DOE/SC-0095

BreakingtheBiologicalBarriers
toCellulosicEthanol:
AJointResearchAgenda
AResearchRoadmapResultingfromthe
BiomasstoBiofuelsWorkshop
December79,2005
Rockville,Maryland
WorkshopSponsored
bythe
U.S.DepartmentofEnergy
OfceofEnergyEfciency OfceofScience
andRenewableEnergy
OfceofBiologicalandEnvironmentalResearch
OfceoftheBiomassProgram Genomics:GTLProgram
PublicationDate:June2006
i
ii BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

ExecutiveSummary
Wellalsofundadditionalresearchincutting-edgemethodsofproducing
ethanol,notjustfromcorn,butfromwoodchipsandstalksorswitchgrass.
PresidentGeorgeW.Bush,StateoftheUnionAddress,January2006*
A
robustfusionoftheagricultural,industrialbiotechnology,and
energyindustriescancreateanewstrategicnationalcapabilityfor
energyindependenceandclimateprotection.InhisStateofthe
UnionAddress(*Bush2006),PresidentGeorgeW.Bushoutlinedthe
AdvancedEnergyInitiative,whichseekstoreduceournationaldepen-
denceonimportedoilbyacceleratingthedevelopmentofdomestic,
renewablealternativestogasolineanddieselfuels.Tepresidenthasseta
nationalgoalofdevelopingcleaner,cheaper,andmorereliablealternative
energysourcestosubstantiallyreplaceoilimportsinthecomingyears.
Fuelsderivedfromcellulosicbiomassthebrous,woody,andgenerally
inedibleportionsofplantmatteroeronesuchalternativetoconven-
tionalenergysourcesthatcandramaticallyimpactnationaleconomic
growth,nationalenergysecurity,andenvironmentalgoals.Cellulosic
biomassisanattractiveenergyfeedstockbecauseitisanabundant,domes-
tic,renewablesourcethatcanbeconvertedtoliquidtransportationfuels.
Tesefuelscanbeusedreadilybycurrent-generationvehiclesanddistrib-
utedthroughtheexistingtransportation-fuelinfrastructure.
TeBiomasstoBiofuelsWorkshop,heldDecember79,2005,was
convenedbytheDepartmentofEnergysOceofBiologicalandEnvi-
ronmentalResearchintheOceofScience;andtheOceoftheBio-
massProgramintheOceofEnergyEciencyandRenewableEnergy.
Tepurposewastodenebarriersandchallengestoarapidexpansionof
cellulosic-ethanolproductionanddeterminewaystospeedsolutions
throughconcertedapplicationofmodernbiologytoolsaspartofajoint
researchagenda.Althoughthefocuswasethanol,thescienceappliesto
additionalfuelsthatincludebiodieselandotherbioproductsorcoproducts
havingcriticalrolesinanydeploymentscheme.
Tecorebarrieriscellulosic-biomassrecalcitrancetoprocessingtoethanol.
Biomassiscomposedofnaturesmostreadyenergysource,sugars,butthey
arelockedinacomplexpolymercompositeexquisitelycreatedtoresist
biologicalandchemicaldegradation.Keytoenergizinganewbiofuel
industrybasedonconversionofcellulose(andhemicelluloses)toethanolis
tounderstandplantcell-wallchemicalandphysicalstructureshowtheyare
synthesizedandcanbedeconstructed.Withthisknowledge,innovative
energycropsplantsspecicallydesignedforindustrialprocessingto
biofuelcanbedevelopedconcurrentlywithnewbiology-basedtreatment
andconversionmethods.Recentadvancesinscienceandtechnological
capabilities,especiallythosefromthenascentdisciplineofsystemsbiology,
promisetoaccelerateandenhancethisdevelopment.Resultingtechnologies
*www.whitehouse.gov/stateoftheunion/2006/
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy iii

Executive Summary
willcreateafundamentallynewprocessandbioreneryparadigmthatwill
enableanecientandeconomicindustryforconvertingplantbiomassto
liquidfuels. Tesekeybarriersandsuggestedresearchstrategiestoaddress
themaredescribedinthisreport.
Astechnologiesmatureforaccomplishingthistask,thetechnicalstrategy
proceedsthroughthreephases:Intheresearchphase,within5years,an
understandingofexistingfeedstocksmustbegainedtodevisesustainable,
eective,andeconomicalmethodsfortheirharvest,deconstruction,and
conversiontoethanol.Researchiscenteredonenzymaticbreakdownof
cellulosicbiomasstocomponent5-and6-carbonsugarsandlignin,usinga
combinationofthermochemicalandbiologicalprocesses,followedby
cofermentationofsugarstospeciedendproductssuchasethanol.Proc-
esseswillbeintegratedandconsolidatedtoreducecosts,improveecacy,
reducegenerationofandsensitivitytoinhibitors,andimproveoverall
yieldsandviabilityinbioreneryenvironments.
Tetechnologydeploymentphase,within10years,willincludecreationof
anewgenerationofenergycropswithenhancedsustainability,yield,and
composition,coupledwithprocessesforsimultaneousbreakdownof
biomasstosugarsandcofermentationofsugarsvianewbiologicalsystems.
Teseprocesseswillhaveenhancedsubstraterange,temperatureand
inhibitortolerance,andthecapabilitytofunctionincomplexbiorening
environmentsandovertimescalesthatareeconomicallyviable.
Tesystems-integrationphase,within15years,willincorporateconcur-
rentlyengineeredenergycropsandbioreneriestailoredforspecic
agroecosystems.Employingnewandimprovedenzymesforbreaking
biomassdowntosugarsaswellasrobustfermentationprocessesjointly
consolidatedintoplantsormicrobes,thesehighlyintegratedsystemswill
accelerateandsimplifytheend-to-endproductionoffuelethanol.Inmany
ways,thesenal-phasetechnologieswillstrivetoapproachtheoretical
conversionlimits.Tenewgenerationofbiotechnologieswillspurengi-
neeringofexiblebioreneriesoperableindierentagriculturalregionsof
thecountryandtheworld.
Tesuccessofthisprogramforeectivelyconvertingcellulosicbiomassto
ethanolwillbebasedoncouplingsophisticatedengineeringwithfunda-
mentalbiologicalresearch.Tenewgenerationofbiologicalresearch
systemsbiologyisbuiltonthenationalinvestmentingenomics.Systems
biologyinvolvesnewtechnologiesforincreasinglydetailedhigh-through-
putanalysesandcomputingtomaketransparentthecomplexitiesof
biologyandallowpredictiveunderstandingandrationaldesign.Multidis-
ciplinaryteamresearchapproacheswillacceleratescienticprogressandits
translationtonewbioreneryprocesses.Comprehensivesuitesoftech-
nologies,someinresearcherslaboratoriesandsomeconsolidatedin
facilities,willenhancetechnicalperformance,improveproductivity,and
reducecoststoallowaordableandtimelyprogresstowardthesegoals.
Newresearchcapabilitiesandfacilitieswillserveasanengineforfunda-
mentalresearch,technologydevelopment,andcommercialization.
iv BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Contents
ExecutiveSummary..............................................................................................................................................................................iii
Introduction.............................................................................................................................................................................................1
JointWorkshopChallengesBiofuelScienceandTechnologyCommunities.........................................................................................1
AmericasEnergyChallenges...................................................................................................................................................................2
ThePromiseofBiofuels.........................................................................................................................................................................2
AGrowingMandateforBiofuels:Policy,Legislative,andOtherDrivers..............................................................................................3
BeneftsofBiofuels..................................................................................................................................................................................5
NationalEnergySecurityBenefts.........................................................................................................................................................6
EconomicBenefts.................................................................................................................................................................................6
EnvironmentalBenefts.........................................................................................................................................................................7
FeasibilityofBiofuels...............................................................................................................................................................................8
LandAvailability....................................................................................................................................................................................8
AgriculturalSustainabilityofBiomassProduction...............................................................................................................................12
TodayFuelEthanolProductionfromCornGrain(StarchEthanol).....................................................................................................12
TomorrowBiorefneryConcepttoProduceFuelEthanolfromCellulosicBiomass.............................................................................13
Ethical,Legal,andSocialIssues(ELSI).................................................................................................................................................17
EEREOBPPlatformforIntegratedBiorefneries..................................................................................................................................17
DOEOfceofSciencePrograms.............................................................................................................................................................19
BiomasstoBiofuelsWorkshop:CreatingaCommonResearchAgendatoOvercomeTechnologyBarriers.....................................22
CitedReferences.....................................................................................................................................................................................24
BackgroundReading..............................................................................................................................................................................28
TechnicalStrategy:DevelopmentofaViableCellulosicBiomasstoBiofuelIndustry...................................................29
ResearchPhase(within5years)............................................................................................................................................................29
FeedstockUseandOptimization..........................................................................................................................................................29
Deconstruction....................................................................................................................................................................................30
FermentationandRecovery.................................................................................................................................................................31
TechnologyDeploymentPhase(within10years)................................................................................................................................32
Feedstocks...........................................................................................................................................................................................34
Deconstruction....................................................................................................................................................................................34
FermentationandRecovery.................................................................................................................................................................34
SystemsIntegrationPhase(within15years).......................................................................................................................................34
IntegrationandConsolidation.............................................................................................................................................................35
SYSTEMSBIOLOGYTOOVERCOMEBARRIERSTOCELLULOSICETHANOL................................................................................37
LignocellulosicBiomassCharacteristics.......................................................................................................................................39
StructureandAssemblyofCellWalls....................................................................................................................................................41
FactorsinRecalcitranceofLignocelluloseProcessingtoSugars.........................................................................................................44
PlantArchitecture...............................................................................................................................................................................45
Cell-WallArchitecture..........................................................................................................................................................................45
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy v

CONTENTS
MolecularStructure.............................................................................................................................................................................45
OptimizationofPlantCellWalls............................................................................................................................................................47
UnderstandingCell-WallStructureandFunction.................................................................................................................................47
ControlofLigninSynthesisandStructure............................................................................................................................................49
ImprovedMethods,Tools,andTechnologies........................................................................................................................................50
TechnicalMilestones..............................................................................................................................................................................51
CitedReferences.....................................................................................................................................................................................56
FeedstocksforBiofuels.......................................................................................................................................................................57
CreationofaNewGenerationofLignocellulosicEnergyCrops...........................................................................................................60
MaximizingBiomassProductivity.......................................................................................................................................................61
TechnicalMilestones...........................................................................................................................................................................67
EnsuringSustainabilityandEnvironmentalQuality............................................................................................................................68
TechnicalMilestones...........................................................................................................................................................................70
ModelSystemsforEnergyCrops............................................................................................................................................................71
TechnicalMilestones...........................................................................................................................................................................73
TheRoleofGTLCapabilitiesforSystemsBiology.................................................................................................................................74
ProteinProductionCapabilities...........................................................................................................................................................74
MolecularMachinesCapabilities.........................................................................................................................................................75
ProteomicCapabilities.........................................................................................................................................................................75
CellularSystemCapabilities.................................................................................................................................................................75
DOEJointGenomeInstitute.................................................................................................................................................................76
OtherNeededCapabilities...................................................................................................................................................................76
OtherBiofuelOpportunities:DevelopmentofHigh-ProductivityBiodieselCrops............................................................................77
CitedReferences.....................................................................................................................................................................................80
ForFurtherReading...............................................................................................................................................................................81
DeconstructingFeedstockstoSugars............................................................................................................................................85
DeterminingFundamentalPhysicalandChemicalFactorsintheRecalcitranceofLignocellulosicBiomasstoProcessing...........86
ResearchGoals....................................................................................................................................................................................88
TechnicalMilestones...........................................................................................................................................................................89
TheRoleofGTLandOBPFacilitiesandCapabilities.............................................................................................................................90
CrosscuttingTools,Technologies,andScience......................................................................................................................................91
DevelopingBetterEnzymaticSystemsforBiologicalPretreatment:LigninasesandHemicellulases.............................................91
ResearchGoals....................................................................................................................................................................................92
TechnicalMilestones...........................................................................................................................................................................94
CrosscuttingTools,Technologies,andScience......................................................................................................................................97
UnderstandingtheMolecularMachineryUnderpinningCelluloseSaccharifcation:CellulasesandCellulosomes.......................97
ResearchGoals....................................................................................................................................................................................98
TechnicalMilestones.........................................................................................................................................................................101
CrosscuttingTools,Technologies,andScience....................................................................................................................................103
TheRoleofGTLandOBPFacilitiesandCapabilities...........................................................................................................................104
HarvestingtheBiochemicalPotentialofMicroorganismsThroughMetagenomics.......................................................................105
ResearchGoals..................................................................................................................................................................................106
vi BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

TechnicalMilestones.........................................................................................................................................................................106
TheRoleofGTLandOBPFacilitiesandCapabilities...........................................................................................................................107
CharacterizingCellWallsUsingHigh-ThroughputMethods..............................................................................................................108
TechnicalMilestones.........................................................................................................................................................................110
TheRoleofGTLandOBPFacilitiesandCapabilities...........................................................................................................................111
CrosscuttingTools,Technologies,andScience....................................................................................................................................112
Breakthrough,High-PayofOpportunity:SimplifyingtheBioconversionProcessbyUnderstandingCell-Wall
DeconstructionEnzymesExpressedinPlants....................................................................................................................................113
ScientifcChallengesandOpportunities............................................................................................................................................113
ResearchGoals..................................................................................................................................................................................113
References............................................................................................................................................................................................115
CellulosomeReferences.......................................................................................................................................................................117
CellulosomeReviews.........................................................................................................................................................................117
DesignerCellulosomes......................................................................................................................................................................117
SugarFermentationtoEthanol.....................................................................................................................................................119
OptimizingMicrobialStrainsforEthanolProduction:PushingtheLimitsofBiology.....................................................................122
ScienceChallengesandStrategy.......................................................................................................................................................124
TechnicalMilestones.........................................................................................................................................................................130
TheRoleofGTLCapabilities...............................................................................................................................................................131
AdvancedMicroorganismsforProcessSimplifcation.......................................................................................................................132
ScienceChallengesandStrategiesforProcessSimplifcation............................................................................................................135
TechnicalMilestones.........................................................................................................................................................................137
TheRoleofGTLCapabilities...............................................................................................................................................................138
EnablingMicrobiologicalToolsandTechnologiesthatMustbeDeveloped.....................................................................................138
Breakthrough,High-PayofOpportunities.........................................................................................................................................140
MicrobialCommunitiesforRobustEnergyProduction.......................................................................................................................140
Model-DrivenDesignofCellularBiocatalyticSystemsUsingSystemsBiology...................................................................................142
DirectBioproductionofEnergy-RichFuels.........................................................................................................................................147
OptimalStrains:FermentativeProductionof40%EthanolfromBiomassSugars..............................................................................149
AnAlternativeRouteforBiomasstoEthanol:MicrobialConversionofSyngas...................................................................................152
CitedReferences...................................................................................................................................................................................154
Crosscutting21stCenturyScience,Technology,andInfrastructureforaNewGeneration
ofBiofuelResearch............................................................................................................................................................................155
OpportunitiesandChallenges............................................................................................................................................................155
AnalyticalToolstoMeettheChallengesofBiofuelResearch............................................................................................................156
Genomics..........................................................................................................................................................................................156
Transcriptomics:High-ThroughputExpressionAnalyses....................................................................................................................158
Proteomics........................................................................................................................................................................................158
Metabolomics...................................................................................................................................................................................159
GlycomicsandLignomics..................................................................................................................................................................160
Fluxomics..........................................................................................................................................................................................161
EnzymeStructureandFunction.........................................................................................................................................................162
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy vii


CONTENTS
ImagingTechnologies..........................................................................................................................................................................163
ImagingNeedsforFeedstockResearch..............................................................................................................................................163
ImagingNeedsforMicrobialCommunitiesinDeconstructionandConversionofBiomasstoEthanol...............................................164
MicrobialCultivation............................................................................................................................................................................165
DataInfrastructure...............................................................................................................................................................................166
ComputationalModeling.....................................................................................................................................................................168
Modeling:GenomeSequenceAnalysis..............................................................................................................................................168
Modeling:Molecular.........................................................................................................................................................................170
Modeling:PathwaysandNetworks...................................................................................................................................................171
Modeling:BiorefneryProcess...........................................................................................................................................................171
CapabilitySuitesforBioenergyResearchandFacilityInfrastructure..............................................................................................172
DOEJointGenomeInstitute...............................................................................................................................................................173
ProductionandCharacterizationofProteinsandMolecularTags......................................................................................................173
CharacterizationandImagingofMolecularMachines......................................................................................................................175
AnalysisofGenomeExpression:TheOmics.......................................................................................................................................176
AnalysisandModelingofCellularSystems.......................................................................................................................................178
CitedReferences...................................................................................................................................................................................180
BioprocessSystemsEngineeringandEconomicAnalysis.....................................................................................................181
ResearchGoals......................................................................................................................................................................................181
Milestones.........................................................................................................................................................................................182
TheRoleofGTLandOBPFacilitiesandCapabilities...........................................................................................................................183
APPENDICES.........................................................................................................................................................................................185
AppendixA.ProvisionsforBiofuelsandBiobasedProductsintheEnergyPolicyActof2005..................................186
AppendixB.WorkshopParticipants.............................................................................................................................................188
AppendixC.WorkshopParticipantBiosketches......................................................................................................................192
viii BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Introduction
I
nhis2006StateoftheUnionaddress(Bush2006),thepresident
outlinedthenewAdvancedEnergyInitiative(AEI)tohelpovercome
Americasdependenceonforeignsourcesofenergy(AEI2006)and
theAmericanCompetitivenessInitiativetoincreaseR&Dinvestments
andstrengtheneducation(ACI2006).Heseekstoreduceournational
dependenceonimportedoilbyacceleratingthedevelopmentofdomestic,
renewablealternativestogasolineanddieselfuels.
WithAmericaonthevergeofbreakthroughsinadvancedenergytech-
nologies,thebestwaytobreaktheaddictiontoforeignoilisthrough
newtechnologies. WhiteHousePressReleaseontheStateoftheUnion
AddressandAEI( January31, 2006)
Breakthroughtechnologiestorealizethepotentialofcellulosicbiofuels
canbeexpeditedbyapplicationofanewgenerationofbiologicalresearch
createdbythegenomerevolution.Overcomingbarrierstodevelopmentof
thesefuelsonanindustrialscalewillrequirehigh-performanceenergyfeed-
stocksandmicrobialprocesses,bothtobreakdownfeedstockstosugarsand
tofermentsugarstoethanol.Afocusedsetofinvestmentslinkingrevolu-
tionarybiofueltechnologieswithadvancesfromthebiological,physical,
computational,andengineeringscienceswillquicklyremovebarrierstoan
ecient,economic,andsustainablebiofuelindustry.
JointWorkshopChallengesBiofuelScienceand
TechnologyCommunities
TwoDepartmentofEnergy(DOE)ocesareteamingtoadvancebiofuel
developmentanduse:TeOceofBiologicalandEnvironmentalResearch
(OBER)withintheOceofScience(SC)andtheOceoftheBiomass
Program(OBP)withintheOceofEnergyEciencyandRenewable
Energy(EERE)(seedescriptionsofthetwoDOEprograms,pp.17and
19). Teseocesarechallengingtheircommunitiestoidentifycriticalsci-
enceneedstosupportasubstantialandsustainableexpansionofbiomass-
derivedfuels,specicallycellulosicethanol.Inthejointlysponsored
BiomasstoBiofuelsWorkshopheldDecember79,2005,inRockville,
Maryland,morethan50scientistsrepresentingawiderangeofexpertise
convenedtodenebarriersandchallengestothisnewbiofuelindustry.Te
workshopconcentratedonimprovementofbiomasscropsandtheirpro-
cessingtotransportationfuels.Althoughthefocuswasethanol,thescience
appliestoadditionalfuelsthatincludebiodieselandtootherbioproductsor
coproductshavingcriticalrolesinanydeploymentscheme.
References:p.24
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 1

INTRODUCTION
Tecurrentapproachtointroducingbiofuelsreliesonanevolutionary
businessandeconomicdriverforasteadybutmoderateentryintothe
market.Technologiesforimplementingthisnewindustryarebeingtested
eitherbyproducinghigher-valueproductsfromrenewables(suchaslactic
acid)orasincrementaladditionstocurrentcorn-ethanolreneries(suchas
theconversionofresidualcorn-kernelberstoethanol).
Tisreportisaworkshop-producedroadmapforacceleratingcellulosic
ethanolresearch,helpingmakebiofuelspracticalandcost-competitiveby
2012($1.07/galethanol)andoeringthepotentialtodisplaceupto30%
ofthenationscurrentgasolineuseby2030.Itarguesthatrapidlyincorpor-
atingnewsystemsbiologyapproachesviasignicantR&Dinvestmentwill
spuruseofthesetechnologiesforexpandedprocessingofenergycropsand
residues.Furthermore,thisstrategywilldecreaseindustrialriskfromuse
ofarst-of-a-kindtechnology,allowingfasterdeploymentwithimproved
methods.Ultimately,theseapproachesfostersettingmoreaggressivegoals
forbiofuelsandenhancethestrategyssustainability.
AmericasEnergyChallenges
Tetripleenergy-relatedchallengesofthe21stCenturyareeconomicand
energygrowth,energysecurity,andclimateprotection.TeUnitedStates
importsabout60%ofthepetroleumitconsumes,andthatdependencyis
increasing.*SincetheU.S.economyistiedsocloselytopetroleumproducts
andoilimports,disruptionsinoilsuppliescanresultinsevereeconomicand
socialimpacts.Conventionaloilproductionwillpeakinthenearfuture,and
theresultingenergytransitionwillrequireaportfolioofresponses,includ-
ingunconventionalfossilresourcesandbiofuels.Environmentalqualityand
climatechangeduetoenergyemissionsareadditionalconcerns.Annual
U.S.transportationemissionsofthegreenhousegas(GHG)carbondioxide
(CO
2
)areprojectedtoincreasefromabout1.9billionmetrictonsin2004
toabout2.7billionmetrictonsin2030(EIA2006).
ThePromiseofBiofuels
Fuelsderivedfromcellulosicbiomass**thebrous,woody,andgenerally
inedibleportionsofplantmatteroeranalternativetoconventional
energysourcesthatsupportsnationaleconomicgrowth,nationalenergy
security,andenvironmentalgoals.Cellulosicbiomassisanattractiveenergy
feedstockbecausesuppliesareabundantdomesticallyandglobally.Itisa
renewablesourceofliquidtransportationfuelsthatcanbeusedreadilyby
current-generationvehiclesanddistributedthroughtheexistingtranspor-
tation-fuelinfrastructure.Ethanolfromcorngrainisanincreasingly
importantadditivefuelsource,butithaslimitedgrowthpotentialasa
primarytransportationfuel.***TeU.S.starch-basedethanolindustry
willjumpstartagreatlyexpandedethanolindustrythatincludescellulosic
ethanolasamajortransportationfuel.
Celluloseandhemicelluloses,foundinplantcellwalls,aretheprimary
componentofbiomassandthemostplentifulformofbiologicalmaterialon
earth.Teyarepolysaccharidesmadeupofenergy-richsugarsthatcanbe
convertedtoethanol(seesidebar,UnderstandingBiomass,p.53).Current
2 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

methodstobreakdownbiomassintosimplesugarsandconverttheminto
ethanolareinecientandconstitutethecorebarriertoproducingethanol
atquantitiesandcostscompetitivewithgasoline.
Biologicalresearchisundergoingamajortransformation.Tesystems
biologyparadigmbornofthegenomerevolutionandbasedonhigh-
throughputadvancedtechnologies,computationalmodeling,andscientic-
teamapproachescanfacilitaterapidprogressandisareadilyapplicable
modelforbiofueltechnology.SystemsbiologyisthecoreoftheOBER
Genomics:GTLprogram,whosegoalistoachieveapredictiveunderstand-
ingofthecomplexnetworkofinteractionsthatunderpinthebiological
processesrelatedtobiofuelproduction.BiologicalchallengestowhichGTL
canapplysystemsbiologyapproachesincludeenhancingtheproductivity
ofbiomasscropsoptimizedforindustrialprocessing,improvingenzyme
systemsthatdeconstructplantcellwalls,andincreasingtheyieldofetha-
nol-producingmicroorganisms.Systemsbiologytoolsandknowledgewill
enablerationalengineeringofanewgenerationofbioenergysystemsmade
upofsustainableenergycropsforwidelyvaryingagroecosystemsandtai-
loredindustrialprocesses.Tisresearchapproachwillencouragethecritical
fusionoftheagriculture,industrialbiotechnology,andenergysectors.
AGrowingMandateforBiofuels:Policy,Legislative,
andOtherDrivers
Aprimarygoalofthepresidents2001NationalEnergyPolicy(NEP)
istoincreaseU.S.energysupplies,incorporatingamorediversemixof
domesticresourcestosupportgrowthindemandandtoreducenational
dependenceonimportedoil(NEPDG2001).AEIacceleratesand
expandsonseveralpolicyandlegislativemandates(AEI2006).Itaimsto
reducethenationsrelianceonforeignoilintheneartermandprovidesa
22%increaseinclean-energyresearchatDOEforFY2007,accelerating
progressinrenewableenergy.
AccordingtoAEI,theUnitedStatesmustmovebeyondapetroleum-
basedeconomyanddevisenewwaystopowerautomobiles.Tecountry
needstofacilitatedomestic,renewablealternativestogasolineanddiesel
fuels.Teadministrationwillaccelerateresearchincutting-edgemethods
ofproducingsuchhomegrownrenewablebiobasedtransportationfuels
asethanolfromagriculturalandforestryfeedstocksincludingwoodchips,
*Gasolineanddieselconstituted98%ofdomestictransportationmotorfuelsin2004,
withethanolfromcorngrainsupplyingmostoftheremaining2%.Annualgasoline
consumptionin2004wasabout139billiongallons,and3.4billiongallonsofethanol
wereusedprimarilyasafuelextendertoboostgasolineoctanelevelsandimprove
vehicleemissions.
**Cellulosicbiomass,alsocalledlignocellulosicbiomass,isacomplexcompositematerial
consistingprimarilyofcelluloseandhemicellulose(structuralcarbohydrates)bondedto
lignininplantcellwalls.Forsimplication,weusethetermcellulosicbiomass.
***In2004,11%oftheU.S.cornharvestyielded3.4billiongallonsofethanol(NRDC
2006),roughly1.7%ofthe2004fueldemand.Tusifallcorngrainnowgrowninthe
UnitedStateswereconvertedtoethanol,itwouldsatisfyabout15%ofcurrenttrans-
portationneeds.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 3

INTRODUCTION
stalks,andswitchgrass.AEIwouldfostertheearlycommercializationof
advancedbiofueltechnologies,enablingU.S.industrytoleadindeploying
biofuelsandchemicalsinternationally.
Achievingthegoalofdisplacing30%ofthenationscurrentgasolineuseby
2030wouldrequireproductionlevelsequaltoroughly60billiongallonsa
year(Bgal/year)ofethanol(seeTable1.Comparisonsof2004Gasolineand
EthanolEquivalents,thispage).Anannualsupplyofroughlyabilliondry
tonsofbiomasswillbeneededtosupportthislevelofethanolproduction.
ArecentreportbytheU.S.DepartmentofAgriculture(USDA)andDOE
ndspotentialtosustainablyharvestmorethan1.3billionmetrictonsof
biomassfromU.S.forestandagriculturallandsbymid-21stCentury
(Perlacketal.2005).InvestmentsinR&Dandinfrastructureareneededto
realizethisfeedstockpotential.
TeU.S.EnergyPolicyActof2005(EPAct;AppendixA,Provisionsfor
BiofuelsandBiobasedProductsintheEnergyPolicyActof2005,p.186)
hasestablishedaggressivenear-termtargetsforethanolproduction.Akey
provisionrequiresmixing4Bgalofrenewablefuelwithgasolinein2006.
Tisrequirementincreasesannuallyto7.5Bgalofrenewablefuelby2012.
For2013andbeyond,therequiredvolumewillincludeaminimumof250
milliongallons(Mgal)ofcellulosicethanol.AnothersectionoftheEPAct
authorizesfundsforanincentiveprogramtoensuretheannualproduction
of1Bgalofcellulosicbiomass-basedfuelsby2015.Ethanolisthemost
commonbiofuelproducedfromcellulose,butotherpossiblebiofuelcom-
poundscanbeproducedaswell.
OtherimportantlegislativedriverssupportingbiofuelsaretheBiomassR&D
Actof2000andTitleIXoftheFarmBill2002(U.S.Congress2000;U.S.
Congress2002). TeBiomassR&DActdirectedthedepartmentsofEnergy
andAgriculturetointegratetheirbiomassR&DandestablishedtheBiomass
ResearchandDevelopmentTechnicalAdvisoryCommittee(BTAC),which
advisestheSecretaryofEnergyand
theSecretaryofAgricultureonstra-
Table1.Comparisonsof2004GasolineandEthanolEquivalents
2004
Gasoline
(billiongallons)
EthanolEquivalents
(billiongallons)
U.S.consumption,2004 139 200
About60%fromimports 83 120
Requirementstodisplace30%of2004U.S.
consumption
42 60
Biomassrequirementsat80gal/ton 750Mton
Landrequirementsat10ton/acreand
80gal/ton
75Macre
Numbersofreneriesat100Mgal/
renery
600(each
requiring
160miles
2
netor
125,000acres)
tegicplanningforbiomassR&D.As
aprecedenttothecurrentpresidential
initiative,in2002BTACsetagoal
requiringbiofuelstomeet20%ofU.S.
transportationfuelconsumptionby
2030aspartofitsvisionforbiomass
technologies(BTAC2002). TitleIX
supportsincreaseduseofbiobasedfuels
andproductsandincentivesandgrants
forbiofuelandbioreneryR&D.
Inadditiontolegislativemandates,sev-
eralindependentstudieshaveacknowl-
edgedthegreatpotentialofbiofuels
inachievingamorediversedomestic
energysupply(NCEP2004;Greeneet
al.2004;Lovinsetal.2005).Growing
4 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

supportfordevelopingbiomassasakeyenergyfeedstockiscomingfroma
varietyofnationalandinternationalorganizations(GEC2005;AgEnergy
WorkingGroup2004;IEA2004).Althoughthesereportsdierinthe
amountsofgasolinethatcouldbereplacedbyethanolfrombiomass,theyall
agreeonthreekeyissues:(1)Currenttrendsinenergyusearenotsustainable
andareasecurityrisk;(2)Nosinglesolutionwillsecuretheenergyfuturea
diverseportfolioofenergyoptionswillberequired;and(3)Biofuelscanbea
signicantpartofthetransportationsectorsenergysolution.
Initsevaluationofoptionsfordomesticproductionofmotorfuels,the
NationalCommissiononEnergyPolicy(NCEP)recommendedcellulosic
biomassasanimportanttopicfornear-termfederalresearch,development,
anddemonstrationandfoundthatcellulosicethanolhasthepotentialto
makeameaningfulcontributiontothenationstransportationfuelsupply
inthenexttwotothreedecades(NCEP2004).
TeNaturalResourcesDefenseCouncil(NRDC)hasprojectedthatan
aggressiveplantodevelopcellulosicbiofuelsintheUnitedStatescould
producetheequivalentofnearly7.9millionbarrelsofoilperdayby2050
morethan50percentofourcurrenttotaloiluseinthetransportation
sectorandmorethanthreetimesasmuchasweimportfromthePersian
Gulfalone(Greeneetal.2004).Tiscorrespondstoroughly100Bgal/year
ethanol.NRDCalsorecommends$1.1billioninfundingbetween2006
and2012forbiomassresearch,development,anddemonstrationwith45%
ofthisfundingfocusedonovercomingbiomassrecalcitrancetoethanol
processing. Tisleveloffundingisexpectedtostimulatearegularowof
advancesneededtomakeethanolcost-competitivewithgasolineanddiesel.
AnindependentanalysisfromtheRockyMountainInstitutefoundthat
signicantgainsinenergyeciencyandthelarge-scaledisplacementof
oilwithbiofuels,mainlycellulosicethanol,wouldbekeycomponentsof
itsstrategytoreduceAmericanoildependenceoverthenextfewdecades
(Lovinsetal.2005).
Toillustratethewidespreadsupportforfuelethanol,theGovernorsEtha-
nolCoalition,anorganizationdevotedtothepromotionandincreaseduse
ofethanol,nowincludes32memberstatesaswellasinternationalrepre-
sentativesfromBrazil,Canada,Mexico,Sweden,andTailand.Inarecent
report,thecoalitioncalledforrapidexpansionofethanoltomeetatleast
10%oftransportationfuelneedsassoonaspracticableandfordevelop-
mentoflignocellulosic-basedfuelsforexpansionbeyondthoselevels
(GEC2005).Teuseofethanol,particularlybiomass-derivedethanol,
canproducesignicantsavingsincarbondioxideemissions.Tisapproach
oersano-regretspolicythatreducesthepotentialfuturerisksassociated
withclimatechangeandhastheaddedbenetofeconomicdevelopment.
BeneftsofBiofuels
Biofuels,especiallycorn-derivedandcellulosicethanol,constitutetheonly
renewableliquidtransportationfueloptionthatcanbeintegratedreadily
withpetroleum-basedfuels,eets,andinfrastructure.Productionanduse
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 5

INTRODUCTION
ofbiofuelscanprovidesubstantialbenetstonationalenergysecurity,
economicgrowth,andenvironmentalquality.
NationalEnergySecurityBenefts
Nationalsecurityislinkedtoenergythroughthedependenceofthis
countryandmanyothersonimportedoilmuchofitlocatedin
politicallytroubledpartsoftheglobe.Assuch,thepotentialforlarge-
scalefailuresintheglobalproductionanddistributionsystempresents
arealthreat. GovernorsEthanolCoalition(GEC2005)
TodaytheUnitedStatesisdependentonoilfortransportation.Alterna-
tive,domesticallybased,andsustainablefuel-developmentstrategies,
therefore,areessentialtoensuringnationalsecurity.Americaaccountsfor
25%ofglobaloilconsumptionyetholdsonly3%oftheworldsknown
oilreserves.About60%ofknownoilreservesarefoundinsensitiveand
volatileregionsoftheglobe.Increasingstrainonworldoilsupplyis
expectedasdevelopingcountriesbecomemoreindustrializedanduse
moreenergy.AnystrategytoreduceU.S.relianceonimportedoilwill
involveamixofenergytechnologiesincludingconservation.Biofuelsare
anattractiveoptiontobepartofthatmixbecausebiomassisadomestic,
secure,andabundantfeedstock.Globalavailabilityofbiomassfeedstocks
alsowouldprovideaninternationalalternativetodependenceonan
increasinglystrainedoil-distributionsystemaswellasareadymarketfor
biofuel-productiontechnologies.
EconomicBenefts
Abiofuelindustrywouldcreatejobsandensuregrowingenergysupplies
tosupportnationalandglobalprosperity.In2004,theethanolindustry
created147,000jobsinallsectorsoftheeconomyandprovidedmorethan
$2billionofadditionaltaxrevenuetofederal,state,andlocalgovernments
(RFA2005).Conservativeprojectionsoffuturegrowthestimatetheaddi-
tionof10,000to20,000jobsforeverybilliongallonsofethanolproduc-
tion(Petrulis1993).
In2005theUnitedStatesspentmorethan$250billiononoilimports,
andthetotaltradedecithasgrowntomorethan$725billion(U.S.Com-
merceDept.2006).Oilimports,whichmakeup35%ofthetotal,could
riseto70%overthenext20years(EthanolAcrossAmerica2005).
Amongnationaleconomicbenets,abiofuelindustrycouldrevitalize
strugglingruraleconomies.Bioenergycropsandagriculturalresiduescan
providefarmerswithanimportantnewsourceofrevenueandreducereli-
anceongovernmentfundsforagriculturalsupport.Aneconomicanalysis
jointlysponsoredbyUSDAandDOEfoundthattheconversionofsome
croplandtobioenergycropscouldraisedepressedtraditionalcroppricesby
upto14%.Higherpricesfortraditionalcropsandnewrevenuefrombio-
energycropscouldincreasenetfarmincomeby$6billionannually(DeLa
TorreUgarte2003).
6 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.1.ReducedCarbon
DioxideEmissionsofEthanol
fromBiomass.Whencom-
paredwithgasoline,ethanol
fromcellulosicbiomasscould
dramaticallyreduceemissions
ofthegreenhousegas,carbon
dioxide(CO ).Althoughburn-
2
inggasolineandotherfossil
fuelsincreasesatmospheric
CO concentrations,thepho-
2
tosyntheticproductionofnew
biomasstakesupmostofthe
carbondioxidereleasedwhen
bioethanolisburned.[Source:
AdaptedfromORNL Review
(www.ornl.gov/info/ornlreview/
v33_2_00/bioenergy.htm)]
EnvironmentalBenefts
ClimateChange
Whenfossilfuelsareconsumed,carbonsequesteredfromtheglobal
carboncycleformillionsofyearsisreleasedintotheatmosphere,where
itaccumulates.BiofuelconsumptioncanreleaseconsiderablylessCO
2
,
dependingonhowitisproduced.Tephotosyntheticproductionofnew
generationsofbiomasstakesuptheCO releasedfrombiofuelproduction
2
anduse(seeFig.1.ReducedCarbonDioxideEmissionsofEthanolfrom
Biomass,thispage).Alife-cycleanalysisshowsfossilCO
2
emissionsfrom
cellulosicethanoltobe85%lowerthanthosefromgasoline(Wang2005).
Teseemissionsarisefromtheuseoffossilenergyinproducingcellulosic
ethanol.NonbiologicalsequestrationofCO
2
producedbythefermentation
processcanmakethebiofuelenterprisenetcarbonnegative.
Arecentreport(Farrelletal.2006)ndsthatethanolfromcellulosic
biomassreducessubstantiallybothGHGemissionsandnonrenewable
energyinputswhencomparedwithgasoline.Telowquantityoffossil
fuelrequiredtoproducecellulosicethanol(andthusreducefossilGHG
emissions)isduelargelytothreekeyfactors.Firstistheyieldofcellu-
losicbiomassperacre.Currentcorn-grainyieldsareabout4.5tons/acre.
Starchis66%byweight,yielding3tonstoproduce416galofethanol,
comparedtoanexperimentalyieldof10drytonsofbiomass/acrefor
switchgrasshybridsinresearchenvironments(10drytonsatafuture
yieldof80gal/ton=800galethanol).Useofcorngrain,theremaining
solids(distillersdriedgrains),andstovercouldyieldethanolatroughly
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 7

