzymatic and microbial 

hydrolysis of cellulosic materials, and conversion of “real-world”

cellulosic materials such as waste sludge produced from paper 

mills. A particular focus of the Lynd group is “consolidated bioprocessing,” a widely applicable potential breakthrough in which 

production of cellulase enzymes, hydrolysis of biomass fiber, and 

fermentation of resulting sugars are accomplished in one process 

step by a single microbial community. 

Lynd is a recipient of the National Science Foundation’s Presidential Young Investigator Award and a two-time recipient of 

the Charles A. Lindbergh Award for his efforts to promote balance between technological progress and preservation of natural 

and human environments. Professional activities include service 

as associate editor for Biotechnology and Bioengineering, member 

of a presidential advisory committee on reducing greenhouse 

gas emissions from personal vehicles, and organizing committee 

member for the Annual Symposium on Biotechnology for Fuels 

and Chemicals. Lynd has authored more than 60 peer-reviewed 

manuscripts and holds 5 patents. 

George Mchaels 

In May 2004, George Michaels was named associate laboratory 

director of the newly formed Computational and Information 

Sciences Directorate at Pacific Northwest National Laboratory 

(PNNL). This new directorate delivers innovative solutions to 

address national and global problems by enabling large-scale 

scientific discoveries through R&D in science-driven computing. He holds a doctorate in biochemistry and molecular biology 

and a bachelor’s degree (1974) in microbiology, all from the 

University of Florida. 

He joined PNNL in April 2003 as director of bioinformatics 

for the Biomolecular Systems Initiative. He is an internationally 

recognized pioneer in bioinformatics and in the practical development of biotechnological approaches for discovery. During 

his career spanning nearly 30 years, he has provided increasingly 

significant technical and leadership contributions to his field. He 

holds patents in methods for designing DNA-binding proteins 

and for morphological reconstruction. 

Most recently, Michaels held leadership positions at Monsanto 

in St. Louis, Missouri, where he designed an integrated expression-profiling program. He also cofounded and served as vicepresident and chief scientist of Genome Dynamics, a Maryland 

biotechnology startup company. While an associate professor 

at George Mason University in Fairfax, Virginia, he initiated 

one of the nation’s first doctoral programs in bioinformatics and 

computational biology. He also has served as a special expert to 

the office of the director of the National Institutes of Health. 

Coln Mtchnson 

Colin Mitchinson earned his bachelor’s degree in biochemistry 

from the University of Edinburgh and doctorate in biochemistry 

from the University of Newcastle. He has extensive research 

experience in the study of structure-function relationships in 

proteins ranging from (Ca++/Mg+) ATPase of muscle sarcoplasmic reticulum, ribonuclease, and subtilisin. He also has performed protein engineering on starch-processing enzymes and 

cellulases and was project leader and principal investigator for a 

multidisciplinary effort to develop a new cellulase. He currently 

serves as senior staff scientist at Danisco Genencor, where his 

research focuses on development of new cellulase products for 

biomass conversion. His publications include reviews on protein 

folding, substrate binding, and active site characterization using 

molecular genetic, biochemical, and biophysical techniques. A 

representative recent publication is M. Sandgren, J. Stahlberg,

and C. Mitchinson, “Structural and Biochemical Studies of GH 

Family 12 Cellulases: Improved Thermal Stability, and Ligand 

Complexes,” Progress in Biophysics and Molecular Biology 89, 

246–91 (2005). 

Mark Morrson 

Mark Morrison is a professor in the Department of Animal 

Science at Ohio State University and also holds a nonsalaried 

appointment in the Department of Microbiology. He has a longstanding interest in gastrointestinal microbiology and bacterial 

physiology. Much of his research has focused on the ecophysiology of plant biomass degradation in herbivores and the molecular biology underpinning cellulose degradation and bacterial 

adhesion to plant structural polysaccharides. He serves as project 

leader for the North American Consortium for Genomics of 

Fibrolytic Bacteria, involving scientists from The Institute for 

Genomic Research, Cornell, University of Illinois, and University of Guelph. The consortium’s activities include sequencing of 

four rumen bacterial genomes (Fibrobacter succinogenes, Prevotella 

bryantii, Prevotella ruminicola, and Ruminococcus albus) as well as 

comparative and functional genomic studies with these bacteria. 

