EBI Annual Report 2008

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TABLE OF CONTENTS

About the Energy Biosciences Institute

An Introduction to Biofuels

Feedstock: Agronomy, Engineering, and the Environment

Bioconversion: Attacking CellulosicDegradation on Several Fronts

Biofuels Production: Transforming Feedstockto Fuel with Microbes

Societal and Economic Impacts of Biofuels

The EBI at 1 Year Olda BP Perspective

Education and Outreach

EBI Research Programs, Projects, and Research Personnel

Responding to increasing evidence that continued dependence on fossil fuels iscausing climate change, with consequences both uncertain and unwelcome, BPsteps up to create a visionary collaboration of academia and industry.

Understanding the complex issues surrounding biofuels is key for the EBI in orderto ensure a successful research agenda. Challenges span the entire life cycle ofthe fuel, from the first seed in the ground to its use in transportation.

EBI scientists are at the literal ground level of the search for the most productivebiofuel crops, seeking feedstock that grow in difficult environments, usingsustainable fertilizers, and maximizing cultivation and harvest techniques.

Research at the EBI is addressing several of the major bottlenecks impeding thebreakdown of lignocellulosic feedstock into fermentable sugars.

The routes to biological production of fuel molecules are numerousbut one ofthe most effective is through microbial fermentation and synthesis.

EBI scientists are developing modeling frameworks to project the potentialimpacts of a biofuels industry on factors such as land use, food production,carbon emissions, the global economy, and environmental stability.

EBI Associate Director Paul Willems reflects upon the energy companys goalsand how the Institute has fared in year one: We are positioned for successfulcollaboration and delivery of great innovation in years ahead.

The EBI recognizes its role in educating and training young researchers so thatthey are prepared for the coming bio-revolution, as well as sponsoring a broadrange of activities that facilitate information sharing and staff enrichment.

A listing of the first 50 funded projects in the Institute, including the teamsof principal investigators, research associates, postdocs, graduate, andundergraduate students.

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Message from the DirectorEBI Director Chris Somerville offers a personal perspective on the development

of the Institute: We can look back at having successfully navigated a myriad oftypical startup challenges as well as a few that were unusual, if not unique.

Exploring the Applications of Modern Biologyto the Energy Sector


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of the EBI is to develop an

integrated holistic understanding

of the research topics related

to energy biosciences.

An overriding goal

ABOUT THE ENERGY BIOSCIENCE

Societal and Economic Impacts of Biofuels

A major goal of the EBI is to understand the potential environmental, economic, and societalmpacts of meeting a growing portion of the worlds energy needs through cellulosic or

lgal biofuels. Many in the world are concerned that the demand for energy is so large thatnrestrained conversion of land to biofuel production could have negative envi ronmentalects and could further disadvantage many poor people by increasing prices for food, feed,nd ber.erefore, the EBI is working to understand how land is used around the world ando model the impacts of growing bioenergy crops on land that is not used for food productionr is providing key ecosystem services, such as carbon storage or biodiversity.

BI investigators are also testing the environmental impacts of various bioenergy crops andeveloping economic models that may help to understand the feasibility of bioenergy croproduction around the world. An important aspect of understanding the environmentalects of cellulosic biofuels will include development of complete life cycle models thatncorporate both direct and indirect eects of the biofuels.

eedstock: Agronomy, Engineering, and the Environment

Work in this area seeks to identif y and characterize plant species that can maximize cellulosiciomass production in various regions around the world, and to learn how to grow andarvest them sustainably. A primary goal is to discover plants that maximize the productionf cellulosic biomass, using minimal land, water, and energy. Because of the importance ofoil carbon in the global greenhouse gas balance, the EBI is particularly interested in identify-ng species that can be grown on the large amounts of minimally productive land around the

world.ese considerations favor the use of perennial grasses and certain woody species.However, the possible utility of algal species is also being explored.

Bioconversion: Attacking Cellulosic Degradation on Several Fronts

e main constituents of t he body of higher plants are polysaccharides and lignin. Fashioninguel from plants requires conversion of the polysaccharides to sugars by severing the chemicalond that holds them together, among the most critical and dicult steps in the process.

Todays practices are costly and i necient. EBI scientists are investigating natures methodsf releasing these sugars to achieve an eective and less costly method of breaking down

hese substances.is will be key to ensuring that biofuels can be reasonably priced.e EBIexamining the processes that take place in cow rumen, termites, compost heaps, and other

e primary initial thrust of EBI research is an exploration of the feasibili ty of commerciallyiable, sustainable and environmentally benign transportation fuels from biomass.e mostromising opportunities are currently thought to be cellulosic biofuels , but the EBI is alsoupporting a study on the feasibility of algal biofuels. e development of cellulosic fuelsnvolves identifying the most suitable species of plants for use as energy crops; improving

methods of breeding, propagation, planting, harvesting, storage and processing; and ensuringhat this is done in a sustainable way without negative impacts on food production or thenvironment. Production of biofuels also involves the development of biomass-to-liquiduels technologies that yield major benets in regard to both net energy output and netreenhouse gas balance based on consideration of all inputs.

To accomplish this, research is div ided into several areas of inquiry:

The Areas of Study

NERGY BIOSCIENCES INSTITUTE // 2008 ANNUAL REPORT

environments where biomass degradation takes place. In paralleexploring the development of new synthetic catalysts that can athe degradation of polysaccharides and lignin.

Biofuels Production: Transforming Feedstockto Fuel with Microbes

In order to convert sugars to liquid fuels, the proportion of oxygmust be reduced.is can be accomplished by bioconversion, sfermentation, or by chemical transformations. With no clear froat the present time, the EBI is exploring several methods in para

Methods used for production of biofuels today are similar to thefermentation practices used to make beer and wine, but these trmethods are not optimized for the large-scale, energy-ecient ption of cellulosic biofuels. EBI researchers are exploring ways ofing bioconversion of sugars to next-generation fuels by using th

of systems biology to characterize new types of microbes and bygenetic modications of promising organisms.ey are particulinterested in exploring ways of producing biofuels that will not rmajor changes in the transportation infrastructure.is involveing chemical and fermentation routes to products more hydrophethanol and butanol.e EBI is also interested in exploring altebioconversion technologies, such as the use of non-biological catransform biologically derived chemicals into fuels.

Microbiology of Fossil Fuel Reserves

During the past several decades, it has become apparent that sigpopulations of microorganisms are found in both coal and petroreservoirs deep underground.ese microbial populations can to the properties of the reservoirs in deleterious ways, such as thcatalyzing the souring of petroleum, but they may also contribupositively by activities such as altering the porosity of the reservcould allow more ecient recovery of oil. In order to understaneects of these microbial populations, the EBI intends to suppointo characterization of the organisms found in various reservoithe tools of modern biolog y, such as high-throughput DNA seqand analysis. By understanding the genomics of the reservoir miit may be possible to infer how their activities can be beer conttoward useful purposes.e collaboration with BP provides a ra

opportunity for academic scientists to access deep-earth samplereservoirs that have been geologically characterized.


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The close association within the EBof university academics and industrmanagers from BP offers a rare oppto accelerate the processes that arassociated with conversion of acaddiscovery into real-world application

ABOUT THE ENERGY BIOSCIENCES

THE RESEARCH ENTERPRISE

PUBLIC AND PRIVATE

Openness of the research enterpriseand theacademic freedom of its faculty, graduate studeuniversity researchersis paramount for the thpublic institutions in the EBI. Inventions madethe course of research within the EBI are ownethe academic institutions according to U.S. patand BP receives an automatic non-exclusive licin return for funding the research. All four parthave representation in the EBIs two managempanels, with none having a majority or veto powon the governing board, ensuring consensus indecisions.e Executive Commiee, which prscientic direction and operational oversight, icomposed of professors from the academic par

e Licensing Executives Society, a professionorganization for intellectual property specialistthe EBI for its 2008 Deal of Distinction awarbeing an innovative model for collaboration bacademia, government labs, and industry.

Collaborative research between universities anindustry yields new ideas and more eective pafor moving discoveries from the laboratory int

mercial use.

ese collaborations also help prestudents for non-academic careers and addressneed for real-world evaluation and implementasolutions science.

In the EBI, intellectual resources of leading resinstitutions are being brought to bear on the qusustainable, aordable, renewable energy.e tise of an international corporation is being emto ensure that commercialization and applicatihappen as rapidly as the discoveries allow.

PROGRAMS AND PROJECTS

e EBI is a mission-oriented research organization. One approach torealizing the EBI mission is to develop a consortium of comprehensiveexpertise and research activities concerning energy biosciences withinthe partner organizations. To achieve adequate breadth of scope, researchfunds are allocated to various topics based on predened targets.Workshops are scheduled throughout the year to share data and to helpdene key questions to be answered. Topical proposals that addressproblems dened by the EBI are solicited from faculty and scientists inthe partner institutions.us, the EBI mandate denes the problem,but the EBI investigators propose the research approaches to solutions.A peer-review process narrows proposals drawn from solicitations to afocused set of projects and programs for funding.