INTRODUCTION
700gal/acre.Currentyieldfornonenergy-cropbiomassresourcesis
about5drytons/acreandroughly65gal/ton.Tegoalforenergycropsis
10tons/acreat80to100gal/tonduringimplementation.Second,peren-
nialbiomasscropswilltakefarlessenergytoplantandcultivateandwill
requirelessnutrient,herbicide,andfertilizer.Tird,biomasscontains
ligninandotherrecalcitrantresiduesthatcanbeburnedtoproduceheat
orelectricityconsumedbytheethanol-productionprocess.
Energycropsrequireenergyinputsforproduction,transportation,and
processingaviablebioenergyindustrywillrequireasubstantialpositive
energybalance.Figure2.ComparisonofEnergyYieldswithEnergyExpen-
ditures,thispage,comparesresultsforcellulosicandcornethanol,gasoline,
andelectricity,demonstratingasubstantiallyhigheryieldforcellulosicetha-
nol.Overtimeamaturebioenergyeconomywillsubstitutebiomass-derived
energysourcesforfossilfuel,furtherreducingnetemissions.
Fig.2.ComparisonofEnergyYieldswithEnergyExpendi-
tures.Tefossilenergyreplacementratio(FER)compares
energyyieldfromfourenergysourceswiththeamountoffos-
silfuelusedtoproduceeachsource.Notethatthecellulosic
ethanolbiorenerysprojectedyieldassumesfuturetechno-
logicalimprovementsinconversionecienciesandadvances
thatmakeextensiveuseofabiomasscropsnoncellulosic
portionsforcogenerationofelectricity.Similarassumptions
wouldraisecornethanolsFERif,forexample,cornstover
weretoreplacecurrentnaturalgasusage.Tecornethanol
industry,alreadyproducingethanolasanimportantadditive
andfuelextender,isprovidingafoundationforexpansion
tocellulosicethanol.[Source:Figure,basedontheArgonne
NationalLaboratoryGREETmodel,isderivedfromBrink-
manetal.2005.Otherpapersthatsupportthisstudyinclude
Farrelletal.2006andHammerschlag2006.]
OtherEnvironmentalBenefts
Perennialgrassesandotherbioenergycropshave
manysignicantenvironmentalbenetsovertradi-
tionalrowcrops(seeFig.3.MiscanthusGrowthover
aSingleGrowingSeasoninIllinois,p.9).Peren-
nialenergycropsprovideabetterenvironmentfor
more-diversewildlifehabitation.Teirextensive
rootsystemsincreasenutrientcapture,improvesoil
quality,sequestercarbon,andreduceerosion.Etha-
nol,whenusedasatransportationfuel,emitsless
sulfur,carbonmonoxide,particulates,andGHGs
(Greeneetal.2004).
FeasibilityofBiofuels
TeUnitedStatescouldbenetsubstantiallyby
increasingitsuseofdomestic,renewablefuels
inthetransportationsector,butcanbiofuelsbe
producedatthescaleneededtomakearealdier-
enceintransportationconsumptionoffossilfuels?
Morespecically,isthereenoughlandtoprovide
theneededlarge-scalesupplyofbiomass,istheuse
ofbiofuelssustainableagriculturally,canbiofuels
becomecost-competitivewithgasoline,andiscel-
lulosic-biofuelproductiontechnicallyfeasiblefor
energy?Teshortanswertoallthesequestionsis
yes,andthissectionsummarizesrecentreportsthat
supportthisview.
LandAvailability
Amajorfactorinuencingtheextenttowhich
biofuelswillcontributetoAmericasenergyfutureis
theamountoflandavailableforbiomassharvesting.
8 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Arebiomassresourcessucient
tomeetasignicantportionof
transportation-fuelconsump-
tion,andhowwouldharvest-
ingbiomassforenergyaect
currentagriculturalandforestry
practices?
In2005,astudyjointlysup-
portedbyDOEandUSDA
examinedwhetherland
resourcesintheUnitedStates
aresucienttosustainproduc-
tionofover1billiondrytons
ofbiomassannually,enough
todisplace30%ormoreofthe
nationscurrentconsumption
ofliquidtransportationfuels.
Byassumingrelativelymodest
changesinagriculturalandfor-
estrypractices,thisstudyproj-
ectsthat1.366billiondrytons
ofbiomasscouldbeavailable
forlarge-scalebioenergyand
bioreneryindustriesbymid-
21stCenturywhilestillmeeting
demandforforestryproducts,
food,andber(Perlacketal.
2005)(seesidebar,ABillion-TonAnnualSupplyofBiomass,p.10).Tis
supplyofbiomasswouldbeasevenfoldincreaseoverthe190milliondry
tonsofbiomassperyearcurrentlyusedforbioenergyandbioproducts.
Mostofthisbiomassisburnedforenergy,withonly18milliondrytons
usedforbiofuels(primarilycorn-grainethanol)and6milliondrytons
usedforbioproducts.
Tebiomasspotentialdeterminedbythebillion-tonstudyisonescenario
basedonasetofconservativeassumptionsderivedfromcurrentpracticesand
shouldnotbeconsideredanupperlimit.Crop-yieldincreasesassumedin
thisstudyfollowbusiness-as-usualexpectations.Withmoreaggressivecom-
mitmentstoresearchonimprovingenergycropsandproductivity,thebio-
masspotentialcouldbemuchgreater.Energy-cropyieldisacriticalfactorin
estimatinghowmuchlandwillbeneededforlarge-scalebiofuelproduction,
andthisfactorcanbeinuencedsignicantlybybiotechnologyandsystems
biologystrategiesusedinmodernplantbreedingandbiomassprocessing.
Manypotentialenergycrops(e.g.,switchgrass,poplar,andwillow)are
essentiallyunimprovedorhavebeenbredonlyrecentlyforbiomass,
comparedtocornandothercommercialfoodcropsthathaveundergone
substantialimprovementsinyield,diseaseresistance,andotheragro-
nomictraits.Amorecompleteunderstandingofbiologicalsystemsand
Fig.3.MiscanthusGrowthover
aSingleGrowingSeasonin
Illinois.Miscanthushasbeen
exploredextensivelyasapoten-
tialenergycropinEuropeand
nowisbeingtestedintheUnited
States.Tescaleisinfeet.Tese
experimentsdemonstrateresults
thatarefeasibleindevelopment
ofenergycrops.[Imagesource:
S.Long,UniversityofIllinois]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 9

INTRODUCTION
ABillion-TonAnnualSupplyofBiomass:SummaryofPotentialForestand
AgriculturalResources
I
n2005,astudyjointlysupportedbyDOEandUSDAexaminedwhetherlandresourcesintheUnited
Statesaresucienttosustainproductionofover1billiondrytonsofbiomassannually,enoughtodis-
place30%ormoreofthenationscurrentconsumptionofliquidtransportationfuels(Perlacketal.2005).
Assumingrelativelymodestchangesinagriculturalandforestrypractices,thisstudyprojectsthat1.366billion
drytonsofbiomass(368milliondrytonsfromforestand998milliondrytonsfromagriculture)couldbe
availableforlarge-scalebioenergyandbioreneryindustriesbymid-21stCenturywhilestillmeetingdemand
forforestryproducts,food,andber(seeFig.A.PotentialBiomassResources,below).Tissupplyofbiomass
wouldbeasevenfoldincreaseoverthe190milliondrytonsofbiomassperyearcurrentlyusedforbioenergy
andbioproducts.Mostofthisbiomassisburnedforenergy,withonly18milliondrytonsusedforbiofuels
(primarilycorn-grainethanol)and6milliondrytonsusedforbioproducts.
LandareaintheUnitedStatesisabout2billionacres,with33%forestlandsand46%agriculturallandscon-
sistingofgrasslandsorpasture(26%)andcroplands(20%).Oftheestimated368milliondrytonsofforest
biomass,142milliondrytonsalreadyareusedbytheforestproductsindustryforbioenergyandbioproducts.
Severaldierenttypesofbiomasswereconsideredinthisstudy.Residuesfromtheforestproductsindustry
includetreebark,woodchips,shavings,sawdust,miscellaneousscrapwood,andblackliquor,aby-productof
pulpandpaperprocessing.Loggingandsite-clearingresiduesconsistmainlyofunmerchantabletreetops
andsmallbranchesthatcurrentlyareleftonsiteorburned.Forestthinninginvolvesremovingexcesswoody
materialstoreducerehazardsandimproveforesthealth.Fuelwoodincludesroundwoodorlogsburnedfor
spaceheatingorotherenergyuses.Urbanwoodresiduesconsistprimarilyofmunicipalsolidwaste(MSW,
e.g.,organicfoodscraps,yardtrimmings,discardedfurniture,containers,andpackingmaterials)andconstruc-
tionanddemolitiondebris(seeTableA.PotentialBiomassResources,thispage,andFig.B.BiomassAnalysis
fortheBillion-TonStudy,p.11).
Fig.A.PotentialBiomassResources:ATotalof
Morethan1.3 BillionDryTonsaYearfromAgri-
culturalandForestResources.
BiomassResources
Million
DryTonsperYear
ForestBiomass
Forestproductsindustryresidues 145
Loggingandsite-clearingresidues 64
Forestthinning 60
Fuelwood 52
Urbanwoodresidues 47
SubtotalforForestResources 368
AgriculturalBiomass
Annualcropresidues 428
Perennialcrops 377
Miscellaneousprocessresidues,manure 106
Grains 87
SubtotalforAgriculturalResources 998
TotalBiomassResourcePotential 1366
TableA.PotentialBiomassResources
10 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.B.BiomassAnalysisfortheBillion-TonStudy[Source:Multi Year Program Plan, 20072012,OBP,EERE,

U.S.DOE(2005)]
Severalassumptionsweremadetoestimatepotentialforestbiomassavailability.Environmentallysensitive
areas,landswithoutroadaccess,andregionsreservedfornontimberuses(e.g.,parksandwilderness)were
excluded,andequipment-recoverylimitationswereconsidered.Asannualforestgrowthisprojectedtocon-
tinuetoexceedannualharvests,continuedexpansionofstandingforestinventoryisassumed.
Amongagriculturalbiomassresources,annualcropresiduesaremostlystemsandleaves(e.g.,cornstoverand
wheatstraw)fromcorn,wheat,soybeans,andothercropsgrownforfoodandber.Perennialcropsconsidered
inthestudyincludegrassesorfast-growingtreesgrownspecicallyforbioenergy.Grainprimarilyiscornused
forethanolproduction,andmiscellaneousprocessresiduesincludeMSWandotherby-productsofagricul-
turalresourceprocessing.
Atotalof448millionacresofagriculturallands,largelyactiveandidlecroplands,wereincludedinthisstudy;
landsusedpermanentlyforpasturewerenotconsidered.Otherassumptionsforagriculturalbiomassresources
includea50%increaseincorn,wheat,andsmall-grainyield;doublingtheresidue-to-grainratioforsoy-
beans;recoveryof75%ofannualcropresidueswithmoreecientharvestingtechnologies;managementofall
croplandwithno-tillmethods;55millionacresdedicatedtoproductionofperennialbioenergycrops;average
biomassyieldforperennialgrassesandwoodyplantsestimatedat8drytonsperacre;conversionofallmanure
notusedforon-farmsoilimprovementtobiofuel;anduseofallotheravailableresidues.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 11

INTRODUCTION
applicationofthelatestbiotechnologicaladvanceswillacceleratethe
developmentofnewbiomasscropshavingdesirableattributes.Tese
attributesincludeincreasedyieldsandprocessability,optimalgrowthin
specicmicroclimates,betterpestresistance,ecientnutrientuse,and
greatertolerancetomoisturedecitsandothersourcesofstress.Fur-
thermore,manybiotechnologicaladvancesforgrowingbetterbiomass
cropswillbeusedtoimprovefoodcrops,easingthepressureonland
areaneededtogrowfood.Jointdevelopmentofthesebiotechnological
advanceswithothercountrieswillhelpmoderatetheglobaldemandfor
crudeoil.Inanidealizedfuturescenariowithgreaterper-acreproductiv-
ityinenergy,food,andbercropsanddecreaseddemandfortransporta-
tionfuelsresultingfrommoreecientvehicles,theUnitedStatescould
havesucientlandresourcestoproduceenoughbiomasstomeetallits
transportation-fuelneeds.
AgriculturalSustainabilityofBiomassProduction
Sustainablepracticesforgrowingandharvestingbiomassfromdedicated
cropswillbeessentialtothesuccessoflarge-scalebiofuelproduction.
Capitalcostsofreneriesandassociatedfacilitiestoconvertbiomass
tofuelswillbeamortizedoverseveraldecades.Tesecapitalassetswill
requireasteadyannualsupplyofbiomassfromalargeproportionof
surroundingland.Terefore,athoroughunderstandingoftheconversion
pathwayandofbiomassharvestingslong-termimpactsonsoilfertility
isneededtoensuresustainability.Vitalnutrientscontainedinprocess
residuesmustbereturnedtothesoil.Perennialcropsexpectedtobe
usedforbiofuelsimprovesoilcarboncontentandmakehighlyecient
useofmineralnutrients(seesidebar,TeArgumentforPerennialBio-
massCrops,p.59).Additionalinformationaboutthecompositionand
populationdynamicsofsoilmicrobialcommunitiesisneeded,however,
todeterminehowmicrobescontributetosustainingsoilproductiv-
ity(seesection,EnsuringSustainabilityandEnvironmentalQuality,p.
68).Mixedcultivarsofgeneticallydiverseperennialenergycropsmay
beneededtoincreaseproductivityandpreservesoilquality.Because
conventionalannualfoodandbercropsaregrownasmonocultures,
relativelylittleresearchhasbeencarriedoutonissuesassociatedwith
growingmixedstands.
TodayFuelEthanolProductionfromCornGrain(StarchEthanol)
In2004,3.41Bgalofstarchethanolfuelwereproducedfrom1.26billion
bushelsofcorn11%ofallcorngrainharvestedintheUnitedStates.
Tisrecordlevelofproductionwasmadepossibleby81ethanolplants
locatedin20states.Completionof16additionalplantsandotherexpan-
sionsincreasedethanol-productioncapacityto4.4Bgalbytheendof
2005;additionalplannedcapacityisonrecordforanother1Bgalfrom
2006to2007(RFA2005).Althoughdemandforfuelethanolmorethan
doubledbetween2000and2004,ethanolsatisedlessthan2%ofU.S.
transportation-energydemandin2004.
12 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

IntheUnitedStates,ethanolisproducedincornwetordrymills.Corn
wetmillsfractionatethecorngrainforproductslikegermandoilbefore
convertingthecleanstarchtosugarsforfermentationorforsuchvalu-
ablefoodproductsashigh-fructosecornsyrupandmaltodextrins. Te
cornberby-productusuallyissoldasanimalfeed.Incorndrymills,
thegrainisground,brokenintosugarmonomers(saccharied),and
fermented.Sincethegrainisnotfractionated,theonlyby-productis
theremainingsolids,calleddistillersdriedgrainswithsolubles,ahighly
nutritiousproteinsourceusedinlivestockfeed.Abushelofcornyields
about2.5galethanolfromwet-millprocessingandabout2.8galfrom
drygrind(BothastandSchlicher2005).Some75%ofcornethanolpro-
ductionisfromdry-millfacilitiesand25%fromwetmills.
TomorrowBiorefneryConcepttoProduceFuelEthanolfrom
CellulosicBiomass
Cellulosicethanolhasthepotentialtomeetmost,ifnotall,transportation-
fuelneeds.However,duetothecomplexstructureofplantcellwalls,cellu-
losicbiomassismoredicultthanstarchtobreakdownintosugars.Tree
keybiomasspolymersfoundinplantcellwallsarecellulose,hemicellulose,
andlignin(seeLignocellulosicBiomassCharacteristicschapter,p.39).
Tesepolymersareassembledintoacomplexnanoscalecomposite,not
unlikereinforcedconcretebutwiththecapabilitytoexandgrowmuch
likealiquidcrystal.Tecompositeprovidesplantcellwallswithstrength
andresistancetodegradationandcarriesoutmanyplantfunctions.Teir
robustness,however,makesthesematerialsachallengetouseassubstrates
forbiofuelproduction.
Traditionalcellulosicbiorenerieshavenumerouscomplex,costly,and
energy-intensivestepsthatmaybeincompatibleorreduceoverallprocesse-
ciency. Tecurrentstrategyforbiochemicalconversionofbiomasstoethanol
hasitsrootsintheearlydaysofwoodchemistry.Developedinthe1930sfor
wartimeuseinGermany, itisusedinRussiatoday. Tisprocessinvolvesthree
basicsteps,eachelementofwhichcanbeimpactedbycellulosicbiomass
research(seeFig. 4. TraditionalCellulosicBiomassConversiontoEthanol
BasedonConcentratedAcidPretreatmentFollowedbyHydrolysisand
Fermentation,p.14).Afteracquisitionofsuitablecellulosicbiomass,bioren-
ingbeginswithsizereductionandthermochemicalpretreatmentofraw
cellulosicbiomasstomakecellulosepolymersmoreaccessibletoenzymatic
breakdownandtofreeuphemicellulosicsugars,followedbyproductionand
applicationofspecialenzymepreparations(cellulases)forhydrolysisofplant
cell-wallpolysaccharidestoproducesimplesugars.Finalstepsintheprocess
includefermentation,mediatedbybacteriaoryeast,toconvertthesesugarsto
ethanolandothercoproductsthatmustberecoveredfromtheresulting
aqueousmixture.Recentresearchanddevelopmenthasreduceddramatically
thecostofenzymesandhasimprovedfermentationstrainstoenablesimulta-
neoussaccharicationandfermentation(SSF),inwhichhydrolysisof
celluloseandfermentationofglucosearecombinedinonestep.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 13

































INTRODUCTION
Figure5.ABioreneryConceptIncorporatingAdvancedPretreatment
andConsolidatedProcessingofCellulosetoEthanol,p.15,depictskey
targetsforsimplifyingandimprovingthebioreneryconcept.Feedstock
researchseeksrsttoincreasebiomassyieldsandenhancebiomasschar-
acteristicstoenablemoreecientprocessing.Advancedbiocatalystswill
augmentorreplacethermochemicalmethodstoreducetheseverityand
increasetheyieldofpretreatment.Morerobustprocessesandreduction
ofinhibitorswouldalloweliminationofthedetoxicationandseparation
steps.Developingmodiedenzymesandfermentationorganismsulti-
matelywillallowincorporationofhydrolysisenzymeproduction,hydro-
lysis,andfermentationintoasingleorganismorafunctionallyversatile
butstablemixedculturewithmultipleenzymaticcapabilities.Termed
consolidatedbioprocessing(CBP),thiscouldenablefourcomponents
comprisingsteps2and3(greenboxes)inFig.4tobecombinedinto
one,whichinFig.5iscalleddirectconversionofcelluloseandhemicel-
lulosicsugars.Furtherrenementwouldintroducepretreatmentenzymes
(ligninasesandhemicellulases)intotheCBPmicrobialsystemsaswell,
Fig.4.TraditionalCellulosicBiomassConversionto
EthanolBasedonConcentratedAcidPretreatmentFollowed
byHydrolysisandFermentation.Treestepsintheprocessare
(1)sizereductionandthermochemicalpretreatmentofrawcel-
lulosicbiomasstomakecellulosepolymersmoreaccessibletoenzy-
maticbreakdownandfreeuphemicellulosicsugars(blueboxesonleft);
(2)productionandapplicationofspecialenzymepreparations(cellulases)
thathydrolyzeplantcell-wallpolysaccharides,producingamixtureofsimple
sugars(greenboxes);and(3)fermentation,mediatedbybacteriaoryeast,to
convertthesesugarstoethanolandothercoproducts(yellowdiamonds).Recent
research and development has reduced dramatically the cost of enzymes and has improvedfermentationstrains
toenablesimultaneoussaccharicationandfermentation(SSF,greenboxessurroundedbyorange),inwhichhydro-
lysisofcelluloseandfermentationofglucosearecombinedinonestep.Cellulosicbiomassresearchistargetingthese
stepstosimplifyandincreasetheyieldofbiomassproductionandprocessing(seeFig.5.p.15).[Source:Adaptedfrom
M.HimmelandJ.Sheehan,NationalRenewableEnergyLaboratory]
14 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig5.ABioreneryConceptIncorporatingAdvancedPretreatment
andConsolidatedProcessingofCellulosetoEthanol.Testrategies
discussedinthisroadmaparebasedonrstdevelopingtechnologiesto
allowmoreenergy-ecientandchemicallybenignenzymaticpretreat-
ment.Saccharicationandfermentationwouldbeconsolidatedintoasimplestepandultimatelyintoasingleorgan-
ismorstablemixedculture(consolidatedbioprocessing),thusremovingmultiplewholestepsinconvertingbiomass
toethanol.AlsoseeFig.6.p.16.[Source:AdaptedfromM.HimmelandJ.Sheehan,NationalRenewableEnergy
Laboratory]
reducingtoonesteptheentirebiocatalyticprocessingsystem(pretreat-
ment,hydrolysis,andfermentation).Teseprocesssimplicationsand
improvementswilllessenthecomplexity,cost,andenergyintensityofthe
cellulosicbiorenery.
Inadditiontopolysaccharidesthatcanbeconvertedtoethanol,thelignin
inplantcellwallsisacomplexpolymerofphenylpropanoidsubunits
thatmustbeseparatedfromcarbohydratesduringbiomassconversion.
Energy-richlignincanbeburnedforheat,convertedtoelectricitycon-
sumablebyotherstepsintheethanol-productionpathway,orgasied
andconvertedtoFischer-Tropsch(FT)fuels(seeFig.6.MatureBiomass
ReningEnergyFlows:ExampleScenario,p.16,andTableA.Sum-
maryofEnergyFlowsinMatureBioreneryConcept,p.16).Formore
information,seeDeconstructingFeedstockstoSugars,p.85,andSugar
FermentationtoEthanol,p.119.Foranoverviewofhowgenomicscan
beappliedtodevelopingnewenergyresources,seemegasidebar,From
BiomasstoCellulosicEthanol,p.26.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 15

INTRODUCTION
Fig.6.MatureBiomassReningEnergyFlows:ExampleScenario.Amatureintegratedcellulosicbiomassbioren-
eryencompassesbiologicalandthermochemicalprocesses,demonstratingthe
ecienciespossiblewithafullyintegrateddesign.Tisscenarioincorporates
theconsolidatedbioprocessing(CBP)concept,inwhichallbiologicalprocesses
areincorporatedintoasinglemicrobeormicrobialcommunity.Energyderived
fromfeedstocksischemicallyandphysicallypartitionedtoethanolandother
products.Dottedarrowsfromaboveindicateenergyinputsneededtorun
machinery.Tethermochemicalportionreleasesenergythatcanbeused,for
example,tosustainnecessarytemperatures,bothheatingandcooling,andto
powerpumpsandotherancillaryequipment.TableAisasummaryofenergy
owsinthisbioreneryconcept.[Source:AdaptedfromL.Lyndetal.,Envi-
sioningMatureBiomassReneries,presentedatFirstInternationalBioren-
erySymposium,Washington,D.C.(July20,2005).]
TableA.SummaryofEnergy
FlowsinMatureBiorefnery
Concept
Products
54%ethanol
5%power(electricity)
10%diesel
6%gasoline

ProductionInputs
21%capturedforprocessenergy
orlost
<5%agriculturalinputs(e.g.,
farmingcosts,feedstocktransport)

16 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Ethical,Legal,andSocialIssues(ELSI)
Usingbiomasstoproducebiofuelsholdsmuchpromise
forprovidingarenewable,domesticallyproducedliquid
energysourcethatcanbeaviablealternativetopetroleum-
basedfuels.BiofuelR&D,therefore,aimstoachievemore
thanjustscienticandtechnologicaladvancesperse.Itis
conductedtoaccomplishimportantsocietalneeds,with
thebroadergoalsofbolsteringnationalenergysecurity,
economicgrowth,andtheenvironment.Analyzingand
assessingthesocietalimplicationsof,andresponsesto,this
researchlikewiseshouldcontinuetobeframedwithinthe
contextofsocialsystemsandnotsimplyintermsoftech-
nologicaladvancesandtheirecacy(seesidebar,Ethical,
Legal,andSocialIssuesforWidespreadDevelopmentof
CellulosicBiofuels,thispage).
EEREOBPPlatformforIntegrated
Biorefneries
TeDepartmentofEnergysstrategicplanidenties
itsenergygoal:Toprotectournationalandeconomic
securitybypromotingadiversesupplyanddeliveryof
reliable,aordable,andenvironmentallysoundenergy.
Oneofseveralstrategiesidentiedtoachievethisgoalis
toresearchrenewableenergytechnologieswind,hydro-
power,biomass,solar,andgeothermalandworkwiththe
privatesectorindevelopingthesedomesticresources.
TedepartmentsOceofEnergyEciencyandRenew-
ableEnergy(EERE)OceoftheBiomassProgram
(OBP)elaboratesonthatgoal:Improveenergysecurity
bydevelopingtechnologiesthatfosteradiversesupplyof
reliable,aordable,andenvironmentallysoundenergyby
providingforreliabledeliveryofenergy,guardingagainst
energyemergencies,exploringadvancedtechnologiesthat
makeafundamentalimprovementinourmixofenergy
options,andimprovingenergyeciency.
Majoroutcomessoughtincludethefollowing.
By2012,completetechnologydevelopmentneces-
sarytoenablestartupdemonstrationofabiorenery
producingfuels,chemicals,andpower,possiblyatan
existingornewcorndrymillmodiedtoprocesscorn
stoverthroughasidestream.
By2012(basedonAEI),completetechnologyintegra-
tiontodemonstrateaminimumsugarsellingpriceof
$.064/lb,resultinginaminimumethanolsellingpriceof
$1.07/gal.Ethanolwouldbeproducedfromagricultural
residuesordedicatedperennialenergycrops.
Ethical,Legal,andSocialIssues
forWidespreadDevelopmentof
CellulosicBiofuels
S
ocietalquestions,concerns,andimplications
clearlymayvaryaccordingtotheevolution-
arystageofbiofueldevelopment.Acceptance
andsupportfromdiversecommunitieswillbe
needed.Further,societalandtechnologicalinterac-
tionsatearlierphasesofresearch,development,
demonstration,deployment,anddecommission-
ing(RDDD&D)willaectinteractionsatlater
phases.Withinthecontextofsocialsystems,three
overarchingquestionsemerge.
Whatarethepossiblelong-termimplications
ofbiofueldevelopmentanddeploymentfor
socialinstitutionsandsystemsifthestrategy
worksasanticipatedandifitdoesnot?
Howareindividuals,organizations,andinsti-
tutionslikelytorespondovertimetothis
developmentandthechangesintegraltoits
deployment?
Whatactionsorinterventions(e.g.,regulations)
associatedwithbiofueldevelopmentanditsuse
anddeploymentwillprobablyorshouldbetaken
atlocal,regional,andnationallevelstopromote
sociallydeterminedbenetsandtoavoid,mini-
mize,ormitigateanyadverseimpacts?
Broadtopicsraisedattheworkshopincludedthe
following:
Sustainabilityofthetotalintegratedcycle.
Competinginterestsforlanduse.
Creationanduseofgeneticallymodied
plants.Whocreatesandusesthem,who
decidesbasedonwhatcriteria,andhowmight
orshouldtheyberegulated?
Creationanduseofgeneticallymodiedmicro-
bialorganismsinacontrolledindustrialsetting.
Individualsandgroupsthathavetheauthority
topromoteorinhibitR&D,demonstration,
anduse.
Groupsmostlikelytobeaected(positively
ornegatively)bybiofuelsatallevolutionary
stagesofRDDD&Donthelocal,national,
andgloballevels.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 17

INTRODUCTION
By2030,helpenabletheproductionof60billiongallonsofethanol
peryearintheUnitedStates.Areportelaboratingonthisgoalwillbe
releasedsoon.
TeBiomassProgramalsoisalignedwithrecommendationsintheMay
2001NEPtoexpandtheuseofbiomassforwide-rangingenergyappli-
cations.NEPoutlinesalong-termstrategyfordevelopingandusing
leading-edgetechnologywithinthecontextofanintegratednational
energy,environmental,andeconomicpolicy.
Fig.7.DOEEnergyEciencyandRenewableEnergyStrategicGoalsasTeyRelatetoDevelopmentofBiofuels.
[Source:Multi Year Program Plan 20072012,OBP,EERE,U.S.DOE(2005)]
18 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Teprogramsoverarchingstrategicgoalistodevelopbiorenery-related
technologiestothepointthattheyarecost- andperformance-competitive
andareusedbythenationstransportation,energy,chemical,andpower
industriestomeettheirmarketobjectives. Tenationwillbenetbyexpand-
ingclean,sustainableenergysupplieswhilealsoimprovingitsenergyinfra-
structureandreducingGHGsanddependenceonforeignoil. Tisgoalisin
alignmentwithDOEandEEREstrategicgoalsasshowninFig.7.DOE
EnergyEciencyandRenewableEnergyStrategicGoalsasTeyRelateto
DevelopmentofBiofuels,p.18.
PlanningdocumentsofEEREsOBPdescribeadvancestheprogramseeks
forfourcriticalobjectives:(1)Alterfeedstocksforgreateryieldandforcon-
vertinglargerportionsofrawbiomassfeedstockstofuelethanolandother
chemicals;(2)decreasecostsandimproveenzymeactivitiesthatconvert
complexbiomasspolymersintofermentablesugars;(3)developmicrobesthat
canecientlyconvertall5- and6-carbonsugarsreleasedfromthebreak-
downofcomplexbiomasspolymers;and(4)consolidateallsaccharication
andfermentationcapabilitiesintoasinglemicrobeormixed,stableculture.
Acommercialindustrybasedoncellulosicbiomassbioconversionto
ethanoldoesnotyetexistintheUnitedStates,butseveralprecommercial
facilitiesareindevelopment.TeCanadiancompany,IogenCorporation,a
leadingproducerofcellulaseenzymes,operatesthelargestdemonstration
biomass-to-ethanolfacility,withacapacityof1Mgal/year;productionof
cellulosicethanolfromwheatstrawbeganatIogeninApril2004.OBP
hasissuedasolicitationfordemonstrationofcellulosicbioreneries(U.S.
Congress2005,Section932)aspartofthepresidentialBiofuelsInitiative.
DOEOfceofSciencePrograms
TeDOEOceofScience(SC)playskeyrolesinU.S.research,includ-
ingthecontributionofessentialscienticfoundationstoDOEsnational
energy,environment,andeconomicsecuritymissions(seeFig.8.DOE
OceofScienceProgramsandGoalsasTeyRelatetoDevelopment
ofBiofuels,p.20).Otherrolesaretobuildandoperatemajorresearch
facilitieswithopenaccessbythescienticcommunityandtosupportcore
capabilities,theories,experiments,andsimulationsattheextremelimits
ofscience.AnSCgoalfortheOceofBiologicalandEnvironmental
Research(OBER)istoharnessthepowerofourlivingworldandprovide
thebiologicalandenvironmentaldiscoveriesnecessarytocleanandprotect
ourenvironmentandoernewenergyalternatives.SCsgoalforitsOce
ofAdvancedScienticComputingResearch(OASCR)istodelivercom-
putingforthefrontiersofscience(U.S.DOE2004).
Toaddressthesepriorities,OBERandOASCRaresponsoringthe
Genomics:GenomestoLife(GTL)program.Establishedin2002,GTL
usesgenomedataastheunderpinningsforinvestigationsofbiologi-
calsystemswithcapabilitiesrelevanttoDOEenergyandenvironmental
missions. TeGTLscienticprogramwasdevelopedwithinputfrom
hundredsofscientistsfromuniversities,privateindustry,otherfederal
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 19

INTRODUCTION
Fig.8.DOEOceofScienceProgramsandGoalsasTeyRelatetoDevelopmentofBiofuels.[DerivedfromOce
ofScienceStrategicPlanandGenomics:GTLRoadmap]
agencies,andDOEnationallaboratories.Providingsolutionstomajor
nationalproblems,biologyandindustrialbiotechnologywillserveasan
engineforeconomiccompetitivenessinthe21stCentury.DOEmissions
inenergysecurityaregrandchallengesforanewgenerationofbiological
research.SCwillworkwithEEREtobringtogetherbiology,computing,
physicalsciences,bioprocessengineering,andtechnologydevelopmentfor
thefocusedandlarge-scaleresearcheortneededfromscienticinves-
tigationstocommercializationinthemarketplace.Researchconductedby
thebiofuelR&DcommunityusingSCprogramsandresearchfacilities
willplayacriticalroleindevelopingfuturebioreneriesandensuringthe
successofEEREOBPsplans.
Tenationsinvestmentingenomicsoverthepast20yearsnowenables
rapiddeterminationandsubsequentinterpretationofthecompleteDNA
sequenceofanyorganism.Becauseitrevealstheblueprintforlife,genom-
icsisthelaunchingpointforanintegratedandmechanisticsystems
understandingofbiologicalfunction.Itisanewlinkbetweenbiological
researchandbiotechnology.
20 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy



































Fig.9.UnderstandingBiologicalCapabilitiesatAllScalesNeededtoSupportSystemsBiologyInvestigationsofCellu-
losicBiomass.Capabilitiesareneededtobringtogetherthebiological,physical,computational,andengineeringsciences
tocreateanewinfrastructureforbiologyandtheindustrialbiotechnologyinthe21
st
Century.Tisguredepictsthefocus
ofGTLonbuildinganintegratedbodyofknowledgeaboutbehavior,from
ing.DOE
genomicinteractionsthroughecosystemchanges.Simultaneouslystudying
multiplesystemsrelatedtovariousaspectsofthebiofuelproblemispower-
fullysynergisticbecauseenduringbiologicalthemesaresharedandgeneral
principlesgoverningresponse,structure,andfunctionapply
throughout.Accumulatingdataastheyareproduced,theGTL
KnowledgebaseandGTLcomputationalenvironmentwill
interactivelylinkthecapabilitiesandresearcheorts,allowing
thisinformationtobeintegratedintoapredictiveunderstand-
stechnologyprogramscanwork
withindustrytoapplysuchcapabilities
andknowledgetoanewgenerationofproc-
esses,products,andindustries.
GTLsgoalissimpleinconceptbutchallenginginpracticetorevealhow
thestaticinformationingenomesequencesdrivestheintricateanddynamic
processesoflife. Troughpredictivemodelsoftheselifeprocessesandsup-
portingresearchinfrastructure,GTLseekstoharnessthecapabilitiesof
livingsystems.GTLwillstudycriticalpropertiesandprocessesonfoursys-
temslevelsmolecular,cellular,organismal,andcommunityeachrequiring
advancesinfundamentalcapabilitiesandconcepts. Tesesameconceptsand
capabilitiescanbeemployedbybioprocessengineerstobringnewtechnolo-
giesrapidlytothemarketplace.
AchievingGTLgoalsrequiresmajoradvancesintheabilitytomeasurethe
phenomenologyoflivingsystemsandtoincorporatetheiroperatingprin-
ciplesintocomputationalmodelsandsimulationsthataccuratelyrepresent
biologicalsystems. TomakeGTLscienceandbiologicalresearchmore
broadlytractable,timely,andaordable,GTLwilldevelopcomprehensive
suitesofcapabilitiesdeliveringeconomiesofscaleandenhancedperformance
(seeFig.9.UnderstandingBiologicalCapabilitiesatAllScalesNeededto
SupportSystemsBiologyInvestigationsofCellulosicBiomass,thispage).In
verticallyintegratedbioenergyresearchcenters,thesecapabilitieswillinclude
theadvancedtechnologiesandstate-of-the-artcomputingneededtobetter
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 21