Art Ragauskas 

Art Ragauskas received his doctorate in chemistry from the University of Western Ontario in 1986, with subsequent postdoctoral research at the University of Alberta and Colorado State 

University. He is a fellow of the International Academy of Wood 

Science and the Technical Association of the Pulp and Paper 

Industry (known as TAPPI). His research program at Georgia 

Institute of Technology is seeking to understand and exploit 

innovative sustainable lignocellulosic materials and develop 

new and improved applications for nature’s premiere renewable 

biopolymers including cellulose, hemicellulose, and lignin. 

Ragauskas’s research is directed toward innovative processes 

for converting lignocellulosic biomass into innovative biomaterials and biofuels. Achieving this goal requires research in 

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Appendix C. Participant Biosketches 

several fields of study, including lignocellulosic fiber chemistry 

and physical properties; carbohydrate, lignin, and extractive 

chemistry; nanobiomaterials; biotechnology; and material and 

polymer science. These studies are supported by expertise in 

advanced spectroscopy, imaging, nanotechnology, chemoenzymatic biotechnology, cold plasma, composites, bleaching, and 

pulping technologies. His research is sponsored by a consortium 

of industrial partners, Defense Advanced Research Projects 

Agency, National Science Foundation, U.S. Department of 

Agriculture, Department of Energy, and Georgia Traditional 

Industries Program (TIP3). 

Ragauskus has been a Luso-American Foundation Teaching 

Fellow at the Universidade da Beira Interior, Portugal; an invited 

guest teaching professor at Chalmers University of Technology, Sweden and South China University of Technology; and 

an invited research professor at Royal Institute of Technology,

Stockholm. He has published 185 papers, patents, and conference proceedings. He is an associate editor for the Journal of 

Pulp and Paper Science, Holzforschung, and the Journal of Chemistry and Technology and has served on several advisory boards 

and review panels. 

John Ralph 

John Ralph received his bachelor’s degree with honors in chemistry in 1976 from the University of Canterbury, New Zealand, and 

his doctorate in chemistry and forestry in 1985 from the University of Wisconsin, Madison (UWM). He is a research chemist 

at the U.S. Dairy Forage Research Center and a professor in the 

UWM Department of Forest Ecology and Management. 

An organic chemist and nuclear magnetic resonance (NMR) 

spectroscopist specializing in cell-wall model compound 

syntheses and monolignol polymerization reactions, Ralph is 

involved principally in studies aimed at detailing the mechanisms of lignin-polysaccharide cross-linking and their effect on 

limiting cell-wall degradability. He developed NMR methods 

for cell-wall structural analysis, including an NMR database of 

model compounds for lignin and related wall components, as 

well as methods for analyzing lignin structure and nondegradatively solubilizing the entire cell-wall faction of finely divided 

plant cell walls. He has additional expertise in synthetic organic 

chemistry, specifically in the synthesis of cell-wall model compounds, lignin oligomers, enzyme precursors, and products. 

Smo Sarkanen 

Simo Sarkanen is a professor in lignin chemistry and biochemistry at the Department of Biobased Products, University of 

Minnesota. He received his undergraduate training at King’s 

College, Cambridge (England), and was awarded a doctorate 

in chemistry from the University of Washington, Seattle. His 

first publications were in theoretical (computational) chemistry, but his doctoral dissertation was in bioorganic chemistry 

(enzyme kinetics). At the postdoctoral level in the Department 

of Chemical Engineering at the University of Washington, he 

embarked on a journey into various controversial aspects of 

lignin chemistry. Currently, his research interests range from 

lignin biosynthesis and biodegradation to new formulations for 

lignin-based thermoplastics. 

Most lignin chemists and biochemists have thought that configurations of lignin macromolecules are random (or combinatorial), but Sarkanen’s group is trying to develop an explicit 

working hypothesis for replicating specific lignin primary 

structures during lignin biosynthesis. The first step in lignin biodegradation generally has been considered under the control of 

lignin peroxidase, manganese-dependent peroxidase, or laccasemediator systems. Sakanen and his coworkers actively promote 

the view that a completely different kind of lignin depolymerase 

may be responsible for cleaving lignin macromolecules in vivo.

Finally, they have produced the first series of thermoplastics with 

promising mechanical properties composed predominantly or 

entirely (85 to 100%) of simple lignin derivatives. Previous work 

in the field typically had encountered incorporation limits of 25 

to 40% for lignins in potentially useful polymeric materials. 