During the startup phase of the EBI in the summer of 2007, a verybroad solicitation was announced. From an initial list of more t han 250

pre-proposals from researchers at the three partner institutions, EBImanagement and advisors requested 85 full research proposals and,following external peer review, narrowed the eld to 50 high-priorityresearch eorts that received the rst round of funding. A secondsolicitation in the summer of 2008 was more narrowly focused. Awardsare divided into two categories: programs and projects. Programs aretypically large integrated multi-investigator eorts with broad goals,funded at anywhere from about $400,000 per year up to about $1 millionper year, and may continue for the 10-year life of the institute. Projectsare smaller activities of 2-3 years in duration that are usually narrower inscope.ese average about $150,000 per year.

Program research is conducted mostly within EBI space so that post-doctoral, support, and graduate student researchers from dierentdisciplines will work side-by-side, and so that space constraints will notlimit the ability of EBI investigators to participate in the EBI mission. iswill facilitate synergy across elds and will provide a training environmentand a broad appreciation of the scientic, technological, environmental,economic, and policy issues that must all be addressed to achieve theInstitutes goal of environmentally sustainable bioenergy.



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ABOUT THE ENERGY BIOSCIENCE

BP

BP, one of the worlds largest energy companies, ishe leading producer of oil and natural gas in the

United States, and the largest investor in U.S. energyevelopment. BP provides its customers with fuel for

ransportation and energy for heat and light, employ-ng more than 100,000 people worldwide and morehan 35,000 in the U.S. BP was the rst major energyompany to acknowledge the need for precautionaryction to reduce greenhouse gas emissions, and todayt continues to lead the eort to meet the worldsrowing demand for sustainable, environmentallyesponsible energy.

The University of Illinoisat Urbana-Champaign

e University of Illinois at Urbana-Champaign is aworld-class public university whose faculty, student,

nd alumni honors have brought internationalistinction. Home of the largest public universitybrary collection in the world, Illinois is also a

eader in supercomputing design and applicationnd boasts multi-disciplinary research excellencen dozens ofelds.e University has a pioneeringistory of sustainability research as the originator ofo-till agriculture, and is home to the longest running

nvestigation of the impacts of varied crop manage-ment methods on soi l quality (1876, Morrow plots)

utside of Europe. Founded in 1867, Illinois enrollsver 29,000 undergraduates in more than 150 eldsf study, and over 11,000 graduate and professional

tudents in over 100 programs. It is among the topve universities in the United States in the numberf annual doctorates awarded. Among its array of

aculty honors, the U of I is one of only 11 campusesworldwide to have been awarded two separate NobelPrizes in one year (2003).

The University of California, Berkeley

Founded in 1868, the University of California, Berkeley,is the nations top-ranked public university and the ag-ship of the 10-campus University of California system. Itenrolls over 24,000 undergraduates, distributed among80 degree programs, and more than 10,000 graduatestudents each year.e campus not only produces morePhDs than any other university in the country, but agreater number of its graduates go on to earn a PhD atBerkeley or elsewhere than do graduates of any otherinstitution.e university is distinguished by its researchprograms, which were funded in scal year 2006 by $469million in contract and grant awards from outside spon-sors. Berkeley faculty and researchers have won 20 NobelPrizes, 6 Pulitzer Prizes, 30 National Medals of Scienceand 29 MacArthur genius Awards. Of its academicsta, more than 130 are current members of the NationalAcademy of Sciences, and 85 belong to the NationalAcademy of Engineering.

Lawrence Berkeley National Laboratory

Lawrence Berkeley National Laboratory (Berkeley Lab)has been a leader in science and engineering researchfor more than 75 years. Located on a 200-acre site in thehills above UC Berkeleys campus, the Lab is the oldestof the U.S. Department of Energys national laboratories.Managed by the University of California, it operateswith an annual budget of more than $550 million anda staof about 3,800 employees, including more than500 students and 250 principal investigators with jointappointments at UC Berkeley. It employs a team

concept to its research, as developed by founder ErnestOrlando Lawrence, and boasts a legacy that has yieldedrich dividends in basic knowledge and applied technol-ogy, and a profusion of awards. Berkeley Lab conductsunclassied research across a wide range of scienticdisciplines, with key eorts in fundamental studies of theuniverse, quantitative biology, nanoscience, new energysystems and environmental solutions, and the use ofintegrated computing as a tool for discovery. Its uniqueuser facilities include the Advanced Light Source, theMolecular Foundry, the National Center for ElectronMicroscopy, and the Joint Genome Institute.

The Partners


t is an enticing formulatake the technical and intellectual strengths of threenternationally known public research institutions, add the successful commercialegacy of one of the worlds leading energy companies, and blend together in a uniqueartnership in which collaborations are forged and innovation is maximized. The sum isreater than all of these impressive parts.


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e EBI operates as one of several research centers within t hepartner institutions.e faculty and students that design andcarry out the research activities of the EBI have appointmentsand academic responsibilities within the various academicdepartments of the partners.e EBI administers the nancialand material resources and facilities supported by fundingfrom BP.

e EBI is managed on a day-to-day basis by a Director and asmall team of colleagues and advisors from the four partners(the Executive Commiee).is administrative team imple-ments processes for deciding what research opportunities tofund, for providing or facilitating research and administrativesupport to EBI investigators, and for facilitating communica-tions within the EBI and between EBI investigators and variousstakeholders and interested parties around the world.

e Governance Board has the responsibility to de ne, overseeand review the implementation of EBI programs in the opencomponent of research. It also appoints the EBI Director and

Deputy Director.e Board has eight voting members, fourfrom the research partnersat least one each from the Berkeleyand Illinois campuses and one from Berkeley Laband fourappointed by BP.e EBI Director, Associate Director, andDeputy Director are ex-ocio members.

e EBI Director, Chris Somerville, manages the conresearch projects and EBIs public communications, eand outreach activities. He works with the Executive Ctee to develop an annual program plan with goals and stones, and he prepares the annual budget request. Deputy Director, Steve Long, manages research condat the Illinois EBI site and its integration into the Instias a whole.e Associate Director, Paul Willems, is threpresentative on the EBI management team. He alsoteam of BP employees who are located at the EBI.

e Executive Commiee is t he EBIs program manabody, with Director Somerville as chair. He, the deputassociate directors, and ve other professors from the institutions (currently Adam Arkin, Dan Kammen, DZilberman, Evan DeLucia, Michael Marlea) compricommiee membership.is panel proposes the annustrategic work plan, including priority research projecinstitute funding, for approval by the Governance Bo

Governance and leadership are critical components of the EBI agreement. Completed in 20EBI contract combines the resources of the partner institutions into an agreement that prova non-exclusive, royalty-free license to an i nvention.

EBI Leadership

ABOUT THE ENERGY BIOSCIENCES

Research in the Energy Biosciences Institute is being conducted at two primarylocationsin California, at the historic Calvin Laboratory and the nearby HildebrandHall chemistry building at UC Berkeley; and at the new Institute for Genomic Biologybuilding in the heart of the University of Illinois campus. A 320-acre Energy Farm, thelargest of its type (just south of the Illinois campus), includes land for demonstrations,large-scale production, plant breeding and storage. In 2008, 120 acres of this farm wereplanted, and another 160 are slated for development in the near future.

e Berkeley center includes dedicated biotechnology laboratories and specializedfacilities for high-throughput chemical synthesis and assays of many types. eIllinois program is housed in a building specically designed for integrated researchand development eorts, with a complete suite of microscopy, imaging, plant growth,microfabrication, and bioanalysis facilities and tools. In addition, ind ividual research-ers have access to the oces, technical laboratories and user facilities of their homecampuses.

In 2013, the EBI is planning to move its permanent headquarters into a 144,000-square-foot facility dedicated to renewable energy research to be built on UC Berkeleyland adjacent to Berkeley Lab.e Helios building, as it will be called, will haveoce and laboratory space for 150 co-located institute sta, including the EBIinvestigators, BP scientists, and laboratory personnel. e building will have spaceand amenities that will promote a collegial and collaborative research environment,including meeting and seminar rooms, lounges, and a caf.

he Facilitiesm the hills of Berkeley tofarm fields of Illinois, EBIearch spans the nation

d the world.


Architect's design of

proposed Helios Energy

Research Facility in

Berkeley, future home of

the EBI


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CORN AND CANE ETHANOL

At present, most biofuel is produced from coand from sugar extracted from sugarcane usinrelatively mature technology and establishedproduction practices. Research within the EBgenerally not concerned with these sources obut is directed toward next-generation oppobased on using the lignocellulose that comprbody of plants. But an understanding of the pinvolved in current biofuel production is impif improvement is to be achieved in the secongeneration.