INTRODUCTION
Fig.10.Creatinga
CommonResearch
Agenda.TeEERE
OceoftheBio-
massProgramsMulti
YearProgramPlan
20072012contains
aroadmapforbiofuel
developmentthat
identiestechnological
barrierstoachieving
goalsdenedinFig.7,
p.18.Tesechal-
lengesincludetheneed
fornewfeedstocks,
theirdeconstruc-
tiontofermentable
sugars,andfermen-
tationofallsugars
toethanol.Within
theDOEOceof
Science,OBERand
OASCRsroadmapfor
theGenomics:GTL
programoutlinesscienticgoals,technologies,computingneeds,andaresourcestrategytoachievetheGTLgoalof
apredictiveunderstandingofbiologicalsystems.Tisdocumentisaroadmapthatlinksthetwoplans.
understandgenomicpotential,cellularresponses,regulation,andbehaviorsof
biologicalsystems.Computingandinformationtechnologiesarecentralto
theGTLprogramssuccessbecausetheywillallowscientiststosurmountthe
barrierofcomplexitynowpreventingthemfromdeducingbiologicalfunction
directlyfromgenomesequence.GTLwillcreateanintegratedcomputational
environmentthatwilllinkexperimentaldataofunprecedentedquantityand
dimensionalitywiththeory,modeling,andsimulationtouncoverfundamental
biologicalprinciplesandtodevelopandtestsystemstheoryforbiology.
BiomasstoBiofuelsWorkshop:CreatingaCommon
ResearchAgendatoOvercomeTechnologyBarriers
AproductoftheBiomasstoBiofuelsWorkshop,thisroadmapanalyzes
barrierstoachievingOBPgoals(asdescribedherein)anddetermines
fundamentalresearchandcapabilities(asdescribedintheGTLRoad-
map)thatcouldbothaccelerateprogressinremovingbarriersandallowa
morerobustsetofendpoints(seeFig.10.CreatingaCommonResearch
Agenda,thispage).Relatinghigh-leveltopicalareasandtheirgoalstokey
scienticmilestonesidentiedbyworkshopparticipantscouldhelpachieve
progresstowardOBPgoalsincollaborationwithSC(seeTable2.Over-
comingBarrierstoCellulosicEthanol:OBPBiologicalandTechnological
ResearchMilestones,p.23).
22 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Table2.OvercomingBarrierstoCellulosicEthanol:OBPBiologicalandTechnologicalResearchMilestones
OfceoftheBiomass
Program(OBP)BarrierTopic
TechnologyGoals ScienceResearchMilestones
Feedstocks Bettercompositionsandstructuresforsugars Cell-wallarchitectureandmakeuprelativetoprocessability
Developsustainable
production
Genomesequenceforenergycrops
technologiesto
supplybiomassto
bioreneries
Domestication:Yield,tolerance
Betteragronomics
Sustainability
Domesticationtraits:Yield,tolerance
Cell-wallgenes,principles,factors
Newmodelsystemstoapplymodernbiologytools
Soilmicrobialcommunitydynamicsfordetermining
sustainability
Feedstock PretreatmentEnzymes Cell-wallstructurewithrespecttodegradation
Deconstructionto
Sugars
Reducedseverity
Reducedwaste
Modicationofthechemicalbackboneofhemicellulose
materialstoreducethenumberofnonfermentableand
derivatizedenzymes
Developbiochemical
conversion
Highersugaryields Cell-wallcomponentresponsetopretreatments
technologiesto
Reducedinhibitors Principlesforimprovedcellulases,ligninases,
producelow-cost
sugarsfrom
Reductioninnonfermentablesugars
hemicellulases
lignocellulosic
biomass
EnzymeHydrolysistoSugars
Higherspecicactivity
Higherthermaltolerance
Reducedproductinhibition
Broadersubstraterange
Cellulasesandcellulosomes
Understandingofcellulosomeregulationandactivity
Actionofenzymesoninsolublesubstrates(fundamental
limits)
Fungalenzyme-productionfactors
Nonspecicadsorptionofenzymes
Originofinhibitors
SugarFermentationto CofermentationofSugars Fullmicrobialsystemregulationandcontrol
Ethanol
C-5andC-6sugarmicrobes
Rapidtoolsformanipulationofnovelmicrobes
Developtechnologies
toproducefuels,
chemicals,and
Robustprocesstolerance
Resistancetoinhibitors
Utilizationofallsugars
Sugartransporters
powerfrombiobased
sugarsandchemical
Marketableby-products
Responseofmicroorganismstostress
buildingblocks
Newmicrobialplatforms
Microbialcommunitydynamicsandcontrol
ConsolidatedProcessing EnzymeProduction,Hydrolysis,and Fundamentalsofmicrobialcelluloseutilization
Reduceprocesssteps
CofermentationCombinedinOneReactor
Understandingandcontrolofregulatoryprocesses
andcomplexityby
integratingmultiple
processesinsingle
Productionofhydrolyticenzymes
Fermentationofneededproducts(ethanol)
Engineeringofmultigenictraits
Processtolerance
reactors
Processtolerance
Stableintegratedtraits
Allprocessescombinedinasinglemicrobeor
stableculture
Improvedgene-transfersystemsformicrobialengineering
Understandingoftransgenichydrolysisandfermentation
enzymesandpathways
Teworkshopwasorganizedunderthefollowingtopicalareas:Feedstocks
forBiofuels(p.57);DeconstructingFeedstockstoSugars(p.85);Sugar
FermentationtoEthanol(p.119);andCrosscutting21stCenturyScience,
Technology,andInfrastructureforaNewGenerationofBiofuelResearch
(p.155).AcriticaltopicdiscussedinseveralworkshopgroupswasLigno-
cellulosicBiomassCharacteristics(p.39).Tesevetopicsandplanswould
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 23

INTRODUCTION
tiethetwoocesroadmapstogetherandalsoserveasakeydriverfor
implementingthecombinedroadmapsinpursuitofahigh-levelnational
goal:Createaviablecellulosic-biofuelindustryasanalternativetooilfor
transportation.Tesetopicsandtheirrelationshipsarediscussedinsub-
sequentchaptersoutliningtechnicalstrategyanddetailedresearchplans
developedintheworkshop.
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BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 25

INTRODUCTION
linktoFrom
BiomasstoBio-
fuelds
26 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 27

INTRODUCTION
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28 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

TechnicalStrategy:Development
ofaViableCellulosicBiomass
toBiofuelIndustry
I
nnovativeenergycropsplantsspecicallydesignedforindustrialpro-
cessingtobiofuelscanbedevelopedconcurrentlywithnewbiorenery
treatmentandconversionprocesses.Recentadvancesinscienceand
capabilities,especiallythosefromthenascentdisciplineofsystemsbiology,
promisetoaccelerateandenhancethisparadigm.Resultingtechnologies
willallowthefusionofagriculture,industrialbiotechnology,andenergy
valuechainstoenableanecientandeconomicallyviableindustryforcon-
versionofplantbiomasstoliquidfuels.Displacingupto30%ofthenations
currenttransportationfuelby2030willrequiresignicantpolicysupport
andtechnicaladvancementoverthenext5to15years.Researchandtech-
nologydevelopmentdescribedinthisroadmapwilloccurinthreephasesto
enableindustrytomeetthe2025and2030goals(seeFig.1.Phased
DevelopmentofBioenergySystems,p.32).IntheResearchPhase(this
page),applicationofgenome-basedsystemsbiologywillprovidethebasis
ofknowledge,concepts,andtoolsforconcertedresearchanddeployment
oftechnologymodulesintheTechnologyDeploymentPhase(p.32).In
theSystemsIntegrationPhase(p.34),bothfundamentalandapplied
researchandtechnologydevelopmentwillsupportmultiplebioenergysys-
temsthroughconcurrentdevelopmentofcropsandbioreneryprocesses
forvariousU.S.agroecosystems.Driversforeachphaseandtheresearch
targetsandgoalsforFeedstocks,FeedstockDeconstruction,andFermenta-
tiontoEthanolandRecoveryareoutlinedinTable1.TechnicalStrategy
Timeline,p.33.
ResearchPhase(within5years)
Optimizingcelluloseprocessingbyreningbiomasspretreatmentand
convertingcropresidues,rst-generationenergycrops,andothersources
toliquidfuelswillbethenear-termfocus.Tiswillentailreducingcost,
enhancingfeedstockdeconstruction,improvingenzymeactionandstabil-
ity,anddevelopingfermentationtechnologiestomoreecientlyusesugars
resultingfromcellulosebreakdown.Onegoalistodecreaseindustrialrisk
fromarst-of-a-kindtechnology,allowingmorerapiddeploymentof
improvedmethods.
FeedstockUseandOptimization
Arangeofplantmaterials(e.g.,cornstoverandhardwoods)withwidely
varyingphysicalandchemicalcharacteristicscouldbemadeavailableas
feedstocksforconversiontoethanolinbioreneries.Teselegacyfeedstocks
areexpectedtosatisfyone-fourthtoone-thirdofthenationsanticipated
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 29

TECHNICAL STRATEGY
transportationbiofuelneeds.Toachievehigherproductiongoals,new
energycropswithgreateryieldperacreandimprovedprocessabilityare
needed.Advancedgenome-basedcapabilitieswillhelpdeterminehowsoil
microbialcommunitiesfunctionandhowmuchcarbonfromcropresidues
anddedicatedenergycrops,aswellasothernutrients,isneededtosustain
soilecosystemfunctionandproductivity.
Toestablishanewgenerationofplantsasenergycropsanddevelopstable
agroecosystems,biologicalandchemicaltoolsareneededtoprovide
detailedunderstandingofplantcellwalls,theirrolesinplantfunction,and
factorscontrollingrecalcitranceandoptimizationofprocessesforfermenta-
tionofsugars.Genome-basedcapabilitieswillidentifygenesinvolvedinthe
synthesisofcell-wallpolymersandhigherstructures;reactionsperformed
bythemultitudeofenzymesinvolved;designprinciplesofcellwalls;and
factorscontrollingtheamounts,composition,andstructureofpolymers
andpolymermatrices.Tecomplexstructuresofplantcellwallsperform
numerouscriticalfunctionsintheplantsgrowthandmaintenance.Only
someofthesefunctionsarenowunderstood.Plantengineeringsendgoal
istouserationaldesignforpreservingcriticalplantfunctionstomaximize
yieldandagroeconomicfactors,whileoptimizingplantbiomassmakeup
andstructureforcreatingbiofuelsandotherproducts.Oncedesirable
cell-walltraitsareestablishedforenergycrops,modiedvarietiesmustbe
domesticatedforrobustnessandyield,andbioprocessingstepsmustbe
adaptedtothesuperiorpropertiesofthesevarieties.
Plantdesign,bioprocessengineering,andbiomass-processingstrategies
areintimatelylinked.Plantshaveevolvedcomplexmechanismsforresist-
ingassaultontheirstructuralsugars(wallpolymers)fromthemicrobial
andanimalkingdoms.Cell-wallpolymerorganizationandinteractions
areformidablebarrierstoaccessbydepolymerizingenzymesandmustbe
deconstructedinthepretreatmentsteptoobtainadequateratesofrelease
andsugaryields.
Deconstruction
Understandingfactorsgoverningplantcell-wallrecalcitrancetoprocess-
inganddeconstruction-enzymeinteractionswiththecell-wallmatrixis
criticaltoachievingtheintegratedbioreneryconcept.Currenttechnolo-
giesforbiomasspretreatment(breakingdownligninandhemicelluloses
andfreeingcrystallinecellulose)relyonthermochemicalprocessing,which
degradestheresultingbiomassandresultsinaccumulationofinhibitors
thataretoxictoensuingbioreningprocesses.Teseharshandenergy-
intensivepretreatmentsmustbereplacedbymorebenignprocedures,
includingthosethatwillenhanceexistinghydrothermalandmechanical
methodsandfreecellulosemicrobrils.Economicalenzymaticprocedures
thatwouldallowgreaterpotentialforintegratedbiorenerystrategiesalso
willbeincorporated.
Genomicsandtheapplicationofsuchrelativelynewtoolsasproteomics,
metabolomics,andimagingwillaidinvestigatorsinminingburgeoning
genomicdatabasestounderstandhowmicroorganismsattackbiomass
30 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

andtakeadvantageofnaturalenzymediversity.Availableenzymeshave
relativelylowspecicactivities,soenzymeinteractions,mechanismsof
action,andfundamentallimitsmustbeexplored.Weneedtodetermine
whetherlowactivitiesreectafundamentallimittothehydrolysisrateof
certainsubstratesorifratescanbeimprovedbyrationaldesignthrough
experimentationandmodeling.Newclassesofligninasesandhemicellu-
laseswillbeidentied,theirmechanismsofactionunderstood,andtheir
performancerenedtoallowintroductionofenzymaticpretreatment
thatwillfreecellulosemicrobrilsforenzymaticsaccharication(break-
downtosugars).
Bettermethodswillproduceinexpensiveandmorerobustcellulaseswith
higheractivityandlongerlifetimesforbreakdownofcellulosemicro-
brilstosugars.Mechanisticprinciplesofcellulose-degradingenzymeswill
beevaluatedusingarangeofgenesandproteinsfoundinthebiosphere
tounderstandbasicdesignprinciplesandallowrationalredesignfor
enhancingproperties.Tediversityofcellulaseandcellulosomefunctional
schemeswillbemodeledandoptimizedforspecicbiomasssubstrates
(feedstocks).Temicrobialcellulosomeisauniquetypeofmolecular
machinethatcanecientlysolubilizenativeandpretreatedlignocellulose
substrates.Cellulosomescancontainthefullrepertoireofplantcell-wall
polysaccharide-degradingenzymes,andasinglecellulose-bindingmodule
presentstheentirecomplementofenzymestothesubstratesurface(see
sidebar,TeCellulosome,p.102).
FermentationandRecovery
Useofmicrobesforfermentationisthemostcommonmethodforcon-
vertingsugarsproducedfrombiomassintoliquidfuels. Todevelop
commerciallyviableprocessesforcellulosebioconversiontoethanol,an
organismisneededthatusesallsugars(cofermentationofC-5andC-6
sugars)producedfrombiomasssaccharicationatratesandinhigh-
alcoholconcentrationsthatmatchorsurpasscurrentyeast-basedglucose
fermentations.Tesecapabilities,alongwithprocess-toleranttraits,involve
multiplegenesandpathwaysthatarenotreadilyresolved.Today,thecapa-
bilitytointroduceandcontrolmultiplegenechangessimultaneouslyinan
organismislimited.
Newclassesoffermentationorganismsandenzymescapableofmetaboliz-
ingbothC-5andC-6sugarsresultingfrombiomassdeconstructionare
requiredtoadvancebioprocessing.Vastandlargelyuntappedbiochemical
potentialinthemicrobialworldmaybeaccessiblethroughthesequenc-
ingofnewmicrobialgenomesandcommunitymetagenomes.Asrst-
generationorganismsarebeingtestedandimproved,thefocuswillbeon
advancesthatalloweliminationofwholestepsintheconversionprocess.
Forexample,thermophilicmicroorganismswillbeexaminedfortheirabil-
itytofermentbiomasssugarsatelevatedtemperatures,allowingdevelop-
mentofoptimalandsimultaneoussaccharicationandcofermentation
(SSCF)andthusincreasingoverallprocesseciency.Metabolicengineer-
ingwithadvancedbiologicaldiagnosticswillbeusedtodevelopstrains
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 31

TECHNICAL STRATEGY
Fig.1.PhasedDevel-
opmentofBioenergy
Systems.Overthenext5
to15years,researchand
technologyadvancement
willoccurinthreegeneral
phases.IntheResearch
Phase,applicationof
genome-basedsystems
biologywillprovide
thebasisofknowledge,
concepts,andtoolsfor
implementationinthe
TechnologyDeployment
Phase.IntheSystems
IntegrationPhase,both
fundamentalandapplied
researchandtechnol-
ogywillsupportmul-
tiplebioenergysystems
throughtheconcurrrent
developmentofcropsand
bioreneryprocessesfor
thevariousU.S.agro-
ecosystems.Details are
outlinedinTable1,p.33.
withhightolerancetoprocessstresses,inhibitorscreatedinpretreatment,
andhigh-alcoholconcentrations.Genomic,proteomic,metabolomic,and
imagingtechnologies,coupledwithmodelingandsimulation,willelucidate
theregulationandcontrolofmicrobialmetabolismandprovideapredic-
tiveunderstandingofcell-designprinciplestosupportsystemengineering
ofintegratedbioprocessing(seeFig.1.PhasedDevelopmentofBioenergy
Systems,thispage,andTable1.TechnicalStrategyTimeline,p.33).
TechnologyDeploymentPhase(within10years)
Anewgenerationofdedicatedenergycropswithcompositionandstruc-
turebettersuitedforbreakdowntosugarsforfermentation,highyield,and
robustnesswillbeessentialinachievingenergysecurity.Newlyengineered
deconstructionenzymeswillenhanceorsupplantthermochemicalprocess-
ingtodeconstructmoreecientlyabroadrangeofbiomassfeedstocks.
Ultimately,thiswillleadtoenhancedenergy-ecientandenvironmentally
attractiveprocessesforcarryingouttraditionalpulpingprocessesandother
wood-processingtechniques.
32 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Table1.TechnicalStrategyTimeline
ResearchPhase(0to5years) TechnologyDeploymentPhase (5to10years) SystemsIntegrationPhase (10to15years)
LegacyResourcesandEarlyEnergyCrops(E-Crops) Transition:ModularTechnologyDeployment IntegrationandConsolidation
Drivers Drivers Fusionofeconomicvaluechains
Expandbiomassresourcebaseandincrease Needfornewenergycropstoreachor Drivers
utilization exceed1billiontonsofbiomass
Consolidatedbioenergysystems
EarlyE-crops
Cellulose-processingviability

Processsimplicationandimprovement
inmodules

Technologiestailoredforvarious
regions

Costreduction
Useofsystemsbiology,chemistry
Bioprocessengineering
Rationalsystemsdesign

Buildout
Feedstocks
E-cropsustainability
Impactsonsoilecosystems,nutrients
E-cropmodeldevelopment
Genes,principles,E-cropsubsystem
controls
Cell-wallmakeupandstructure
Tiestodeconstructionand
fermentation

Feedstocks
Understandplantassystem
DomesticateE-crops
Enhancesugars,minimizeligninand
toxicinhibitors
Increaseyieldandsoilsustainability

End-to-EndConcurrentlyEngineered
BiofuelSystems(Biome-ECropProcessing)
Systemstailoredtoregionsandfully
consolidatedprocessing
IncludesE-cropswithenhanced
composition
Toolkitsforplantengineering
Consolidatedprocessingtiedto
biofuelsystems
Tailoreddeconstructionenzyme
mix
Engineeredmicrobialmetabolic
systems
Stressandprocesstolerance
Fullsystemcontrol

FeedstockDeconstruction
Reduceenzymecosts
Understandenzyme-lignocellulose
interactions

Deconstruction
Deploy
Improvedenzymes(rate,specicity)
Broadenedsubstraterange

Cell-wallrecalcitrance
Surveynaturalenzymediversity
Establishfundamentalenzymelimits
Developligninasesandhemicellulases
Developgene-transfersystemsfor
cellulolyticmachinesandentire
pathways

Reducedinhibition
E-cropconcept
Toolstodiagnoseandmanipulateenzyme
substrateinteractions
Toolstodesignandimproveenzymes

Toolkitsforrapidmanipulationand
diagnosis

FermentationtoEthanolandRecovery FermentationandRecovery
Studyuseofallsugars,includingdirect Deploy
celluloseutilization
CofermentationofC-5andC-6sugars
Studystressresponseandinhibitors
Newstrains(multiple)
High-alcoholand-sugar
concentrations

Stresstolerance
Understandregulationandcontrol
Hightemperature
Surveynaturaldiversity
Toolsforfullregulatorycontrol
Toolsforrapidanalysisandmanipulation
Testingofconsolidatedorganisms
possessingcelluloyticandethanologenic
properties

BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 33

TECHNICAL STRATEGY
Mid-termtechnologywillbegintheconsolidationofprocesssteps.For
example,improvedorganismswillbeengineeredwiththeabilityto
fermentmixedsugars,demonstrateresistancetotoxicsubstances,and
produceeectivedeconstructionenzymes.Systemsbiologyandanew
generationofsyntheticandanalyticalorganicchemistrywillbecriticalfor
understandingthesebioenergysystemsandforpredictingandmodifying
theirfunction.
Feedstocks
Plantsintendedforbiomassproductionanddownstreamprocessesinvolv-
ingconversiontosugarsand,ultimately,ethanolwillbeunderstoodand
designedasasystem.Newbreedsofenergycropswillbeintroducedwith
enhancedsugarcontentandoptimizedcell-wallstructuresforprocessing,
includingminimizationofligninandinhibitorprecursors.Plantdomesti-
cationandsustainableagroecosystemsbasedonperennialsengineeredto
increaseyield,productivity,andtolerancetosuchstressorsasdroughtand
salinitywillreachamaturestate.Multiplecropswillbedevelopedforvari-
ousregionalandglobalagroecosystems.
Deconstruction
Tisphasewillresultindeploymentofimprovedpretreatmentprocedures
andsaccharifyingenzymeswithenhancedcatalyticrateandsubstrate
specicity,abroaderrangeofapplications,andreducedinhibitorproduc-
tion.Improvedunderstandingofcell-wallrecalcitranceandenzymeaction
willprovidedesignspecicationsfornewenergycrops.Advancedhigh-
throughputbiologicalandchemicaltoolswillbeavailabletodiagnoseand
manipulateenzyme-substrateinteractions.Improvedbiocatalystswith
desirabletraitscanberationallydesignedforspecicfeedstocksandincor-
poratedintomolecularmachinessuchascellulosomes.
FermentationandRecovery
Newstrainsofindustrial-processingorganismswithsuchnovelcapabilities
ascofermentationofC-5andC-6sugarsandhightolerancetoinhibitors,
alcoholendproduct,andtemperaturewillcontributetoamoreenergy
andproductecientbioprocess.Systemsbiologyinvestigationswillhave
producedapredictiveunderstandingofcellularmetabolismandregulatory
controlsinkeyfermentationmicrobes.Tisknowledgewillserveasafoun-
dationforrationaldevelopmentofnewstrainswithconsolidatedsubsetsof
pretreatment,hydrolysis,andfermentationcapabilities.High-throughput
biologicalandchemicaltools,includingcomputationalmodelingforrapid
analysisandmanipulationinthelaboratoryandinproductionenviron-
ments,willbeavailable.
SystemsIntegrationPhase(within15years)
Bioenergysystemswillincludeaconcurrentlyengineeredsetofdesigner
energycropsforspecicagroecosystems,deconstructionandsaccharica-
tionenzymes,androbustfermentation.Incorporatedasmultipleprocesses
34 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

inplantsormicrobes,thesemethodswillaccelerateandsimplifytheend-
to-endproductionoffuelethanol,enablingexiblebioreneriesthatcan
operateonaregionalscale.
IntegrationandConsolidation
Creationoffullyconsolidatedbioenergysystemstailoredforspecic
regionalclimateandsoilcharacteristicswillallowbuildoutofthesesus-
tainablebioenergyeconomies.Tesesystemswillmeldfeedstocks,bio-
massdeconstruction,bioprocessengineering,andfermentationresearch
anddevelopment,yieldingoptimaltwo-stepprocesses.Terststepis
basedonconsolidatedfeedstocktraits,andthesecondisbasedoncon-
solidatedmicrobialtraits.Aconcurrentlyengineeredend-to-endbiofuel
systemusingadvancedsystemsbiologyandchemical-analysiscapabili-
tieswillbepracticable.Toolkitsforplantandagroecosystemengineer-
ingwillsupportsystemstailoredtoregionsandconsolidatedprocessing.
Companionconsolidatedbioprocessengineeringwillbetiedtoagroeco-
systems;withtailoredenzymemixes,engineeredmicrobialmetabolicsys-
temswillincorporatestressandprocesstoleranceandpermitfullsystem
control.Instrumentationavailablebothinfacilitiesandintheeldwill
enablerapiddiagnosisandmanipulationofallcriticalaspectsofthe
integratedbiorenery.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 35

TECHNICAL STRATEGY
36 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

SYSTEMSBIOLOGYTOOVERCOME
BARRIERSTOCELLULOSICETHANOL
LignocellulosicBiomassCharacteristics:Makeup,Structure,
andProcessability.............................................................................39
Sidebar
UnderstandingBiomass:PlantCellWalls....................................................53
FeedstocksforBiofuels......................................................................57
DeconstructingFeedstockstoSugars.................................................85
SugarFermentationtoEthanol........................................................119
CrosscuttingScience,Technology,andInfrastructure........................155
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 37
38 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

LignocellulosicBiomass
Characteristics
Makeup, Structure, and Processability
L
ignocellulosicbiomasshaslongbeenrecognizedasapotentiallow-
costsourceofmixedsugarsforfermentationtofuelethanol.Plant
biomasshasevolvedeectivemechanismsforresistingassaultonits
structuralsugarsfromthemicrobialandanimalkingdoms.Tisproperty
underliesanaturalrecalcitrance,creatingtechnicalbarrierstothecost-
eectivetransformationoflignocellulosicbiomasstofermentablesugars.
Moderateyieldsandtheresultingcomplexcompositionofsugarsand
inhibitorycompoundsleadtohighprocessingcosts.Severaltechnologies
havebeendevelopedoverthepast80years,ofteninwartime,thatallow
thisconversionprocesstooccur,yettheclearobjectivenowistomakethe
processcost-competitiveintodaysmarkets.
Cellwallsinlignocellulosicbiomasscanbeconvertedtomixed-sugarsolu-
tionspluslignin-richsolidresiduesbysequentialuseofarangeofthermo-
chemicalpretreatmentsandenzymaticsaccharication.Telowrateat
whichbiomassisconvertedtosugarsandthecoproductionoffermenta-
tioninhibitorsincreaseequipmentsizeandresultinhighpretreatmentand
enzymecosts.Newapproachesfordesigningimprovedenergyfeedstocks,
deconstructingplantcellwalls,andtransformingtheirpolysaccharides
tofermentablesugarsareneeded.Asystematicunderstandingofenzyme
interactionswithplantcellarchitectureandhierarchy,aswellascellulose,
hemicellulose,andligninstructureduringchemicalandenzymatichydrol-
ysis,willallowthepredictionofplant-tissueresponsetohydrolyticattack
andthecreationofnewsystems.
Signicanttechnologydevelopmentwillbeneededforcreationoflarge-
scalebioenergyandbioreneryindustriesthatcanhandleabilliontons
madeupofavarietyofbiomasseachyear.IntheDOE-USDABillion-
TonStudy,cornstoverandperennialcropssuchasswitchgrassandhybrid
poplarmakeupabouthalfthepotential1.3billiontonsofbiomassthat
couldbeavailablebythemid-21
st
Century(Perlacketal.2005).Under-
standingthestructureandfunctionoftheseandotherbiomassresources
willbecriticaltoenhancingtheirprocessability.
Teresultofanalysisandresearchdescribedherewillbetoincreasethe
eciencywithwhichthesolid(substrate)interactswithlargeprotein
macromolecules(enzymes)atitssurfacewhilethesurfaceitselfisbeing
erodedintosolubleoligosaccharides[seesidebar,ImageAnalysisofBio-
energyPlantCellSurfacesattheOBPBiomassSurfaceCharacterization
Lab(BSCL),p.40].Tisknowledge,combinedwithdevelopmentofnew
proteinsthatcatalyzethesetransformationsaswellasmicrobialsystems
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 39

LIGNOCELLULOSIC BIOMASS
ImageAnalysisofBioenergyPlantCellSurfacesattheOBPBiomassSurface
CharacterizationLab(BSCL)
M
anyaspectsofcurrentbiomassconversiontechnologyarebecomingbetterunderstood,andanascent
biomassprocessingindustryisemergingforsomenichemarkets.Toreachthemid-andlong-term
goalsstatedintheDOEOceoftheBiomassProgramsMultiYearProgramPlan:2007-2012,
however,enhancedfundamentalunderstandingoffeedstocksandallbioreneryprocessesiscritical.Forexam-
ple,detailedknowledgeaboutplant
cell-wallultrastructureandfunc-
tiontoformulateimprovedenzyme
mixturesandpretreatmentswill
reducethecostofproducingsug-
ars.(Seeimages,right.)
Inmanycases,weknowhowto
describebiomasscompositionally.
Tatis,wecanconductchemi-
calorspectroscopicanalysesand
determinethepercentagesof
individualsugars,protein,uronic
acids,andlignin.Whenwestudy
biomassconversionofcornsto-
ver,hardwoods,orricestraw,for
example,weareinfactworking
primarilywiththeplantsstruc-
turalparts,mostofwhicharecell
wall.Terefore,moreknowledgeis
neededaboutthenaturalorganiza-
tionandstructureofpolymersand
chemicalsinplanttissuethataect
chemicalpretreatment,enzymatic
digestibility,andthegenerationof
compoundsinhibitingfermentative
microorganismsusedtoproduce
thenalfuelorchemical.Testudy
ofplantcellwallsatthesubmi-
cronormacromolecularscaleis
challenging.Imagingandimage
analysisareatthecuttingedgeof
botany,molecularbiology,bio-
chemistry,chemistry,andmaterial
andcomputersciences.Descrip-
tionsofmicroscopiesimportant
forultrastructureimagingarein
theImagingTechnologiessection
oftheCrosscuttingTechnologies
chapter,p.163,andsidebar,Some
ImagingTechnologiesRelevantto
FeedstockCharacterization,p.163.
Fig.A.CollageofScanningElectronMicroscopyImagesShowinga
RindandAdjacentPithSectionCutfromaField-DriedCorn-Stem
CrossSection.Terindshowsahigherdensityofvascularelementsmade
fromthick-walledcells.Tepithsection(shownlongitudinally)showsa
greaternumberofthin-walledparenchymacells.Overall,mostcellulose
neededforbiomassconversionislocatedintherind,althoughthepithrep-
resentsmostofthestemvolume.Closeupsofacell-wallpitalsoareshown
(~150,000).Tesestructuresarethoughttoaidtransferofchemicalsand
enzymesusedinprocessingwithinthebiomassbulk.
Fig.B.AFM:CornParenchymaCellWall.
40 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

forfermentationandconsolidation,willenablethedesignofprocedures
andhardwarethatdramaticallyspeeduptheprocess,improveyield,and
lowercosts.
StructureandAssemblyofCellWalls
Plantcellwallsareacomplexanddynamicmixtureofcomponentsthat
performmanyfunctions(seeFig.1.SimpliedCellWallandFig.2.
ConceptualIllustrationofCell-WallBiogenesis,p.42;andsidebar,Under-
standingBiomass,pp.53to55).Tecellwallsareintricateassemblagesof
celluloses,hemicelluloses(i.e.,xyloglucans,arabinoxylans,andglucoman-
nans),pectins(i.e.,homogalaturonans,rhamnogalacturonanIandII,and
xylogalacturonans),lignins,andproteoglycans(e.g.,arabinogalactan-pro-
teins,extensins,andproline-richproteins).Mostmassintheplantcell
wallisintheformofpolysaccharides(celluloseandhemicelluloses).Te
nextmostabundantpolymerislignin,whichiscomposedpredominantly
ofphenylpropanebuildingblocks.Ligninsperformanimportantrolein
strengtheningcellwallsbycross-linkingpolysaccharides,thusproviding
supporttostructuralelementsintheoverallplantbody.Tisalsohelpsthe
plantresistmoistureandbiologicalattack.Tesepropertiesoflignin,how-
ever,interferewithenzymaticconversionofpolysaccharidecomponents.
Additionally,sinceligninisnotconvertedreadilytoethanol,wemustnd
otherusesintheprocessifwearetomaximizeenergyyieldfrombiomass.
Severalthousandgeneproductsareestimatedtoparticipateinsynthesis,
deposition,andfunctionofcellwalls,butveryfewassociatedgeneshave
beenidentiedandverylittleisknownabouttheircorrespondingenzymes.
Manyquestionsremain,forexample,regardinghowpolysaccharidesand
ligninaresynthesized,howwallcompositionisregulated,andhowcom-
positionrelatestocell-wallbiologicalfunctions.Toanswerthesequestions,
weneedtodiscoverthefunctionsofmanyhundredsofenzymes,where
proteinsarelocatedwithincells,whetherornottheyareincomplexes,
whereandwhencorrespondinggenesareexpressed,andwhatfactorsand
genescontrolexpressionandactivitiesoftheproteinsinvolved.Applica-
tionofneworimprovedbiological,physical,analytical,andmathemati-
caltoolswillfacilitateadetailedmechanisticunderstandingofcellwalls.
Tatknowledgewillpermitoptimizationofvariousprocessesinvolvedin
producingbiomassandconvertingittofuels.
Productivityandconversion-processecienciescanbeincreasedbyalter-
ingfundamentalaspectsofplantgrowth,development,andresponseto
bioticandabioticstress.Alteringcell-wallcompositiontoincreasethe
relativeamountofcelluloseanddecreaselignin,forexample,couldhave
signicanteects(seesidebar,OptimizingLigninCompositionforMore
EcientBioethanolProduction,p.43).Eventually,asystemscell-wall
modelincorporatingbiophysicalaspectswithstructuralpropertiesand
knowledgeofproteinsinvolvedinsynthesiswillaidinrationaldevelop-
mentofhighlyproductivefeedstockspecieswhosecellwallsareopti-
mizedforconversion.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 41

LIGNOCELLULOSIC BIOMASS
Fig.2.ConceptualIllustrationofCell-WallBiogenesis.TeGolgiapparatusparticipatesinhemicelluloseandlignin
biosynthesis.Cellulosemicrobrils(CMF)arelaidseparatelyintheswollengelofhemicelluloses.Asligninisdeposited,
thecellwallbecomeshydrophobic.Waterremovalfromtheswollengel,togetherwithperoxidase(PO)andcalcium,
causesanisotropicshrinkageperpendiculartotheCMFs.Tisshrinkagedrivesfurtheroligolignolpolymerizationby
orientingtheligninaromaticringparalleltothecell-wallsurface.[AdaptedfromN.Terashimaetal.,Comprehensive
ModeloftheLigniedPlantCellWall,pp.24770inForage Cell Wall Structure and Digestibility,ed.H.G.Jungetal.,
American
Societyof
Agronomy,
CropSci-
enceSociety
ofAmerica,
andSoilSci-
enceSociety
ofAmerica
(1993).]
Fig.1.SimpliedCell
Wall.Formoredetails,see
sidebar,Understanding
Biomass,p.53.[Adapted
withpermissionfrom
C.Somervilleetal.,
Science 306,220611
(2004);2004AAAS.]
42 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Plantscanhavetwotypesofcellwalls,primaryandsecondary.Primary
cellwallscontaincellulose,whichconsistsofhydrogen-bondedchainsof
thousandsof-1,4-linkedglucosemolecules,inadditiontohemicelluloses
andothermaterialswovenintoananoscalenetworkwiththecellulose.
Celluloseinhigherplantsisorganizedintomicrobrils,eachmeasuring
about3to6nmindiameterandcontainingupto36cellulosechains.Each
OptimizingLigninCompositionforMoreEfcientBioethanolProduction
Plantlignin(guaiacylandsyringyl)interfereswiththereleaseandhydrolysisofcell-wallpolysaccharides.Meta-
bolicengineeringoftheligninbiosyntheticpathwayhasbeensuggestedasamethodformodifyinglignincon-
tentinfeedstocks.StudiesinArabidopsisdemonstratedthatoverexpressionoftheenzymeferulate5-hydroxylase
(F5H)increasesligninsyringylmonomercontentandabolishesthetissuespecicityofitsdeposition(Fig.A).
Todeterminewhetherornotthisenzymehasasimilarregulatoryroleinwoodyplants,F5Hwasoverexpressed
inpoplartreesusingacinnamate4-hydroxylasepromotertodriveF5Hexpression.Transgenictreesdisplayed
enhancedligninsyringylmonomercontent,indicatingthatF5Hoverexpressionisaviablemetabolicengineer-
ingstrategyformodifyingligninbiosynthesis.Tesehigh-syringylligninpoplarsdemonstratedasignicant
increaseinchemicalpulpingeciency.[R.Frankeetal.,ModiedLignininTobaccoandPoplarPlantsOver-
ExpressingtheArabidopsisGeneEncodingFerulate5-Hydrosylase,PlantJ.22(3),22334(2000).]Similar
metabolicengineeringstrategiesholdpromisefordevelopingimprovedfeedstocksforbioethanolproduction.
Manyaspectsofligninbiosynthesisremainmattersofdebate.Althoughmostgenesinvolvedinthebiosyn-
theticpathwayhavebeenclonedandfunctionsassigned,mechanismsthatregulatethepathwaystillarelargely
unknown,asisitsrelationshipwithothercell-wallbiochemicalpathwaysandplantdevelopment.Topicsto
bestudiedincluderegulationoflignindepositionandtissuespecicity,identityofproteinsinvolvedinmono-
lignoltransportandpolymerization,andwaysinwhichlignincontentandcompositioncanbemodied(see
Fig.4.PhenylpropanoidPathwayLeadingtoLigninBiosynthesisinPlants,p.49).Alsoneededisadetailed
understandingoflignin-biodegradationmechanisms,includingthataccomplishedbywhiterotfungi,which
breakdownyellowligninandleavebehindcrystallinewhitecellulose(seesidebar,WhiteRotFungus,p.93).
Comprehensiveexplorationsofligninbiosynthesisanddegradationarerequiredtomaximizeenergyyield
frombiomasscrops.
Fig.A.LigninCompositionControlledbyGeneticManipulationandMonitoredvia
HistochemicalStainingforLigninMonomerCompositioninArabidopsisStemCross
Sections.Intheligniedcellsofwild-typeArabidopsisstems,thepresenceofsyringyl(S)
orguaiacyl(G)monomerscanbevisualizedbyhistochemicalstainingofSlignin(red)
andGlignin(yellow).
Histochemicalstainingallowsadiagnosisoftheeectsofexperimentstomanipulate
lignincomposition.Forexample,eliminatingoneenzymeoftheligninbiosyntheticpath-
wayinanArabidopsismutant(fah1-2)leadstoapureGlignin,andoverexpressionofthe
sameenzymeleadstoahomogeneousdepositionofSlignin(35S-F5H).Te fah1gene
encodesferulate-5-hydroxylase,acytochromeP450dependentmonooxygenasethat
catalyzeshydroxylationofconiferaldehydeandconiferylalcoholinthepathwayleadingto
syringyllignin.[FigurespublishedinC.Chappleetal.,LigninMonomerComposition
isDeterminedbytheExpressionofaCytochromeP450DependentMonooxygenasein
Arabidopsis,Proc. Natl. Acad. Sci. USA 95,661923(1998);1998NationalAcademyof
Sciences,U.S.A.]
Slignin
Slignin
Glignin
Glignin
Slignin
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 43