Chrstophe Schllng 

Christophe Schilling is a cofounder of Genomatica, Inc. He 

received his doctoral degree in bioengineering under Bernhard 

Palsson at the University of California, San Diego, where he was 

a Powell Foundation and Whitaker Foundation Fellow. He also 

holds a bachelor’s degree in bomedical engineering from Duke 

University, where he was a Howard Hughes Undergraduate 

Research Fellow. 

As president and chief scientific officer of Genomatica, he currently directs all the company’s business and scientific efforts 

toward applying advanced modeling and simulation technologies to a number of metabolism-driven products. This includes 

overseeing development of Genomatica’s integrated computation and experimental platform to drive the metabolic engineering of microbes to support next-generation bioprocesses being 

developed by the company’s industrial partners. 

He is coauthor of numerous scientific articles in systems biology 

and metabolic modeling, and he is an inventor on a number of 

patent filings surrounding Genomatica’s core technologies. He 

was featured in the February 2001 edition of Genome Technology 

as one of 16 “up and comers” who have the talent and drive to 

make great strides in science, technology, and business. In 2003 he 

was named one of the top 100 young innovators under 35 whose 

innovative work in technology will have a profound impact on the 

world, as selected by MIT’s Technology Review magazine. 

John Shankln 

John Shanklin is a senior biochemist in the Biology Department 

at Brookhaven National Laboratory and an adjunct professor in 

the Biochemistry Department at the State University of New 

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APPENDICES 

York at Stony Brook. He received his bachelor’s degree in physiology from the University of Lancaster, United Kingdom, in 1981 

and his master’s in forestry from the University of Wisconsin,

Madison (UWM), in 1984. He was awarded a doctorate in horticulture from UMW in 1988, working on the ubiquitin system. 

Shanklin’s current interests focus on plant lipid biochemistry 

and developing plant oils as renewable industrial feedstocks.

Specifically he studies structure and function in lipid modification enzymes. This class of enzymes performs high-energy 

chemistry on a wide variety of substrates with various chemical 

outcomes. In addition to studying how specific fatty acids are 

synthesized, he also conducts metabolic engineering experiments 

on how modulations in pathway components affect storage oil 

accumulation for both usual and unusual fatty acids. 

Shanklin is the winner of several awards, including the Office 

of Energy Research Young Scientist Award, a Presidential Early 

Career Award, and the Terry Galliard Medal for Plant Lipid 

Biochemistry. He was chair of the Scientific Program Committee 

for the National Plant Lipid Cooperative meetings from 2001 to 

present. He has served on panels for the Department of Energy 

and the National Science Foundation and is on the Scientific 

Advisory Board for Genetic Engineering, Principles and Methods 

and Advances in Plant Biochemistry and Molecular Biology. He currently is a member of the Council for Energy Biosciences on the 

Basic Energy Sciences Committee of Visitors (2005). 

Sharon Shoemaker 

Sharon Shoemaker joined the University of California, Davis 

(UCD), in 1991 as founder and executive director of the California Institute of Food and Agricultural Research (known as 

CIFAR). She also is research leader at the UC Forest Products 

Laboratory and site director of the National Science Foundation–UCD Center for Advanced Processing and Packaging.

Shoemaker holds a bachelor’s degree in chemistry, master’s 

in food science, and doctorate in biochemistry and nutrition 

from Virginia Tech. After postdoctoral training in biochemical 

engineering, she joined Cetus Corporation in Berkeley, California, the first U.S. biotechnology company. After 7 years, she 

left Cetus to join Genencor, a company more aligned with her 

interests in applying fermentation and enzymology to problems 

in food and agriculture. 

Shoemaker’s industrial experience led to patents on novel yeast 

strains to convert biomass to ethanol and on novel bacterial 

strains to produce new forms of cellulose. She also led team 

efforts to improve enzyme systems for converting biomass to 

sugars and subsequently fermenting them to chemicals and to 

characterize and develop ligninase systems for use in the pulp 

and paper industry. Her research interests focus on cellulose 

applications in biomass conversion (e.g., rice straw, wood,

and mixed waste paper); integration of various unit operations in biomass-conversion processes (membrane filtration 

and enzymes); and development of new analytical methods for 

quantifying specific cellulase activities. Shoemaker is active in 

regional, national, and international task forces, review panels,

and programming on new and emerging biobased processing 

technologies, carbon sequestration, and cellulase R&D. 