Sugarcane (Saccharum sp.) is a highly productropical grass that accumulates sucrose in thetissues.e stalks are crushed to produce a ssolution that can be fermented to produce a

ethanol solution.e crushed stalks or bagcomprise the body of the plants are currentlyto produce heat that is used to distill the ethafrom the fermentation broth and to produceelectricity. It is possible that, with the develoecient technologies for conversion of lignoto fuels, a large proportion of the sugarcane bwill also be used to produce liquid fuels in th

In Brazil, where land suitable for growing sugis abundant, about 4.2 billion gallons of canewas produced in 2005 on less than four millihectares (a hectare is 2.47 acres) of land. Ethcomprises about 40 percent of all liquid tranfuel used in Brazil.e automobile eet is lacomposed of ex-fuel vehicles that can utilvarying ratios of ethanol and gasoline. By cononly about 2 percent of the eet in the Uniteex-fuel vehicles; the remainder cannot use gasoline mixtures containing more than 10 pethanol without mechanical modications.

Corn (Zea mays) is the largest U.S. crop with81 million acres planted in 2005, yielding ab11.1 billion bushels of corn seed. Approxima

percent of the mass of corn seed is starch.is released by grinding the seed in either a drprocess, cooked to gelatinize the starch, hydrwith enzymes to glucose, and fermented. Folfermentation and separation of ethanol by dithe residual slurry of insoluble ber, protein,called distiller dry grains with solubles (DDused as animal food. e U.S. is expected to pabout eight billion gallons of ethanol from abpercent of the corn crop in 2008.

...an understanding of the processes

involved in current biofuel production

is important if improvement is to be

achieved in the second generation.

AN INTRODUCTION T

...it would be possible to produce about

half of all transportation fuels by growing a

plant like Miscanthus on about 1 percent

of the terrestrial surface area.

WHY BIOFUELS?

e global energy market provides humans with about 370 exajoules ofnergy per year, which is equivalent to the energy content of about 170

million barrels of oil per day. Approximately 87 percent of energy pur-hased globally comes from fossil fuels. Although humans may eventuallyeplete reserves of fossil fuels, that moment is quite far o. In addition

o substantial remaining reserves of oil and gas, abundant coal depositsre projected to be adequate to meet human energy needs for severalundred years. Coal can be converted into a wide variety of liquid fuels

hat can substitute for petroleum. us, if concerns about greenhouseas-induced climate change and energy are ignored, there is not a pressingeed to develop biofuels.

e linkage between climate change and biofuels arises from the fact thatome types of biofuels can be substantially less carbon-intensive sourcesf energy than fossil fuels. Energy from sunlight is collected by the photo-ynthetic system of plants and used to reduce and condense atmospheric

CO2 into the chemicals that comprise the body of plants. When plantsre burned, the energy is released as heat that can be used for work, suchs generating electricity, and the CO2 is recycled. With highly productivelants such asMiscanthus giganteus growing on good soils with adequateainfall and favorable mean temperature, such as is found in central Illi-ois, more than 1 percent of annual incident solar insolation (exposure tounlight) is retained as chemical energy in biomass. If one uses a value forotal solar insolation of 120,000 TW (terawas), 1 percent solar conver-on eciency, and an energy recovery value of 50 percent, it would beossible to produce about half of all transportation fuels by growing alant like Miscanthus on about 1 percent of the terrestrial surface area.



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AN INTRODUCTION T

OTHER BIOFUELS

Ethanol is not an ideal f uel in several respects and may not be thmajor biofuel in 20 years.e main problem is its ability to mix water, which imposes an energy cost for disti llation, creates proin transporting the fuel via pipelines, and leads to poisoning of tmicroorganisms that produce it. us there is interest in developbiofuels that are more hydrophobic and spontaneously partitionthe aqueous phase. For instance, butanol dehydrates spontaneoabout 9 percent solution, has very low vapor pressure, and has a heat similar to octane so that f uel-air mixing at low temperatureproblem. When added to ethanol-gasoline mixt ures, small amobutanol depress the vapor pressure, reducing the hazards of explduring fuel handling. Unfortunately, butanol is toxic to organismproduce it at concentrations very much lower than 9 percent sol

Several companies have recently announced plans to produce bfermentation of sugars from biomass. Some microorganisms havreported to secrete alkanes, which do not mix with water. Plantsother organisms have a wide array of lipid synthetic pathways, rathe opportunity for engineering t hese pathways into bacteria orIt seems likely that additional types of biofuels with physical prosimilar to those in current use will be developed.

Although much of the current research on cellulosic fuels i s focuon bioconversion technologies, other approaches are being explwithin the EBI. In particular, it is possible to convert biomass tofuels by direct chemical conversions using either gasication folre-forming of the gaseous products to fuels, or by chemically caconversion of biomass to fuels. Because the mandate of the EBIexplore biological technologies, it is not pursuing research basegasication technologies. However, a number of EBI research grexploring the development of new catalysts for hydrolysis of biosoluble molecules and conversion of such molecules to liquid fu

COORDINATION, INTEGRATION

e major opportunity to expand the use of biofuels will be fouimproving the various components of cellulosic biofuels producmiracles are required to develop cost-eective cellulosic biofuel

of two-fold improvements in the e

ciency of various steps coulbiofuels less expensive than liquid fossil fuels. However, implemrational improvements in the overall process is challenging. Manthe various components will have to be coordinated, integratingknowledge from many scientic and engineering disciplines. Intis encouraged through the placement of researchers from dieredisciplines in shared space. is is what the EBI was established

No miracles are

required to develop

cost-effective cellulosic

biofuels; however,

implementing rational

improvements in the

overall process is

challenging.

Many components of the cellulosic ethanol bioconver-on process are not yet optimized for commercialroduction. For instance, the strains of yeast that aresed for industrial fermentations do not normallytilize sugars other than glucose. Strains of yeastSaccharomyces cerevisiae) andEschericia coli have beenngineered to ferment xylose to ethanol, but additional

work needs to be done to adapt such lines to industrialonditions, to optimize metabolic control of the path-

ways, and to enable fermentation of other sugars.

Another problem is that large amounts of cellulasere required to hydrolyze cellulose. Process improve-

ments during the past decade have reduced the cost ofellulase per gallon of ethanol from about $5 to about0 cents but that is still considered too expensiveompared to other enzyme-based processes.ere is

widespread interest in nding enzymes with higherctivity than current cellulases by surveying the prop-rties of enzymes from poorly explored sources suchs termite guts, rumen, compost heaps, and tropicalorests. Alternatively, it may be possible to improve thectivity of industrial cellulases by protein engineering.t is also important to understand the structure andunction of cellulosomes, extracellular enzyme com-lexes that catalyze hydrolysis of cellulose and otherolysaccharides. Industrial ethanol production hassed yeasts that ferment glucose. To make use of theugars derived from hemicelluloses, yeasts and otherrganisms are being engineered to use these sugarss well as glucose. e holy grail of cellulosic ethanolroduction is to incorporate improvements in bothellulases and fermentation into a single organism that

would secrete all of the necessary enzymes and utilizell of the available sugars in a process referred to asintegrated bioprocessing.

Many other problems may have multiple solutions, butelatively lile progress has been made. For instance,

many plant polysaccharides, during biomass hydroly-s, release acetic acid that inhibits the growth of the

ermentative organisms. Similarly, furfural, an organicquid compound produced by a side reaction duringcid-catalyzed polysaccharide hydrolysis, inhibits

microbial growth. In principle, these and relatedroblems may be overcome by developing resistant

organisms, by altering the chemical composition of thebiomass, or by process improvements.

e development of a biofuel industry is only feasiblein regions where land and water resources are availableto support the growth of plant biomass that is excessto other needs.e Departments of Energy and Agri-culture conducted a study of biomass availability andconcluded that approximately 1.3 billion dry metrictons of excess cellulosic biomass is available eachyear in the U.S.is includes half the corn stover (theleaves and stalks of the corn plant) and wheat straw,and about 40 million acres of set-aside land to growperennial grasses such as switchgrass and Miscanthus.At a conversion value of about 100 gallons of ethanolper ton of lignocellulosic biomass, this would beequivalent to about 130 billion gallons of ethanol, orabout 40 percent of U.S. liquid fuel consumption on anenergy-equivalent basis.

Based upon the proportion of transportation fuelalready produced by Brazil, it seems likely that SouthAmerica could meet all its needs for transportationfuels with biofuels. A recent analysis of the 15 coun-tries in the European Union concluded that Europecould produce approximately 11.7 exajoules (units ofenergy) per year of biofuels, an amount similar to theU.S. goal of 30 percent of transportation fuels (11.6exajoules).

A recent study by ecologists at Stanford concluded thatabout a billion acres of land around the world that wasfarmed in the past has been abandoned. It seems likelythat much of this land could be used for productionof energy crops without impacts on food productionand without incurring production of greenhouse gasesfrom land conversion. Although cellulosic fuels willnot completely meet global needs for transportationfuels anytime soon, they are expected to become asignicant component worldwide.e widespreadimplementation of trading in carbon credits could

accelerate progress toward that goal and could encour-age best practices in terms of land management andfuel production.



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Most fuel ethanol today is producedfrom cornstarch.e EnergyBiosciences Institute is lookingbeyond the major food crops as the

feedstock for biofuels, with a focuson sustainable crops that could begrown on land unsuited for foodproduction.