LIGNOCELLULOSIC BIOMASS
cellulosechainisalinearcollectionofthousandsofglucoseresidues.Pairs
ofglucoseresidues(cellobiose)makeuptherepeatingunitofcellulose.
Likesteelgirdersstabilizingaskyscrapersstructure,themechanical
strengthoftheprimarycellwallisduemainlytothemicrobrilscaold.
Amicrobrilscrystallineandparacrystalline(amorphous)cellulosecore
issurroundedbyhemicellulose,abranchedpolymercomposedofpentose
(5-carbon)and,insomecases,hexose(6-carbon)sugars.Inadditionto
cross-linkingindividualmicrobrils,hemicellulosesinsecondarycellwalls
alsoformcovalentassociationswithlignin,acomplexaromaticpolymer
whosestructureandorganizationwithinthecellwallarenotcompletely
understood(seeFig.3.AssociationofLigninwithPolysaccharides,this
page).Tecrystallinityofcelluloseanditsassociationwithhemicellulose
andligninaretwokeychallengesthatpreventtheecientbreakdownof
celluloseintoglucosemoleculesthatcanbefermentedtoethanol.
Fig.3.AssociationofLigninwithPolysaccharides.Tesche-
maticdiagramshowspossiblecovalentcross-linksbetween
polysaccharidesandligninincellwalls.Ligninisbondedtothe
celluloseandhemicellulosepolysaccharidesandservesasasti-
eningandhydrophobicagent,complicatingbiomassbreakdown.
[Source:AdaptedfromK.Iiyama,T.Lam,andB.Stone,Cova-
lentCross-LinksintheCellWall,Plant Physiol. 104(2),318
(1994).ReprintedwithpermissionofAmericanSocietyofPlant
Biologists,ASPB1994.]
Manyenzymesinvolvedincell-wallsynthesis
ormodicationarethoughttobelocatedin
proteincomplexes.Withintheplasmamem-
branearerosettescomposedoftheenzyme
cellulosesynthase;theseproteincomplexes
movelaterallyalongthemembranetosynthe-
sizecellulosemolecularchains(36perrosette),
whichcrystallizeintomicrobrils.Movement
oftherosettemolecularmachineisassoci-
atedwithcorticalmicrotubulesthatunderlie
themembrane,butthatlinkagealsoispoorly
understood.Teinteractionofcellulosesyn-
thasewiththecytoskeletonhasanimpacton
cellulosebrilorientationandperhapslength.
Understandingthefunctionofthesecom-
plexesandtheirinteractionswithmetabolic
pathwaysthatproducesugarswillbeimpor-
tantforeventuallycontrollingcell-wallcom-
position.Anumberofcellulose-synthasegenes
havebeenclonedforavarietyofplants.(See
sidebar,UnderstandingBiomass,beginningon
p.53,foranillustratedexplanation.)
FactorsinRecalcitranceof
LignocelluloseProcessingtoSugars
Organizationandinteractionsamongpolymers
ofthecellwallconstructedforstrengthand
resistancetobiological,physical,andchemi-
calattackconstituteabarriertoaccessby
depolymerizingenzymesandmustbepartially
deconstructedinthebioconversionpretreatment
stepbeforesaccharicationcanoccur.Although
44 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

variouspretreatmentshavebeendeveloped,westilldonothaveadetailed
understandingoffundamentalphysicalandchemicalfeaturesoflignocel-
lulosicbiomassthatlimititsbreakdowntosugarsandultimatebioconversion
eciency.Improvedmethodsmustbedevelopedtocharacterizebiomassand
itsinteractionwithvariouschemicaltreatments,aswellaswithdeconstruc-
tionandsaccharicationenzymes.
Naturalfactorsbelievedtocontributetolignocellulosicfeedstocksrecalci-
trancetochemicalsorenzymesincludeplantandcell-wallarchitectureand
molecularstructure.
PlantArchitecture
Teorgansofaplant(leaf,stemortrunk,androot)arecomposedof
myriadsofcellswithdierentfunctionsintheplantseconomy.Eachhas
itsownparticulartypeofcellwallwhosecompositionisrelateddirectlyto
cellfunction[e.g.,support(bers),protection(epidermis),andtransport
(xylemandphloem)].Leaf,stem,androottissuesinvariablycontaincells
ofmorethanonetype.Intissues,individualcellsarecloselyassociatedat
theircell-wallinterfacestogiveacompacttissuestructure.Tisstructure
mustbedisassembledbymilling(comminution)toallowliquidaccessto
cellwalls.
Tewaxybarriercomprisinggrasscuticleandtreebarkimpedespen-
etrationofenzymes.
Evenmilledplantstemsandwoodytissueslimitliquidpenetrationby
theirnature.
Cell-WallArchitecture
Tenanoscalecompositenatureoftheplantcellwallrestrictspenetration
ofchemicalsandenzymestotheirsubstrates.Telignin-hemicellulose
coatingonthecellwallscellulosicmicrobrilsaectsthefollowing:
Conformationofcelluloseandnoncellulosicpolysaccharidesmaking
upthemicrobrillimitsaccessibilityofhydrolyticenzymestotheir
substrates.
Lignin-carbohydratecomplexeslimitenzymatichydrolysisofbiomass
polysaccharides.
MolecularStructure
Cellulosecrystallinityseverelyrestrictscellulaseattacks.Cellulasesmust
physicallyreleaseindividualcellulosechainsfrommicrobrilcrystalsfor
subsequentcatalytichydrolysistosugars.Limitingfactors:
Inherentdicultyofenzymesinactingonpoorlyhydratedcellulosesurfaces.
Amountandcomposition(includingheterogeneity)oflignin.
Chemicalheterogeneityandstrengthofcovalentinteractionsbetween
ligninandnoncellulosicpolysaccharides.
Robustnessofhydrogenbondingincellulosemicrobrilsarisingfrom
extendedhydrogen-bondperiodicity.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 45

LIGNOCELLULOSIC BIOMASS
Inaddition,allnativecellulosesundergophysicalmodicationsthatcan
inhibitsaccharicationastheyaredehydratedintraditionalmethodsof
isolationorstorageafterharvestandinpretreatmentprocesses.Charac-
terizingtheeectsofconditionsandstorageenvironmentswouldpoint
tomodicationsinharvestingandstorageforbiomassresources.Under-
standingphysicochemicalcharacteristicsofthecell-wallpolysaccharide
systemwouldguidegenomicmodicationsofbioenergycropstofacilitate
processingandresistdeleteriousphysicalmodicationasmuchaspossible.
Forexample,structuralelementsofmanylignocellulosicmaterialsreactto
pretreatmentinwaysthatreduceenzymaticdigestibility.
Highmechanicalpressuresuchasthatfromplugfeederscollapsesthe
naturalvascularstructure.
Dilute-acidchemicalpretreatmentsmaypermitcellulosetoreanneal,
leadingtohornicationofcelluloseinmicrobrils.
Ambientorelevatedtemperaturesmayacceleratedenaturation(e.g.,the
tendencyofmost(beta-1,4)-pentosansandhexosanstohaveinverse
water-solubilityrelationshipswithtemperature).
Somepretreatmentsmaypermitlignintobecomesolubleandplate
outoncellulosesurfacesduringthecool-downphase.
Teseprocess-inducedcausesofrecalcitrancemustbeunderstoodand
overcomethroughprocessmodicationorbiomassdesign.
Aftercellulose,hemicelluloseis
OptimizingHemicelluloseAcetylationinCellWalls
thenextmostabundantpolysac-
charideinnativebiomassfeed-
Hemicellulose Acetylation Degradation Products Are Toxic to Microbes
stocks.Structuralinformation
onthesepolymericsubstratesis
A
cetylsidegroupsfromhemicellulosebiomasspolymersarereleased
duringcurrentpretreatmentsteps.Tesesmallacetylmolecules
necessary,andmechanisticmodels
oftenaretoxicandinhibitthemicrobialactivitythatconvertssug-
mustbedevelopedtoidentify
arstoethanol.Hemicellulosiccomponentssuchasxyloglucanandgluc-
bottlenecksinhemicellulose
uronoarabinoxylanandpecticcell-wallcomponentsoftenareO-acetylated.
bioconversion(seesidebar,Opti-
Forinstance,O-acetylgroupsmaybepresentontheglucanbackboneof
mizingHemicelluloseAcetylation
xyloglucanorongalactoseorarabinoseresiduesofsidechains.Tedegree
inCellWalls,thispage).
ofsugar-residueO-acetylationofpectinsvariesfrom0to90%depend-
Asystematicapproachtounder-
ingonthetissue,species,andmethodofpreparation.TeroleofO-acetyl
standingthesefactorswill
substituentsinvivoisnotknown,butinvitroexperimentssuggestthatone
promotemoreeectiveuseof
functionmaybetheirinvolvementinhinderingenzymaticpolysaccharide
lignocellulosicbiomassinbiocon-
breakdown.O-acetylsubstituentsalsoaectpolysaccharidesolubilityand
versionsystems.Fortunately,we
pectinsgelationproperties(PaulyandScheller2000).
nowhavenewbiological,physi-
Plantgenesexhibitweaksequencesimilaritytoputativebacterialacetyl-
cal,analytical,andmathematical
transferasegenes.GenetictoolsinplantssuchasArabidopsiswillenable
toolsthatcanhelpinreliably
theidenticationofgeneproductscatalyzingpolysaccharideacetylation
identifyingandquantifyingthe
andthedeterminationofacetylationsroleincell-wallstructureandfunc-
relativeimportanceofvarious
tion.Suchstudieswillprovideinsightsintothepossibilityofdeveloping
potentiallylimitingfactors.We
biomasscropvarietieswithsignicantlyreducedpolysaccharideacetyla-
alsohavetoolstoidentifyand
tionandthusimprovingthefermentationprocess.
optimizefacilitatingfactors,for
example,throughplantbreeding.
46 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Tegoalistoprovidearationalbasisfordesignofpractical,eective,and
economicalpretreatments,includingcontrollingthephysicalmodication
ofnativecellulosesandrelatedcell-wallpolysaccharidesduringthermal
andchemicaltreatments.Currentthermochemicaltreatmentsultimately
willbereplacedwithmorebenignenzymatictreatmentstothedegree
feasible.Necessarydetailedanalysesarediscussedinthechapter,Decon-
structingFeedstockstoSugars,p.85.
OptimizationofPlantCellWalls
Optimaleciencyofbiofuelproductiondependsonmaximizingfuelyield
fromaunitofbiomassandminimizingenergyinputs.Teplantcellwall,a
complexassemblythatplaysaprimarily,butnotexclusively,structuralrole
duringplantgrowthanddevelopment,maybeparticularlyamenabletothe
applicationofengineeringprinciplesinredesigningthecellwalltomeet
energyneeds.Tobreedplantsinwhichcell-wallcompositionisoptimized
forconversioneciency,understandinghowcellwallsaremade,how
compositionisregulated,andtherolesofvariouspolymersinsupporting
plantgrowthanddevelopmentwillbenecessary.Telong-termgoalisto
developasystems-levelunderstandingtofacilitaterationalimprovement
ofplantcell-wallcompositionindedicatedenergycrops.Suchknowledge
ofplantcellwallsisinaveryprimitivestagebecauseofscienticand
technicalchallengesthathaveimpededscienticprogress.Futureresearch
oncell-wallsynthesisandfunctionrequiresinterdisciplinaryapproaches
rangingfromgenomicstosyntheticcarbohydratechemistryandbiophys-
ics.Modelorganismsareimportantinfacilitatingadvancesinbasicbiology
andinbringingthemostsophisticatedbiologicaltoolstotheproblem.
Severalnewplantmodelscloselyrelatedtospeciesselectedforenergy
cropsareadvocated.ApowerfulrststepistoobtaincomprehensiveDNA
sequencesfortheseorganisms.
UnderstandingCell-WallStructureandFunction
Increasingtheproductionofbiofuelsbeginswithincreasingbiomasspro-
ductivity,eitherbymakingmorecellwallsormakingcellwallswithmore
carbon.Inaddition,changesincell-wallcompositioncouldhavemajor
eectsontheeciencywithwhichbiomasscanbeconvertedtofuels;rela-
tiveamountsofcertainsugarscouldbeincreasedorwallpolymerscouldbe
mademoreamenabletoenzymatichydrolysis,thusimprovingtheyieldof
sugarsdeliveredtothebioreneryasrawfeedstock.
Importantquestionsremainaboutthestructuresofcell-wallpolymers,how
theyaremade, andtheirfunctionsinplantgrowthanddevelopment. To
optimizetheamount,composition,andstructureofwallsforbiofuelproduc-
tion,wemustidentifythegenesinvolvedinsynthesisofcell-wallpolymers,
thedesignprinciplesforcellwalls,andfactorsthatcontroltheamountsand
organizationofvarioustypesofenzymesandresultantpolymers.Prelimi-
naryevidencesuggeststhatcell-wallbiophysicalpropertiesimportantto
plantgrowthanddevelopmentmaybeachievedinmanydierentwayswith
regardtochemicalcomposition. Tus, cell-wallcompositionofenergycrops
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 47

LIGNOCELLULOSIC BIOMASS
probablycanbealteredsotheyarebettersuitedforfuelproduction.Desir-
ableimprovementsincludeincreasingtheamountofsuchusefulpolysac-
charidesascelluloseorcertainhemicellulosesandminimizingthecontentof
suchundesirablecomponentsasligninoracetylgroups.
EvidenceindicatesthatphotosyntheticCO
2
xationisregulatedbyplants
inresponsetodemandforxedcarbon,sounderstandingphotosynthate
uxintocell-wallpolymersrelativetootherpathwaysofprimarycarbon
metabolismandstorageisimportant.Understandingmechanismsthat
regulatecarbonuxandsynthesisofvariouspolysaccharidesmaymake
possiblethedevelopmentofplantsthataccumulatesignicantlymore
polysaccharidepercell.Expectedsignicantincreasesintheratioof
carbontonitrogenandmineralnutrientswouldhaveabenecialeecton
agriculturalinputs(e.g.,planting,fertilizing,cultivating,andharvesting),
costs,andsustainability.
Progressinthisarearequiresbroadapproachestoachieveafoundation
ofknowledgeaboutcell-wallstructureandfunctionthatwillbethebasis
forasystemsapproachtopredictingandcontrollingbiomasscomposi-
tion.Beforeasystemsapproachcanbeimplemented,acomprehensive
understandingisneededaboutwhatreactionsareperformedbythemany
hundredsofenzymesinvolvedincell-wallsynthesisanddeposition,where
andwhenrelevantgenesareexpressed,andwhatgenescontrolexpression
andactivityofproteinsinvolvedinpolysaccharideandligninsynthesis
andmodication.Indeed,onegrandchallengeinsystemsbiologymay
beunderstandinghowtoengineercellwallsthatmeettheneedofchemi-
calbioreneriesforoptimizedfeedstocksyetstillmeettheplantsneedfor
development,robustness,andmaximalratesofgrowth.
GTLcapabilitiescouldprovideextensivesupportforresearchoncell-wall
synthesis,structure,andfunction.Sequencingsupportformodelorganisms
(seebelow)andforidentifyingrelevantgenesinenergycropsisanimme-
diategoal.Tedevelopmentofpopulationsoftransgenicexperimental
plantswithepitope-taggedproteinswouldgreatlyfacilitatethedetermina-
tionofsubcellularproteinlocalizationandtheapplicationofproteomic
techniquestoidentifyproteincomplexes.DNAchips,inconjunctionwith
advancedgenetictechnologies,canbeusedforasystems-levelunderstand-
ingoftranscriptionalcontrolofcell-wallsynthesisandmodicationpath-
ways.EpitopetaggingalsomaybeusedtofacilitatemRNApurication
fromsinglecells,facilitatinginsightsintoprocessesspecictocelltypes.
Ultimately,GTLcapabilitiesinsystemsanalysiswillpermitanintegrated
systemsmodelthatcanbeusedtosupportdirectedmodicationofcell
wallsforspecicapplications.
Eortstounderstandandmodifycellwallsneedtobecoordinatedwith
bioconversionandplantcell-walldeconstructioninitiativestooptimize
feedstockcompositionbasedonpretreatmentandconversionmethods
andeects.Teseobjectivesalsoneedcoordinationtodevelopanalytical
andvisualizationmethods,computationalfacilities,andorganic-chemistry
methodsforproductionofenzymesubstratesandstandardsusedinphe-
notypingandgenecharacterization.
48 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

ControlofLigninSynthesisandStructure
Althoughligninisnotconvertedreadilytoethanol,ligninbiomassmaybe
amenabletochemicalorthermalprocessingtoachievesuchliquidfuelsas
low-gradedieselorfueloil.Oneaspectofoptimizingbiomasscomposi-
tionforethanolproductionisminimizinglignincontent.Alternatively,
developingplantswithmodiedligninthatcanberemovedeasilyduring
biomassprocessingmaybepossible.
Ligninisacomplexaromaticpolymerassociatedwithpolysaccharidesin
secondarycellwalls(seeFig.3,p.44,andFig.4.PhenylpropanoidPathway
LeadingtoLigninBiosynthesisinPlants,thispage).Ligninconstitutes
asignicantbarrierinbiomassconversiontofuelsbyinhibitingenzyme
accesstopolysaccharidesandbyreleasingtoxinsduringdegradationthat
inhibitorganismgrowthduringfermentationofcell-wallhydrolysatesto
ethanol.Geneticstudieshaveindicatedthatligninreductionsmaycause
deleteriouschangesinplantgrowthanddevelopment.However,lignin
possiblymaybereducedwithorwithoutharmfuleectsonplantgrowth
ifcompensatingchangescouldbemadeintheamountofcell-wallpoly-
saccharides.Someearlyexperimentsareunderway.Tedegreetowhich
Fig.4.Phenylpro-
panoidPathway
LeadingtoLignin
Biosynthesisin
Plants.Horizontal
reactionsarering
modications;
verticalreactionsare
side-chainmodi-
cations.[Figure
source:C.Fraser
andC.Chapple,
PurdueUniversity]
4CL,4-(hydroxy)cinnamoylCoAligase;C3'H,p-coumaroylshikimate/quinate3'-hydroxylase;C4H,cinnamate4-hydroxylase;CAD,
cinnamylalcoholdehydrogenase;CCoAOMT,cafeoylCoAO-methyltransferase;CCR,cinnamoylCoAreductase;COMT,cafeicacid/
5-hydroxyferulicacidO-methyltransferase;F5H,ferulate5-hydroxylase;HCALDH,hydroxycinnamaldehydedehydrogenase;HCT,
hydroxycinnamoyl-CoAshikimate/quinatehydroxycinnamoyltransferase;PAL,phenylalanineammonia-lyase.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 49








































LIGNOCELLULOSIC BIOMASS
celluloseamountcanbeincreasedwithorwithoutsimultaneouschangesin
hemicellulosecontentandcompositionmustbeascertained.
Exploringligninbiosynthesisanditsregulationinacomprehensivefash-
ionmaymakepossibletheformulationofmethodsforlimitingandalter-
inglignicationtomaximizebiomass-to-energyconversion.Forinstance,
thegeneforferulate-5-hydroxylasehasbeenusedtoincreasethesyringyl
monomercontentofpoplarlignin(seesidebar,OptimizingLigninCompo-
sition forMore Ecient BioethanolProduction, p. 43). Teresulting trees
hadnormalgrowthanddevelopment,butthepulpingtimewasreduced
bymorethan60%(Huntleyetal.2003).Similarly,opportunitiesexistto
modifylignicationcellspecicitysoitsimpactonenergyconversioncan
beminimized.Agoalistocreatealignicationtoolboxtomanipulate
polymerdepositionsgeneticallyandanalyzetheimpactofthosemanipula-
tionswithadvancedanalyticalorganicchemistry.Suchdetailedknowledge
couldcreatenovelopportunitiesforfundamentallychanginghowbiomass
issynthesizedandsubsequentlyprocessedforbiofuels.Forinstance,novel
monomersmightbeincorporatedtogenerateligninswithunique,useful
chemistriesreadilycleavablelinkagesthatcouldfacilitatelignindepoly-
merizationundermorebenignconditions(i.e.,withenzymes).
ImprovedMethods,Tools,andTechnologies
Newanalyticalmethods,tools,andtechnologieswillacceleratetheunder-
standingofcell-wallsynthesis,makeup,structure,andfunctionandwill
speedbreedingorrationalmodicationofenergycropvarieties.
Atthebasicresearchlevel,newandimprovedmethodsareneededtoana-
lyzewallcompositionandnanoscalestructure.Ideally,thesemethodscould
beappliedtoanalysisofasmallnumberofcells.Moleculesincellwalls
rangefrom2to5angstroms(0.2to0.5nm)indiameter(i.e.,apolysac-
charidechain)andtomanymicronsinlength.Primarycellwallsarefrom
50to100nminthicknessand,insomecells,arethoughttobechemically
dierentiatedfromonesidetoanother.Newimagingmodalitiesthattake
advantageofvariouschemicallyspecicimagingtagswillsupportthelong-
termvisionofinsituimagesoflivingplantcellwalls.Imageswillrevealkey
molecularprocessesoccurringinrealtimeduringthefulllifecycleofcell-
wallformation,maturation,transformation,dehydration,andprocessing
intosimplefeedstocks.Teunderstandingobtainedthroughresearchusing
suchimagingisexpectedtoresultinquantitative,predictivemodelingasa
guidetoformulatingadvancedfeedstocksandtheirsubsequentprocessing.
Asystematicapproachisrequiredtoidentifyplantbiomarkersandspecic
antibodiesorothermoleculartagsusefulinfeedstockimprovement.
Poorlyunderstoodnow,thenestructureofintactwallsmustbestudiedto
determinehowthepartsttogethertocomprisethewholewallsphysical
properties.Someaspectsofthegeneralproblemmayberesolvedsimplyby
encouragingtheapplicationofsuchexistingmethodsasveryhighresolution
electronandscanningprobemicroscopy(seesidebar,ImageAnalysisofPlant
CellSurfacesattheOBPBiomassSurfaceCharacterizationLab,p.40).
50 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Similarly,greateruseofnuclearmagneticresonance(NMR)andmag-
neticresonanceimagingmayallowthedevelopmentof2Dand3Dmaps
ofcell-wallcompositionfromimportantexperimentalandproduction
speciessuchasArabidopsisandpoplar.UseofNMRmaybeexpandedby
isotopiclabelingandfurtherdevelopmentofsolventscapableofdissolv-
ingcell-wallcomponents.Completeannotationof2Dmapscould
facilitategreatlytheanalysisofgeneticvariationincell-wallcomposition
andtheassignmentoffunctiontogenesimplicatedinwallbiosynthesis
andmodication.
Otherapproachesmeritinginvestmentincludeexpandedcollectionsof
antibodiesandaptamerstocell-wallcomponents,theuseofenzyme-
basedpolysaccharidengerprinting,pyrolysisgaschromatographymass
spectrometry(GC-MS),andrelatedmethods.Achallengeisassociated
withcharacterizingenzymesthatsynthesizecell-wallpolysaccharides:
Manyenzymesaddsugarstopreexistingpolysaccharides(i.e.,accep-
torsorprimers)thatarenotreadilyavailableasstandardsandreagents.
Focusedinvestmentsincarbohydratechemistrywillberequiredto
constructsubstratesincludinglabeledsubstratesformeasuringthe
activityofmanywallbiosyntheticenzymes.Expertiseincarbohydrate
syntheticchemistryalsowouldbeaneededcomplementtoproteomic
andmetabolomiccapabilitiesenvisionedinGTLcapabilitysuites.
Expandedcapabilitiesinsyntheticcarbohydratechemistrycouldopen
upnewhigh-throughputmethodsforcharacterizingcarbohydrate-active
enzymesbasedonhigh-densityandhigh-diversityglycochips.Inthis
method,theactivityorbindingofatargetproteincouldbeevaluated
simultaneouslywithhundredsorthousandsofpotentialsubstratesand
verysmallamountsofreagent.
High-throughputmethodsofcell-wallanalysisareneededforplant
breedingandimprovement,allowingtimelyanalysiswiththemost
sophisticatedanalyticaltechniques.Ultimately,ineldcharacterization
isrequiredtosupportbreeding,molecularmarkermapping,andstudies
involvingsuchenvironmentalvariablesasfertilizersandvariousbiotic
andabioticstresses.Methodsmustbeaccurateandrelativelyinexpen-
siveforthelargenumbersofsamplestypicallyhandledduringabreed-
ingprogram.Additionally,theyshouldbeapplicabletoawidevariety
ofmaterials,fromcornstovertowood.Inprinciple,ahigh-throughput
sampleanalysismaybeenabledbydetailedanalysisoftherelationship
betweencell-wallcompositionandfeaturesofFouriertransforminfrared
spectroscopyspectraorpyrolysisGC-MSchromatograms,combined
withcomputationalmethods.
TechnicalMilestones
Within5years
Developrapid,accessibletoolsandmethodsforconsistentbiomass
compositionalanalysisinbulkandfractions(seesection,Characterizing
CellWallsUsingHigh-TroughputMethods,p.108).
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 51

LIGNOCELLULOSIC BIOMASS
Identifygenesforenzymesthatcatalyzesynthesisofmajorpolysaccha-
ridebackbones.
Identifyasubstantialfractionofenzymesthatcatalyzesynthesisof
polysaccharidesidechainsanddeterminesidechainbiologicalfunction
inmodelplantspecies.
Identifyenzymesthatacetylatepolysaccharides,andestablishbiological
functionforsuchmodications.
Identifygeneticregulatoryfactorsthatcontrolligninsynthesisand
deposition.
Within10years
Clarifyregulationofpolysaccharidebiosynthesis,includingkeysteps
thatregulatecarbonowfromphotosynthesisintocell-wallpolymers.
Denemechanismsthatcontrolcelluloseamountandbrillengthand
angle.
Modifycelluloseswithalterednumbersofglycanchainsinsecondary
walls,andproduceandtesttheminmodelspecies.
Makeavailablefortestingbiomasscropplantswithdecreasedlignin
andincreasedamountsofcelluloseorotherpolysaccharides.
Developnewtoolsandmethodstohelpusunderstandcell-wallstruc-
ture,includinghighlyparallelcomputationalsimulationsandhigh-
sensitivity2DNMRandMSinstrumentationforanalysisofligninin
smalltissuesamples.
Identifyallgenesthatcatalyzesynthesisofpolysaccharidesidechains.
Within15years
Determineregulatorygenesthatcontrolamountsofmajorpolysaccha-
rides,includingcellulose.
Developmethodsformanipulatingpolysaccharidecompositionof
anyparticularcelltypewithinaspecictissue.
Makeavailableplantswithimprovedwallcomposition.Teseplantswill
haveincreasedyieldsoffermentablesugars,requiringlesscostlyprepro-
cessing;cell-walldegradationwillresultininsignicantlevelsofinhibi-
torycompounds(inthefermentationprocess).
Developadetailedmodelofligninmonomertransport,polymerization
initiation,andtheinteractionsofligninpolymerswithpolysaccharide
componentsoftheplantcell.
52 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy




































A First Step to Optimizing Feedstocks
for Fuel Production
O
ptimizingplantbiomassformoreecientprocessingrequiresabetterunderstandingof
plantcell-wallstructureandfunction(seenexttwopages).Plantcellwallscontainlong
chainsofsugars(polysaccharides)thatcanbeconvertedtotransportationfuelssuchas
ethanol.Tesaccharicationprocessinvolvesusingenzymestobreakdown(hydrolyze)thepoly-
saccharidesintotheircomponentsugarsforfermentationbymicrobestoethanol(seesidebar,From
BiomasstoCellulosicEthanol,p.26).Signicantchallengesforecientconversionarepresented
byboththelargenumberofenzymesrequiredtohydrolyzediversesugarlinkagesandthephysical
inaccessibilityofthesecompoundstoenzymesbecauseothercell-wallcomponentsarepresent.
Plantcellwallscontainfourdierentpolymertypescellulosemicrobrils,hemicelluloses,
pectins,andlignins.Microbrilsperformanimportantroleinstrengtheningcellwalls,thus
providingsupporttotheoverallplantbody.Somepropertiesoflignin,however,interferewith
enzymaticconversionofpolysaccharidecomponents.Additionally,sinceligninisnotreadily
convertedtoethanol,wemustndotherwaysitcanbeusedifwearetomaximizetheyieldof
energyfrombiomass.
Severalthousandgenesareestimatedtoparticipateincell-wallsynthesis,deposition,andfunc-
tion,butveryfewgeneshavebeenidentiedandverylittleisknownabouttheircorresponding
enzymes.Manyquestionsremain,forexample,regardinghowpolysaccharidesandligninare
synthesized,howwallcompositionisregulated,andhowcompositionrelatestothebiological
functionsofcellwalls.Toanswerthesequestions,weneedtodiscoverthefunctionsofmany
hundredsofenzymes,whereproteinsarelocatedwithincells,whetherornottheyareincom-
plexes,whereandwhenthecorrespondinggenesareexpressed,andwhichgenescontrolthe
expressionandactivitiesofproteinsinvolved.Applicationofneworimprovedbiological,physi-
cal,analytical,andmathematicaltoolswillfacilitateadetailedmechanisticunderstandingof
cellwalls.Tatknowledgewillpermitoptimizationofvariousprocessesinvolvedinproducing
biomassandconvertingittofuels.
Majoropportunitiesexisttoincreaseproductivityandconversion-processecienciesbyalter-
ingfundamentalaspectsofplantgrowth,development,andresponsetobioticandabioticstress.
Alteringcell-wallcompositiontoincreasetherelativeamountofcelluloseandtodecreaselignin,
forexample,couldhavesignicanteects(seesidebar,OptimizingLigninCompositionfor
MoreEcientBioethanolProduction,p.43).Eventualdevelopmentofacomprehensivephysi-
ologicalcell-wallmodelincorporatingbiophysicalaspectswithstructuralpropertiesandknowl-
edgeofproteinsinvolvedwillaidinrationaldevelopmentofhighlyproductivefeedstockspecies
whosecellwallsareoptimizedforconversion.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 53
54 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 55

LIGNOCELLULOSIC BIOMASS
CitedReferences
Huntley,S.K.,etal.2003.SignicantIncreasesinPulpingEciency
inC4HF5HTransformedPoplars:ImprovedChemicalSavingsand
ReducedEnvironmentalToxins,J.Agric.FoodChem.51(21),617883.
Pauly,M.,andH.V.Scheller.2000.O-AcetylationofPlantCellWall
Polysaccharides:IdenticationandPartialCharacterizationofaRhamno-
galacturonanO-Acetyl-TransferasefromPotatoSyspension-Cultured
Cells,Planta210(4),659-67.
56 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

FeedstocksforBiofuels
O
necriticalfoundationfordevelopingbioenergycropsandtheir
processingtechnologiesisamelioratingcell-wallrecalcitranceto
breakdown.Understandingcellwallsisessentialforoptimizing
theirsynthesisandtheprocessesusedtodeconstructthemtosugarsfor
conversiontoethanol(asdiscussedinthepreviouschapter,Lignocellulosic
BiomassCharacteristics,p.39).Aprerequisiteforacompetitivebiofuel
industryisthedevelopmentofcropsthathavebothdesirablecell-wall
traitsandhighbiomassproductivityundersustainablelow-inputcondi-
tions.Majoragriculturalcropsgrowntodayforfood,feed,andberin
theUnitedStateshavenotbeenbredforbiofuels.Tus,manycarefully
selectedtraitsinfoodandfeedcrops,suchasahighratioofseedtostraw
production(harvestindex),aredisadvantageousinbiofuelproduction.A
suiteofnewcropsandnewvarietiesofexistingcropsspecicallybredfor
biofuelsandadaptedtoarangeofdierentsoiltypesandclimaticcondi-
tionsisrequired.
Duringthepastcentury,improvementofagriculturalcropswassupported
byfederalinvestmentinmanyaspectsofbasicplantscience,agronomy,
plantbreeding,pathology,agriculturalengineering,andsoilscience.How-
ever,manytopicsparticularlyimportantinbiofuelproductionhavenot
beenemphasizedandarepoorlydevelopedasaresult.Arecenteditorial
inSciencenoted:
Tere are major technological challenges in realizing these goals.
Geneticimprovementofenergycropssuchasswitchgrass,poplar,and
jatropha has barely begun.It will be important to increase the yield
andenvironmentalrangeofenergycropswhilereducingagricultural
inputs. Plant development, chemical composition, tolerance of biotic
andabioticstresses,andnutrientrequirementsareimportanttraitsto
bemanipulated.Tecombinationofmodernbreedingandtransgenic
techniques should result in achievements greater than those of the
GreenRevolutioninfoodcrops,andinfarlesstime.(Koonin2006)
StevenE.Koonin,ChiefScientist,BP,London
TeDepartmentofEnergy(DOE)mission-orientedresearchprogram
envisionedhereinisdesignedtosupplementcurrentinvestmentinplants,
withthefocusonfacilitatingrapidprogressinformulatingbiomassfeed-
stockcrops,alsoreferredtoasenergycrops.Oneworkshopgoalwasto
identifyspecicareasinwhichafocusedresearchinvestmentwouldspeed
progresstowardanoptimizedfeedstocksupplyforconversiontobiofuels.
Ingeneralterms,thegoaloffeedstockdevelopmentistoobtainmaximumusable
Large and cost-effective
energy production on a scale
that signif icantly impacts
petroleum use calls for new
crops with yield and produc-
tivity not currently avail-
able. (EERE 2003).
References:p.80
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 57

FEEDSTOCKS FOR BIOFUELS

organiccarbonperacreinanenvironmentallyandeconomicallysustainable
way.Manypreviousstudieshaveindicatedthatminimizingsuchinputs
asannualeldpreparationandfertilizationimpliestheuseofsuchperen-
nialsasswitchgrassandpoplar,thusdecreasingcosts(seeFig.1.Switch-
grassBalesfroma5-Year-OldFieldinNortheastSouthDakota,this
page).AjointdocumentofDOEandtheU.S.DepartmentofAgriculture
(USDA)BiomassasaFeedstockforaBioenergyandBioproductsIndus-
try:TeTechnicalFeasibilityofaBillion-TonAnnualSupply(Perlacketal.
2005)calledforperennialcropstoprovideaboutone-thirdofbiomass-
derivedfuelsfortheinitialphaseofbioethanoldevelopment.
Inaddition,becausetransitiontolarge-scalecultivationofdedicated
energycropsmaytakeyearsifnotdecades,researchimperativesmustbe
exploredforoptimizingtheuseofcurrentlyavailableagriculturalcrop
andforestryresidues.Sustainabilitywillbeakeyissueinimplementing
theuseofcropandforestryresiduesforbiofuel,sincetheremovalofcrop
residuescanreduceorganiccarbonandnutrientlevelsinthesoilandaect
soilmicrobialcommunityhealth(seesection,EnsuringSustainabilityand
EnvironmentalQuality,p.68).Moreinformationaboutthecomposition
andpopulationdynamicsofsoilmicrobialcommunitiesisneededtofacili-
tatemodelingoflong-termeectsonsoilfertility.
Currentknowledgeindicatesthatperennialspeciesexpectedtobeused
forbiofuelproductionimprovesoilcarboncontentandmakehighly
ecientuseofmineralnutrients.Developmentofperennialenergycrops
alsomayfacilitateuseofgeneticallydiversemixedstandsratherthan
monoculturesofsinglecultivars.Becauseconventionalcropsaregrown
asmonocultures,relativelylittleresearchhasbeencarriedoutonissues
associatedwithgrowingmixedstands(seesidebar,ABillion-TonAnnual
SupplyofBiomass,p.10,andsidebar,TeArgumentforPerennialBio-
massCrops,p.59).
Fig.1.SwitchgrassBalesfroma5-Year-
OldFieldinNortheastSouthDakota
in2005.Each1200-lb.balerepresents48
gallonsofethanolataconversionrateof
80gallonsperton.Tecultivarusedinthis
eldhasayieldpotentialof5to6tonsper
acre(correspondingto400to500gallons
peracre)becauseitwasbredforuseasa
pasturegrass.Inexperimentalplots,10tons
peracrehavebeenachieved.Processing
goalstarget100gallonspertonofbiomass,
whichwouldincreasepotentialethanol
yieldto1000gallonsperacre.[Source:
K.Vogel,UniversityofNebraska]
58 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