Yuval Shoham 

Yuval Shoham has been head since 2004 of the Department 

of Biotechnology and Food Engineering at the Israel Institute 

of Technology (Technion) in Haifa. He received his bachelor’s 

degree in biology from Tel Aviv University in 1980, his master’s in 

microbiology in 1982, and his doctorate in biochemical engineering from MIT in 1987. In 1988 he joined the Technion, where he 

is director of the Otto Meyerhoff Minerva Center for Biotechnology and holds the Erwin and Rosl Pollak Chair in Biotechnology.

He is a fellow of the American Academy of Microbiology. 

Shoham’s research focuses on the catalytic mechanisms and 

structure-function relationships of industrial enzymes, especially 

glycoside hydrolases, and on gene regulation of the hemicellulolytic system in Geobacillus stearothermophilus and cellulosomerelated genes in Clostridium thermocellum. He has been involved in 

several industrial projects, including the development of a largescale process for bleaching paper pulp with alkaline thermostable 

xylanases and an enzymatic process for making complex lipids 

with novel SN-2 lipases. He has authored more than 120 articles 

and book chapters and holds several patents. 

Lloyd M. Smth 

Lloyd M. Smith is John D. MacArthur Professor of Chemistry 

and director of the Genome Center at the University of Wisconsin, Madison (UWM), where he has been since 1988. He 

received a bachelor’s degree in biochemistry from the University 

of California, Berkeley, in 1977 and a doctorate in biophysics 

from Stanford University in 1981. In 1982 he moved to the 

California Institute of Technology, where he developed the first 

fluorescence-based automated DNA sequencing instrument. 

Smith has been named one of Science Digest’s Top 100 Innovators and has received the Presidential Young Investigator 

Award, Eli Lilly Analytical Chemistry Award, Association of 

Biomolecular Resource Facilities Award for the development 

of automated DNA sequencing, and the American Chemical 

Society Award in Chemical Instrumentation. He has served on 

the NIH National Human Genome Research Institute Advisory Council and the NIH Human Genome Study Section,

has authored more than 165 scientific papers, and is inventor 

on 20 issued U.S. patents. He is a cofounder of the biotechnology company, Third Wave Technologies, and is a member of 

the board of directors of GWC Technologies, Inc., and GenTel 

Biosurfaces,Inc., where he also is chair of the Scientific Advisory 

Board. His primary area of research is the development of new 

technologies for analysis and manipulation of biomolecules. 

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Appendix C. Participant Biosketches 

Chrs Somervlle 

Chris Somerville is director of the Carnegie Institution Department of Plant Biology and professor in the Department of Biological Sciences at Stanford University. He has published more 

than 170 scientific papers and received patents in plant and 

microbial genetics, genomics, biochemistry, and biotechnology.

His current research interests are focused on the characterization of proteins such as cellulose synthase, which is implicated 

in plant cell-wall synthesis and modification. He is a member 

of the senior editorial committee of Science magazine and of the

scientific advisory boards of numerous academic institutions 

and private foundations in Europe and North America. He is a 

member of the U.S. National Academy of Sciences, Royal Society of London, and Royal Society of Canada. He has received 

numerous scientific awards and several honorary degrees. He is 

chairman of the board of Mendel Biotechnology, a private plant 

biotechnology company in the San Francisco Bay area. 

Gregory Stephanopoulos 

Since 1985, Gregory Stephanopoulos has been a professor of 

chemical engineering at Massachusetts Institute of Technology 

(MIT). He received his bachelor’s degree from the National 

Technical University of Athens, master’s from the University 

of Florida, and doctorate from the University of Minnesota, all 

in chemical engineering. Upon finishing his doctorate in 1978,

he joined the faculty of the California Institute of Technology, where he served as assistant and associate professor until 

1985. He was associate director of the Biotechnology Process 

Engineering Center between 1990 and 1997 and was appointed 

Bayer Professor of Chemical Engineering and Biotechnology.

He also is the Taplin Professor of Health Sciences and Technology (2001–), instructor of bioengineering at Harvard Medical 

School (1997–), member of the international faculty of the 

Technical University of Denmark (2001–), and fellow of the 

Singapore–MIT Alliance (2000–). 

Stephanopoulos’s current research focuses on metabolic engineering and its applications to the production of biochemicals 

and specialty chemicals, the rigorous evaluation of cell physiology 

using advanced isotopic methods, the metabolism and physiology 

of mammalian cells with emphasis on obesity and diabetes, and 

bioinformatics and functional genomics whereby new genomicsbased technologies are applied to the elucidation of cell physiology and metabolic engineering. He has coauthored or coedited 5 

books and published some 250 papers and 19 patents. 