Dried stems, roots and leaves ofmost plants are made up largely ofthree types of polymers: cellulose,hemicelluloses and lignin.ismixture of the three polymers,termed lignocellulose, comprisesmost of the structural mass of allplants.

Tree trunks, crop residues, andfall-harvested shoots of perennialssuch as switchgrass and Miscanthusare predominantly lignocellulose.If the sugars of the cellulose andhemicellulose can be ecientlyreleased, then these can befermented or chemically convertedto ethanol and other fuels.ismakes any plant material a potentialfeedstock for the manufacture ofcellulosic biofuels.

e ideal cellulosic biofuelfeedstock will vary with location,but as a general rule they will allprovide high productivity with aminimum of inputs. Such crops willminimize the footprint required

to achieve sucient feedswhile ensuring environmeconomic sustainability.

If the use of lignocellulosup all plants as potential fhow has the EBI narrowedown to a manageable tasExamination of yields in tconversion of incident sointo biomass has shown aof grasses, the Androponoappear particularly eciegroup includes sugarcaneand Miscanthus.e EBIProgram, and those lookiutilization, have chosen thas their rst targets for deinvestigations.ese specalso of primary interest toFeedstock Genomics grothe University of Illinois Moose, since sorghum habeen sequenced and manlar markers have been devsugarcane.is allows thea jump-start with Miscansince the DNA sequenceprotein-coding regions aphighly homologous with relatives, sorghum and suGenetic similarity amongmay also facilitate mechastudies by Berkeley scienMcCormick of self-incommechanisms that impedein Miscanthus and other g

0

FEEDSTOCK: AGRONOMY, ENGINEERING, AND THE EN

Can microbes be superheroes? Can crops grownfor biofuels actually help the environment? Can agiant Asian grass really power a biofuels revolutioPerhaps. EBI scientists are quite literally on the grlevel of the search for the most productive biofueplants, conducting field trials, studying essential famachinery, searching for sustainable fertilizers, aexploring the issues surrounding agro-ecosystemdiversity that may make second-generation biofueboon for the environment.

Agronomy, Engineering, and the Environment

FEEDSTOCK:



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It all begins with the feedstock, thesource plant material. Commercialbiofuel production will require lots

of it, preferably grownwhere food crops cant,and in quantities thatyield huge amounts ofbiomass.

Meet Steve Long. Hewas looking through

old family photographsthe other day, and hecame upon one in which he wasteaching a college course in 1978.e subject of his lecture? Biofuels.I was surprised it was that longago, he says.

Since that prescient moment, Longhas become one of the worldsforemost experts on the so-calledC4 perennial grasses like Miscan-thus, which has emerged as a majorbioenergy crop in the United King-dom, where he conducted some ofthe rst analyses of this crop. Hiswork in the U.S. has now shownit to be a promising candidate asa dedicated biofuel feedstock. Heis deputy director of the EBI andthe Robert Emerson Professor ofCrop Sciences and Plant Biology atthe University of Illinois Urbana-Champaign. And he is passionateabout plants, in particular about

the impacts of global atmosphericchange on photosynthesis.

Ive always been fascinated abouthow plants work, says the so-

spoken, British-born Long. AndIve been curious about how we canmaximize the eciency with whichthey convert solar energy intobiomass, to achieve more yield peracre. When the opportunity aroseto apply his expertise to a potentialsolution to the world energy crisis,he seized it.

Aer a productive 23-year career as

scientist and professor at EnglandsUniversity of Essex, he decided topull up roots in 1999 and headedoverseas, to Illinois, where therewas a much larger concentrationof plant biologists, as beed theland-grant schools in the UnitedStates.ere were broad intel-lectual resources, and access to thefarm.

e farm is UIUCs vast acreageof experimental croplands, inwhich he could test cultivationand nutrition techniques on thecarbon-xing grasses he had beenstudying in EuropeMiscanthusx giganteus, found naturally in thehighland areas of Japan and China,and switchgrass, a variety on whichAmerican agronomists were focus-ing for fuel conversion.

When Lawrence Berkeley NationalLaboratory Director Steve Chu

invited him in 2006 to join a panelof experts to brainstorm alternativefuels, he traveled to Berkeley andlistened to Chris Somerville talkabout the potential of biology to

enable the production of based transportation fueltwo colleagues had neverwhen the Berkeley leaderintroduced the prospect oing for the BP-funded EBthey jumped at the chanc

Long, the feedstock expealready reported researchthat indicate Miscanthus

rior crop in terms of biomand sustainability in poorthe search is far from over

What the EBI allows us to explore options, to loomore plants than we havehe says. We are open to tsibility that there might bpossible feedstocks.erbe one solution. For examdrier areas, switchgrass mthe beer option, and in tAgaves.

Or, he says, the answer mbe found in crops not curknown. With others he wlooking at over 20 diereat the EBI Energy Farm inand many more variants wthose species. He is also iin pursuing salt-tolerant pthat could thrive on salinsalinated through geology

irrigation practices or hydwith sea water.


Passionate About Plants and Their PotentialSTEVE LONG:

costs?e Agricultural EngineeringProgram, led by KC Ting at Illinois,is developing and testing machin-ery that can improve eciencyin production and transport. Forexample, harvesting can todayrequire three operations.e EBIprogram has tested a machine thatmay integrate the three into one. Itis also looking at the cost/benetof on-farm compaction to facilitatetransport.

All these options are evaluated in asystems analysis context to identifythe key bolenecksengineering,environmental and economic

from planting to delivery tobiofuel synthesis. While Miscan-thus, switchgrass, sorghum andsugarcane are the current programfocus, it will certainly expand intotrees, such as poplar and willow,for locations where trees will bemore appropriate feedstockse.g.on terrain that is too irregular forconventional machine harvesting.A wide range of other perennials isalso being tested on the EBI EnergyFarm at Illinois.

e Engineering, Agronomy,and Genomics groups are alsocollaborating on the developmentof remote sensing methods of track-ing plant growth and production,which will allow rapid assessmentof thousands of dierent geneticlines non-destructively. Methodsare currently being tested thatpromise far more rapid identica-tion of the most promising materi-

als. In parallel, Michael Dietzein Illinois is building a databaseof the broad basket of potentialcellulosic feedstocks coupled withthe development of mechanisticmodels of growth, production andecosystems services.


uch as switchgrass.e develop-ment of transformation technologyor grasses by Jack Widholm, Don

Duvick and colleagues in Illinois isxpected to facilitate direct tests ofypotheses about the mechanistsasis of important traits such aself-incompatibility

e Agronomy Program led byTom Voigt at Illinois is in the

rocess of establishing comparativerials of Miscanthus and switch-rass around North America.is includes comparisons withorghum and sugarcane at moreouthern locations and with prairie

lants at other locations. Aboutalf of the spectacular gain inields of the major food crops haseen achieved through improvedgronomy, and it is expected thatgronomy will be similarly impor-ant for optimizing production ofnergy crops.e agronomy groupexamining how plant spacing,

weed control and fertilizers can beptimized to increase yields and

minimize inputs. A project lead byGerman Bollero at Illinois is devel-

ping a computer model that wille able to predict how energy cropsespond to factors such as climate,oils and nutrients.e plants usedn the sustainability trials are alsoeing used by the Biotic Stressrogram, under Illinois Mike Gray,

o identify the diseases and pestshat may emerge with these newrops, and strategies for protectinghem.e group is monitoringnsects, nematodes, fungi, bacteriand viruses.

New crops present on-farm engi-eering challenges. In particular,ow can these crops be planted,arvested, stored and transportedciently, and without large energy

The answer might be found

in crops not currently known.

and many more variants within

those species.

He will be looking at over 20

different grasses at the farm,


17/34

In three years, we should have a pretty good idea of the in

of microbes that work on Miscanthus, and we will be able

demonstrate whether nitrogen fixation plays an important

FEEDSTOCK: AGRONOMY, ENGINEERING, AND THE ENNERGY BIOSCIENCES INSTITUTE // 2008 ANNUAL REPORT

orgive Angela Kent if shes biased, but sheelieves that microbes are the most importantrganisms in the world. She is especially highn bacteria, the most abundant group ofrganisms on the planet. And she thinks theyould be key to the ecient productivity ofuture biofuel feedstocks.

Microbes have the greatest reservoir ofenetic diversity on earth, says the young

microbial ecologist at the University of Illinois,Urbana-Champaign. ey can do incredible

hings. Its almost like they have superpowers.ey can nd a way to make a living with anyind of compound.

e EBI believes in Kent and her bacterialroops, supporting a project that seeks toxploit the nitrogen-xing capabilities of

microbes to enhance the sustainability ofioenergy crops.