TheArgumentforPerennialBiomassCrops
M
anymajoragriculturalcropstodayareannualplantspropagatedfromseedorcuttingsatthebegin-
ningofeachgrowingseason.Bycontrast,cropsdevelopedandgrownspecicallyforbiofuelpro-
ductionareexpectedtobebasedonperennialspeciesgrownfromrootsorrhizomesthatremainin
thesoilafterharvestingtheabove-groundbiomass.Perennialspeciesareconsideredadvantageousforseveral
reasons(seeFig.2.AttributesofanIdealBiomassCrop,p.61).First,inputcostsarelowerthanforannuals
becausecostsoftillageareeliminatedonceaperennialcropisestablished.Additionally,long-livedrootsof
perennialsmayestablishbenecialinteractionswithrootsymbiontsthatfacilitateacquisitionofmineralnutri-
ents,therebydecreasingtheamountoffertilizerneeded.Someperennialsalsowithdrawasubstantialfraction
ofmineralnutrientsfromabove-groundportionsoftheplantattheendoftheseasonbutbeforeharvest(see
Fig.A.NitrogenUseEciencyTeoryforPerennials,below).
Perennialplantsintemperatezonesalsomayhavesignicantlyhighertotalbiomassyieldperunitofland
areathancomparableannualspecies.Perennialsestablishaphotosyntheticallyactivecanopymorequicklyin
thespringandmaypersistlongerinthefall(seeFig.B.ComparingNetPhotosynthesisofCornandSeveral
PerennialSpecies,below).Tus,theirannualsolar-energyconversioneciencyishigherthanthatofannual
plantswithsimilarcapabilities.
Perennialspecieshavemuchlowerfertilizerrunothandoannuals.Forinstance,comparingthenative
perennialswitchgrasswithcornindicatesthatswitchgrasshasaboutone-eighththenitrogenrunoandone-
hundredththesoilerosion.Perennialgrassesharvestedforbiomassexhibitincreasedsoil-carbonlevelsand
providehabitatforuptovetimes
asmanyspeciesofbirds.Finally,in
contrasttoannualrowcropsthat
typicallyaremonocultures,increas-
inghabitatdiversitybygrow-
ingseveralintermixedspeciesof
perennialsmayprovemorefeasible.
Fig.A.NitrogenUse
EciencyTeory
forPerennials.
[Source:S.Long,
UniversityofIllinois]
Fig.B.ComparingNetPhotosynthesisofCornand
SeveralPerennialSpecies.Annualizednetphotosyn-
thesisisproportionaltotheareaunderthecurve.Tus,
ifmaximalratesofphotosynthesisaresimilar,theperen-
nialcrops(yellow)havemuchhigherannualizednet
photosynthesisthantheannualcrop,corn(Zea mays,
blue).[Source:S.Long,UniversityofIllinois]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 59

FEEDSTOCKS FOR BIOFUELS
Amongmanyfactorsinplantproductivity,severalarethoughttobeof
centralimportanceinbiofuelproduction.Becauseofevidencethatmost
plantsdonotroutinelyachievemaximalphotosyntheticCO xationrates,
2
understandingfactorslimitingtheoverallprocessisimportant.Emphasis
shouldbeplacedondetermininghowplantsallocaterecentlyxedcarbon
toproductssuchasstoragepolysaccharides(e.g.,starch)andstructural
polysaccharidessuchascellulose.Energycropswillbegrownonmarginal,
excess,orsurplusagriculturallands,soidentifyingfactorsthatfacilitate
toleranceandsurvivalduringexposuretodrought,freezing,andotherabi-
oticstresseswillbevital.Issuesforperennialsmaybequitedierentfrom
thoseforannuals,whichhavebeenthesubjectofmostresearchandcrop
experimentation.Teseissuesmaybeparticularlyacuteinregardtopres-
surefrompestsandpathogensthatcanbecontrolledtosomeextentby
croprotationinannualspecies.Duringthepast25years,DOEsocesof
EnergyEciencyandRenewableEnergy(EERE)andBasicEnergySci-
enceshavebeenaprimarysourceofresearchsupportoncentrallyimpor-
tanttopicsindevelopingbioenergyfeedstocks.Researchonkeyissues
identiedhereconstitutesacompellingopportunity.Uniquecapabilities
developedinboththeBiomassandGenomics:GTL(GTL)programs
canbebroughttobearonremainingpracticalandfundamentalproblems
inproducingfeedstocks.Tefollowingsectionsoutlinetheseissuesand
opportunitiesinthecontextoftangiblegoals,timelines,andmilestones.
CreationofaNewGenerationofLignocellulosic
EnergyCrops
Treedistinctgoalsareassociatedwithdevelopmentofbiofuelfeedstocks:
Maximizingthetotalamountofbiomassproducedperacreperyear,
Maintainingsustainabilitywhileminimizinginputs,and
Maximizingtheamountoffuelthatcanbeproducedperunitofbiomass.
Exactvaluesforeachoftheseparameterswillvaryfromonetypeofenergy
cropandonegrowingzonetoanother.Ayieldof20drytonsperacreper
yearmaybeconsideredareasonabletargetinareasofthecountrywith
adequaterainfallandgoodsoils,whereas10drytonsperacreperyearmay
beacceptableindrierorcolderzones.
Tus,theoverallobjectiveofdevelopingfeedstocksmustbefocusedon
broadlyusefulinsightsapplicabletoavarietyofplantspeciesgrown
undervariousgrowingconditionsandexhibitingbenecialattributes
(seeFig.2.AttributesofanIdealBiomassCrop,p.61).Tisisbest
accomplishedbyworkingtowardsystems-levelpredictivemodelsthat
integratedeepknowledgeofunderlyingmechanismsforguidingcultivar
andprocessdevelopment.Tisambitiousgoalisonlynowbeginningto
berealizedbycompaniesthathavebredadvancedcultivarsformajor
agriculturalcommodities.
Tissystems-levelapproachisfeasiblebecauseofthelastdecadesbiology
revolutioningenomicsequencingofhigherplantsandmicrobes.Sequencing
60 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

providesameansforconnecting
knowledgeaboutallorganisms
intoacommonframeworkfor
understandingallformsoflife.
CurrentandfutureDOEinvest-
mentsinplantDNAsequencing
aordtheopportunitytocreate
themechanisticknowledgeof
energycropsneededforcost-
eectiveandpracticalfeedstocks.
Tisenablinginformationmust
beelaboratedfurtherbystrategic
investmentsinunderstanding
aspectsofbasicbiologyspeci-
callyrelevanttoenergycrops(see
sidebar,EnhancingPoplarTraits
forEnergyApplications,p.62).
MaximizingBiomass
Productivity
DomesticationofEnergyCrops
TecontinentalUnitedStatesis
composedofanumberofgrow-
ingzonesoragroecoregionsthat
varywithsuchfactorsasmean
temperature,rainfall,andsoil
quality.Nosingleplantspecies
isoptimalforallzones,sousing
dierentspeciesasenergycrops
willbenecessary.PreviousDOE
studieshaveidentiedanumber
ofpromisingplantspecies,and
academicstudieshavesuggested
additionalones(seeFig.3.Geo-
graphicDistributionofBiomass
Crops,thispage).Ingeneral,
energycropscanbedividedinto
twotypes:Tose,suchasmaize,
whichareusedforagricultural
foodandfeedproductionbut
producesubstantialamountsof
usablebiomassasaby-product
(TypeI),andthoseusedonlyfor
energy(TypeII).TypeIplants
arehighlydevelopedfrommany
decadesofresearchandstudyfor
anotherpurpose(foodorber).
Fig.2.AttributesofanIdealBiomassCrop.[TableadaptedfromS.Long,
UniversityofIllinois]
Fig.3.GeographicDistributionofBiomassCrops.Multipletypesdesignedfor
variousagroecosystemsprobablywillberequiredtoobtainenoughbiomassfor
large-scaleproductionofliquidfuels.[Source:AdaptedfromORNLBiomass
Program]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 61

FEEDSTOCKS FOR BIOFUELS

EnhancingPoplarTraitsforEnergyApplications
G
ainingabetterunderstandingofgenesandregulatorymechanismsthatcontrolgrowth,carbonalloca-
tion,andotherrelevanttraitsinthepoplartree(Populustrichocarpa)mayleadtoitsuseasamajor
biomassfeedstockforconversiontobioethanol.AninternationalteamledbytheDOEJointGenome
Instituterecentlycompletedsequencingthepoplargenome,makingitthersttree(andfourthplant,afterthe
mustardweedArabidopsisthaliana,rice,andthealgaChlamydomonas)tohaveitscompletegenomesequenced
(Tuskanetal.,inpress).Tesedatanowoerthemolecularaccessneededtodene,quantify,andunder-
standatamechanisticlevelbasicbiologicalprocessesthatimpactimportanttraits.
Poplarwaschosenforsequencingbecauseofitsrelativelycompactgenome(500millionbases),only2%that
ofpine.Moreover,manyspeciesareavailableworldwide,andtheirrapidgrowthallowsmeaningfulmeasures
ofimportanttraitswithinafewyears.Extensivegeneticmapsalreadyavailableincludeinitialidenticationof
markersassociatedwithsuchtraits.
EarlycomparativesequenceanalysesofpoplarandArabidopsisgenomesareprovidinginsightsintogenome
structureandgene-familyevolution;biosyntheticprocessessuchascell-wallformation,diseaseresistance,and
adaptationtostress;andsecondarymetabolicpathways.Comparisonsofgene-familysizesshowsubstantial
expansionofpoplargenesinvolvedincarbontocelluloseandligninbiosynthesis.
Movingfromadescriptivetopredictiveunderstandingofpoplargrowth,development,andcomplexfunc-
tionwillrequireintegrationofsequenceinformationwithfunctionaldata.Tesedatawillbegeneratedby
suchnewtoolsandapproachesasgeneandproteomeexpressionstudies,metabolicproling,high-throughput
phenotypingandcompositionalanalysis,andmodelingandsimulation.Ultimately,thisinformationwilllead
totheengineeringoffaster-growingtreesthatproducemorereadilyconvertiblebiomass(seebelow,FigA.
VisionfortheFuture).TeInternationalPopulusGenomeConsortiumhasproducedascienceplantoguide
postsequencingactivities
(TePopulusGenome
SciencePlan20042009:
FromDraftSequenceto
aCatalogueofAllGenes
TroughtheAdvancement
ofGenomicsTools,www.
ornl.gov/ipgc).
Otherareastobe
addressedforpoplarand
otherpotentialbioen-
ergycropsincludesus-
tainabilityforharvesting
biomass,harvesting
technologiestoremove
biomassatlowcosts,and
infrastructuretechnolo-
giesthatallowbiomass
tobetransportedfrom
harvestlocationstocon-
versionfacilities.
Fig.A.VisionfortheFuture:DesiredTraitsoftheDomesticatedEnergyPoplar.
62 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy
















































Teywerebredasmonoculturesunderintensiveagriculture.Asnoted
elsewhereinthisdocument,researchprioritiesregardingproductionof
energyfromthesecropsareconcernedlargelywiththreeissues. Tese
issuesaresustainabilityfollowingremovalofpreviouslyunderutilized
straworstover,processingtheresidues,andthepossibilityofmaking
improvementsincell-wallcompositionforenhancedconversiontofuels.
Bycontrast,TypeIIplantsarerelativelypoorlydevelopedasagronomic
cropsandgenerallyareperennials.Perennialherbaceousandwoodyplants
haveseveralpropertiesthatmakethembettersuitedforbiofuelproduc-
tionthanareannualcrops(seesidebar,TeArgumentforPerennialBio-
massCrops,p.59).First,becausetheytypicallyretainasignicanttissue
massbelowground,theyrapidlyformacanopyinthespringandaccumu-
latebiomasswhenmanyannualsarestillseedlings.Tus,theymayexhibit
higherratesofnetphotosyntheticCO
2
xationintosugarswhenmea-
suredannually,resultinginhigheramountsoftotalbiomassaccumulation
peracreperyear.Second,perennialsrequirelittleifanytillage,saving
energyandlaborandsignicantlyreducingsoilerosionandnutrientloss.
PerennialssuchasswitchgrassandMiscanthuscanbeharvestedannually
withoutreplanting.Tird,perennialstypicallywithdrawmineralnutrients
intorootsattheendofagrowingseason,therebyreducingfertilizercosts.
Perennialherbaceousandwoodyplantsrepresentacriticalcomponentof
ourbioenergyfuture.
Advancesinconventionalbreeding,coupledwithmoleculartoolsandhigh-
throughputtransformationsystems(Busovetal.2005)willberequiredto
acceleratedomesticationofspecieshavingpromiseasenergycrops.Asso-
ciatinggenotypeswithphenotypeswillrequirehigh-throughputtechnolo-
giesforgenotyping(i.e.,identifyinggenesandalleles)andphenotyping
awidearrayofrelevanttraits(e.g.,biomassyield,cell-wallcomposition)
withmoleculartechniquessuchasgeneexpression(transcriptomics),
proteomics, andmetabolomics.Availabilityofwhole-genomesequences
anddeepexpressedsequencetag(EST)librariesofallpotentialenergy
cropswillbenecessarytoprovidegeneticreagentsformarker-aided
selection,associationgenetics,andtransformationstudies(seesidebar,
Marker-AssistedBreeding,p.64).Associationgeneticsidentiesallelic
variants[e.g.,singlenucleotidepolymorphisms(SNPs)]withempirically
determinedphenotypiceectsontraitsofinterestandallowsselection
forfavorableallelesusingmolecularmarkers(Rafalski2002;Nealeand
Savolainen2004;Remingtonetal.2001).Tisformofmarker-assisted
selectionisdirect(asopposedtoindirectonquantitativetraitloci)and
allowssimultaneousselectionofmanygenes.Teresultisarational,
marker-aidedbreedingandselectionapproachwiththeexpectationof
signicantlyenhancedgeneticgainandanaccelerateddevelopmentpro-
cess(Brownetal.2003).
FormostTypeIIbiomasscrops,signicantlyenhancedbreeding,testing,
andselectionpopulationswillbenecessary,alongwithappropriateinfra-
structuretobreedfordesiredtraitsandadaptabilityacrossawidearrayof
environmentsinmultiplephysiographicregions.Inadditiontotargeted
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 63

FEEDSTOCKS FOR BIOFUELS
Marker-AssistedBreeding
O
neofthewaysinwhichDNAsequenc-
ingofbiomassspeciesimpactsbiofuel
feedstockdevelopmentisbyenabling
acceleratedbreedingmethodstobeapplied
toplantsusedforbiomassproduction.Te
goalinplantbreedingistorstidentifyuse-
fulgeneticvariationfortraitsofinterest(e.g.,
diseaseresistanceanddroughttolerance)by
screeningnaturalormutagenizedpopulationsof
individuals.Manysuchtraitsarecontrolledby
multiplegenes.Individualswithusefulvariations
areintercrossedtoproduceprogenywithnew
combinationsoftheusefulvariation.Usually,
manytraitsareofinterestandmanysourcesof
variationareused,makingtheoverallbreed-
ingprocessverytimeconsumingandexpensive.
Teavailabilityoflargeamountsofsequence
informationfacilitatesidenticationofDNA
polymorphismssmalldierencesintheDNA
sequencesofindividualswithinaspecies.Having
completegenomicsequencesalsomakespossible
theidenticationofgeneslocatednearpolymor-
phismsonchromosomes.
Tisknowledgehaspracticalapplicationsin
marker-assistedbreeding,amethodinwhich
aDNApolymorphism,closelylinkedtoagene
encodingatraitofinterest,isusedtotrackthe
traitamongprogenyofsexualcrossesbetween
plantlines.Temethodallowsbreedersto
monitorplantsforatraitthatmaybeexpressed
onlyincertaintissuesordevelopmentalstages
ormaybeobscuredbyenvironmentalvaria-
tion.Similarly,bycorrelatingtraitsandDNA
polymorphismsinindividualsfromgenetically
diversenaturalpopulations,associatingaparticu-
larchromosomalregionwithatraitofinter-
estfrequentlyispossible.Ifalargenumberof
polymorphismsareavailable,theamountoftime
requiredtobreedanimprovedplantcultivaris
greatlyreduced.
Inprinciple,plantswithoptimalcombinations
ofparentalgenesmaybeidentiablewithinthe
rstseveralgenerationsfollowingasexualcross
ratherthaneightgenerationsormorefollowing
conventionalbreeding.Inthecaseofspeciessuch
astrees,marker-assistedbreedingcouldelimi-
natemanydecadesofexpensivestepstodevelop
morehighlyproductiveplants.
breeding,manycropswillrequirefertilitycontrolinthe
eld,eithertoensureparentageorpreventgeneow
towildpopulations.Aholisticapproachforimproving
feedstocksincludesmolecularresources,high-throughput
screeningtools,andwell-characterizedbreedingpopula-
tions.GTLresourcesandtechnologiesarewellsuitedto
creatingcomprehensivesetsofmolecularmarkers(i.e.,
SNPsandsinglesequencerepeats)andhigh-through-
put,low-costphenotypingtools.Teseecientdeliver-
ableswillbeusedtodevelopappropriatecultivarsand
varietiestomeettargetgoals.GTLgenomicandother
resourceswillplaycriticallyimportantrolesviaimple-
mentationofdeepESTsequencing,markeridentica-
tion,high-throughputgenotyping,anddevelopmentand
applicationofanalyticaltools.Tesetoolswillbeused
forhigh-throughputmolecularphenotypingofbiomass
compositionandplantstructureandahigh-throughput
transformationprocessformajorbiomassenergyspe-
cies.Suchcapabilitysuitesalsowillprovidecentralized
bioinformaticssupportforanalysisandarchivingof
genomedata.
Bydenitioncultivardevelopment,eldandplantation
establishmentandgrowth,andbiomassconversionto
biofuelsnecessitateaholistic,systemsbiologyapproach.
Integratingthesmallersubcomponentswillbechal-
lenging,requiringacoordinatedandfocusedprogramto
facilitateexchangeofinformationandgeneticmaterials
acrossorganizationsandinstitutions.
EnhancingtheYieldofBiomassCrops
Teyieldofbiomasscropscanbedenedastheamount
ofxedcarbonperacreperyear.Achievingthemaximal
yieldofadedicatedenergycrop(TypeII)isasigni-
cantlydierentgoalfrommaximizingtheyieldofmost
existingcropspecies(TypeI),whereonlythenumberof
reproductiveorstorageorgansisconsidered.Teyield
ofaTypeIIspeciesisafunctionofthetotalnumber
ofcellsperacremultipliedbythemeanamountof
accumulatedcarbonpercell.Tus,biomassyieldcanbe
enhancedbyincreasingthenumberofcellsperacreper
year,theamountofcarbonpercell,orboth.Achieving
eithertypeofenhancementisacomplexsystemsprob-
lem.Atthecoreoftheproblem,however,istheneed
tomaximizephotosyntheticCO xationtosupport
2
carbonaccumulation.Additionally,xedcarbonmustbe
directedintoeithercell-wallpolymersorstoragecarbo-
hydratesorusedtosupportextracelldivision.Cell-wall
64 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

polymersincludecellulose,hemicellulose,andlignin;storagecarbohy-
dratesincludesugarsandstarches.
Plantsareregulatedtoxonlythecarbonneededfornormalgrowthand
development.Tisgenerallyisreferredtoassource-sinkregulation,a
poorlyunderstoodphenomenon.Plantscanxconsiderablymorecarbon,
however;theactualphotosyntheticCO
2
xationrateofmostorallplants
issignicantlybelow(i.e.,~50%)therateobservedfollowingexperimental
partialdefoliation.Tatis,plantsappeartoaccumulatemorecarbonper
unitofleafareafollowingdefoliationthantheynormallywouldwithout
anychangesinarchitectureorphotosyntheticelectrontransport.When
mechanismsunderlyingthisregulationareunderstood,plantscanbe
developedthatexhibitsignicantlyhigherratesofnetphotosyntheticCO
2
xationandhigheramountsoftotalcarbonaccumulationperacreperyear.
Terefore,ahigh-priorityresearchgoalistounderstandmechanismsthat
regulatenetphotosyntheticCO
2
xation.Acloselyrelatedpriorityisto
identifyfactorsthatlimitcarbonuxintocell-wallpolysaccharidesand
storagepolymers.
Acomplementaryapproachistoidentifyfactorsthatregulateplantgrowth
rateandduration.Dierentplantspeciesvarywidelyingrowthrates,sug-
gestingthatgrowthratesareundergeneticcontroland,therefore,subject
tomodication.Recently,severalgeneshavebeenidentiedinfunctional
genomicsscreensthatcausesignicantincreasesingrowthratesofdif-
ferenttypesofplants.Identifyingothergenesthatcontrolgrowthand
developmentandunderstandinggeneactionmaycreatenewopportunities
todevelophighlyproductiveenergycrops(seeFig.4.GrowthRateModi-
cation,thispage).
EnhancingAbioticStressToleranceofBiomassSpecies
Wateravailabilityisamajorlimitationtoplantproductivityworldwide,
generallyintwoways.First,becausewaterescapesfromplantleaves
throughstomatawhenCO
2
enters,acertainamountofwaterisrequired
tosupportaunitofphotosyntheticCO xation.PlantswithC4pho-
2
tosynthesis(e.g.,corn,sugarcane,switchgrass,andMiscanthus)typically
requirelesswaterperunitofCO
2
xedthandoC3species(e.g.,wheatand
soybean)becauseC4
plantscanachieve
highratesofCO
2
xationwithpar-
tiallyclosedstomata.
Otherplantssuch
ascacticlosetheir
stomataduringthe
daytoreducewater
lossbutopenthem
atnighttotakein
CO
2
forphotosyn-
thesisthenextday.
Tisphenomenon
Fig.4.GrowthRate
Modication.Te
Arabidopsis planton
therighthasbeen
modiedbyalter-
ingtheexpression
ofregulatorygenes
controllinggrowth.
[Source:Mendel
Biotechnology]
Unmodifed
Modifed
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 65

FEEDSTOCKS FOR BIOFUELS
alsoinhibitscarbonlossbyphotorespiration.Exceptforthepossibilityof
enhancingtheseadaptations,currenttheoryimpliesnootheroptionsfor
signicantlyreducingaplantswaterrequirementtoobtainmaximalyields.
Tewaterproblemssecondcomponent,however,concernstheeectsof
temporalvariationinsoil-watercontent.Inrain-fedagriculture,periods
oflowsoil-watercontentarefrequentbecauseofirregularitiesinrainfall.
Teabilityofplantstosurviveextendedperiodsoflowsoilwatercanbe
acriticalfactorinacropsoverallyield(seeFig.5.CornYieldonaMis-
souriExperimentStation,thispage).Furthermore,dierentplantsexhibit
widelydierentabilitiestosurviveextendedperiodsofdrought,indicating
thatdrought-tolerantenergycropsmaybepossible(seeFig.6.Modica-
tioninDrought-StressTolerance,thispage).
Currently,themostproductivefarmlandisusedforfoodproduction,with
anamountheldinreserve[ConservationReserveEnhancementProgram
(CREP)lands].Whileinitialenergycropswouldbegrowninhighly
productiveCREPland,asubstantialproportionofbiomasscropswillbe
grownonmarginallandthatissuboptimalinwateravailability,soilquality,
orboth.Recentprogressinunderstandingthemechanisticbasesofplant
drought,salt,andcoldtolerancehasraisedthepossibilityofmodifying
plantstoenhanceproductivityundertheseandotherstressconditions.A
priorityindedicatedenergycropsistounderstandmechanismsbywhich
plantssurvivedroughtandadaptthisknowledgetoimprovingenergycrops.
Fig.5.CornYieldonaMissouriExperimentStation.
Tisgureillustrateswidedierencesinyieldobserved
invariousannualgrowingconditions,including
periodsofdrought.Testrongupwardtrendinyield
reectsadvancesinbreedingandagronomicalpractices
forcorn.[FigureadaptedfromQ.HuandG.Buy-
anovsky,ClimateEectsonCornYieldinMissouri,
J. Appl. Meteorol. 42(11),162335(2003).]
Fig6.Modicationin
Drought-StressTolerance.
Teplantsontheleftofeach
photographareunmodied,
andtheplantsontheright
havebeenalteredgenetically
fordrought-stresstolerance.
Allplantsweresubjectedto
aseveredrought.[Photos
2003MonsantoCompany.
Usedwithpermission.]
66 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

UnderstandingandEliminatingUndesirableBiomassCropCharacteristics
Researchshouldbeconductedtounderstandandeliminatesuchundesir-
ableplanttraitsasinappropriateresiduesandinvasivenessofnon-native
energy-cropspecies.Twoexamplesaredescribedhere.
Relativelylargeamountsofsilicainplantssuchasriceleadtoaccumula-
tionsofashwhentheplantbiomassisburnedforenergy.Whenthebio-
massisconvertedtoliquidfuels,unusablesolidsareproduced.Sincemany
plantsdonotaccumulatesilica,thetraitappearstobedispensable;devel-
opingcultivarswithreducedsilicaaccumulationbygeneticmethodsmay
bepossible.Terolesofsilicainplantgrowthanddevelopment,however,
arepoorlyunderstoodandneedfurtherinvestigation.
Manyfeaturesconsideredidealforabiomasscrop(seeFig.2,p.61)are
characteristicofinvasiveweeds,particularlyperennialC4grasses.Tus,
akeyconsiderationinadaptingthesegrassesforuseasdedicatedenergy
cropsistoensurethatthespeciescanbecontainedandwillnotbecome
aproblem.Somehighlyproductiveperennialgrasses,suchasMiscanthus
giganteus,havebeenstudiedintensivelyinEuropeformorethanadecade
andarethoughtnottoexhibitinvasivecharacteristics.Allcandidateenergy
crops,however,shouldbestudieddirectlyforpotentialinvasivenessat
diverselocationswithintheUnitedStates.Tesestudiesalsocanprovide
insightsintopestsandpathogensthatmightposeathreattoproductivity.
TechnicalMilestones
Within5years
Facilitatetheinitiationandadvancementofbiomassbreedingprograms
inkeyU.S.geographicregionsincooperationwithUSDA,privatecom-
panies,anduniversities.
Developappropriatetestpopulationsforconductingassociationgenet-
icsandquantitativetraitlocusidentication(QTL,directandindirect
marker-assistedselection).
Identifyandtargetforselectionandimprovementkeytraitsthataect
biomassyieldandconversioneciency.
Insupportofmarkerdevelopmentforgenediscovery,carryouttargeted
sequencing(SNPs,SSRs,ESTs,andcDNAs)forpotentialbiomass
specieshavinglargeandcomplexgenomes;sequencewholegenomesfor
specieswithmodestgenomesizes.
Developmarkersthatcandierentiatesuperiorparentsandospring.
Initiateandvalidatemethodsforhigh-throughputscreeningforspecic
traitssuchasimprovedcell-walldigestibility.
Formulatetheinfrastructureforfunctionalscreeningofthousandsof
potentiallyusefulgenesinselectedspecies.
Within10years
Applyhigh-throughputphenotypingtoolstointegratedconventional
andmolecularbreedingprograms.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 67

FEEDSTOCKS FOR BIOFUELS
Gainnewunderstandingofgenomestructureandgeneexpressioninbio-
energycrops,includingthegeneticbasisofheterosis(hybridvigor).Major
barrierstofaciletransformationofselectgenotypeswillbeovercome.
Within15years
Identifyandintegratemajornewdomesticationgenesintoenergycrops.
Integrateenablingtechnologieswithbreedingprogramstoallowfor
deploymentofgeneticallysuperiormaterialsoverlargelandbasesdedi-
catedtobiomassproduction. Timetocommercialdeploymentwillbe
shorteneddramaticallythroughhighlyreliablescreeningmethods,genetic
controlofoweringandsexualreproduction,andeectiveearly-selection
modelsforpredictingperformanceandyield.Geneticallyenhancedcul-
tivars,hybrids,andvarietiescapableofmeetingtargetedgoalsforethanol
yieldperacreperyearwillbeavailableformajorbiomasscropspecies.
EnsuringSustainabilityandEnvironmentalQuality
Toensuretheviabilityofbioethanoltomeetthelargenationalneedfor
transportationfuels,wemustunderstandtheeectsoflong-termbiomass
harvestingonsoilfertilityandotheraspectsofsustainability.Objectives
forthisworkaretodeterminehowtomaintainsoilecosystemfunction
andproductivity.Afurtherobjectiveistodevelopmanagementpractices
thatcanoptimizesustainability.Becauseverylongperiodsoftimemaybe
requiredfordirectlytestingtheeectsofbiomassremovalonsoilquality,
amechanisticunderstandingofthisissueisessentialtoobtainingpredic-
tivemodelsandmonitoringprocedures.Tecriticalquestioninusingcrop
residuesanddedicatedcropsforbiomassenergyis,Howmuch,ifany,of
above-groundbiomassneedstobeleftonaeldtoprotectsoilfromero-
sionandsustainsoilfunction(soilquality)?
Inthepast30years,cropandsoilresearchhasemphasizedusingcrop
residuesinminimumandno-tillfarmingoperationstoimprovesoilorganic
carbon(SOC)andsoilquality.Researchhasdemonstratedthatcornstover
removalinsomeproductionsystemscanreducegrainyield(Wilhelmetal.
2004). Teamountofstoverremovalandloweryieldwereassociatedwith
theamountofSOC(Maskinaetal.1993).Atanytime,SOCcontentisthe
balancebetweentheratesofinputanddecomposition(Albrecht1938).Ifall
otherculturalpracticesareunchanged,removalofcropresiduewillfurther
decreasecarboninputsandSOCwilldecline(Follett2001)(seeFig.7.Soil
CarbonAlterationswithManagementChanges,p.69).LossofSOC
typicallyhasdetrimentaleectsonsoilproductivityandquality,presum-
ablybecausemicrobialcommunitiesareimpactednegatively.Forexample,
considerablecarbonistranslocatedtomycorrhizalfunguscommunitiesthat
playasignicantroleinsoilnutrientcycling(Fitteretal.2005).
Tisresearchmustbecompletedforeachmajoragroecosysteminthe
UnitedStateswherecropresiduesanddedicatedcropsareafeasiblesupply
sourceforbiomassenergy.Sustainabilityanalysisofbothdedicatedenergy
cropsandofstoverorstrawremovalfromagriculturalcropsshouldbecar-
riedoutsincetheeectsmaybesignicantlydierent.
68 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Soilsarecomplexecosystemscomposedofunknownnumbersoforgan-
isms.Teeectsofcrop-residuequantityandcompositiononsoilecosys-
temsarelargelyunknown.Soilmicrobiologistsneedaccesstolarge-scale
genomicsandotheranalyticalfacilitiestoconductsoilmicrobialresearch.
Systemsbiologyanalysesofhighlyproductivesoils,includingtheirecosys-
tem-scalegenomiccharacterization,areaccessiblewithcurrentsequencing
capabilities.Questionsincludethefollowing.
Whatcharacteristicsandfunctionsofsoilmicrobialcommunitiesare
neededtomaintainsoil-ecosystemfunctionandproductivity?
Howdosoilmicrobialcommunitiesfunction?
Whataretheinteractions,positiveandnegative,amongmicrobes,fungi,
androotsintherhizosphere?
Howdonutrientlevelsaectmicrobialcommunities,andhowdo
microbialcommunitiesaectnutrientavailability?
Howcanmanagementofmicrobialcommunitiesimproveproductivity?
Howmuchcarbonfromcropresiduesanddedicatedenergycropsis
neededtomaintainsoilecosystemfunctionandproductivity?
Doesthecompositionofresiduehaveaneect?
Whatishappeningbothphysicallyandbiologicallyinsoilsasvari-
ouslevelsofplantbiomassareremoved?
Canmanagementfactors,notillage,modiedminimumtillage,orspe-
cialpracticesaecttheamountofneededresidue?
Howdoessoilcompositionvarybetweenannualandperennialcropsin
responsetovaryinglevelsofbiomassremoval?
Whatarethemicrobial-communitycharacteristicsofmarginaland
severelydepletedlands,andhowcantheyberestoredtosupportenergy
crops?
Canenergycropsbeusedto
restorelandsforfoodandber
cropsbybuildingupcarbonand
nutrientsinmarginallands?
Finally,howmightthesepro-
cessesbeutilizedforreduction
ingreenhousegasemissionsor
carbonsequestration?
GTLfacilitycapabilitiescanbe
usedinmanywaystoanswerthese
generalquestions.Inparticular,
GTLfacilitiescouldsupporteco-
system-analysisstudies,including
sequencingofmicrobialcommuni-
tiesandgenomicsapproachesfor
analysisofmicrobial-community
functioning.GTLcouldsupport
Fig.7.SoilCarbonAlterationswithManagementChanges.Cultivation
generallyleadstoreductionofsoilorganiccarbon,which,withoutosetting
practices,isexacerbatedbycorn-stoverremoval.Amechanisticunderstanding
oflong-termharvestingeectsonsoilfertilityandotheraspectsofsustain-
abilityisessentialforpredictivemodelsandmonitoringprocedures.[Source:
W.W.Wilhelm,USDAAgriculturalResearchService,Lincoln,Nebraska.]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 69

FEEDSTOCKS FOR BIOFUELS
technologiestoevaluatehowmicrobialcommunitiesfunctionby,forexam-
ple,developinggenechipswithmillionsofdiagnosticprobes.
Determinationofmicrobial-communityphysiologymustbelinkedwith
analysisofsoilphysicochemicalstatesfacilitatinganalysisofsoilemis-
sionsorcarbonsequestrationinsoils.Improvedmethodsofsoilcarbon
analysesneedtobedevelopedandmadeavailableinhigh-throughput
format.Currentmethodsareextremelylaborious.Improvinganalytical
methodsforanalyzingcompositionofplantcellwalls,describedbelow,
alsomayfacilitateanalysisofsoilcarboncomposition.
TechnicalMilestones
Within5years
Determinetheeectsofcorn-stoverandothercrop-residueremovalon
soilproductivityineachmajoragroecosystemoftheUnitedStates.
Useexistingstudysiteswherestoverhasbeenremovedfor5ormore
yearstoinitiatecomprehensiveanalysisofmicrobialcommunities(and
soilcarbonandmineralcontent)inunderlyingsoils,incomparison
withlandwherestoverhasnotbeenremoved.
Establishlong-termstudysitesforfuturesustainabilitystudiesonpro-
spectiveenergycropsinlandsinallmajoragroecosystems.
Pursuelong-termcontractswithprivateorpublicproviders.Te
EuropeanMiscanthusProductivityNetwork,supportedbytheEuro-
peanCommission,providesausefulmodel.SequenceanalysisofDNA
extractedfromsoilsshouldbecarriedouttosurveydiversity(Handels-
manetal.1998).Sequenceanalysisshouldbeusedtodevelopdiagnos-
ticmethodsforexaminingthedynamicsofsoilmicrobialcomposition
andabundance.
Within10years
Conductadditionalstudieswithvaryingharvestratesandmanagement
practices,includingcroppingsystemsandanalysesofmicrobialcommu-
nitiesandofcarbonlevelsinthesoils.
Performcomprehensiveecologicalsystemanalysesofsoilsasaliving
medium.
Includenotonlymicroorganismspresentbutalsohowtheircomplex
interactionscontributetotheirnetfunctionasacommunity.Markers
ofoptimallyfunctioningmicrobialcommunitiesshouldbedeveloped
toenableidenticationofsustainabilityrequirementsforsoilsineach
majoragroecosystem.Inadditiontoitsvaluetotheenergy-production
functionofcroplands,thisresearchshouldenhancethesustainabilityof
foodproduction.
Within15years
Formulatemanagementguidelinesontheamountofbiomassthatcanbe
removedineachmajorU.S.agroecosystem.
70 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