Stephanopoulos currently is editor-in-chief of the journal Metabolic Engineering and serves on the editorial boards of seven scientific journals. In 1992 he chaired the Food, Pharmaceutical, and 

Bioengineering Division of the American Institute of Chemical 

Engineers (AIChE) and was elected a founding fellow of the 

American Institute for Medical and Biological Engineering. In 

2002 he received the Merck Award in Metabolic Engineering 

and was elected to the board of directors of AIChE. In 2003, he 

was elected to the National Academy of Engineering and in 2005 

was awarded an honorary doctorate (doctor technices honoris 

causa) by the Technical University of Denmark. 

He has taught a variety of undergraduate and graduate courses 

in the chemical engineering curricula at California Institute of 

Technology and MIT. He also has developed a number of new 

courses, including Metabolic Engineering, Metabolic and Cell 

Engineering, and, more recently, Bioinformatics. He coauthored 

the first textbook on metabolic engineering and has taught a 

number of biotechnology courses in the summer sessions since 

1985. He introduced and directed two such courses, Metabolic 

Engineering (1995–99) and Bioinformatics (2000–). 

Bruce Stone 

Bruce Stone received his bachelor’s degree from the University 

of Melbourne in 1948 after majoring in chemistry and biochemistry. In 1951 he was seconded for training in mycology 

to the Commonwealth Mycological Institute, Kew, England.

In 1952 he commenced doctoral studies in the Department of 

Biochemistry at University College, London. After graduating 

in 1954, he held postdoctoral appointments in Ottawa (National 

Research Council Fellow) and London (Imperial Chemical Industries Fellow). He returned to the Russell Grimwade 

School of Biochemistry, University of Melbourne, as a lecturer 

in 1958 and was appointed reader in agricultural biochemistry in 

1966. From 1972 until his official retirement in 1995, he served 

as foundation professor of biochemistry at La Trobe University. 

Stone twice held the position of dean of the School of Biological Sciences at La Trobe (1976–78, 1987-91), was president 

of the Australian Biochemical Society (1988–90), and was chairman of the Royal Australian Chemical Institute, Cereal Chemistry Division (1978–79). He currently is editor-in-chief of the 

Journal of Cereal Science and assistant director of the Australian 

Academy of Science and Technology’s Crawford Fund, an organization supporting international agricultural research. 

His major research interest in the chemistry and biochemistry 

of plant polysaccharides arose during his first appointment 

through an investigation of cellulose-breakdown enzymology.

His specific studies on polysaccharide components of the cell 

walls of cereals and grasses encompassed their structure, biosynthesis, depolymerization, and their interactions with lignins and 

proteins. Their outcomes have been applied to the solution of 

agricultural and horticultural problems, especially in relation to 

cereal-grain quality and processing behavior and in human and 

ruminant nutrition. Stone has a special interest in the biology 

and chemistry of callose and related(1→3)-ß-D-glucans, and,

with Adrienne Clarke, published a treatise on the subject in 

1993. Currently he is investigating the biology and biosynthesis 

of a bacterial (1→3)-ß-D-glucan, curdlan. 

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APPENDICES 

Bob Tabta 

Bob Tabita is professor of microbiology and plant biology 

and an Ohio Eminent Scholar at Ohio State University. His 

doctoral work in the late Don Lundgren’s laboratory at Syracuse 

University introduced him to the metabolism and biochemistry 

of autotrophic bacteria; his postdoctoral research was in Bruce 

McFadden’s group in the Chemistry Department at Washington State University. A key experiment, in which he discovered 

that growth with a reduced electron donor upregulates Rubisco 

synthesis, has been the foundation for his entire career with 

photosynthetic bacteria, enabling studies on the enzymology of 

Rubisco and other pathway enzymes. His laboratory continues 

this strong interest in molecular regulation, biochemistry, and 

enzymology of carbon dioxide assimilation and the control of 

Rubisco synthesis. 

All organisms require CO2.It is used in many enzyme-catalyzed 

reactions in processes as important and varied as carbohydrate 

metabolism, lipid biosynthesis, and production of vital metabolic intermediates for the cell. With the realization that many 

microorganisms use CO2

 to elicit pathogenesis, CO2 metabolism 

and its control are recognized as having great health relevance.