Production of nitrogen fertilizer requires a lotf energy input from fossil fuel, Kent says. It

akes chemicals to make and farm machineryo apply. But microbes can do that essentiallyor free. If we can nd conditions that favoracterial nitrogen xation in the plants, we cannuence their long-term sustainability.

e rst step is to understand how thessociation of certain bacteria with targetediofuel grasses will work, and then how thatssociation is aected by environmentalactorswater, climate, soil texture andertility, etc.en there is the task of analyzinghe characteristics of individual microbes

hrough genetic phenotyping, searching forhe genes that are involved in the nitrogenroduction. Considering that there can be

millions of microbes in a gram of soil (suchs the samples seen at right), Kents task isaunting.

eres no guarantee that inoculations ofaturally occurring nitrogen-xing bacteria will

mprove the growth of crops like Miscanthus,

but Kent thinks the prospectsare high. With the assistanceof two postdocs and twoundergraduate students in herlaboratory, she hopes to builda library of relevant bacteriaand then apply them toMiscanthus and other plantsin the EBI Energy Farm.She says she suspects that both growth andsustainability will be improved.

Kents early studies at the University ofWisconsin focused on a medical career,her fascination about microbes leading her

into the study of pathogens and their role incausing disease. But applications to humanhealth were long-term, and she admits tolooking for something more immediate.Environmental microbiology felt muchmore applied to me, looking at microbesinvolved in plant health. I knew I could makea dierence, she recalls.

Now that dierence might be the applicationof microbiology, and her super microbes,to the development and nurturing of a newclass of plant-based transportation fuels.

In three years, Kent says, we should

have a prey good idea of the inventory ofmicrobes that work on Miscanthus, and wewill be able to demonstrate whether nitrogenxation plays an important role, as well aswhat traits contribute to the colonization ofthe plant.

Looking to Microscopic Superpowersto Sustain Plant Growth

NGELA KENT:

They can do incredible things. Its

almost like they have superpowers.

They can find a way to make a living

with any kind of compound."

"


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Biofuels are oen touted as a solution to rising oil prices and global climate changebut they could alsolve agricultural problems caused by centuries of land abuse.at possibility is being put to the test inresearch program being conducted by University of Illinois Professor Evan DeLucia, who is heading an

gation of the impact and sustainability of feedstock production. I am hopeful, says Dthat not only will biofuel crops provide sustainable energy, but that they will diversifimprove the health of our agricultural ecosystem.

Such an improvement is long overdue. Yearssoybean rotation in the Midwest have erodedpolluted water, and disrupted carbon and nitcycles. Continuing that cycle could spell disafarmland, while introducing new, low-maintecrops like Miscanthus or switchgrass could r

the nutrients that have been leached from th

DeLucias team is tryi ng to turn these hypothinto hard facts.ey have planted plots of foumass crops at Illinois new EBI Energy FarmMiscanthus, switchgrass, and mixed restoredgrass. is is the rst time that side-by-side sons of the ecology of dierent biofuel feedsbe conducted under realistic eld conditionsDeLucia. As the crops continue to grow overthree-year establishment period, high-tech intation will carefully monitor the plants eecenvironment.

And that eect is complex, to say the least. Ta full sense of the crops impact, tests range fcarbon measurements to energy exchange reto analysis of arthropod activity.e variety requires researchers with all kinds of specialtEBI team includes ecologists, entomologistsgists, and many others. eir program reectunique interdisciplinary nature of EBI researcia, a plant biologist, calls working with sciensuch diverse elds invigorating and excitingthis breadth of experience can provide deep

into the sustainability of biof uel crops.

is isnt the rst foray into environmental rfor DeLucia, who has spent his career studyicarbon cycles, climate change, forest preservand pollution. His previous research has shothe pressing need for sustainable biofuels, anhe hopes that biofuels could solve more thanenvironmental problem.

Reviving Depleted Agricultural LandA Biofuel Bonus?

EVAN DELUCIA:


e Environment Program is using the trials established by theAgronomy Program to provide the rst actual measurements, as

pposed to projections, of greenhouse gas balances and impactsn water availability and quality.

n 2008, large-scale (up to 10 acres each) replicated trials ofMis-anthus x giganteus, switchgrass, mixed species restored prairie,nd continuous corn were established on the energy farm abovenstrumented eld drains that monitor the volumes of water,utrients and carbon draining out of each crop. Simultaneously,sing a technique known as eddy-covariance, the net uxes ofases containing carbon and nitrogen emied and absorbedy these plots are being monitored continuously. is will be a

nique data set, critical to constraining Life Cycle Assessmentmodels and greenhouse gas balance models of t he ecosystemservices provided by dierent biofuel cropping systems. It willlso test the predictions that the group has already made usinghe state-of-the-art models of soil carbon balance in conjunction

with consultant Bill Parton.

ese studies will also be vital to gaining informed Life CycleAssessments (LCAs), an activity that is the focus of research byArpad Horvath andomas McKone at Berkeley. A key motiva-

on for developing second-generation biofuels is to provideruly renewable liquid transportation fuelfuel that may be

made without degrading the ability of the land to produce theeedstock, and with a minimum of greenhouse gas emissions inheir production, utilization and indirect impacts compared toossil fuels. Any new activity that is likely to impact the environ-

ment, for beer or worse, is typically subject to an LCA. AnCA examines, in the case of a second-generation biofuel, thenvironmental costs and benets at each step from the conver-on of land and planting of the crop through the combustion of

he fuel. What are the inputs and outputs of carbon, energy andther resources at each stage? Projections around farm opera-ons are currently hypothetical, because there have been no

horough measurements of greenhouse gas balances over theseew cropping systems and conversion operations.is is further

omplicated by the fact that, as with any new technology in itsnfancy, there is uncertainty as to the appearance of the matureechnology.e experience gained on the energy farm will beritical to adding more substance and reality to LCAs

Two major tasks of the Feedstock Programs on Agronomy,ngineering, and Environment are 1) to actually measurereenhouse gas and energy balances; and 2) to dene the

most sustainable farm operations and opportunities for landonversion that would maximize opportunities for s equesteringarbon from the atmosphere, as opposed to releasing carbon.

A complementary project led by Ryan Stewart at Illinois isfocused on studying the eects on soil quality of stands ofMiscanthus in Japan that are more than 1,000 years old.

One of the key env ironmental issues surrounding productionof biofuels concerns eects on water quality and availability.Plants transpire enormous amounts of water during growth,and such water emissions can aect soil moisture and theamount of water runothat may support streams and lakes. epotential eects of cellulosic crops on water are being modeledusing climate and soil data in a collaboration between CarlBernacchi at Illinois and Tracy Twine at the University of Min-nesota. In addition, a detailed analysis of the potential eects

on the well-characterized Lake Bloomington watershed is beingcarried out by Ximing Cai, John Braden, Wayland Eheart andGeorge Czapar at Illinois.

Concerns about water and land use have stimulated interest inthe possibility of using algae to produce biofuels. Preliminaryanalyses of the costs of fuels produced from algae indicatethat there are challenges associated with bringing the costs ofproduction in line with alternatives. To understand the oppor-tunity, the EBI is supporting a f easibility study by a large groupof algal biologists led by Berkeley Lab scientist Nigel Quinn.

Environmental and LifeCycle Analysis



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VISUALIZING LIGNOCELLULOSELignocellulose is a composite material made up of a variety of pthat are tightly bound to one another. Because the polymers areto be seen by most types of microscopy, the exact molecular struof the cell walls that comprise plant biomass is not known. Berkscientist Manfred Auer and colleagues are using new method s omicroscopy to visualize biomass at nanometer resolution. His coPaul Adams and Jim Schuck are building a novel Raman microsthat they expect will provide spatially resolved chemical informaabout cell walls that will be complementary to other types of imUsed together, these types of imaging provide insights into howtreatments aect the structure of biomass and facilitate improvethe overall conversion process.

BIOPROSPECTING AND DISCOVERY OF NOVE

ORGANISMS AND ENZYMES FOR BIOMASS

DEPOLYMERIZATION AND CONVERSION TO

BIOFUELS

e ability to carry out biomass processing or fermentation at rehigh temperatures has several advantages. In addition to lower rmicrobial contamination, a higher temperature accelerates enzy

lyzed reactions and would reduce cooling costs and facilitate ethbutanol) removal and recovery. To enable translation of these adto practice, EBI investigators are seeking to isolate and characteenzymes from several extremely thermophilic bacterial strains sadapted for cellulose and hemicellulose degradation.

For example, Doug Clark, Harvey Blanch, and co-workers at UCley, in collaboration with the Frank Robb laboratory at the UnivMaryland, are cultivating communities of organisms from hot swith cellulosic substrates with the aim of enriching for, and isolatithermophiles that produce biofuels and/or thermostable cellulol

0Bioconversion research at the EBI is addressing sethe major bottlenecks impeding the practical prodof biofuels, such as ethanol and butanol, from lignolulosic feedstocks. These programs have several inrelated componentsthe discovery and characterof fungi and thermophiles that produce new enzymlignin and cellulose deconstruction; protein engineeand kinetic modeling of improved cellulases; new odiscovery and cellular engineering for enhanced bioproduction and improved tolerance of the biofuel pand bioprocess engineering to optimize fermentat

BIOCONVERSION: ATTACKING CELLULOSIC DEGRADATION ON SEVER

Attacking Cellulosic Degradation on Several Fronts

BIOCONVERSION:



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PROTEIN ENGINEERING

OF IMPROVED CELLULASES

Protein engineering has proven to be a powerful toolin creating enzymes with new and improved proper-ties. However, designing and employing methods toscreen or select cellulase mutants using solid cellulosicsubstrates remains a largely unmet challenge. Res earchby Clark, Blanch, and co-workers aims to overcome

this challenge as well as that of developing more cost-eective cellulases, by developing high-throughputsolid substrate assays and applying them in the directedevolution of thermophilic cellulases and of cellulaseswith high activity in ionic liquids. e methodologydeveloped will be applicable to the generation andstudy of improved cellulases that can be used in variousprocess congurations for the production of biofuelsfrom cellulosic biomass.