ModelSystemsforEnergyCrops
Applicationofmodelsystemstowardthestudyofbothbasicandapplied
problemsinplantbiologyhasbecomeroutineandcanquicklybringthe
techniquesof21
st
Centurysystemsbiologytobearonthecomplexprob-
lemsassociatedwithdomesticatingenergycrops(seesidebar,Translational
Research: TePathfromDiscoverytoApplications, p.72).Researchers
usingthemodeldicotArabidopsisthalianahavemadetremendousstrides
inunderstandingareasofplantbiologyrangingfromnutrientuptakeand
metabolismtoplant-pathogeninteractions.Unfortunately,asanannual
dicot,Arabidopsisisnotanoptimalmodeltostudyquestionsuniqueto
potentialwoodyandgrassyperennialenergycrops(e.g.,woodformation
intreesorcell-wallcompositioningrasses).Despiteitssequencedgenome
andgeneticresources,ricealsoisnotanidealmodelforgrassyperennial
energycropsbecauseitisaspecializedsemiaquatictropicalgrass.Inaddi-
tion,itslargesize,longgenerationtime,anddemandinggrowthrequire-
mentsmakeexperimentsexpensive.UsingBrachypodiumandPopulusas
modelsystemswouldprovideresearchersworkingtodomesticateenergy
cropswithsomeofthemostpowerfultoolsdevelopedbythehighlysuc-
cessfulArabidopsiscommunity.Tesemodelsystemswouldhelpidentify
genescontrollingtraitsrelevanttoenergy-cropproductivityandquality,
includingsuchglobalprocessesascell-wallbiosynthesis,nutrientuptake,
carbonux,andplantarchitecture.Temodelscanbeusedforrapidtest-
ingofstrategiestoimprovetheusefulnessofgrassesandtreesasenergy
crops.Suchtoolswouldallowscientiststousebothforwardandreverse
geneticapproachesandmodernmoleculargeneticmethodstoidentify
genescontrollingtraitsrelevanttothedesignofsuperiorenergycrops.Tis
isimportantbecauseoftheproblems(i.e.,diculttransformation,large
size,longgenerationtime,andself-incompatibility)associatedwithwork-
ingdirectlywithenergycrops.
Brachypodiumdistachyonisasmalltemperategrasswithallattributes
neededtobeamodernmodelorganism,includingsimplegrowthrequire-
ments,fastgenerationtime,smallstature,smallgenomesize,andself-
fertility(Draperetal.2001).Brachypodiumalsoistransformedreadilyby
Agrobacterium(Vogeletal.2006)orbiolistics(Christiansenetal.2005),
thusfacilitatingmanybiotechnologicalapplications.Brachypodiumis
nowscheduledtobesequencedbytheDOEJointGenomeInstitute.
TePopulusgenomehasbeensequenced(Brunner,Busov,andStrauss
2004;Tuskan,DiFazio,andTeichmann2004),assembled,andannotated
throughinvestmentsfromDOE, NSF, andalargeinternationalcon-
sortium(Tuskanetal.,inpress).Tisresourceisavailabletoresearch-
ersworkingonadedicatedwoodycropforbiofuelsandbiomassenergy
applications.Despitetheseadvances,manymoleculartoolsandresources
currentlynotavailablewouldgreatlyenhancethediscoveryanduseof
genesandgenefamiliesrelatedtoenergytraitsandthedevelopmentof
poplarasanenergycrop(seesidebar,EnhancingPoplarTraitsforEnergy
Applications,p.62).Aswithothermodelorganisms,researchonpoplar
wouldbefacilitatedbyorganism-specictoolssuchasfull-genomeDNA
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 71

FEEDSTOCKS FOR BIOFUELS

TranslationalResearch:ThePathfromDiscoverytoApplications
M
odelorganismsplayanimportantroleinbothbasicandappliedresearch.Temosthighlydevel-
opedplantmodelisArabidopsisthaliana,asmallplantinthemustardfamily(Fig.4.GrowthRate
Modication,p.65).Itwasadoptedasamodelintheearly1980sbecauseofitsrapidlifecycle,
simplegenetics,smallgenome(125Mb),easytransformability,andtraitstypicalofoweringplantsinmost
respects.Genomesequencingwascompletedin2000byaninternationalconsortium,andabout13,000
researchersworldwidecurrentlyusetheplantforstudiesofplantbiology.
TeArabidopsiscommunityhasdevelopedasuiteofverypowerfulexperimentalresourcesthatincludeseveral
hundredthousandsequencedinsertionmutationsavailablethroughstockcenters,afull-genomeDNAchip
thatcanbeusedtomeasuretheexpressionofmostgenes,ahigh-densitypolymorphismmap,andaheav-
ilycurateddatabase(arabidopsis.org)thatprovidesaccesstogenomicinformation.Teseandotherresources
haveresultedinanexplosionoffundamentaldiscoveriesaboutallaspectsofplantgrowthanddevelopment.
Inaddition,severalcompanies,foundedtoexploitknowledgegleanedfromArabidopsis,havetranslatedthe
newinformationintosuchcropspeciesascorn,soybean,andcanola(seeFig.A.DroughtResistanceinCanola
CausedbyDirectedModicationofaSingleGene,below).Plant-improvementgoalsthatwereunattainable
byappliedresearchoncropplantshavebeenrealizedbytranslatingbasicresearchonmodelspeciestocrops.
Muchaccumulatedknowledgeaboutcropspeciesandmodelorganismswillbeapplicabletoimprovingspecies
suitedforuseasdedicatedbiomasscrops.However,sometraitsofinterestinthatregardhavenotbeenaprior-
ityincropresearchandarepoorlyunderstood.Forinstance,knowledgeaboutcell-wallstructure,function,and
synthesisisveryunderdevelopedandwillneedextensiveresearch.Oneimportantspeciessuitedforuseasa
biofuelispoplar,amodelwoodyspeciesthathasarelativelysmallgenome.Poplarwassequencedrecentlyat
theDOEJointGenomeInstitute(seesidebar,EnhancingPoplarTraitsforEnergyApplications,p.62).
TeDNAsequenceofmanypoplargenesissimilartothatofcorrespondingArabidopsisgenes.Tiscom-
parabilitywillhelplinkmechanisticknowledgeaboutthesespeciestoaholisticunderstandingofcommon
processes.Poplarisrelativelyeasytotransform,whichgreatlyfacilitatesexperimentaltestsoftheoriesabout
proteinandgenefunction.Mostimportant,poplarhasseveralimportanttraits,suchasalonglifecycleandthe
formationofwood,thatcannotbestudiedinherbaceousplants.
Brachypodiumdistachyonisanotherpotentiallyimportantmodelforsuchhighlyproductivegrassesasswitchgrass
andMiscanthus.InterestinBrachypodiumarisesbecauseitsverysmallgenomehasaDNAcontentabout2.5
timeslargerthanthatofArabidopsis.Additionally,itssimplegrowthrequirements,smallstature,self-fertility,and
readytransformabilitymakeitwellsuitedtobecomeamodelorganism.BecauseArabidopsisisdistantlyrelated
toanddiersfromgrassesinanumberofimportantrespects(e.g.,cell-wallcomposition),Brachypodiumcould
becomeapowerfulnew
modelforcellandmolecu-
larbiologicalstudiesof
grasses.Ahighpriorityin
facilitatingthedevelop-
mentofBrachypodiumis
sequencingitsgenome.
Fig.A.DroughtResis-
tanceinCanolaCausedby
DirectedModicationofa
SingleGene.[Photo2003
MonsantoCompany.Used
withPermission.]
72 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

chips,sequence-indexedmutations,faciletransformationmethods,and
specializedlibrariesformethodsincludingtwo-hybridscreens.
BrachypodiumandPopulusmustbeacceleratedintopowerfulmodelsys-
temsbyrstsequencingBrachypodiumandthencreatingextensiveEST
databases(i.e.,1to2millionperspecies)fromdiverse(includingfull-
length)cDNAlibrariesforbothorganisms(Sterkyetal.2004).Agrobac-
terium-mediatedtransformationofbothBrachypodiumandPopulushas
beendevelopedbutmustbeoptimizedforhigh-throughputapproachesto
genediscovery.High-throughputcapacityfortransformingdiversegeno-
typeswouldenableforwardandreversegeneticapproachesbygenerating
collectionsofsequence-indexedinsertionalmutants,includingbutnot
limitedtoactivationtagging(Grooveretal.2004,Busovetal.2005),and
byallowingtargetedsilencingoroverexpressionoflargenumbersofgenes.
Transposon-basedmethodsforgeneratinginsertionalmutantsalsoshould
bedeveloped.High-throughputresequencingcapacitiesshouldbeusedfor
identifyingmolecularmarkers(e.g.,SNPs)associatedwithbiomassand
bioenergytraits.Similarly,phenotyping(transcriptandproteinproling)
capacitiesthatidentifygenotypesassociatedwithrelevantcell-wallcompo-
sitionoragronomictraitswouldbeuseful.
Usingthissuiteoftools,thefunctionofallBrachypodiumandPopulusgenes
willbeevaluatedfortheircontributiontobiomassenergy-relevanttraits.
Genesdeemedrelevantwillbeusedtocreateadvancedgenotypesinenergy
crops.Publicorganism-specicdatabasesshouldbecreatedandcuratedin
parallelwithadvancedexperimentalcapabilitiesfortheseorganisms.
Tosetresearchpriorities,theDOEOceofSciencesGTLprogramis
interactingwiththeconversionprogrammanagedthroughEEREsOce
oftheBiomassProgramandotherinterestedparties(e.g.,USDA,NSF,
andprivateindustry).
TechnicalMilestones
Within5years
CompleteBrachypodiumgenomesequencing,plusESTsequencingand
molecular-markeridenticationforbothBrachypodiumandPopulus.
OptimizetransformationmethodsfordiversegenotypesofBrachy-
podiumandPopulus,andgeneratesequence-indexedinsertionmutant
populationsof>100,000eventsforbothorganisms.
Developtoolsfortranscriptionalandproteinprolingandcarryout
rst-stageprolingofkeytissues(e.g.,cambialdevelopmentinPopulus).
Developorganism-specicdatabasesaroundannotatedgenome
sequences.
Within10years
Identifygenesrelevanttobiomassproduction(e.g.,thosethatcontrol
cell-wallcomposition,nutrientuptake,carbonpartitioning,ower-
ing,stresstolerance,anddiseaseresistance)usingforwardandreverse
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 73

FEEDSTOCKS FOR BIOFUELS
geneticscreensandnaturalpopulationsbasedonhigh-throughput
phenotyping[e.g.,useFouriertransforminfraredspectroscopy(called
FTIR)orothertechniquesthatcanbeautomatedtoidentifygeno-
typeswithalteredcell-wallcompositionandadvancedimageanalysisto
examineplantarchitecture].
Developandteststrategiesusingthesegenestoimproveenergycropsin
BrachypodiumandPopulus.
Within15years
Inbreedingprograms,usegenesandstrategiesshowntoaectbiomass
productionandqualityinBrachypodiumandPopulustoimproveenergy
crops,eitherbytransgenicapproachesorbymarker-assistedselectionof
naturallyoccurringvariabilityinorthologousgenes.
Transferimprovedenergycropsandgeneticinformationtotheconver-
sionprogrammanagedthroughEEREandtheOceoftheBiomass
Programandtootherinterestedparties(e.g.,USDA,NSF,andprivate
industry).
TheRoleofGTLCapabilitiesforSystemsBiology
CapabilitiesofGTL(withinDOEsOceofBiologicalandEnvironmen-
talResearch)willsupportorenabletheattainmentoflignocellulosicchar-
acteristics(seeLignocellulosicBiomassCharacteristicschapter,p.39)and
feedstockobjectivesinnumerousways.Potentialcontributionsofeachof
these,whetherdistributedorconsolidatedinafacility,aredescribedbelow.
ProteinProductionCapabilities
Mostenzymesofinterestincell-wallbiosynthesisarethoughttobemem-
braneassociatedand,therefore,diculttopurifyandcharacterizeby
conventionalmethods.Basedonpreliminaryexperimentswithmembrane-
localizedglycosyltransferases,apotentiallypowerfultoolincharacterizing
thefunctionofallplantglycosyltransferasesistoexpresstheminsuitable
hostssuchasinsectcells,inwhichendogenousactivitieswillnotinterfere
withassays. Tiscapabilitycouldundertakethehigh-throughputexpression
ofallglycosyltransferasesandothercellwallactiveenzymes(e.g.,peroxi-
dasesandlaccases)fromhigherplants(e.g.,Arabidopsis,Populus,Brachypo-
dium,orrice)orspecicbiomassspeciesinoneormoresuitablesurrogate
hosts.Suchexpressionsystemswillgreatlyfacilitatefunctionidentication
ofenzymesthatcatalyzesynthesisofcellwalls.Heterologouslyexpressed
proteinswouldbetestedfortheirsubstratespecicityandcatalyticactivity
andalsowillbeusedasaresourceforgeneratingantibodiesandtags.
Antibodyresourceswouldhavemultipleapplications.Acollectionof
uorescent-orepitope-taggedproteinsinaplantsuchasArabidopsisor
Brachypodiumalsowouldbeveryusefulforavarietyofreasons.First,the
taggedproteinscouldbeusedtorecovercomplexesforassaybymassspec-
trometricandimagingmethods.Fluorescenttagscouldbeutilizedforcel-
lularlocalizationorcollocation[e.g.,confocalmicroscopyoruorescence
74 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

resonanceenergytransfer(calledFRET)].Additionally,ifcertainproteins
arenotactiveinasurrogate,thetaggedversioninaplanthostmightbe
usedtoidentifyfunctionbyinvitroassaysofanity-puriedproteins.
Developingsucharesourcewouldrequireahigh-throughputtransforma-
tioncapabilitythatalsowouldbeusefulforgeneratinginsertionmutants
inBrachypodiumandPopulus.
MolecularMachinesCapabilities
Celluloseissynthesizedbyanintricatemultienzymecomplexinthe
plasmamembrane,andmatrixpolysaccharidesprobablyaresynthesized
bymultienzymecomplexesintheGolgi.Understandingthefunctionof
thesecomplexesandtheirinteractionswithmetabolicpathwaysthatpro-
ducesugarnucleotidesinthecytosolwillbeimportantforunderstanding
thecontrolofpolysaccharidebiosynthesis.Inaddition,cellulosesynthase
interactswiththecytoskeletontocontroltheorientationofcellulose
brilsandpossiblyinuencebrillength.Manyquestionsaboutthisand
relatedprocesseswillbenetfromtheapplicationofnewtechnologies
forimagingandmanipulatingproteincomplexes.Sincemostorallsuch
complexesaremembranebound,specializedmethodsmustbedeveloped
toworkwiththecomplexes.Also,becauseplantcellwallsareintricate
mechanicalassemblies,toolsdevelopedforinvestigatingproteinmachines
maybeusedtogainnewinsightsintoassemblyandfunctionofthelarge
polysaccharidecomplexesthatcomprisecellwalls.
ProteomicCapabilities
Tesecapabilitieswillfacilitateidenticationofproteinsthatmakeup
complexesinsuchmodelplantsasArabidopsis,Populus,andotherbiomass
crops.Sincemanycomplexesaremembranebound,innovativemethods
willbeneededtopurifymembranecomplexesusinganarrayofcapa-
bilities.Also,becauseofpoorcorrelationbetweenmRNAandprotein
abundance,proteomiccapabilitieswillenabledocumentationofthe
proteomeoflivingcelltypesfoundinvascularandothertissues,includ-
ingrayparenchymaandphloem,asafunctionoftimeandconditions.
Proteomicanalysisofplantswithfullysequencedgenomeswillidentify
post-translationallymodiedproteins.Preliminaryevidenceindicates
thatsomekeyproteinsinvolvedincell-wallsynthesisareregulatedby
modicationssuchasphosphorylation.Finally,comprehensiveproteomic
analysiscoupledwithimaginganalysisisrequiredtoidentifyproteins
locatedinsuchcompartmentsasthecellwall,nuclearmembrane,Golgi,
andendoplasmicreticulum.
CellularSystemCapabilities
Tesecapabilitiesareenvisionedtohelpinvestigatorsunderstandhow
complicatedmicrobialcommunitiesinsoilsrespondtovariouscropping
regimesbydevelopingbaselineanalysesofspeciescompositionandabun-
danceinsuitableexperimentalplots.Alsoneededarecost-eectivetools
(e.g.,diagnosticDNAchips)formonitoringtheseandotherplotsover
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 75

FEEDSTOCKS FOR BIOFUELS

longtimeperiodsandforintegratingresultingdataintoaviewofhow
biomasscroppingalterssoilecology.
Inadditiontoanalyzingsoilmicrobialcommunities,acquisitionandanaly-
sisoftranscriptomicandproteomicdatafrommodelandcropplantswillbe
apowerfultoolforassigningprobablefunctiontogenesimplicatedincell-
wallsynthesis.Asafunctionforeachenzymeemerges,thecellularsystems
capabilitythroughimaginganddynamicalanalyseswillbeusedtodevelop
asystemsmodeltoincorporatethewallsbiophysicalaspectswithstruc-
turalpropertiesandknowledgeofthefunctionsofproteinsinvolvedinwall
synthesis.Tismodelwillfacilitatetherationaldevelopmentoffeedstock
speciesbasedondesignprinciples,inwhichthewallschemicalcomposi-
tionandstructureareoptimizedwhile,atthesametime,notcompromising
maximalplantproductivity.Tecellularsystemsanalyticalandmodeling
resourceandproteomiccapabilitieswillcarryoutasystematicapproachfor
identifyingplantbiomarkerstoguideproteinproductionandthegenera-
tionofspecicmoleculartagsutilizingproteinproductioncapabilities.
Telong-termvisionistomakeavailableinsituimagesoflivingplantcell
wallsthatshowallkeymolecularprocessesoccurringinrealtime.Studies
willcoverthefulllifecycleofcell-wallformation,maturation,transfor-
mation,dehydration,andprocessingintosimplefeedstocksforconversion
intoethanolorproductionofotherfuelsandchemicals.Teunderstand-
ingobtainedthroughresearchthatusessuchimagingisexpectedtoresult
inquantitative,predictivemodelingasaguidetodevelopingadvanced
feedstocksandprocessingthemintofuels.Tesetoolswilltakeadvantage
ofvariouschemicallyspecicimagingtagscreatedwithproteinproduc-
tionresources.
DOEJointGenomeInstitute
DOEJGIwillsequence,assemble,andannotatethegenespaceorentire
genomesofmodelandemergingbioenergycropsandwillresequence
additionalcultivarsorecotypesformarkerdiscovery.Identicationof
genesthatcontrolcell-wallpolysaccharidesynthesisormodications
inbiomassspeciesandthedevelopmentoftoolssuchasgenechipswill
dependontheavailabilityofnucleotidesequences.Micro-RNAsare
expectedtoplayaroleinexpressioncontrolofmanyrelevantgenes,so
DNAsequencingmustbedeepenoughtoidentifytheminthesespecies.
JGIalsowillsequencecommunitygenomes,includingthoseoftherhizo-
sphereassociatedwithkeyproposedenergycropsandtrees.
OtherNeededCapabilities
GTLcapabilitiesascurrentlyenvisioneddonotencompassallresources
neededfordevelopmentofenergycrops.Toaccomplishobjectivesassoci-
atedwiththeenergymission,thecurrentvisionshouldbeexpandedto
includethethreecapabilitiesdescribedbelow.
76 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

TransformationServices
Substantialcapabilitieswillbeneededtocarryouttransformationofmodel
andappliedspeciesimportanttofeedstockdevelopmentonbehalfofthe
R&Dcommunityandtowarehousegeneticresourcesforthesespecies.
ChemicalPhenotypingServices
Feedstockimprovementforincreasedyieldandprocessingwillrequire
manipulationandassessmentofmultiplegenesincandidateorganisms
ormodelsystems.Newtoolsareneededtodetect,quantify,andcompare
changesincell-wallcompositionand3Darchitectureinprimaryand
secondarywallsduringassemblyandbeforeandduringprocessing.Te
toolswouldfacilitatefeedstockdevelopmentforimprovedperformance
inbiomassconversion.Detailedanalysisofcell-wallcompositionby
2Dnuclearmagneticresonance(NMR)imagingorothermethodsis
beyondthescopeofroutineprocessinginmostresearchlaboratories.
Capabilitiesforsuchanalyseswouldbroadlyfacilitateresearch.
SyntheticCarbohydrateChemistry
Analysesofcellwallsintheirnativestateandasafunctionoftreat-
mentandprocessingrequireadvancedsyntheticcarbohydratechem-
istrycapabilitiestocreatestandardsandmodels.NMRandmass
spectroscopyfacilitieswillbenecessarytosupportthesyntheticchem-
istrywork.
OtherBiofuelOpportunities:Development
ofHigh-ProductivityBiodieselCrops
Tomaximizesolarenergyuseandstorage,anidealbiomasscrop
wouldcarryoutphotosynthesisatthetheoreticalmaximumeveryday
oftheyearandwouldstorexedcarboninadirectlyusefulandeasily
harvestableform,evenwithoutmineralnutrients.Incarryingoutthis
process,manyplantsaccumulatelargeamountsofoilsorwaxesin
suchspecializedstoragetissuesasseedsormesocarptissues.Insome
cases,oilaccountsformorethanhalfthedryweightofthesetissues.
Mostoilseedspecies,however,arenotasproductiveasotherssuchas
maizethatstoreprimarilystarchratherthanoil.Intheory,obtain-
inghigheryieldsofoilshouldbepossiblebygeneticallymodifying
alreadyhighlyproductivespecies(e.g.,sugarbeet,potato,andmaize)
sotheywillaccumulateoilsorwaxesinsteadofcarbohydratesintheir
storageandvegetativetissues.
Otherplantsproducedbygeneticmodicationsofdevelopmentalproc-
essesaccumulateoilintheirroots(Ogasetal.1997),suggestingthefea-
sibilityofthisstrategy.Acetyl-CoAcarboxylaseistherstcommitted
stepinfatty-acidbiosynthesisinpotatotubers,whichaccumulatestarch
almostexclusively.Overexpressionofthisenzymeresultedinavefold
increaseintriacylglycerolaccumulationandprovidedanexampleofoil
depositioninacarbohydrate-storagetissue(Klausetal.2004).However,
DecodingtheDNA
ofSoybeanASource
ofBiodiesel
D
OEandtheU.S.Depart-
mentofAgriculturewill
supportgenomesequenc-
ingofthesoybeanastherst
projectinanagreementtoshare
resourcesandcoordinatestudiesof
plantandmicrobialgenomics.Te
soybean,Glycinemax,istheworlds
mostvaluablelegumecropandthe
principalU.S.sourceofbiodiesel,a
renewablealternativefuel.Diesel
enginesinherentlyaremore
thermodynamicallyecientthan
combustionengines.Biodieselhas
thehighestenergycontentofany
alternativefuelandissignicantly
moreenvironmentallyfriendlythan
comparablepetroleum-basedfuels,
sinceitdegradesrapidlyinthe
environment.Italsoburnsmore
cleanlythanconventionalfuels,
releasingonlyhalfthepollutants
andreducingtheproductionof
carcinogeniccompoundsbymore
than80%.Sequencingwilltake
placeattheDOEJointGenome
Institute,supportedbyDOEs
OceofScience.Tesoybean
genomeisabout1.1billionbase
pairsinsize,lessthanhalfthesize
ofmaizeandhumangenomes.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 77

FEEDSTOCKS FOR BIOFUELS
understandinghowtoreprogramplantcellstostoreoilratherthancarbo-
hydrateisverychallenging.Itrequiresnotonlylarge-scalechangesinthe
complementofexpressedmetabolicenzymesbutalsochangesincellular
structuretypicallyassociatedwithcellidentity.
Becauserecoveryofoilfromplantsistechnicallysimpleandecient,
processingoil-accumulatingplantmaterialonthefarmmaybepossible
somineralnutrientsandsoiladjuvantscanberetainedatthefarmfor
returntothesoil(seesidebar,DecodingtheDNAofSoybeanASource
ofBiodiesel,p.77).Inprinciple,oilcouldberecoveredduringharvestby
inexpensivescrew-presstechnology,greatlyreducingtransportandpro-
cessingcostsandenhancingsustainability.
Telipidsthatcomprisemostplantoilsarehighlyreducedformsofcar-
bonandthereforerepresentthemostenergy-denseplant-storagecom-
pounds.Derivativessuchasbiodieselcanbeproducedeasilybyinexpensive
andecientconversionofplant-derivedoiltofattyacidmethylesters.
Productionofhigh-yielding,oil-accumulatingplantscouldresultinanew
biofuelsourcethatwouldreducelossofcarbonassociatedwithfermenting
sugarstoethanolaswellascostsassociatedwithconvertinglignocellulosic
feedstockstoliquidfuels.Teplantthatmostnearlymeetsthisdescription
isoilpalm(seesidebar,OilPalm,thispage).
OilPalm:AnImportantBiofuelPlant
O
ilpalm,ElaeisguineensisJacq.,isatropical
treespeciesthatproducesbunchesofoil-
richfruitresemblingavocados.Teplants
growinlowlandsofthehumidtropics(15Nto
15S),whererainfallof1800to5000mmisevenly
distributedthroughouttheyear.Oilpalmsmature
slowlybut,onceestablished,yieldasmuchas10.6
tonnesofoilperhectare(ha)peryear,althoughthe
averageislessthanhalfthatamount.Teybeginto
bearfruitafterabout3yearsandremaininusefor
some25years,soannualmaintenancecostsarelow.
Palmoilfromthemesocarp,whichcontains45to
55%oil,issimilarincompositiontosoyoil.Oil
frompalmkernels,extractedfromtheendosperm,
containsabout50%oilrichinmedium-chain
fattyacidsandwellsuitedforbiodieselapplica-
tions.Malaysiaisthemajorsourceoftheseoils,
producing13.4billionpoundsofmesocarpoil
and3.5billionpoundsofkerneloilfromabout
3.8millionha.Plantsareharvestedbyhand,but
typicalplantingdensityisonly150plants/ha,and
laborcostsarenotamajorfactorinproduction.
Becauseoftherelativelystraightforwardconver-
sionofpalmoilstodieselandfooduses,palm
acreageinthetropicsisexpectedtoexpandsigni-
cantly.[Picturesource:C.Somerville,Stanford
University]
78 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Somequestionstobeaddressedbybasicresearch:
Howiscellidentitycontrolled?Morespecically,whathigh-level,regu-
latorygeneticcontrolsprogramacelltoexpressgenesinvolvedinlipid
synthesisandaccumulation?
Whatregulatescarbonuxfromphotosynthetic-sourceleavestostor-
ageorgans?
Whatregulatesmetabolicpartitioningofcarbonamongproteins,starch,
andoilproduction?
Whatspeciesaremostpromisingforconversiontooilaccumulation?
Forinstance,isoilpotatoabettercandidatethanoilbeetorveryhigh
oilmaize,orshouldplantstemsbeengineeredtodepositextremely
thickwax-richcuticles?
Whataretheopportunitiesfordevelopingnewhigh-yieldingoilseed
speciestailoredtoenergyproductionbyuncouplingoilaccumulation
fromseedcarbohydrateorproteinaccumulation?
Whatistheimpactofhighlipidlevelsonplantcells?Mostcellular
mechanismsoperateinaqueousenvironments.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 79

FEEDSTOCKS FOR BIOFUELS
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84 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

DeconstructingFeedstockstoSugars
T
hischapterdescribingthechallengesofdeconstructingcellulosic
biomasstoethanoliscriticallylinkedtoboththefeedstockand
fermentationareas.Lignocelluloserecalcitrancetobioprocessing
willremainthecoreproblemandwillbethelimitingfactorincreatingan
economybasedonlignocellulosicethanolproduction.Understandingbio-
massrecalcitrancewillhelptodrivecropdesign.Knowledgeaboutfeed-
stockbreakdownmechanismsandproductswilldrivefermentationand
consolidationstrategies,whichultimatelywillincludeconsolidatedbiopro-
cessing(CBP).CBPincorporatesthetraitsfordeconstructionandfermen-
tationofsugarstoethanolintoasinglemicrobeorculture.Teprevious
chapterdescribeshowtoolsofmoderngenomics-basedsystemsbiology
canprovidetremendousopportunitiestoengineerenergy-plantgenomes
fornewvarieties.Toseengineeredplantswillgrowmoreecientlywhile
alsoproducingoptimalpolysaccharidecompositionsfordeconstructionto
sugarsandfermentationtoethanolandotherproducts.Furtheradvance-
mentsinplantengineeringalsocangeneratenewenergycropsandtrees
harboringpolysaccharidestoragestructures(principallyinthecellwalls)
thataredesignedfordeconstruction.Tisachievementwillbeanimportant
outcomeofscienticresearchneededtooptimizedeconstructionofnative
cellwallsinsuchcropresiduesascornstoverandwheatstrawandsuch
energycropsasswitchgrassandpoplar.
Onceweunderstandmoreaboutthechemistryandultrastructureofcell
walls,improvedthermochemicalandbiologicalmeanscanbeusedtodis-
assemblethem.Forexample,thisreportwillshowthatnewandimproved
(existing)enzymescapableofdepolymerizingcelluloseincell-wallmicro-
brilscansignicantlyreducethecostofderivingsugarsfrombiomassa
criticalfactorinloweringtheoverallcostofcellulosicethanolandmaking
itcost-competitivewithgasoline.Currently,thestructureofcelluloseitself
isnotadequatelyunderstood.Forexample,wedonotknowtheactualfaces
ofthecellulosecrystaltowhichcellulasesbind,andsurfaceinteractions
amongexperimentallyproducedenzymesandsubstratesarenotunder-
stood.Inaddition,suchnewndingsaboutligninasitssynthesis,modi-
cation,anddepolymerizationareneededtodevelopconversionprocesses
thatarelessenergyintensive.
Sugarsproducedinoptimalcell-walldeconstructionwillbeusedby
microbesspecializinginconverting(fermenting)thesecompoundsto
ethanol.Inthenearterm,biomassconversionwillproducesomequantities
offermentationinhibitorsthatcanincludeacetate,polyphenolics(aro-
maticcompoundsderivedfromligninbreakdown),andcellobiose.Even
References:p.115
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 85

DECONSTRUCTING FEEDSTOCKS
highconcentrationsofsugarsfromcell-walldeconstructionareinhibitory
tomanyethanologens.Tusthechapter,SugarFermentationtoEthanol,
p.119,describesresearchtodevelopprocessesatthecellularandgenomic
levels(e.g.,evolutionaryengineeringtomodifymicrobialstrainsthat
tolerateelevatedlevelsoftoxinsorsugars).Also,genomicandbioinfor-
matictoolscanassistinthedesignofnewmetabolicpathwaysthatpermit
ecientsimultaneousfermentationofmixedsugars.
Lookingaheadtolonger-termimprovementsinbioreneryproductiv-
ity,thisreportproposesthatneworganismsconsolidatingtraitsforboth
deconstructionandfermentationshouldbethesubjectofconsiderable
research.Forexample,newethanologenstrainsareneededthatalso
areabletoproducemixturesofhighlycompetentcellulasesandother
hydrolyticenzymes.Teapplicationofadvancedtechniquesingenetics,
molecularbiology,high-throughputscreening,imaging,andmathematical
modelingwillacceleratethepaceatwhichviableprocessingcanbeaccom-
plishedforenergysecurity.
DeterminingFundamentalPhysicalandChemical
FactorsintheRecalcitranceofLignocellulosic
BiomasstoProcessing
Lignocellulosicbiomassisacomplexstructurewithcrystallinecellulose,
hydratedhemicellulose,andligninasmajorcomponents.Todate,thebest
enzymecocktailsproposedforsaccharicationofthismaterialaresyn-
ergisticmixturesofenzymeswithdenedactivities,primarilythosethat
degradecellulose.Tesubstratesheterogeneousnatureandenzymecock-
tailscomplexitysuggestthattraditionalstudiesofbulkpropertieswillnot
providethedetailedunderstandingrequiredforknowledge-basedadvance-
mentsinthiseld.
Tequestionthatmustbeansweredis,Howwillstructuralandchemi-
caldetailsofenzymesubstratebindingsitesaectenzymeadsorption
andreactionrates?Fromcellulasekineticsalone,weneverwouldbeable
toanswerthatquestion.Wemustbeabletoimprovecellulasesusingan
informationalapproach,consideringthat20yearsofmixingandtesting
individualcellulaseproteinshasyieldedonlymodestprogresstowardan
improvedcellulasesystem.Enzymekineticsalwayshasbeenameasureof
ensembleaverage resultsfromexperiments, andthussugar-releasevalues
frombiomassnevercandeliverinformationaboutthereactivesiteofthe
individualenzyme.Becauseofthisdilemma,wedonotknowifcellulose
recalcitranceisduetoenzymeinadequacy,enzyme-substratemismatch,or
both.Whileinmanycaseswecanperformbulkcompositionalanalyses,
molecularandstructuralcorrelationsthatarekeytoprocessabilityremain
achallenge.Tescienceandtechnologyfortheseanalysesmustcome
fromfrontiercapabilitiesinmanydisciplinesinthephysical,biological,
andcomputationalsciences[seesidebar,ImageAnalysisofBioenergy
PlantCellSurfacesattheOBPBiomassSurfaceCharacterizationLab
(BSCL),p.40].
86 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Suchenzymesascellulases,hemicellulases,andotherglycosylhydrolases
(GH)synthesizedbyfungiandbacteriaworksynergisticallytodegrade
structuralpolysaccharidesinbiomass.Teseenzymesystems,however,are
ascomplexastheplantcell-wallsubstratestheyattack.Forexample,com-
mercialcellulasepreparationsaremixturesofseveraltypesofGH,eachwith
distinctlydierentsubstratespecicities(celluloseandxylan)andaction
patterns(exoenzymesactingfromthechainends,endoenzymescleav-
ingwithinthechain,andGHcleavingside-chainbranches,forexample,
arabinosefromarabinoxylansandxylosefromxyloglucans).Optimization
oftheseenzymeswillrequireamoredetailedunderstandingoftheirregula-
tionandactivityasatightlycontrolled,highlyorganizedsystem.
Toolsmustbedevelopedrsttoanalyzebiomassatthelevelsandspeci-
citiesneededtodescribe,understand,predict,andcontrolitsbehaviors.
Teseincludemolecular-levelandnanoscaleunderstandingofcriticalcell-
wallphysicalandchemicalproperties,howtheyareformedintheplant,
howtheyfunction,andhowcertainstructuralfeaturesinhibitorfacili-
tateenzymaticinteractionsandsubsequentsaccharication.Duetothe
dynamicandinterconnectednatureoftheseconversionprocesses,wemust
beabletomonitorthemastheyoccurfromnativestructuralbiomassto
freesugars.Criticalinteractionsandreactionsoccuratmanyscalesoftime
andlengththroughoutmultiplestagesofintricatelyinterlinkedprocess-
ing.Allbiomassstructuralandchemicalpropertiesmustbeanalyzedin
thenativestate,invariousstagesofdehydrationandprocessing,andasthe
materialisdeconstructedintocomplexmixturesofreactionintermedi-
ates.Teseanalysesmustsupportresearchforbiomassoptimizationfor
processingandethanolyield,aswellasinvestigationsofrobustgrowthand
toleranceinthecultivar.Pretreatmentoptimizationwillbothmaximize
fermentablesugaryieldwhileminimizinginhibitorsandotherdeleterious
factorsinsubsequentsteps.
Tisproblemfurtherinvolvesstructureandfunctionatboththeplantand
microbiallevels;theirinteractions;andthefunctionsofproteins,enzymes,
andlivingsystems.Teyarestudiedunderaverywiderangeofconditions,
frompretreatmenttemperaturesandchemistrytotheenzymaticbreak-
downofcomplexcellulosicmicrobrilsandotherpolymersintosugars.
Tounderstandreactionpathways,wemustbeabletodiscernintermediate
digestionandreactionproductsphysicalstructuresastheydegradeand
chemicalmoietiesastheyaretransformedandreact.Allthesecapabilities
willenabletheultimateconsolidationofallstepsintooneprocess.
Animportantandnecessarydeparturefromtraditionalapproachesstems
fromourappreciationthatbiologicalsystemsareincrediblycomplex
andinterconnected.Cellularsystemsofplantsandbiomass-degrading
microbesconsistofhighlycoupledsubsystems.Intheselivingsystems,
eachcomponent(moleculesandmacromolecules)aectsandisaectedby
othercomponents.Resolutionofresearchissuesdescribedinthissection
willadvancebiomasspretreatmentandhydrolysisbydevelopingasystems-
level,predictive,andquantitativenewunderstanding.Suchunderstanding
willfacilitaterationaldesignofbiomass-conversionsystems,informthe
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 87