Carbon dioxide also may be employed as the sole source of 

carbon by a large and diverse group of organisms. For this reason, CO2

 fixation is associated with global issues of agricultural 

productivity, carbon cycling, and industrial productivity. Carbon 

dioxide also is recognized as the chief greenhouse gas and has 

been implicated in general warming of the earth’s biosphere.

For all these reasons, research on various aspects of CO2

 fixation 

control, biochemistry, and ecology have attracted wide interest. Microbial systems studied in the Tabita laboratory include 

R. palustris, C. tepidum, and R. sphaeroides. 

Steve Thomas 

Steve Thomas’s research expertise spans work on insect hormones at the University of California, Los Angeles, to plant 

biotechnology research at the ARCO Plant Cell Research Institute in Dublin, California. Through the National Renewable 

Energy Laboratory, Thomas was manager for the DOE Office 

of Fuel Development project entitled, “Production of Cellulases 

in Tobacco and Potato Plant Bioreactors.” He has worked on the 

Sugar Processing Integration Task, surveying the compositional 

variability of geographically and genetically diverse corn stover 

residues to minimize risk associated with commercialization of 

biomass-conversion technology. He recently joined Ceres, Inc.,

as a principal scientist. 

Jerry Tuskan 

Gerald A. Tuskan, a distinguished scientist in Oak Ridge 

National Laboratory (ORNL) Environmental Sciences Division, holds a bachelor’s degree in forest management from 

Northern Arizona University; a master’s in forest genetics 

from Mississippi State University; and a doctorate in genetics 

from Texas A&M University. He also is a research professor 

in the University of Tennessee’s departments of Entomology,

Plant Pathology, Plant Sciences, and Genome Sciences and 

Technology where he advises graduate students, interacts with 

departmental faculty, and provides guest lectures and graduate 

seminars. Before joining ORNL, he served as associate professor 

of horticulture and forestry at North Dakota State University 

and was an instructor in the Forest Science Department at Texas 

A&M University. 

At ORNL, Tuskan is responsible for coordinating the DOE 

effort to sequence the Populus genome. This includes projects 

with carbon allocation and partitioning in woody plants as a 

means to enhance bioenergy conversion and carbon sequestration, genome-enabled discovery of carbon sequestration genes in 

poplar, environmental influences on wood chemistry and density 

of Populus and loblolly pine, formation of an international Populus genome consortium, and creation of a Populus postsequence

science plan. His research helps identify genes associated with 

cell-wall chemistry, genetic mapping in Populus, particularly 

related to carbon allocation and partitioning, and the use of 

genomics information to accelerate domestication of Populus. 

Ed Uberbacher 

Ed Uberbacher is lead scientist for computational biology in 

the Life Sciences Division at Oak Ridge National Laboratory.

He received a bachelor’s degree from Johns Hopkins University 

in 1974 and a doctorate in chemistry from the University of 

Pennsylvania in 1979. He is the codeveloper of GRAIL, the 

first of many gene-finding programs created during the Human 

Genome Project. Next-generation algorithms such as Grail-Exp,

which can use both EST and complete cDNA data, provide 

another level to the analysis of draft data. 

As a member of the Computational Biology Institute, Uberbacher works within a collaborative environment that combines 

the expertise of biologists, computer scientists, and mathematicians with high-performance computing to create and provide 

tools and infrastructure to advance systems and computational 

biology. He is CEO of Genomix Corporation, a provider of 

analysis systems, information resources, and partnering opportunities to guide R&D teams through critical steps in pharmaceutical discovery and development. He also serves as a faculty 

member in computational biology and bioinformatics at the 

University of Tennessee–ORNL Graduate School of Genome 

Science and Technology. 

Fernando Valle 

Fernando Valle is staff scientist for process science at Danisco 

Genencor International, with expertise in microbial pathway 

engineering for the biological production of enzymes, therapeutic proteins, peptides, antibiotics, vitamins, amino acids, and 

organic acids. His significant research accomplishments include 

the optimization and pathway engineering of an E. coli strain 

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Appendix C. Participant Biosketches 

to transform glucose into 1, 3-propanediol, a monomer used in 

producing polyester fibers. 

John Vogel 

John Vogel’s research program is focused on applying molecular biology to improve herbaceous energy crops. Major projects 

include using dsRNA-mediated gene silencing to reduce lignin 

content in switchgrass and studying the model grass Brachypodium 

distachyon to identify genes controlling cell-wall composition.