Enzyme and metabolic pathway engineering are alsoamong the tools being used by the Illinois group

led by principal investigators John Gerlt and JohnCronan in a program directed toward overcomingbiomass recalcitrance as a key obstacle in biofuelproduction.e objectives of their program are toidentify and characterize degradation pathways forlignin, characterize the enzymes that are involved inthose pathways, engineer these enzymes so that theywill have enhanced catalytic properties, and designnew metabolic pathways in organisms so that biofuelproduction can be enhanced.

In addition to soluble cellulases, some organismsdegrade cellulose using cellulosomes, which are com-prised of cellulases organized in a complex assemblyof enzymes and scaolding on the bacterial cell

Understanding the molecular mechanisms (of degradation of cellulosic

biomass) may provide the key i nsights needed to reconstitute "designe

cellulosomes..."

BIOCONVERSION: ATTACKING CELLULOSIC DEGRADATION ON SEVER

wall.e goal of research carried out by JamiMichael Marlea, and co-workers at UC Berdevelop new experimental systems to study csome degradation of cellulosic biomass. eMarlea team aims to develop model systemenable them to study the enzymatic propertilulosomes at a fundamental level. Understanmolecular mechanisms may provide the key needed to reconstitute designer cellulosome

optimized for depolymerization of plant bio

MODELING FOR OPTIMAL CELLU

DESIGN AND CELLULOSE HYDR

Accurate kinetic models of cellulose hydrolycellulases are of critical importance for evalucellulase-component compositions and for dand optimizing processes for cellulose convebiofuels. Such models will also aid in the devand characterization of improved cellulolytic

generated by protein engineering and synthebiology.

Projects under way at UC Berkeley headed bClark, Blanch, and Clayton Radke aim to deva comprehensive model of cellulose hydrolyscan be used to predict cellulase performancecellulase design, and optimize the hydrolysiscellulosic substrates, including those obtaineEBI investigators.

As an important rst step toward modeling chydrolysis and engineering improved cellulaBlanch, and co-workers have isolated the indcomponents of the cellulase mixture secrete

nzymes. Bioprospecting for cellulose/hemicelluloseegradation systems is assisted by whole genomeequencing of novel isolates.

orts to exploit the advantages of thermophilicmicroorganisms for biofuels production are also

nderway at the University of Illinois in a programed by Isaac Cann (photo, page 36), Rob Mackie, ando-workers. Research in their laboratory has resultedn isolation and characterization of three novel ther-

mophilic bacteria with maximum growth temperaturesf about 70C.

ey are also turning to ruminant animals as a promis-ng source of cellulolytic microorganisms that functionciently at more moderate temperatures. Ruminantnimals are specialized in the utilization of grasses assource of feed. Both switchgrass and Miscanthus, in

mesh bags, have been placed in the rumen of stulatedale at the University of Illinois. ese have theneen recovered and the aached microbes investigatedy Eddy Rubin (photo, page 39) and co-workers aterkeley Labs DOE Joint Genome Institute to identifyossible sources of robust cellulolytic enzymes forcient conversion of cellulosic biomass into ferment-ble sugars.ese two complementary programsim to identify enzymes produced by the abundant

microbes responsible for degradation of plant cellwall polymers in the rumen of forage-feeding animalshat can potentially be co-opted for cellulosic biomass

onversion. A related goal is to develop tailor-madenzyme cocktails optimized for saccharication ofpecic bioenergy crops with their subsequent conver-on to alcohol fuels.

Another natural bioconverter that eciently breaksown and transforms plant biomass is the termiteindgut. In a program aimed at discovering novelnzymes capable of degrading wood lignocellulose,hil Hugenholtz and co-workers at the DOE Joint

Genome Institute are performing metagenomic andioinformatic analyses of several species of wood-

eeding and grass-feeding termites. ese studieswill be complemented by functional s creening ofelected enzymes and characterization of the plant cell

wall polymers present in the food sources, hindgut

segments, and fecal pellets of grass-feeding termites.us, todays wood-chomping pests may prove to beimportant players in tomorrows biofuel-producingtechnology.

In addition to cellulose, a particularly problematiccomponent of plant biomass is lignin. Lignin is acomplex, decay-resistant, highly cross-linked aromaticpolymer. Almost all research on lignin and lignocel-lulose biological degradation has so far focused onfungi that decay wood. However, grass cell wallsare very dierent from the walls of conifers, and innature, none of the wood-decaying fungi are knownto decompose grasses. To nd the fungal enzymesbest adapted to deconstruction of grass cell walls,the Berkeley team headed by John Taylor, N. LouiseGlass, and Tom Bruns aims to discover and bring intocultivation the fungi that decompose the lier that

has been accumulated at Illinois under the feedstockof choiceMiscanthus giganteus, a C4 grass closelyrelated to sugarcane.

Using modern high-throughput culture methodsdeveloped by the pharmaceutical industry, amongother techniques, fungi capable of deconstructing Mis-canthus cell walls will be identied and considered forrelevant enzyme production.e Taylor team is alsousing transcriptional proling and lignocellulolyticenzyme characterization of the lamentous fungus,Neurospora crassa, growing on Miscanthus cell walls to

learn how a fungus regulates genes responsible for cellwall deconstructing enzymes.

Berkeley chemist Michelle Chang is taking a dierentapproachin order to explore mechanistic aspects of

how certain organisms degrade lignin, she is exploringhow to modify yeast and bacteria that are normallyunable to degrade lignin so that they are able to carryout the chemical transformations associated withlignin breakdown. In this way she will test whetherour understanding of the pathway is correct andalso whether there may be opportunities to alter thepathway for increased activity in industrial conditions.



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22/34

A number of molecules of biological origin can serve as fuels. Etperhaps the most industrially successful biologically produced f

molecules

ranging from more complex branched-chain alcohacids and hydrocarbonshave a variety of properties that makemore or less aractive as targets for production. And they can beplants and microbes. Some burn cleaner than others; some haveenergy density, possess dierent octane ratings, or can be physictransported or produced more cost-eectively and reliably withenergy input.ose closer in form to what is found in gasoline, jet fuel may prove superior.

e routes to biological production of fuel molecules are numerinclude sources such as plant oils or algae. However, one of the eective routes is through microbial fermentation and synthesisstruction of feedstocks leads to hydrolysates rich in 5- and 6-carsugars such as glucose, xylose, and arabinose along with other cothat can be toxic. Natural or engineered yeast and bacteria can mthe sugars into dierent variants of the possible fuel molecules binhibited to various extents by toxins and the fuel products them

Nonetheless, the relative success of this approach derives from aof factors. Microbes express an amazing array of natural abilitiesconsume simple feed molecules and create complex organic chee revolutions in molecular biology and genomics have enablescientists to discover the genes responsible for these capabilitiestransplant their function into industrially robust host microbes.in quantitative and genome-scale measurement of cellular physi

down to the single-cell level give unprecedented insight into thethat restrict optimal production by limiting metabolic ux and mgrowth.ese tools have only just begun to be applied systematimprove microbial fuel production and, despite some early succthere remains much room for improvement.

e Energy Biosciences Institute has initiated a cuing-edge miengineering program combining both synthetic biology and meengineering approaches to create new routes to fuels in bacteriaas well as systems biology and genomics approaches to measurediagnose function.

0

BIOFUELS PRODUCTION: TRANSFORMING FEEDSTOCK TO FUEL WITH

The current method of converting plant sugars tois similar to centuries-old fermentation practices we have relied on to make beer and wine. While tmethods are successful for spirits, they've proveninadequate in the production of biofuels, especiallylarge scale. Biofuels production researchers at thare searching for ways to boost the concentratioproduced by the biofuel fermentation process by ing yeast, bacteria, and other microbes for indust


Transforming Feedstock to Fuel with Microbes

BIOFUELSPRODUCTION:


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Finding Chemical Keysto Open the Cellulose Lock

ALEX BELL:

Cellulose, that tough organiccomponent of all plant cell walitself constructed of complex cof sugars that hold the key to bdevelopment. Unless cellulose related polysaccharide, hemiceare eectively liberated from thseal, then broken down and dissugars for fuel fermentation wireleased.