DECONSTRUCTING FEEDSTOCKS
Fig.1.ScanningElectronMicroscopy.
(a)Acornstoverparticleshowsasmoothsurface
withafewmicron-sizedporesafterenzymehydro-
lysisconverted11%ofcellulosetoglucosein3h.
(b)Tiscornstoverparticlehasmanymorepores.
Itwaspretreatedinwaterat190Cfor15minand
hydrolyzedbyenzymesat50Cfor3h,resulting
in40%celluloseconversiontoglucose.
Teresultsillustratethatpretreatmentchanges
lignocellulosic-structuresusceptibilitytoattack
byenzymes.Higherresolutioninfutureimaging
techniqueswillfacilitateadeeperunderstanding
ofunderlyingmolecularmechanisms.[Source:
Imagesandconditionsfromunpublishedworkof
M.Zeng,N.Mosier,C.Huang,D.Sherman,and
M.Ladisch,2006.]
(a)EnzymeHydrolysisOnly.
(b)EnzymeHydrolysisFollowingPretreatment.
designofanewgenerationofenergycrops,andaccelerate
adoptionofinnovativebiorenerytechnologies.
ResearchGoals
Biological,mathematical,imaging,andotheranalyticaltools
mustbedevelopedandappliedtoidentifyandquantifythe
relativeimportanceofpotentiallylimiting(orfacilitating)
physicalandchemicalfactorsinbioconversion.Allthese
methodswillbeusedtocharacterizetheeectsofdehydra-
tion,heating,acidity,andcosolvents,amongotherbiomass
treatments.Detailedstudiesusingthesetoolswillhelpus
betterunderstandmechanismsofactionandapplyvarious
pretreatmentsandenzymesystemstoenhancelignocellulose
bioconversiontoethanol.Suchtopicsarediscussedbelow.
MeasurementofBiomassProperties
Teorganizationofpolymercomponentsinbiomassstruc-
turesneedstobenoninvasivelyimagedin3Dtoelucidate
thematerialsorganization.Variousexperimentalapproaches
includingX-rayandneutrondiractionofcrystalsand
drawnbers,enhancedatomicforcemicroscopy(AFM),and
biochemicalprobeenabledandchemicallyspecicimaging
willbeusedtocharacterizethedierentformsoflignocel-
luloseinbothnativeandtreated(storedandprocessed)
biomasscellwalls.
Analyticalmethods[e.g.,nuclearmagneticresonance(NMR),
Fourier transforminfrared (FTIR), and Raman]should
provideinformationonchemicalmoieties,chemicalbonds,
andconformationofwallpolymers.Surfacesthatareenzyme-
bindingsitesshouldbecharacterizedbysuchtoolsasAFM,
scanningelectronmicroscopy,transmissionelectronmicros-
copy,andelectronspectroscopyforchemicalanalysis(see
Fig.1.ScanningElectronMicroscopy,thispage).
Tisinformationcanbecorrelatedusingstatistical-analysis
packagesbasedonprincipalcomponentstoreliablyrelate
changesinlignincarbohydratecomplexes(LCC)andlignin
proles,degreesofcellulosedehydration,andpretreatment
chemistriestotheabilitiesofhydrolyticenzymestoconvert
theresultantmaterialbiomass.
ModelsforDirectEnzymaticInteractions,Action
Mechanisticmodelsofenzyme-systemsubstraterelation-
ships,basedonnewknowledgeaboutplantcell-wallarchi-
tectureandenzymestructureandfunction,needtobe
developed.Teactionofindividualandcombinationsof
enzymesonnativeandmodelsubstratesshouldbeexam-
inedusingsingle-moleculespectroscopyandimaging.
88 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Mechanisticcharacterizationofrelevantenzymes,withattentiontocom-
binedchemicalandbiologicalconversionprocessesofcell-wallmateri-
als,thenwillsupportmodelsforinteraction.Finally,newcomputational
modelsformicrobrilsandcellwallsusingimprovedcodesandpetascale
leadership-classcomputerswillprovidetheneededdelitytosupport
reaction-pathwaycalculations.
TechnicalMilestones
Within5years
Usingintegratedanalyticalandmathematicalmethods,quantitatively
analyzeeectsofselectedbiomasspropertiesandpretreatmentchemis-
triesonenzymatichydrolysis.
Identifyprinciples,genes,andcontrollingfactorsthatinuencebiomass
makeup,assembly,andprocessability,inconjunctionwithplantdesign.
Perfectandextendexistingspectroscopic,surface,andimagingcharac-
terizationmethodsforbiomass.
Developmethodsformonitoringtheprogressofphysicalmodication
duringdehydrationundervariousconditionsofstorageorpretreatment
atelevatedtemperatures.Developimprovedprotocolstobetestedin
eldtrials.
DenetheprimarystructureofligninsandLCCsandtheshape,
dimensions,andheterogeneityoftheirdomains.
Developmodelsconnectinglignocellulosicpropertieswithdeconstruc-
tionandhydrolysisprocesses.
Denitivelycharacterizethedetailedorganizationalstructuresof
principaltypesofplantcelluloseandtheirrelativeenergiesandinter-
relationships.
Understandtheenergeticsofdierentcelluloseformsandwaysin
whichthesestructuresgiverisetocharacteristicsofthenextorgani-
zationlevelinthemesoscopicrangeofsizes,and
Determinehowmicrobrilsinteractwithotherprincipalstructural
components(ligninsandnoncellulosicpolysaccharides).
Within10years
Developanunderstandingofthephysicochemicalbasisofpolysaccha-
rideinteractionswithwater.Tisinformationcanbeusedtorationally
designgenomicvariationsforcell-wallpolysaccharidecompositeswith
moredesirableproperties.
ApplynewbiologicalandchemicaltoolstoalterLCCandotherrele-
vantpropertiesofselectedbiomassspecies.
Subjectthesealteredbiomassspeciesandcontrolstovariouspretreat-
mentchemistriesandtemperatures.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 89

DECONSTRUCTING FEEDSTOCKS
Hydrolyzetheresultingpretreatedbiomasswithvariousspecic
enzymesandcorrelatespectroscopic,surface,andimagedatawith
hydrolysisofstructuralcarbohydrates.
Quantitativelyrelatekeybiomassandpretreatmentpropertieswith
hydrolysisyieldsfordierentbiomassspecies.
Improveenzymesbasedontheknowledgeofsubstrate-imposedlimita-
tionsandanunderstandingofoptimizedcellwalls.
Developmolecularmodelsforenzyme-substratestructure-function
relationships.
Within15years
Developmathematicalmodelsofcellwallsandthetransportofwater,
pretreatmentchemicals,andenzymesthroughthewalls.Suchmodels
shouldhelpindesigningcellwallsthatcanbedigestedmoreeciently
bydevelopedenzymesystems.Designsoptimizedbymodelingcanthen
helpguidegenomicengineeringforplantandenzymeoptimization.
Establishthefoundationforintegratedbiomassprocessingbypredict-
ingandthenverifyingoverallhydrolysisyieldsfornativeandmodied
biomassspeciesusingdierentpretreatmentchemistriesandseverities
(time,temperature,acidity)andarangeofhydrolyticenzymes.
Performrationalengineeringofenzymestospecicrequirements
basedonnewunderstandingsaboutenzymesandtheirmechanismsof
actionandsubstratecellulose-hemicellulose-lignincomplexes.
TheRoleofGTLandOBPFacilitiesandCapabilities
Teprogramoutlinedmostlikelywillneedallfacilityresourcesatone
pointoranother.
ProteinProduction
Proteinproductioncapabilitieswillbeusedforenzymesandtags(biomarkers).
MolecularMachineandCellularSystemAnalysis
Tesecapabilitiescanimageanddiagnoseinteractionsbetweenenzymes
andbiomasssubstrates.Inaddition,BSCL,recentlyestablishedatNREL,
certainlywillproveessential.Imagingfacilitieswouldbeusedtocorrelate
digestibilitytobiomassultrastructure.Tesidebar,ImageAnalysisofBio-
energyPlantCellSurfacesattheOBPBiomassSurfaceCharacterization
Lab,p.40,illustratesthecellwallshighlycouplednatureandhoweach
componentaectsandisaectedbyothercomponents,therebyhighlight-
ingtheneedtostudytheintactsystem.
DOEJointGenomeInstitute
DOEJGIwillmapandidentifygenesconcernedwithdeconstruction
barriersorfacilitatingfactors.
90 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy


















CrosscuttingTools,Technologies,andScience
Bettercharacterizationofcell-wallpolysaccharideassociationproles
inhydratedanddehydratedstatesusingspectroscopicandultramicro-
scopicmethods.
Larger-scalemolecularmechanicsmodelingtoexploretheassociationof
cell-wallpolymerswitheachotherandwithwateratvariouslengthscales.
Robustandrapidmethodsforquantifyingcell-wallpolymersandinter-
polymerlinkages.
Genomicandchemicaltoolstoidentifygenesinvolvedinrate-limiting
LCClinkages.
Newbiomassvariantswithenhancedaccessibilityforstructuralcarbo-
hydratedigestingenzymes.
Newmethodsforspectroscopiccharacterizationofcell-wallpolysac-
charidesinthelivinghydratedstate.Mostpastworkhasbeenbasedon
investigationofisolateddehydratedsamplesandverylittleoncell-wall
constituentsintheirnativestate.
Coupledstructureandprocessingmodelsofcellwallsandcomponents.
Data-analysissoftwaretoidentifyandquantitativelydescribetherela-
tiveimportanceofdierentfactorsgoverningresponsetopretreatment
andenzymatichydrolysisofbiomass.
DevelopingBetterEnzymaticSystemsforBiological
Pretreatment:LigninasesandHemicellulases
Temostecientconditionsforsimultaneoussaccharicationandfer-
mentationoflignocellulosicbiomasswillbethoseinwhichtransforma-
tionsnowperformedduringandfollowingthermochemicalpretreatment
areseamlesslyintegratedintotheoverallprocess.Eectiveenzymatic
breakdownofLCC,toexposecellulosetoenzymeaction,representsan
importantsteptowardthisobjective.
Lignocellulose,anextremelycomplexandwidelyvaryingnanoscalecom-
posite,iswelldesignedtoresistattack.Ligninasesandhemicellulases,for
whichfewexamplesareknown,areinadequatelyunderstood.Technically,
understandingandoptimizingtheseenzymeswillenabletheultimategoal
ofconsolidatingpretreatmentandsaccharication.Consequently,research
isneededtoidentify,characterize,improve,andeconomicallyproducethe
mosteectiveenzymesystemsforbiomasspreconditioning.Teseenzymes
wouldbeappliedbeforeoraftertraditionalpretreatmenttominimizeand,
eventually,replacethermochemicalprocesses,thuslesseningtheeectsof
overallpretreatmentseverityatthemacromolecularlevelandsimplifying
processing. Tosupportimprovements, research mustbe focusedon identify-
ingmoreenzymesofthisclassandcharacterizingtheirprinciplesofaction.
Acrucialcomponentforligninaseidenticationwillbegenomic,func-
tionalgenomic,andproteomiccomparisonsofwhiterotfungiknownor
presumedtoexpresssuchactivities(seesidebar,WhiteRotFungus,p.93).
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 91

DECONSTRUCTING FEEDSTOCKS
Teultimategoalofthisresearchistoproducearecombinantligninase-
hemicellulasemicrobialsystemwithenhancedcatalyticactivityandstability,
industrializedforbioreneryoperations.
ResearchGoals
Techemicalandstructuralnatureofnativeligninispoorlyunderstood.
Developingrobustligninasesrequiresafoundationofknowledgeaboutthe
rangeofligninchemistriesandligninassociationsthatinhibitorfacilitate
freeingcelluloseandhemicelluloseforsaccharication(seesidebar,Ligni-
cation:RandomvsTemplateDirected,p.94).Similarly,despitetheimpor-
tanceofhemicellulosesandhemicellulasesforsaccharication,bothare
poorlyunderstood.Duetothecomplexstructureandcompositionaldiversity
oflignin-hemicelluloseand-cellulosesubstratesthatmustbecontemplated
forfeedstockbiomass,avarietyofenzymaticactivitieswillbeneededto
catalyzedeconstructionforconversiontomonosaccharides(seeFig.2.Com-
plexMixtureofEnzymesforDegradingHemicelluloses,p.96).
HemicellulosesandHemicellulases
Aftercellulose,hemicelluloses(xylanpolymers)arethenextmostabun-
dantpolysaccharidesinnativebiomassfeedstocks(seeTable1.Cellulose,
Hemicellulose,andLigninContentinVariousSourcesofBiomass,p.93).
Althoughconsiderableresearchhasfocusedonconvertingbiomasscellulose
tofermentableglucose,lesshasbeendoneonbioconvertingotherplant
cell-wallcomponents.Abetterunderstandingofthecomplexstructure
andcompositionofthispolysaccharidegroup,whichwilldierintypeand
abundanceamongdierentfeedstocks,willhelptoidentifyandoptimize
themechanisticbasisofrequiredenzymaticactivities.Structuralinforma-
tionandmechanisticmodelsmustbedevelopedtopinpointbottlenecksin
hemicellulosebioconversion.
LigninandLigninases
Despiteitscriticalimportance,theenzymaticbasisoflignindepolymer-
izationinvivohasremainedelusive,ifnotcontroversial.Researchduring
thepast20yearsonputativeligninases,whichcanbothpolymerizeand
depolymerizeligninpreparations,hasnotyieldedreliableinsightsintothe
mechanismsoflignincleavage. Tusthepossibilityofanotherentirelydif-
ferentclassoflignin-degradingenzymescannotbedisregarded,andpoten-
tialcandidateshavebeenidentiedinwhiterotfungi.Genesencodinga
rangeofligninasesmustbeidentied,andgenomicsequencesofwhiterot
fungithatdegradeligninbutdonotexpressknownperoxidaseswillbecom-
paredwiththesequencedP.chrysosporiumgenome.Asuitablehostorganism
willbeneededtoproduceheterologousligninasesinsucientquantities;
possiblehostsincludeE.coli,S.cerevisiae,P.pastoris,andA.oryzae.Heterolo-
gousligninaseexpressionandmanipulationhavehadlimitedsuccess.
Tisresearchwouldincludethefollowinggoals:
Detailedcharacterizationoftherangeofligninandhemicellulosestruc-
turesencounteredinavailablebiomass.Specialattentionmustbepaid
92 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

tothenatureofcovalentbondslinkingLCC.
Modelsystemswillfacilitatetheseanalyses.
Systemsbiologystylesurveyandmolecularand
functionalcharacterizationofthediversityandactiv-
ityofrelevanthemicellulases(glycosidehydrolasesand
esterases),includingdiscoveryofactivitiesfromsuch
novelsourcesassoils,therhizosphere,termitehindgut,
anddecayingbiomass.
Similardiscoveryandmolecularandfunctionalcharac-
terizationofnewlignin-degradingenzymesandtheir
activities.
Reliableexpressionsystemsandhostsforthese
enzymeclasses.
Mechanisticcharacterizationofenzymeswithatten-
tiontopropertiesrelevanttocombinedchemicalandbiologicalconver-
sionprocesses.
Examinationofindividualandcombinationenzymeactivitiesonnative
andmodelsubstrates.
Developmentofmechanisticmodelsofenzymesystemsubstraterela-
tionships,basedonnewknowledgeaboutplantcell-wallarchitecture.
Table1.Cellulose,Hemicellulose,andLignin
ContentinVariousSourcesofBiomass
Feedstock Cellulose Hemicellulose Lignin
Cornstover 36.4 22.6 16.6
Wheatstraw 38.2 24.7 23.4
Ricestraw 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,andD.Johnson,Biomass
FeedstockResourcesandComposition,pp.10518in
HandbookonBioethanol:ProductionandUtilization(Applied
EnergyTechnologySeries),ed.C.E.Wyman,Taylorand
Francis(1996).
WhiteRotFungus:GenomeofKnownLigninDegraderSequenced
L
ignindegradationisthekeytomakingpolysaccharidecomponentsofcellwallsavailableforbreak-
down.Whiterotfungiaretheprimarydegradersoflignin,whichisamongthemostabundantofnatu-
ralmaterialsonearthandplaysapivotalroleinglobalcarboncycling.Teseorganismsalsodegradethe
celluloseandhemicellulosecomponentsofplantcellwalls.
Toaidinunderstandingtheseprocesses,theDOEJointGenome
InstitutesequencedthegenomeofthewhiterotfungusPhanerochaete
chrysosporium.Tisfungusdegradesbrownlignin,theprotective
matrixsurroundingcellulosemicrobrilsofplantcellwalls,leav-
ingbehindcrystallinewhitecellulose.[D.Martinezetal.,Genome
SequenceoftheLignocelluloseDegradingFungusPhanerochaete
chrysosporiumstrainRP78,Nat.Biotechnol.22,695700(2004).]
Analysisofthewhiterotfungusgenomerevealedgenesencoding
oxidases,peroxidases,andotherenzymesthatcontributetodepoly-
merizationoflignin,cellulose,andhemicellulose.Extensivegenetic
diversitywasobservedingenefamiliesencodingtheseenzymes,
possiblyreectingthatmultiplespecicitiesareneededforeective
degradationofcell-wallpolymersfromdierentplantspecies.Elu-
cidatingtheregulationofgenes,proteins,andmetabolitesfromthis
organismandotherswillenhanceunderstandingoftheindividual
andcollectivemechanismsofdegradativeenzymesaswellastheir
interactionswithotherorganismsintheirecosystems.Suchadvances
arenecessaryforgeneratingtheframeworktoengineerlarge-scale
processesforbiomassutilization.
CoverusedwithpermissionfromNature
Biotechnology,www.nature.com/nbt/.

BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 93

DECONSTRUCTING FEEDSTOCKS
Improvementofenzymecatalyticeciencythroughproteinengineering
andofbiomassthroughplantdesign.
Coordinationwithfeedstocksandbiological-conversionresearchto
informplantdesignandtosupportimprovedfermentationandeventual
incorporationofpretreatmentenzymaticprocessesintoconsolidated
processingschemes.
Ligninandhemicelluloseinvestigationswillfollowsimilarresearchpaths
asdescribedbelow.
TechnicalMilestones
Within5years
Initiatemetagenomicsurveysinenvironmentsknowntobreakdownbio-
massnaturally,andidentifyandmolecularlycharacterizeligninasesand
hemicellulases,includingaccessoryenzymes.
Heterologouslyexpressenzymesinsuitableproteinproductionhosts.
Lignifcation:RandomvsTemplateDirected
I
ntermsofenergycontent,ligninsare
thoughttobethemostabundantof
allbiopolymers.Teyarecomposed
ofp-hydroxyphenylpropanoidunitsinter-
connectedthrough8-O-4,8-5,8-8,8-1,
5-5,and4-O-5linkages.Corresponding
substructuresinthepolymerincludealkyl
arylethers,phenylcoumarans,resinols,
tetrahydrofuran-spiro-cyclohexadienones,
biphenyls,dibenzodioxocins,anddiaryl
ethers(seeFig.A,atleft).Teprimary
precursorsthemselvesthethreemono-
lignolsp-coumaryl,coniferyl,andsinapyl
alcoholsdieronlyaccordingtotheiraro-
maticmethoxysubstitutionpatterns.Tese
monolignolsareoxidizedenzymatically
throughsingle-electrontransfertogenerate
therespectivephenoxyradicals.Teactual
couplingofamonolignolradicalwiththe
growingendofaligninchain,however,may
notfallunderdirectenzymaticcontrol.
Accordingly,manyinvestigatorshave
assumedthatligninprimarystructures
mustberandomorcombinatorialasfaras
sequencesofinterunitlinkagesarecon-
cerned.Morerecently,thistheoryhasbeen
reinforcedbyreportsthatcertainkinds
Fig.A.ContemporaryViewofLigninSubstructures. Teoryproposed
byG.Brunowandcoworkersin1998(reproducedwithpermission).
94 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Determinecatalyticpropertiesofenzymes.
Useoverproducedandcommercialenzymestodigestnativehemicellu-
loses,andidentifyintermediateproductsinhemicellulosedecomposition.
Performmolecularevolutionofenzymes,andselectforimproved
kineticeciencyandcompatibilitywithotherenzymesandwith
desiredindustrialapplications.Tisisexpectedtoresultintheidenti-
cationoffactorslimitingenzymaticconversionandthedevelopmentof
enzymecocktailstoovercomethemeciently.
Within10years
Extendmetagenomicsurveystodiscovernovelligninasesandhemi-
cellulases.
Concurrentlyimproveenzymesinthecellwallsofenergycropsinpar-
allelwithknowledgeaboutsubstrate-imposedlimitations.
Developmolecularmodelsforenzyme-substratestructure-function
relationships.
ofnon-nativemonolignolscanbeincorporatedintomacromolecularligninstructures.Ligninsandlignin
derivativesexhibittwofundamentalcharacteristicsthattraditionallyhavebeenviewedasevidenceinfavorof
randomnessintheircongurations:Teyarebothnoncrystallineandopticallyinactive.
1
Nevertheless,anumberofobservationsarethoughtbysometopointintheoppositedirection.Teindividual
molecularcomponentsin(nonpolyionic)ligninpreparationstendtoassociateverystronglywithoneanother
inawell-denedway.Teseprocessesarethoughttobegovernedbyvitalstructuralmotifsderivedfromcor-
respondingfeaturesdisposednonrandomlyalongthenativebiopolymerchain.Moreover,dimericpinoresinol
moietiesarelinkedpredominantlytothemacromolecularligninchainthroughatleastoneoftheiraromatic
C-5positions.
Wedonotknowwhethersuchfeaturescanbeexplainedthroughcombinatorialmechanismsundersimple
chemicalcontrolorifhigher-levelcontrolmechanismsarerequired.Onehypothesisproposesawaytorepli-
catespecicsequencesofinterunitlinkagesthroughadirecttemplatepolymerizationmechanism.According
tothismodel,anantiparalleldouble-strandedlignintemplate,maintainedinadynamicstateattheleading
edgeofeachlignifyingdomain,determinesthecongurationofthedaughterchainbeingassembledonthe
proximalstrandsexposedface.Furthermore,replicationdelitycouldbecontrolledbystrongnonbonded
orbitalinteractionsbetweenmatchingpairsofaromaticringsintheparentandthegrowingdaughterchains.
Teoverallprocessseemstobeconsistentwiththelackofbothcrystallinityandopticalactivityinmacromo-
lecularlignindomains.
Finally,requiredsequenceinformationmaybeencodedinpolypeptidechainsthatembodyarraysofadjacent
lignol-bindingsitesanalogoustothosefoundindirigentpositioningproteins.
2
CitedReferences
1.J.Ralphetal.2004. Lignins:NaturalPolymersfromOxidativeCouplingof4-Hydroxyphenylpropanoids,
PhytochemistryRev.3,2960.
2.S.Sarkanen.1998.TemplatePolymerizationinLigninBiosynthesis,pp.194208inLigninandLignan
Biosynthesis697,ed.N.G.LewisandS.Sarkanen,AmericanChemicalSociety,Washington,D.C.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 95

DECONSTRUCTING FEEDSTOCKS
Improvecatalyticeciencyandstabilityofenzymesthatdegradecell
wallsusingproteinengineering.
Constraintsimposedbythefairlylimitedsetofindustrialhostorganisms
onexpressionofnativeorheterologousGHproteinsmustbeunderstood
(forexample,theroleandmodicationofvariousglycoforms).Greater
understandingintheseareaswouldallowmoreecientimprovementsin
enzymeperformanceinindustrializedhostsandresultantproductioneco-
nomics.Understandingbiomassrecalcitrancewillenableenzymeprepa-
rationstobeengineeredforsuperiorperformanceusingproteomicsand
thesystemsbiologyapproach.Tesechallengeswillbemorepronounced
whendealingwithforeignproteins(suchasligninases)andmultienzyme
complexes(cellulosomes)inheterologoushosts.
Within15years
Performrationalengineeringofenzymestospecicrequirementsbased
onnewunderstandingofenzymesandsubstratecellulose-hemicellulose-
lignincomplexes. Teabilitytodesignspecicenzymestoattackspecic
substratesisexpectedbythistime.
Fig.2.ComplexMixtureofEnzymesforDegradingHemicelluloses.Teexampledepictediscross-linkedglucurono
arabinoxylan.Tecomplexcompositionandstructureofhemicelluloserequiremultipleenzymestobreakdownthepolymer
intosugarmonomersprimarilyxylose,butotherpentoseandhexosesugarsalsoarepresentinhemicelluloses.Avarietyofde-
branchingenzymes(red)actondiversesidechainshangingothexylanbackbone(blue). Tesedebranchingenzymesinclude
arabinofuranosidase,feruloylesterase,acetylxylanesterase,andalpha-glucuronidase(seetablebelow).Asthesidechainsare
released,thexylanbackboneisexposedandmademoreaccessibletocleavagebyxylanase.Beta-xylosidasecleavesxylobiose
intotwoxylosemonomers;thisenzymealsocanreleasexylosefromtheendofthexylanbackboneoraxylo-oligosaccharide.
Tetableshowsthatsomeoftheseenzymesaremulti-
functional,withcatalyticdomainsbelongingtodierent
enzymefamilies.Teirgreatdiversityandthatofother
enzymesinvolvedinhemicellulosedegradationpresenta
remarkablycomplicatedenzymaticsystemwhosemore-
thoroughanalysismayyieldgreaterunderstandingof
hemicellulosicdegradation.
[Source:MolecularstructureadaptedfromL.B. Selinger,
C.W.Forsberg,andK.J.Cheng,TeRumen:AUnique
SourceofEnzymesforEnhancingLivestockProduction,
Anaerobe 2(5),26384(1996).]
GlycosideHydrolase(GH)andCarbohydrateEsterase
(CE)EnzymeFamiliesforDegradingHemicelluloses
Enzyme EnzymeFamilies
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
Acetylxylanesterase CE1,2,3,4,5,6,7
Feruloylesterase CE1
---X14X14X14X14X14X14X14X---
---X14X14X14X14X14X14X14X---
-xylosidase
-Glucuronidase
Acetylxylan
esterase
Arabinofuranosidase
Feruloyl esterase
Lignin
Xylanase
X14X
2X
3

Af
5
Fer
FerOFer
5
Af

3
Ac
3
3
Af
mGu
1
2
96 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Greatlyincreaseunderstandingoffundamentalprotein-secretionlimi-
tationsinhyperproducingstrains.Similarchallengesmustbemetto
enableconsolidatedbioprocessingmicrobes.
Similarly,thecatalyticactivityandthermalstabilityofligninasesmust
beenhancedthroughdirectedevolutionstrategies,whichrequireiden-
ticationofsuitablemodelsubstratesforscreeningmutantenzymesin
variantlibraries.
CrosscuttingTools,Technologies,andScience
Computationalscience,advancedimaging,andhigh-throughputgenomics
toolsmustbeincorporatedtoattaintheobjectivesdescribedhere.Com-
putationalscienceisimportantincoordinatingprocessengineeringand
inexploitingtheproductsofproteinengineering.Structuralimagingof
enzymes,substrates,andenzyme-substratecomplexeswillbeneededto
understandenzymefunctionatthemolecularlevel;alibraryofmodelsub-
stratesforthesekineticandmechanisticstudiesisrequired.
Abetterunderstandingofthestructureandcompositionofspecicxylans
andligninswouldgreatlyimprovetheabilitytodesignoptimalenzyme
mixes.Methodstomonitortheprocessofpolysaccharidehydrolysis(e.g.,
NMRandmassspectroscopy)wouldbebenecial. Transgenicplantsand
treeswithreducedlignincontentsormodiedlignincongurationscould
exhibitdierencesinlignin-depolymerizationrate;understandingenzymatic
mechanismswouldbecrucialinmanipulatingconditionstothebesteect.
Advancedimaging,spectroscopic,andenzymaticmethodsofcharacter-
izingcellwallsbeforeandafterpretreatmentalsorequirenewresourcesfor
producinganddisseminatingchemicalstandards.Tesenewstandardswill
berepresentativeoftherangeofplantcell-wallchemistryandwillpermit
improvedcross-comparisonresearch,notonlyonpolysaccharidedisassem-
blybutalsoonsubsequentsugarmetabolism.
UnderstandingtheMolecularMachinery
UnderpinningCelluloseSaccharifcation:
CellulasesandCellulosomes
Withnewbiologytools,wehavetheopportunitytounderstandenzyme
structure-functionrelationshipsthatgovernthecriticalprocessesofbio-
ethanolproduction.Giventheimportanceofalternativerenewableenergy
sources,theprospectofengineeringimprovedcellulasesisanexcitingand
universallyappealingconcept.Cellulosomes,largemolecularmachinesthat
integratenumerousenzymesandfunctionstobreakdownbiomass,arekey
toreducingtheenzymeloadingrequiredforprocessing.Understanding
cellulosomesandlearninghowtomanipulateandmodifythemforgreater
eciencywillbeimportantinconsolidatedprocessing.Researchrstwill
allowunderstandingandthenimprovementintheperformanceofbothfree
andcomplexed(cellulosomal)cellulasesonbiofeedstocks.Terate-limiting
stepinhydrolysisisnotcatalyticcleavagebutdisruptionofasinglesubstrate
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 97






































DECONSTRUCTING FEEDSTOCKS
chainfromitsnativematrix,therebyrenderingitaccessibletothecatalyti-
callyactivecellulasesite.Tus,wemustbeabletoanalyzeandunderstand
theprocessesandinteractionsthatfacilitatethisdisruptionofinsoluble
cellulose. Toapproachthisprobleminasystematicandrationalfashion,
detailedunderstandingofthestructureandenergeticsofboththemicro-
crystallineandnoncrystallineportionsofcellulosebrilsisrstnecessary.
Howdosolubleenzymesactonaninsolublecrystallinesubstrate?
Hydrolysisofcrystallinecelluloseistherate-limitingstepinbiomass
conversiontoethanolbecauseaqueousenzymesolutionshavediculty
actingonthisinsoluble,highlyorderedstructure.Cellulosemoleculesin
theircrystallineformarepackedsotightlythatenzymesandevensmall
moleculessuchaswaterareunabletopermeatethestructure.
Howdodierentbiomass-degradingenzymesworktogetherasasyner-
gisticsystem?Cellulasesandhemicellulasesaresecretedfromcellsas
freeenzymesorasextracellularcellulosomes.Tecollectiveactivityof
enzymesystemsisbelievedtobemuchmoreecientthantheindivid-
ualactivityofanyisolatedenzyme;therefore,totrulyunderstandhow
enzymesfunction,theymustbestudiedassystemsratherthanindividu-
allyorafewatatime.Inaddition,systemseventuallymustbeanalyzed
underlaboratoryconditionsmorerepresentativeofreal-worldenviron-
ments.Forexample,laboratoriesoftenusepuriedcelluloseasthe
substrateforenzymeanalysisratherthanmoreheterogeneous,natural
lignocellulosicmaterials;thiscanprovideerroneousconclusionsabout
naturalenzymeactivity.Newanalyticalmethodsthatarespatiallyand
chemicallysensitivewillallowrealisticunderstandingofthemechanisms
ofbiomassdegradation.
ResearchGoals
DiscoveringandImprovingFreeCellulases
Newgenerationsofengineeredcellulaseswillprovideenhancedperfor-
mance(activity)requiringlowerproteinloadings,soprocesscostwillbe
reduced.Cellulasecostcurrentlyisestimatedat10to25centspergallon
ofethanolproduced.Tenewtargetwillbesimilartostarchhydrolysis,
or1to2centspergallonofethanol.Forexample,loadingsofabout25-
mgfungalcellulaseproteinsarerequiredtohydrolyzeaboutonegram
ofcelluloseinpretreatedbiomass.Improvedcellulaseswouldconvert
anequivalentamountofcellulosetosugarsusingaroundone-tenththe
enzymeloading.Moreecientcellulasebiomassdigestionalsomayper-
mitreduced-severitythermochemicalpretreatment.
Acquiringnewscienticinsightsintocellulosestructureandthefunction
ofcellulasecocktailsisacriticalobjective(seesidebar,NewCellulase
EnzymesDramaticallyReduceCostsofPlantBiomassBreakdown,p.
100).Teinitialresearchphaseinvolvesanexhaustivesearchforexam-
plesofimportantfamiliesoffreecellulases(thosesecretedintotheextra-
cellularmixture)usinghigh-througputgenomicandenzyme-production
and-characterizationmethods.Celluloseandcell-wallinteractionwith
98 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

degradingenzymesmustbecharacterized.Biochemicalanalysisoffamily
memberswillrevealmuchaboutthenaturaldiversityofsolutionstocel-
lulaseaction.Combinedmutationalanalysisandcomputationalmodel-
ingthenwillbeusedtodenestructure-functionrelationshipsofthese
enzymesandnewlyengineeredmutants.Withthiskineticandthermo-
dynamicunderstandingasaguide,enzyme-engineeringapproacheswill
beusedtotestnovelhypothesesforimprovingcellulaseperformanceon
thecellwallsofplantsconsideredsuitablebiofeedstocks.
UnderstandingandUtilizingCellulosomes
Tecellulosome,auniquetypeofmolecularmachine,caneciently
solubilizenativeandpretreatedlignocellulosesubstrates.Tecellulo-
someservesasamoreecientwayofenzymeloadingandpresentation
tothesubstrate.Inthiscase,nativecellulosomesattachedtothemicrobe
containthefullrepertoireofplantcell-wallpolysaccharide-degrading
enzymes,andasinglecarbohydrate-bindingmodule(CBM)targetsthe
entirecomplementofenzymestothesubstratesurface(seesidebar,Te
Cellulosome,p.102).
Articialdesignercellulosomesthatexhibitaprecisearchitectureand
compositionwillhelprevealvariousprinciplesofcellulosomeconstruc-
tionandaction.Inthiscontext,
Structureandbiochemical-biophysicalaspectsofassortedcel-
lulosomalmodules(i.e.,cohesins,dockerins,CBMs,andcatalytic
domains)fromdierentsubunitsandspecieswillbeexamined.
Recombinantprimaryandadaptorchimericscaoldinswillbecon-
structed,designedtobeardivergentcohesinsforsubsequentincor-
porationofnativeorhybrid(dockerin-tagged)cellulosomalenzymes.
Troughthisapproach,improvedhigh-performance(noncellulo-
somal)cellulasescanbeincorporatedintoacellulosometotake
advantageofenhancedsynergisticpropertiesinherentinthecel-
lulosomecomplex.
Genesencodingforthecomplementofdesignercellulosomalcomponents
alsocanbeclonedintoasuitablehostcellsystemforheterologouspro-
duction,assembly,andsecretionofactivedesignercellulosomesofdesired
compositionandarchitecture.
Severalkeyscienticquestionsandissuesareespeciallyimportantfor
reachingtheproposedgoals.
Wedonotunderstandenoughaboutsubstratemicrocrystallinecel-
lulosesstructureasitexistsintheplantcellwall.Uncertaintiesabout
thecrystallatticestructureinthemicrobrilandthecrystalfacesof
cellulosetargetedbyenzymediversityinthebiosphereareexamplesof
ourlackofinsight.Understandingthesubstratesmoleculararchitec-
turewouldhelpinusingrationaldesigntoproduceimprovedhydro-
lyticmolecularmachines.Withsuchknowledgeitmightbepossible,
forexample,toselectspecicmutationsofcellulose-bindingdomains
thattendtodisruptbrilpackingortousemoleculardynamicsimu-
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 99