Recent accomplishments include creation of transgenic switchgrass plants with silenced lignin biosynthetic genes, development 

of an Agrobacterium-mediated transformation method for Brachypodium, and sequencing of >20,000 Brachypodium ESTs. 

He is a USDA Agricultural Research Service molecular 

biologist at the Western Regional Research Center in Albany,

California. His experience includes extensive research in plant 

pathology as both an assistant professor at the University of 

California, Riverside, and postdoctoral fellow at the Carnegie 

Institution of Washington in Stanford, California. His graduate 

work focused on cytokinin and ethylene signaling in Arabidopsis. 

Ken Vogel 

Kenneth Vogel is a research geneticist at the USDA Agricultural 

Research Service Wheat, Sorghum, and Forage Research Unit 

in Lincoln, Nebraska. He received his bachelor’s and master’s 

degrees from Colorado State University in 1965 and 1967 and a 

doctorate from the University of Nebraska in 1974. 

His responsibilities are in research management and perennial 

grass breeding and genetics. His specific research interest is to 

develop improved perennial grasses, switchgrass in particular, for 

use on marginal lands of the Central Great Plains and midwestern states along with associated management practices. This 

research involves germplasm evaluation and characterization 

of native and introduced grasses and basic and applied genetic 

research in addition to applied breeding work. Since 1990, he 

has been conducting research to develop switchgrass into a 

biomass energy crop for these areas. 

Ncholas Wheeler 

Nicholas Wheeler received an undergraduate degree in forest 

sciences from the University of Washington in 1973, a master’s 

in forest genetics from Michigan State University in 1974, and 

a doctorate in plant breeding and genetics from Wisconsin in 

1981. He has worked as a tree breeder for the government of 

British Columbia for 4 yrs, as a scientist in the forest products 

industry for 22 yrs, and, more recently, as a private consultant in 

his own business (Molecular Tree Breeding Services). 

Wheeler’s research includes projects in physiological and quantitative genetics, genecology, and molecular genetics. He currently 

is an adjunct faculty member in the Department of Forestry and 

Environmental Resources at North Carolina State University, 

working on a National Science Foundation grant focused on 

association genetics in loblolly pine. He also is an affiliate faculty 

member at Oregon State University Department of Forest 

Sciences. He most recently completed a project investigating a 

novel approach to breeding in poplars. 

Davd Wlson 

David Wilson is a professor of biochemistry and molecular and 

cell biology at Cornell University. He received his bachelor’s 

degree from Harvard in 1961 and his doctorate in biochemistry 

from Stanford Medical School in 1966. He did postdoctoral 

work at the Department of Biophysics at Johns Hopkins Medical School from 1966 to 1967 before going to Cornell as an 

assistant professor in 1967. Wilson is a member of the American 

Society of Biological Chemists, American Society of Microbiologists, and American Association for the Advancement of 

Science. He also is a member of the Johns Hopkins Society of 

Scholars and director of the Cornell Institute for Comparative 

and Environmental Toxicology. 

Wilson’s laboratory uses a combination of genomics, protein 

engineering, and molecular biology to study the enzymology 

of plant cell-wall degradation, with a major focus on cellulases.

Enzymes that degrade insoluble substrates have significant 

differences from most enzymes whose substrates are small 

soluble molecules. In addition, cellulases are important industrial 

enzymes and have potential in the production of renewable,

nonpolluting fuels and chemicals. His group has been studying 

the high G-C gram variable soil bacterium Thermobifida fusca, a 

moderate thermophile, for more than 20 years. T. fusca is a major

microorganism degrading plant cell walls in heated plant wastes 

such as compost piles. He also is using a genomic approach 

to compare aerobic and anaerobic organisms to discover novel 

mechanisms for degrading cellulose. 

Research projects involve the biochemistry and chemistry of the 

cell walls of higher plants, with special reference to cereals and 

grasses and the structure-function relationships of wall polysaccharides and proteins: chemistry of lignin-carbohydrate and 

lignin-protein associations in walls of forage plants, with special 

reference to their impact on ruminant digestion; enzymology of 

ß-glucan hydrolases–active site chemistry in relation to specificity and to the design of specific hydrolase inhibitors; molecular 

mechanisms of biosynthesis of ß-glucans in plant cell walls and 

by bacteria; and development of monoclonal antibodies for the 

specific detection of cell-wall polysaccharides. 

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APPENDICES 

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