Enter chemical engineer Alex Bell, whose EBprogram challenge is to pretreat the biomass

that can e

ciently dissolve cellulose and proresidue through depolymerization into sugarthinks the answer might lie in inorganic solveionic liquidssalts in liquid form.ey havebenets of functioning at room temperature,than requiring heat, and they are inert, meaniwont interfere chemically with the reaction.

e challenge is in understanding what propionic uids require to dissolve crystalline cellsays Bell, an expert on the form and functionof catalysts. Hes certain his team will solve thdissolution and depolymerization puzzles; hesure about controlling the chemical transformthe sugars into transportation fuels.

e search is both practical and theoretical. Wa half-dozen lab researchers, mostly postdocgraduate students, employ high-throughput sof candidate solvents (almost 100 reactions atime on automated assay machines), Bells faccolleagues will be looking at the theoretical acellulose and carbohydrate dissolution in ion

ey have a three-year commitment to nd t

answers, in order to determine whether or noprocess will successfully lead to commercialiand industrial application.ats where the Bpartnership comes in handy. Having BPs exin residence, to interact with us, wi ll be invaludetermining which of the approaches will be says Bell.

BIOFUELS PRODUCTION: TRANSFORMING FEEDSTOCK TO FUEL WITH

PRODUCING DIESEL SUBSTITUTES

IN BACTERIA

While ethanol production is an initial target for theEBI, other fuel molecules may prove to have superiorproperties. Biodiesel is an alternative fuel, widelyused in the alternative energy economy. It is usuallycomposed mostly of fay acid alkyl esters (FAAE).ese molecules are oen produced by catalyzedreaction of methanol with fay acids from plants,tallow and used cooking oils. Current estimatespredict that production costs for this fuel will remainsubstantially higher than petroleum-based products.Berkeley Lab scientists led by Nikos Krypides andAthanasios Lykidis are therefore applying biochemicaland metabolic engineering approaches, similar tothose above, augmented with experimental evolutionto generate bacteria with enhanced production of freefay acids, triacylglycerols, or FAAE.

Together these programs and projects are creatingan integrated engineering framework for designingmicrobes for transformation of feedstock into fuels.In the next few years, the EBI will add synergeticprograms to the Biofuels Production eort that willpush the frontiers even farther.

These programs and projects are creating

an integrated engineering framework for

designing microbes for transfromation

of feedstock into fuels.

ENGINEERING YEAST

FOR SUGAR UTILIZATION

Two research groups at the University of Illinoisseek to improve the utilization of the dierent sugarspresent in hydrolysates. Most microbes used for fuelproduction cannot eectively use both the 5 (likexylose and arabinose) and 6-carbon sugars (such asmannose and glucose) that are the most abundantproducts from the feedstock deconstruction.Complete fermentation of all these sugars to biofuelsis necessary to maximize yield and minimize waste inthe production process. Investigators led by chemicalengineer Huimin Zhao and microbial geneticist Yong-Su Jin will identify genes that transport the sugars intothe cell and metabolize them into common precursorsfor fuel synthesis in a number of dierent fungi.ese will be transplanted into industrially importanthost cells starting with the ethanol-producing yeastSaccharomyces cerevisiae. Advanced approaches formetabolic analysis and engineering will be employedto optimize yield and rate of production.

ELUCIDATING THE IMPACT OF

REGULATION AND HETEROGENEITY

ON SUGAR METABOLISM

IN BACTERIA

Engineered pathways are usually dependent upon thenormal metabolism of the host.e endogenous regu-latory machinery oen hampers fuel yields. Metabolicregulation is evolved to sense environmental condi-tions and deploy the right pathways to allow organismsto survive in an uncertain and competitive world.However, they may be triggered by the particular con-ditions found in industrial bioreactors and lead to poor

sugar utilization and production.ese eects arefurther complicated in large-scale fermentations wherecells experience uctuating nutrient conditions dueto imperfect mixing.e populations will be physi-ologically heterogeneous, and thus not all individualswill be producing optimally. EBI scientists in Illinoisunder biochemical engineers Christopher Rao and IdoGolding seek to overcome this problem in Escherichiacoliby creating a quantitative model of the s ystem thatwill facilitate the design of strains capable of homoge-neously, simultaneously and eciently metabolizingthe arabinose, xylose, and glucose.

THE MICROBIAL

CHARACTERIZATION PROGRAM

Researchers at UC Berkeley and Lawrence BerkeleyNational Laboratory have established a high-hroughput genetics and genomics capability andre developing an experimental and computationalrogram to determine the genetic mediators ofptimal fuel production in microbes. Using thethanol-producing bacterium Zymomonas mobilis as a

model system, program scientists headed by Berkeleyioengineer Adam Arkin will screen large-scale geneticnockout libraries and generate high-resolution whole-enome gene expression compendia of the bacteriumxposed to dierent feedstock hydrolysates, theiruried inhibitors and sugars, and the various possible

uel products.is data will allow dissection of themechanisms that impact the ability ofZymomonaso grow and metabolize sugars into fuel and suggestoutes for engineering more ecient production.eomputational framework and experimental facilityreated as part of this program will ultimately scaleo aid the engineering of resistance and fuel moleculeynthesis in this organism and the others beingursued at the EBI.



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25/34

SOCIETAL AND ECONOMIC IMPACTS OF BIOFUELS: IMPLICATIONS OF LAND USE, MARKETS, AND THE EN

LOGISTICS, POLICY, AND INFRASTRUCTURE

e group led by Jrgen Scheran is developing a framework todetermine the optimal capacity and location of bioreneries as of the regional distribution of bioenergy feedstocks in the Midwthe costs of transportation of feedstocks and biofuels, and the dcenters for biofuels. Mathematical programming tools are used location of bioreneries that minimize the costs, including transto and processing of bioenergy crops at reneries, transportatiofuel from reneries to the demand destinations, capital investmreneries, with a net of by-product credits. A multi-year transshand facility location model determines the optimal time to buildplant in the system, water needs and availability, the amount of rmaterial processed by individual plants, and the distribution painputs from crop-producing regions to the reneries and shipmethanol to the demand destinations.

Illinois legal scholars Jay Kesan and Brian Endres are examining

regulatory framework in various regions may impact the establisof a cellulosic fuels industry in the U.S.e group is also investigthe implications of the new Renewable Fuels Standard that theEnvironmental Protection Agency is expected to release early in

Further aeld, UC Berkeleys Dick Norgaard and Alastair Iles arthe structure of the existing biofuel industry in Brazil in order toa basis for evaluating how future development of the industry mBerkeley political scientist Steve Weber is developing an analysihow previous transitions in the energy sector have occurred witto understanding the impacts of current and future energy policemergence of a biofuels industry.

imulated growth and crop yields are within 10 percent of obser vedalues for Illinois and the Midwest, and for a range of sites in Europe.cosystem services, including soil mois ture and carbon balances, areetermined by integrating this model and the CENTURY model usedy the Environment Program in the Integrated Science Assessment

Model (ISAM) developed by Atul Jain as one of the three key modelsor projecting global carbon balances for the Intergovernmental Panel on

Climate Change.

Madhu Khanna and Hayri Onal have developed an economic model thatntegrates these ndings to examine the economically viable land usellocation among various food and fuel crops and the costs of meetingiofuel production targets. But it is also capable of assessing the impactsf systems of crediting carbon storage. Initial application of this model

o Illinois shows that there is considerable spatial heterogeneity in theompetitiveness of various feedstock for biofuel production and thater-acre yields of biofuel crops as well as the cost of the land are keyeterminants of that competitiveness.eynd that a mix of cellulosic

eedstock is likely to be more economically viable than a single feedstock,nd this mix is expected to evolve from greater reliance on crop residuesnitially, then to reliance on energy crops, like Miscanthus, over time.ey are currently working on applying this model to the Midwesterntates and then to other st ates where energy crop production is likely toe viable.

A related approach to a global assessment of land use is being developedy UC Berkeley and Berkeley Lab scientists Norm Miller, Davidunding, Maggi Kelly, and David Zilberman.ey are integratingroduction models for a variety of potential energy crops with spatiallynd temporally dened global geographic and economic data in order toevelop an assessment of global potential for cellulosic fuels. A key featuref such studies is an assessment of how land use is expected to change inesponse to changing climate and population density.


Researchers

are examining

national and global

impacts of biofuelson food and fuel

markets, welfare

of consumers and

producers, and the

environment.


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Work will seek to understand how adoption

of biofuel technologies can incorporate

the interdependencies between farmer,

processor, and oil marketers


FOOD AND FUEL MARKET IMPACTS

David Zilbermans Berkeley team is developinguantitative models and measures to assess the impactf dierent kinds of biofuels on food and energyrices, farmers, consumers, and overall economic

welfare. Analyzing the impact of corn ethanol in 2006-7, theynd that biofuels reduced the average price of

uel between 1 and 3 percent and increased corn pricey between 5 and 20 percent in the U.S.is savedp to $45 billion in fuel costs, increased the food billy up to $20 billion, and raised farm income by up to18 billion.e Berkeley group is also developing a

model to explain global oil prices, which suggests that

OPEC does not behave like an economic cartel thatmaximizes prot but rather as a political cartel thatmaximizes the overall welfare of oil-exporting nations.is will be used to make quantitative assessments

f resource allocation and pricing in fuel, food, andiofuel markets.

ilbermans work analyzing the protability oforn ethanol incorporates stochastic inuences onconomic decisions arising from randomness inatural phenomena and economic processes. Earlyesults indicate that there may be signicant lossesn the biofuel industry during periods of low supplynventories of corn and large prots during periods ofbundant corn supply and high oil prices. His work isurrently analyzing the implications of this boom-ust nature of corn biofuel for the design of contractsnd will be extended to analyze the eect of randomorces on the economics of cellulosic biofuels.

Khanna is also analyzing the eects of liberalizingrade in biofuels with Brazil on food and fuel prices,onsumer and producer well-being, and greenhouseas emissions. She nds that the current biofuelolicy of an import tariand a subsidy on ethanol

mposes an economic cost of over $3 bil lion annuallynd results in signicantly higher corn and ethanolrices in the U.S. with negligible current reductions i narbon emissions as compared to those with no policyntervention.

ROLE OF INNOVATION, INTELLE

PROPERTY RIGHTS, AND

TECHNOLOGY ADOPTION AND

Zilberman and colleagues are analyzing thepotential role of productivity-enhancing agribiotechnology in the growth of the biofuel seEarly results suggest that the capacity of biofumeet energy demand largely depends on theproductivity of traditional crops that may parcompete with biofuel for land, water, and othresources.

UC Berkeley economist Brian Wright is asses

intellectual property (IP) issues relevant to bresearch beyond current ethanol productioninitial assessment of the landscape indicates townership is fragmented and that over 50 assactive in this technology arena, with Genencthe largest portfolio. His analysis shows that tsector is highly active in biofuels research, anto the need to understand public/private relin this area. He plans to measure and assess tevolving eects of intellectual property proteinputs and outputs of research and of some rregulatory constraints, from the viewpoints oscientists involved in activities relevant to theWork is also proceeding on a study of the relbetween the terms of research sponsorship asubsequent patenting and licensing at the UCTechnology Transfer.is has generated intean oer of modest support at the National A

Zilberman is also developing a conceptual frato explain adoption of biofuel technologies wincorporating the interdependencies betweefarmer, processor and oil marketers and to anthe types of production contracts needed to

adoption.

e Illinois team of Anne Heinze SMichael Gray, German Bollero and Maria Vilengaged in surveys of farmers and i nvestors iplants to examine the factors that will inuendecisions to produce and use cellulosic feeds


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Seeking a Biofuels PolicyWhere Everybody Wins

MADHU KHANNA:

Creating a national biofuels policy is t ricky because work for diverse interests. Ideally, such a policy wouimports, curtail greenhouse gas emissions, and be prfor agriculture. And, the biofuels policy cant act as athe economy or limit food production.

ats a tall order. Nonetheless, an EBI research teamwaded into the debate by undertaking a massive studeconomic issues related to biofuel development.

We should be able to assess the benets and unintended conse

of biofuels policies as they stand today, particularly for the envirsaid Madhu Khanna, an environmental economist at t he UniverIllinois at Urbana-Champaign and lead investigator for the EBI And we should be able to determine how those policies might bdesigned to achieve social goals.

To come up with a broadly acceptable biofuels policy, she and hcolleagues must collect huge amounts of research data, much ofwith the logistics of creating a massive new industry. We need thow much switchgrass, Miscanthus, and crop residues can be grcollected in dierent parts of the country, said Khanna. We dobecause weve never done it. We want to know how much carbothose biofuel crops is going to sequester in the soil. And we wanhow much carbon will be produced (by) each crop.

One complicating factor is a federal mandate that requires prod36 billion gallons of biofuel annually by 2022. Of that, 21 billionmust come from advanced biofuelsfuels which produce no mhalf as much greenhouse gas as gasoline. We want to nd the loway we could meet that mandate, Khanna said. e rst step iout where to grow corn, where to grow Miscanthus, and where tswitchgrass in order to meet the 36-billion-gallon mark.

Ultimately, the team expects to give policymakers the informatithey need to help cra a national biofuels policy that works for t

environment, the economy, and farmers.

Farmers wont do this unless they see the right price signals, Kadded. e price signals provided by the market may not reecvarious environmental eects of biofuels produced from dierefeedstocks. So the government may have to provide appropriateincentives so that farmers produce a sustainable mix of biofuel c


ENVIRONMENTAL AND SOCIETAL

MPACTS OF BIOFUELS

everal research teams within the socio-economic program are assessing various aspects ofhe environmental and societal impacts of biofuels from an economic perspective. Atul Jain

using biophysical models to assess the soil carbon sequestration potential and nit rogenequirements of biofuel crops. Khannas team is analyzing data on life cycle emissions inonjunction with detailed spatial data on production methods and land use to determine theotential for biofuel-driven land use changes and displacement of gasoline to mitigate carbon

nd the costs of achieving mitigation.

ey

nd that biofuel production in Illinois, to meet 20ercent of the ethanol mandate by 2022, has the potential to reduce cumulative greenhouseas emissions by 45 percent relative to baseline levels over the next 15 years by displacingnergy equivalent gasoline, but that the en hanced production of corn ethanol to meet a partf the mandate increases nitrogen use by 27 percent over this period.

Using a macro-economic computable general equilibrium model of the U.S., John Bradenslinois group is analyzing the ecient mix of biofuel pathways based on their economic andnvironmental impacts which considers the trade-os between reduced carbon emissionsue to biofuels and water quality degradation due to greater fertilizer use for some feedstock,uch as corn. Zilbermans team is developing ways to integrate economics into the methodsurrently used to assess life cycle carbon emissions of biofuels.e group is developing

measures that modify the standard life cycle analysis to reect the eects of changing marketonditions and policies on choice of production methods and carbon emissions.

One of the complexities of understanding the life cycle aspects of various types of biofuelsthe recent recognition of potential indirect land use eects.e concept is that, because

emand for food is inelastic, if land is converted from food production to fuel production,reviously unused land will be converted to food production with aendant releases ofreenhouse gases. However, the situation is complex because demand for food can also beatised by increasing production on fewer acres in response to price signals. Michael OHarend his Berkeley colleagues are working to develop economic models that can be used tonderstand this issue quantitatively.eir ndings may have important implications for

uture policies that place economic value on greenhouse gas emi ssions.

Another ma

er of potential concern regarding biofuels is the possibility that diversion of lando production of biofuels could negatively impact food availability for disadvantaged peopleround the world.is is a complex issue that appears to be more related to the structurend operation of markets rather than about the availability of land. Berkeley economist Brian

Wright and colleagues are studying this issue, as are Ximing Cai at Illinois and collaboratorsiwa Msangi and Tingju Zhu at the International Food Policy Institute. Much of the otherconomics research in the EBI also impinges on this important question from variouserspectives.is topic is so important that the EBI is developing collaborations with otherrganizations around the world to increase the critical mass of researchers with expertise andccess to data that is relevant to understanding t he core issues.


The EBI is developing collaborations with other organizations

around the world to increase the critical mass of

researchers with expertise and access to data

that is relevant to understanding core issues.


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0Greenhouse Gas Emissions from Biofuels

June 8-9, 2008

In this workshop, participants discussed the currentstate of knowledge of greenhouse gas emissionsassociated with biofuel crops and new research neededto understand mechanisms of greenhouse gas release,sensitivity of that release to land use management andfarming practices, and strategies for extrapolation ofgreenhouse gas emissions to the global scale.

Pan American Congress on Plantsand Bioenergy

June 22-25, 2008

is international conference in Merida, Mexicofeatured plant biologists meeting with government

policy makers, agronomists, microbiologists, econo-mists and ecologists to forge a path toward WesternHemisphere bioenergy security that is sustainable andenvironmentally and economically sound. EBI DeputyDirector Steve Long was co-organizer of the confer-ence and gave the opening lecture.

Transition to a Bioeconomy: Risk, Infrastructure

and Industry Evolution

June 24-25, 2008

Co-sponsored by EBI, this Farm Foundation confer-ence in Berkeley focused on risk and infrastructurefor the biofuel industry of the future. Participantsexamined such issues as nances, business models, andtransportation infrastructure.

EDUCATION AND

Measuring and Modeling the Life Cycle GImpacts of Transportation Fuels

July 1-2, 2008

is Berkeley workshop explored the diereamong the fuel life c ycle Greenhouse Gas (Gestimates from leading models, including sysboundaries and other judgments about land-change. Experts from the academic and fuelscommunities addressed both current and embiofuels and other options for reducing the cfootprint of motor fuels.e EnvironmentalFund was primary sponsor.

Biofuels and Sustainability

October 21-22, 2008

EBI joined with the Illinoi s Sustainable TechCenter to convene this conference in Champdedicated to issues and innovations that canthe sustainability of biofuels. Discussions focthe economic, social, and environmental feaand ethics of biofuels options for meeting enneeds.

Documents

EBI Annual Report 2008