DECONSTRUCTING FEEDSTOCKS
lationstondenvironmentalconditions(temperature,pressure,and
molecularagents)thataectsuchdisruptionbeforethecellulase
attacksthesubstrate.
Tekineticandthermodynamicmechanismofprocessivecellulases
orexoglucanases(GHfamily7)isnotknown.Teseenzymes,protein
machines,conductmostcellulosehydrolysis,withendoglucanasesplay-
ingalesserrole.Tounderstandthismodeofcatalysisatthemolecular
level,amathematicalmodelofthefunctioningofGHfamily7cel-
NewCellulaseEnzymesDramaticallyReduceCostsofPlantBiomass
Breakdown:R&D100Award
Further Advances Needed to Improve Efciency and Economics
C
ellulaseenzymesareusedtobreakdown
thecelluloseofplantcellwallsintosimple
sugarsthatcanbetransformed(fermented)
bymicrobestofuels,primarilyethanol,aswellasto
chemicals,plastics,bers,detergents,pharmaceuti-
cals,andmanyotherproducts.
Likestarchandsugar,celluloseisacarbohydrate
(compoundofcarbon,hydrogen,andoxygen)made
upofsimplesugars(glucose)linkedtogetherin
longchainscalledpolysaccharides.Tesepolymers
formthestructuralportionofplantcellwalls,and
unravelingthemisthekeytoeconomicalethanol
fermentation.Technicalbarrierstolarge-scaleuse
ofcellulosetechnologyincludethelowspecic
enzymeactivityandhighenzyme-productioncosts,
aswellasagenerallackofunderstandingaboutenzymebiochemistryandmechanisticfundamentals.
In2004,theDOENationalRenewableEnergyLaboratory(NREL),workingwithtwoofthelargestindus-
trialenzymeproducers(GenencorInternationalandNovozymesBiotech),achievedadramaticreductionin
cellulaseenzymecosts.Cellulasesbelongtoagroupofenzymesknownasglycosylhydrolases,whichbreak
(hydrolyze)bondslinkingacarbohydratetoanothermolecule.Tenewtechnologyinvolvesacocktailofthree
typesofcellulasesendoglucanases,exoglucanases,andbeta-glucosidases.Teseenzymesworktogetherto
attackcellulosechains,pullingthemawayfromthecrystallinestructureandbreakingocellobiosemolecules
(twolinkedglucoseresidues),splittingthemintoindividualglucosemolecules,andmakingthemavailablefor
furtherprocessing.Tisbreakthroughworkresultedin20-to30-foldcostreductionandearnedNRELand
collaboratorsanR&D100Award(pressrelease:www.nrel.gov/awards/2004hrvtd.html).
Furthercostreductionsarerequired,however,tosupportaneconomicalandrobustcellulosebioreneryindus-
try.Forexample,costsofamylaseenzymesforconvertingcorngrainstarchtoethanolareabout1to2cents
pergallonofethanolproduced,butthemostoptimisticcostestimatesforcellulasepreparationsnowareabout
tenfoldhigherthanthat.Routestoimprovingenzymeecienciesincludethedevelopmentofenzymeswith
moreheattoleranceandhigherspecicactivities,bettermatchingofenzymesandplantcell-wallpolymers,
anddevelopmentofhigh-solidenzymatichydrolysistolowercapitalcosts.Acomprehensiveunderstandingof
thestructureandfunctionoftheseenzymaticproteinmachines,howtheirproductionandactivityarecon-
trolled,andchangestheypromoteonplantcell-wallsurfaceswillbecriticalforsuccess.
(Alsoseesidebar,ImageAnalysisofBioenergyPlantCellSurfacesattheOBPBiomassSurfaceCharacter-
izationLab,p.40.)
CellulaseimagefromM.Himmeletal.,CellulaseAnima-
tion,runtime11min.,NationalRenewableEnergyLabora-
tory(2000).
100 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

lulasemustbedevelopedandtested.Finally,therangeofpermissible
modicationsofthesespecicproteinfoldsisnotknown,norare
constraintsimposedbythefairlylimitedsetofindustrialhostorgan-
ismsonexpressionofwild-typemembersofGHfamiliesornewly
engineeredmutants.
Temajorobjectiveofthedesigner-cellulosomeconceptistorecon-
structimprovedcellulosomesbylinkingessentialandmostecient
enzymestodesiredsubstrates.Numerousscienticissuesandopportu-
nitiesarestillatlarge.
Ourcapacitytocontrolthenaldesigner-cellulosomescomposition
andarchitecturalarrangementwillenableustoposenewhypotheses
regardingenhancedcellulosomeperformanceindegradationofplant
cell-wallmaterial.
Tebestsetofcellulosomalcellulasesmostappropriate(synergistic)
foruseonlignocellulosicsubstratesisstillunknown.
Althoughhybridenzymesderivedfromnoncellulosomalspeciescan
beincorporatedintodesignercellulosomes,whethertheirobserved
synergisticactivityiscomparabletothatofnativecellulosomal
enzymesisstillunknown.
Manyotherscienticquestionshavetobeaddressed,includingthe
optimalstoichiometryandarrangementofcomponentsandwhether
thecombinedactionofcellulosomalandnoncellulosomalsystems
resultsinanadditionalimprovementinperformance.Tisresearch
oersauniqueopportunitytoexploretheexpandinguniverseof
molecularmachinesthatcanbeconstructedbythedesigner-cellulo-
someapproach.
TechnicalMilestones
Within5years
Near-termaccomplishmentsincellulasebiochemistryshouldfocuson
improvingtheenzymes,primarilyfromfungiandactinomycetes,used
inindustrialpreparationsmarketedforthebiorenery.Tisaccom-
plishmentwouldrequiresearchingfornewenzymesfromnewsources
(includingmetagenomicdatabases)andusingdirectedevolutionto
probemutationalspaceforpossibleimprovementsoverwild-type
examples.Teoutcomewouldbebettercocktailsbasedonwild-type
enzymesandenzymesimprovedbynoninformationalmethodsthat
couldbeusedimmediatelybyindustry.
Constructionofdesignercellulosomeswillconcentrateonseveralfronts,
includingincorporationofcurrentlyavailableenzymes;developmentof
novelcellulosomalcomponentsscaoldins,cohesins,dockerins,CBMs,
andlinkersegments;improvementofhybridenzymesandassessment
oftheirenhancedsynergisticactionwithindesignercellulosomes;and
analysisofthecombinedactionofdesignercellulosomeswithhighly
activenoncellulosomalenzymes,includingligninasesandhemicellulases.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 101

DECONSTRUCTING FEEDSTOCKS
Within10years
Temid-termscopepermitsamoresystematicapproachtocellulase
andcellulosomebiochemistry.Suchunderstandingwillrequirecombin-
ingclassicalbiochemistrywithcomputationalscience.
Aparticularlyimportantgoalisidentifyingthermodynamiclimitations
ofimprovement,whichwouldimpactproteinengineering.
Initialstudiestoconvertsuitablehost-cellmicroorganismsintocel-
lulosome-producingstrainswillbepursued.Tedesiredresultwillbe
improvedbiomassdegradationandincreasedunderstandingofthe
structure-functionrelationshipofcellulosomecomponents.
Toachieveheterologousproduction,assembly,andsecretionofactive
designercellulosomes,suitablehostcellsthatcanaccommodatethe
genesforsuchlargeproteinsmustbeidentiedandinherentcloning
andexpressionbarriersovercome.
TheCellulosome:TheSwissArmyKnifeofMolecularMachines
T
hecellulosomeisanextracellularsupramolecularmachinesynthesizedbysomeanaerobicmicroorgan-
ismscapableofdegradingcrystallinecelluloseandotherplantcell-wallpolysaccharides.Eachcel-
lulosomecontainsmanydierentcomplementarytypesofcarbohydrate-activeenzymes,including
cellulases,hemicellulases,andcarbohydrateesterasesthatareheldtogetherbyascaoldinproteintoform
asinglemultienzymecomplex(seeFig.A.SchematicofaCellulosome,below).Tecellulosomeenhances
cell-walldegradationbybringingseveraldierentenzymesintocloseproximitysotheycanworktogetherto
exploitenzyme-accessibleregionsofcellulose.Tevariousproductintermediatesfromoneenzymaticsubunit
canbetransferredreadilytootherenzymaticsubunitsforfurtherhydrolysis(breakdown)ofthecellulose.Te
cellulosomealsopromotescelladhesiontotheinsolublecellulosesubstrate,thusprovidingindividualmicrobial
cellswithadirectcompetitiveadvantageinusingsolublesugarsproducedinthehydrolysis.Cellulosomesneed
notbeassociatedwithcellsforactivity,andtheyfunctionunderbothaerobicandanaerobicconditions.
Eachenzymaticsubunitcontainsadenitivecatalyticmoduleandadockerindomainthatbindstightlytoa
scaoldincohesin.Tus,cohe-
sin-dockerininteractiongoverns
assemblyofthecomplex,while
cellulosomeinteractionwith
Fig.A.SchematicofaCellulosome.
Tescaoldinsubunit,towhichthe
cellulosomalenzymaticsubunits
arebound,ismadeupofdierent
functionalmodulesthatdictateits
architectureandvariousactivities.
TeseincludeasingleCBMand
nineverysimilarrepeatingdomains
calledcohesins,whichbindtheenzy-
maticsubunits.
102 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Within15years
HTPapproachesandcrystallographicandmicroscopictechniqueswill
beusedtoelucidatethethree-dimensionalstructureofcellulosomal
components,theintactnativeanddesignercellulosomes,andtheir
actionwithpure(cellulose)andnative(plantcell-wall)substrates.
Understandingmechanisticprinciplesoftheentireaccessiblerangeof
glycosidehydrolasesandtheirfunctioninthebiospherewillbeevalu-
ated.Tediversityofcellulaseandcellulosomefunctionalschemeswill
bemodeledandoptimizedforspecicbiomasssubstrates(feedstocks).
Tisimprovedunderstandingofcellulaseactionwillprovidenewsac-
charicationparadigmsforthebiorenery.
CrosscuttingTools,Technologies,andScience
Computationalscience,advancedimaging,andHTPgenomictoolsmust
beincorporatedtoattaintheobjectivesdescribedhere.
celluloseismediatedbyscaoldin-bornecellulose-bindingmolecules(CBM).Somescaoldinsalsobeara
divergenttypeofdockerinthatinteractswithamatchingcohesinonananchoringprotein,therebymediating
cellulosomeattachmenttothecellsurface.
TeLEGO-likearrangementofcellulosomalmodulesoersanexcellentopportunitytoengineernewmulti-
enzymecomplexesfordesiredpurposes.Tevariouscohesins(c),dockerins(d),andcatalyticmodules(A,B,
C)arefunctionallyindependentandcanbetetheredtogetherinanycombinationviarecombinantgenetics.
Teresultingcomplexcanbeappliedeitherinthetesttube(bioreactors)orinacellularsetting(fermentors).
Nativescaoldin-bornecohesinsincellulosome-producingbacteriagenerallyrecognizeallenzyme-bearing
dockerinsnonspecically. Tus, reconstructionofacellulosomefromitscomponentpartswouldresultin
randomincorporationofdierentenzymesintocellulosomecomplexes,yieldingaheterogeneouspopulation
ofarticialcellulosomes(Fig.B1.RandomIncorporation,below).
Tocontroltheincorporationofdesiredenzymesintoapreciseposition,distinctivecohesin-dockerinpairs
mustbeused(Fig.B2.ControlledIncorporation,below.).Dockerindomainsthatbindaspeciccohesin
mustbefusedtothedierent
catalyticmodules,andachi-
mericscaoldinisengineered
tocontaincomplementary
cohesinsandasingleCBMfor
substratetargeting.Subsequent
self-assemblyofthemature
designercellulosomecomplex
canthenoccurselectivelyin
vitro,resultinginahomoge-
neouspopulationofcellulo-
somecomplexes.
CellulosomeReferences:p.117
Fig.B1.RandomIncorporation. Fig.B2.ControlledIncorporation.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 103

DECONSTRUCTING FEEDSTOCKS
Computationalscienceisneededforunderstandingcellulasefunctionat
aholisticlevel,specically,newprogramsandcodestohandlemodelsof
ultralargebiologicalmoleculesandsystemsofmorethan1millionatoms.
Newimagingmethodsarecriticalforelucidatingcellulaseactiononthe
plantcellwall.
Finally,toolstoacquire,archive,andinterpretnewcellulasestructures
(bothnativeandmodied)areneededtobetterunderstandtheirnatural
structuralandfunctionaldiversity.
Wemustdevelopnewscienceandtechnicalmethodologiestoexplore
protein-machinefunctionatthemolecularandatomiclevelstoattainthese
objectives.Solutionphysicsandthermodynamicsrulingprotein-domainbio-
logicalfunctionatthenanometerscalearepoorlyunderstood.Inthisrespect,
theneweldofnanosciencemayprovideimportantinsights.Forexample,
describingthemechanismofaprocessivecellulaseatsuchlevelofdetailwill
requiremoreknowledgeaboutthecellulosesurface,water-protein-glycan
dynamics,andbiomolecularmechanicsthanispossibletoday.
TheRoleofGTLandOBPFacilitiesandCapabilities
ProteinProduction
Proteinproductioncapabilitieswillbecriticalforsupplyingsamplesof
wild-typeandrecombinantproteins,including(1)newenzymesfrom
newsources,(2)modiedenzymesfromdirectedevolution,(3)modied
enzymesfromsite-directedmutation,and(4)largequantitiesofenzymes
suitableforpilottesting.Tisresourcewillbecriticalinidentifyingthe
mosteectivenewhemicellulasesandligninaseswithoutrsthavingto
solvehigh-levelexpressionchallengesforallcandidates.
High-throughput(HTP)biochemicalandbiophysicalassayswillbedevel-
opedandcarriedout,andfunctionalannotationwillenrichtherst-pass
sequence-basedannotations.Tisproblemismoredicultandcomplex
forGH,inwhichasingleenzymecanhaveseveralcomparableactivities
ondierentpolysaccharidesubstrates.Tus,amicrochipHTPassayfacil-
ityisneeded.
MolecularMachine
Tesecapabilitiescanprovideanalyticalandcomputationalscienceto
studycellulaseprotein-machinefunctionandadvancedtechniquesforiso-
latingandreconstitutingcomplexinteractionswithinnativeorimproved
hemicellulaseandligninasesystems.Synergisticinteractorsincluding
chaperonins,excretionpaths,andcofactorswillbeisolatedandidentied
innovelcellulases,hemicellulases,andligninases.
Proteomics
Proteomiccapabilitiescanbeusedtodocumenthowsecretomesof
selectedwhiterotfungiandotherorganismssuchasmicroorganismsin
thetermitehindgutrespondtochangingcultureconditionsandoptimize
biodegradationratesoflignocellulosiccomponents.Correlationsbetween
104 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

ligninaseactivityandconcentrationsofenzymesresponsibleforcosub-
strateproductionwillbeofparticularinterestandwillassistresearchersin
designingmoreeectiveexperimentalplansforimprovingtheenergetics
andcarbon-allocationeciencyofcell-walldegradation.Tisalsowillbe
importantinmonitoringandcontrollingtheeectsofsubstrateinducers
andresponsestoheterologousexpression,especiallyofthemostforeign
components.Tisresourcecansupportstudiesaimedatimprovingproduc-
tionofligninaseandhemicellulaseproteinsfrombothnear-termhosts
suchasT.reeseiandlonger-termsystemsincludingbacteriaandyeast.
CellularSystem
Cellularsystemcapabilitieswillvisualizecellulases,ligninases,cellulosomes,
andothermolecularspeciesdirectlyonsubstratesinrealtimeundervary-
ingconditionsandfromvaryingspecies.Modelswillbedevelopedto
optimizehydrolysisunderdierentconditions.
DOEJointGenomeInstitute
DOEJGIcouldembarkonsequencingnewwhiterotfungalgenomes,
includingthosethatdonotencodeligninandmanganese-dependent
peroxidases.Tisworkmayleadtothediscoveryofmore-activeandless-
labileligninasevariants. Afterthat, morebiologicaldiversityfromsuch
bacterialpopulationsasdecayingbiomass,termitehindgut,ruminants,
andtherhizospherecouldbeexamined.
Metagenomics
Metagenomicapproacheswillidentifymechanismsandagentsofeective
hemicelluloseandligninconversionincommunalorunculturablepopula-
tions.Teyalsocanplayakeyroleinsequencingnewgenomesthought
toharborcellulolyticcomponentsacquiredbylateralgenetransferandin
studyingnaturaladaptationsthatallow(initially)heterologousexpression.
HarvestingtheBiochemicalPotential
ofMicroorganismsThroughMetagenomics
Teconceptualfoundationformetagenomicsistherealizationthatmore
than99%ofmicrobesinmosthabitatshavenotyetbeencultured.By
providingdeepinsightintobiologicalcapabilitiesandfunctionwithoutthe
needforculturing,metagenomicseectivelyexpandsourscienticcapabil-
itiesandunderstandingbeyondthesmallpercentageofbacteriathatcur-
rentlycanbecultivatedinthelaboratory.Itfurtherpermitsamoreholistic
andmechanisticanalysisofmicrobialcommunities.Naturalsystemslong
agolearnedhowtoattacklignocelluloseandusetheresultantsugarsand
otherchemicalsfortheirownpurposes.Temetagenomesofcomplex
naturalcommunitiesprovideafertileresourcefordataminingtosearchfor
newexamplesofrelevantenzymes.Teabilitytoconstructandevaluatean
engineeredmetagenome(fromexistinggenomesequences)nowenables
creationofsystemswithsuperiorcapabilities.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 105

DECONSTRUCTING FEEDSTOCKS
Tisresearchhastwoobjectives:(1)employgeneticandbiochemical
studiestocharacterizenovellignocellulose-degradingsystemsand(2)
usemetagenomicstoaugmenttheseeortsbycharacterizingandrecov-
eringthegeneticpotential(includingthatfromunculturedmicrobes)
residentinmicrobialcommunitiescapableofrapidandextensivebiomass
degradation.Outcomesofthesefunctionalandcomparativestudieswill
includearepertoireofnewenzymesandproteinsavailableforengineering
approaches(e.g.,designercellulosomesorfreecellulasesystems).Inthat
context,knowledgeandbioresourcesarisingfromthisresearcharewell
integratedwithintheoverallgoalofimprovingplantcell-walldeconstruc-
tionandconversionoflignocellulosicproductstobioethanol.
ResearchGoals
Toelucidatetherepertoireofgeneproductsnecessarytoeectmorerapid
andextensivehydrolysis(andsolubilization)oflignocellulosicmaterials,
systematicadvancesinourunderstandingofmicrobe-mediatedplantcell-
walldeconstructionmustbeachieved.First,manysequencedmicrobial
genomesencodemoreglycosidehydrolasesforplantcell-walldeconstruc-
tionthanhavebeenidentiedthroughhistoricalbiochemicalandgenetic
studies.Whetherornotthesemultigenefamiliesareanevolutionary
adaptationtoprovidemoretotalactivitydiversity,tocompensatefor
subtletiesinsubstrateconformationandcomposition,oracombination
ofbothisstillunknown.Inaddition,degradativesynergisminmicrobial
communitiesoftenresidesintheconcertedactionsofenzymesfrommul-
tipleorganisms,butmechanisticdetailsandunderstandingofthisconcept
areundeveloped.
Second,aerobicandanaerobicbacteriawithsuperiordegradationcapa-
bilitiesrecentlyhavebeenfoundtolackcanonicalprocessivecellulases
requiredinallotherwell-describedsystemsforsubstratesolubilization.
Tegeneticsandbiochemistryunderpinningtheirlignocellulosedegrada-
tion,whichcouldrepresentanewmechanism,mustbeexplored.
TechnicalMilestones
GTLwillacceleratethedevelopmentofoptimalcellulasesystemsbypro-
vidingresourcesforscreeningthousandsofnaturalandmodiedenzyme
variants,enablingtheHTPproductionandfunctionalanalysisofthese
enzymes,elucidatingregulatorycontrolsandessentialmolecularinterac-
tions,andgeneratingmodelsforanalyzingthestructureandactivityof
naturalandengineeredenzymesystems.
Within5years
Undertakemicrobiomeprojectsforfunctionalandcomparativepur-
poses.TeJGICommunitySequencingProgram,supportedbyDOE,is
nowcharacterizingthemicrobiomeofthetermitehindgut.
Initiatemechanisticcharacterization(atDNA,protein,andorganismal
levels)ofnofewerthanthreenaturalmicrobialcommunitiesdisplaying
rapidandextensivelignocellulosedegradation.Eachshouldbedierent
106 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

intermsofprevailingphysicochemicalconditions(e.g.,temperature,pH,
andsaltconcentration)ordominantfeedstock.Forexample:
SoilsrepresentativeofdominantresiduesintheDOE-USDABil-
lion-Tonstudy.Cornstover,rice,switchgrass,andforestresidues
wouldbeappropriate.
Decayingbiomass(seethesidebar,ExamplesofMetagenomic
Analysis:UnderstandingtheDynamicsofMicrobialColonizationof
DecayingBiomass,p.108).
Determinethemechanismsoflignocellulosesolubilizationbysuch
knownorganismsasF.succinogenesandC.hutchinsoniitohelpguide
metagenomicanalyses,usingacombinationofbiochemicaland
genomicapproaches(seesidebar,SequencingaSoil-CelluloseDegrader,
thispage).
Developheterologousexpressionsystemsforcandidateenzymesand
regulatoryproteins,includingnecessarycofactorsandpost-translational
modications.
BeginHTPproduction,characterization,andintercomparisonofenzy-
maticsystemsdiscoveredinmetagenomicanalyses.Produceappropriate
moleculartagstosupportexperimentationinnaturalandcontrolled
systemsincludingimaging,proteinisolation,anddetectionofinterac-
tions.
Within10years
Completecomprehensiveanalysesofenzymaticsystemstoisolatedif-
ferentfamiliesandhighperformers.
Rationallymodify,express,andcharacterize
nativeenzymestounderstanddesignprinciples
T
andoptimizeproperties.
SequencingaSoil-CelluloseDegrader
heDOEJointGenomeInstituteissequenc-
Buildcomputationalmodelsofbiomass-decay
microbialcommunities,andtestagainstexperi-
ingCytophagahutchinsonii,anaerobicGram-
mentaldata.
negativebacteriumcommonlyfoundinsoil
thatrapidlydigestscrystallinecellulose.Molecular
Within15years analysisofcellulosedegradationbyC.hutchinsoniiis
Deployinnovativeprocessesbasedonthesedis-
nowfeasible,sincetechniquesforgeneticallymanipu-
coveriesandbioresourcestoreduceenzymecosts
latingtheorganismrecentlyhavebeendeveloped.
andloadingbytenfold.Providenewoptionsthat
Tismicrobeexhibitstheabilitytomoverapidlyover
reduceoreliminatetheneedfornonbiological
surfacesbyaprocessknownasglidingmotility,which
feedstockpretreatments.
isthoughttobeimportantinallowingC.hutchinsonii
tocolonizeitsinsolublegrowthsubstrate.Temecha-
TheRoleofGTLandOBPFacilitiesand
nismofglidingmotilityisnotknown,butagellaare
notinvolved.AnalysisoftheC.hutchinsoniigenome
Capabilities
sequencewillfacilitatestudiesofcellulosedegrada-
Inbrief,thefacilitieswillsupportandenable
tionandalsowillrevealmoreaboutbacterialgliding
theattainmentoftheseobjectives.Forinstance,
motility,whichhasremainedanunsolvedbiological
JGIalreadyhascontributedafoundationalset
mysteryforover100years(http://genome.jgi-psf.
ofgenomesequencesforspecialistcellulolytic
org/nished_microbes/cythu/cythu.home.html).
microbes;thisdatasetisaugmentedbygenomes
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 107

DECONSTRUCTING FEEDSTOCKS
ExampleofMetagenomicAnalysis:
UnderstandingtheDynamicsofMicrobial
ColonizationofDecayingBiomass
A
sprimarydecomposers,microbialcommunities
haveevolvedbothascompetitorsandcollabora-
torsinbiomassdeconstruction.Teultimateaim
ofthisresearchistoachievebioprocessesforallstepsin
convertingbiomasstoethanol.Acriticalneedistoreplace
plantbiomassthermochemicalpretreatment,whichisnow
necessarytoconvertrecalcitrantstructurallignoelluloseto
aforminwhichcelluloseismoreaccessibleandamenable
tohydrolyticenzymeaction.Byanalyzingnaturalcom-
munitiesthatcolonizedecayingbiomass,wecanascertain
naturalmechanismsthatcanbeusedtosupplantthermo-
chemicaltreatments.Tegoalistobetterunderstandthe
complexmicrobialcommunitiesresponsibleforlignocel-
lulosicbiomassdeconstruction,harvestkeybiochemical
decaymechanisms,anddeveloppredictivemodelingand
controlofthesecomplexnaturalprocesses.
Metagenomicswillbeusedtodeterminemicrobial-com-
munitycompositionandgeneticdiversity.Comparative
genomictoolswillinitiallydeterminecommunityfunc-
tionalityandidentifygenesofunknownfunction.Coupled
spatialandtemporalmeasurementswillrevealprinciples
ofcommunityformationanddynamics,processessuch
assignaling,andothermicrobialinteractions.Functional
annotationofunknownproteinsthroughproteinproduc-
tionandcharacterizationandotherexperimentationwill
augmentinitialgene-functionassignment.
HTPmethodsfromfacilityandothercapabilitieswillbe
usedformeasuringenzymeactivities:
Determinecellularandbiochemicalfunctionsofgenes
discoveredinunculturedcommunitymembers.
Characterizethetemporalcompositionandfunctional
capabilityofmicrobialcommunitiesWhoisthere,
andwhatmetabolicprocessesarebeingcarriedout?
Characterizeexpressionpatternsofcellulolyticenzymes
andrelatedprocessesandpathwaysusingtranscrip-
tomics,proteomics,andmetabolomics.
Computationaltoolswillpredictthemetabolic,physio-
logic,andbehavioralcharacteristicsofmicrobialcom-
munitiesfromcommunityDNAsequencedata,and
supportingmeasurementswillbedeveloped.Tetools
willallowdesignandengineeringofmicrobialsystems
thatultimatelycanperformallstepsinbiomassprocess-
ingtoethanol.
sequencedatTeInstituteforGenomicResearch
(calledTIGR).Otherresourcescanreadilysup-
porthypothesis-drivenresearchattheRNA,
protein,organismal,andcommunitylevels(see
sidebar,ExampleofMetagenomicAnalysis,this
page). Tisresearchwillvalidateandproducethe
desiredcombinationofproteinsandotherbiolog-
icsnecessarytomakelignocelluloseconversion
comparabletostarchasafeedstockineconomic
andprocesscriteria.
CharacterizingCellWallsUsing
High-ThroughputMethods
Understandingthechemicalnatureandarchi-
tectureofcellwallsatthenano-,micro-,mac-
rochemical,andphysicalscales,aswellastheir
behaviorinpretreatmentandfermentation,is
essentialintakingasystems-levelapproachto
modifyingtheplantgenomeorincreasingthe
biofuelsystemseciency.Knowledgeandinsight
gainedfromHTPcell-wallcharacterization
willdriveseveralareasofbiomass-to-biofuels
research.Teseareasincludeselectingandmodi-
fyingplants,matchingthemicrobialconversion
processtothesubstratespresent,andminimiz-
inginhibitorsduringbiologicalconversionto
valuableproducts.Manynewinstrumentsand
analyticalmethodswillbeneededtocharacterize
arangeofbiomassfeedstocks(e.g.,grasses,crop
residues,short-rotationwoods,andearlyenergy
crops)neededtoachievetheBillion-Tonvision.
Ultimately,manydierenttypesoffeedstocks
willbeatdierentstagesofharvestandhence
willneedtobeanalyzedrapidly.Terobustness
ofthesemethodsandtheirversatilityinaddress-
ingthecompositionofmanydierentmateri-
alsrequiressignicantfurtherresearch.Data
generatedbythesemethodsareusedtovalue
feedstocks,measureconversioneciency,identify
regulatoryissues,andultimatelyestablishproduct
pricingandinvestmentrisk.
Researchmustaddressthedisconnectbetween
thethroughputofgenomicandproteomicanaly-
sisandthatofbiomasschemicalandstructural
characterization.DNAsequencingandmetabolic
prolingareroutinelydoneatthemicroliterscale,
where1000to2000samplescanbeprocessedand
108 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy














































analyzedinoneday.Mostbiomassanalysisrequireshundredsofmilligrams
persampleandveryfewhavebeenautomated,sothroughputisaround20
samplesperweekperperson.Recentadvancementsinbiomassanalysishave
demonstratedhighthroughputusingmultivariateanalysis(MVA)coupled
with analyticalpyrolysis, FTIR, or near infraredspectroscopytoallowchar-
acterizationof200to500samplesperdayperperson. Tesespectroscopic
methods,however,requirecalibrationandvalidationusingslowwetchemi-
calprocedures.MVAoftenisspecictofeedstockandprocess;forexample,
amethoddevelopedforhardwoodanalysiswontaccuratelycharacterizea
softwoodorgrasssample.Tosupportthebroadgoalsofthisroadmap,many
newanalyticalmethodswillneedtobedeveloped.
Researchwillfocusontwoareas:Improvingthroughputoftraditional
calibrationmethodstoaround500samplesperweekandusingthse
datatocalibratenext-generationcapabilitiesofanalyzing1000ormore
samplesperday.Strategiesforimprovingtraditionalmethodswillrequire
theadaptationofanalyticalinstrumentsforbiomassapplicationsand
developmentofnewinstrumentsspecicallyforthiswork.Forexample,
multiplexingroboticsamplepreparationandmultichannelcapillaryelectro-
phoresisincarbohydrateanalysispotentiallycouldoeratenfoldincrease
insamplethroughput,butsuchinstrumentsandmethodsarenotvalidated
forbiomasssamples.Newmethodsandinstrumentswillgeneratedataon
molecular- andgenetic-levelrelationshipsneededto(1)improvefeedstocks
byincreasingbiomassproductionandmakingfeedstocksmoreamenable
tothenextgenerationofbiomass-conversiontechnologies,and(2)develop
datasetsthatenablethedesignofmicrobialcellconversionstrategiesthat
willproducedesiredproductsinhighyieldandpurity.Aspecialcategory
ofanalyticalmethodswithhighsensitivityalsowillbeneededtointerpret
high-resolutionimagesoffeedstocksandprocesssubstrates.
Tecomplexityofthebiomassmatrixpresentssignicantanalyticalchal-
lengesnotfacedinconventionalgenomicswork.Chemicalcharacteriza-
tionmethodswillhavetoassesstherelativeweightpercentofabout14
constituentsineachfeedstock.Structuralconstituentsofpotentialinterest
areprotein,lignin,ash,glucan(cellulose),xylan,mannan,arabinan,galac-
tan,glucuronicacid,ferulicacid,acetylgroups,starch,andgalacturonic
acids(pectin).Somenonstructuralmaterials,whichwillbespecictoeach
feedstock,alsomaybeofinterestinsaccharicationandfermentationstud-
ies.Teseconstituentsincludeinorganicsalts,sucrose,tars,waxes,gums,
lignans,andothers.Analyticalmethodsalsowillbeneededtotrackthe
constituentsandtheirreactionproductsthroughpretreatment,saccharica-
tion,andfermentation.Structuralcharacterizationmethodsmustquantify
functionalgroupsofinterest(e.g.,carboxylicacids,ketones,aldehydes,
esters,andmethoxylandfree-phenolichydroxylgroups).Withtherequire-
mentforsomanymeasurements,aportfolioofcomplementarymethods
willbeneededtoclosemassbalanceoffeedstockandprocesscomponents
acrossmultipleprocessingsteps.
Tisresearchprogramaddressesacriticalcomponentinthefeedstock-
sugarsinterfacebyprovidinganalyticalinstrumentsandmethodswiththe
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 109

































DECONSTRUCTING FEEDSTOCKS
precision,accuracy,andthroughputrequiredtooptimizebiomassselection
anddevelopment,biomasspretreatment,andconversionprocesseslead-
ingtoeconomicalethanolproductioninbioreneries.Biomass-relevant
analyticaltoolsofthistypedonotnowexistfortheseapplications.
Trackingindividualbiomasscomponentsfromfeedstocktoproducts
requireshigh-accuracydataobtainedinanintegratedandconsistent
fashion.Aresearchprogramfocusedonanalyticalmethodsthatcoordinate
datathroughallstageswillimproveprocessintegrationthroughconsistent
dataonfeedstocks,substrates,processinhibitors,andproducts.Integrated
datawillfurnishasystems-levelunderstandingofprocessstreamsand
enablethedesiredcorrelationsbetweenreactivityandgenomeexpression.
Toenablefuturedevelopmentofhigh-energybiomassfeedstocks,method
portfoliosneedtobestandardized;validatedinaconsensusenvironment;
andpublishedinaforumavailabletoplantbreeders,eldscientists,proc-
essengineers,enzymescientists,andfermentationscientists.Technologies
willprovidedataforidentifyingrelationshipsamongplantcell-wallcom-
ponentsandinteractionsamongbiomass-derivedsubstrates,microbes,
andenzymes.Althoughprogresshasbeenmadeinchemicalprocedures
forbiomassanalyses,existingportfoliosofmethodsarelaborintensive,
timeconsuming,expensive,andgenerallynotamenabletotheHTPneeds
ofbreeders,agronomists,feedstockprocessors,andsystemsapproachesto
fermentation.Tobypassthislimitation,HTPtechniquesneedtomeasure
theamountsandstructureofcell-wallcomponentsinprocesses.Correla-
tionsamongprocessdataandgenomicandproteomicdatawillenable
identicationofgenesmostimportantforimprovedbiomassconversion.
Forexample,morethan225,000independentT-DNAinsertionlinesof
Arabidopsishavebeencreatedthatrepresentalmosttheentiregenome
space.HTPanalysismustbeabletoscreentheselargesamplesets,as
wellasthemuchlargernumberofsamplesproducedforotherspecies
withmorecomplicatedgenomes,todeterminetheroleeachgeneplaysin
dierentcell-wallchemistryphenotypes.HTPmethodologiesalsowill
enablescreeningofplant,enzyme,andmicrobeconsortiatoallowfor
deeperunderstandingofdierentplantandmicrobegenomeinteractions.
High-sensitivitymethodswillintegratewithimagingtoolstoenable
spatialcompositionalandfunctionalgroupdeterminationsinindividual
plantcells.Tesestudiesrequiretheanalysisofhundredsifnotthousands
ofsamplesandwouldnotbepossiblewithoutthecostandtimesaving
providedbyrapidbiomass-analysismethods.
TechnicalMilestones
Within5years
Fornear-termdeployment,portfoliosofpreciseandaccurateanalytical
methodsforawiderangeofstructuralandnonstructuralconstituentsand
biomassfunctiongroupswillbedevelopedandvalidatedinaconsensus
modeandpublishedforgeneraluse.Specictechniquesandmethodswill
betailoredtoprovidecomprehensivecharacterizationofbiomassmodel
110 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

substrates(feedstocks).Workingcloselywithcommercialsuppliers,inves-
tigatorswillcreateinstrumentsandtechniquesspecictotheneedsof
biomass-conversiongenomics.Dataobtainedthroughcustomizedanalytical
procedureswillbeusedtocalibrateandvalidaterapidMVAmethodologies
forHTPscreeningoffeedstocksandbiomass-derivedresearchsamples.
Tisscreeningwillhelptoselectsamplesofinterestfromlargepopulations
representingspecieswhosegenomesequencehasbeendetermined.Selec-
tionwillbebasedonidentiabledierencesinphenotypesofmajorplant
cell-wallbiopolymers(lignin,cellulose,hemicellulose,andothers)important
forconversiontofuelsandvaluablechemicals.Datageneratedwillbeused
tosupportthebreedinganddevelopmentofnewplantlines.Inconjunction
withHTPmethods,information-technologystrategieswillcapture,ana-
lyze,manage,anddisseminateresultingdata,enablingcreationofanational
resourceforthebiofuelandplantbiologyresearchcommunity. Totheextent
possible,samplesanddatausedincalibratingnewmethodswillbearchived
safelyforfutureapplications.Archivedsamplesmayberequiredforbench-
marking,newmethoddevelopment,andcrossplatformcomparisons.
Within10years
HTPmethodologieswillbeintegratedwithotherstudiestoprovidea
systems-levelunderstandingofhowtoalterandimproveplantcell-wall
compositionandstructureforecientandeconomicbiofuelproduction.
High-resolution,molecular-specicimagesenabledbytheavailabilityof
moleculartagswillallowspatialdeterminationsofchemicalandstructural
featuresacrossindividualcelltypesandstructures.
Within15years
DeploymentofHTPandhigh-sensitivityanalyticalmethodsandappro-
priatedata-reduction(informatic)techniqueswillbecompletedtointe-
grategenomicandproteomicdatawithcell-wallchemistryinformation.
Asystems-levelunderstandingofinteractionsbetweenplantcell-wall
structureandthemicrobesandenzymesisneededtoconvertbiomass
eectivelytofuelsandchemicals.
TheRoleofGTLandOBPFacilitiesandCapabilities
ManyunderlyingprinciplesandHTPtechniques(e.g.,robotics,informatics,
andpatternrecognition)forHTPchemicalanalysisofplantshavebeenor
arebeingdevelopedbythegenomicandmetabolomiccommunities.Col-
laborationswithresearchersusingtheresourcewillrequiretheadaptation
andvalidationofthesetechniquesforbiomass-conversionapplications.We
expecttechnologiesdevelopedforplants(e.g.,high-sensitivityspatialdetec-
tionofbiopolymers)tohaveapplicationsforstudyingotherorganisms.
Informationobtainedfromtheseanalyticalmethodologieswillfacilitatethe
determinationofplant-genefunctioninsynthesisofplantcellwallsand
biopolymers,especiallygenesrelatedtocarbohydratemetabolism,polymeriza-
tion,andmodication.Analyticalmethodstailoredtobiomassapplications
willbeusedtoidentifyproteinfunctionrelatedtocell-wallconstruction.As
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 111

DECONSTRUCTING FEEDSTOCKS
additionalgenomesaresequenced,thesetoolscanbeusedtocharacterizegene
functioninotherplantlinesofinterestforbiofuelproduction.
CrosscuttingTools,Technologies,andScience
Manyanalyticalmethodsthatsupportasystemsapproachtobiomass
conversionwillrequirebiomass-relevantstandardsandtagsnotreadily
availablefromcommercialsources.Obtainingtheneededarrayofsmall-
moleculestandardswillrequireprep-scaleisolationofmoleculesofinter-
estandadvancedtechniquesofcarbohydrateandnaturalproductorganic
synthesis.Aswelearnmoreabouttheplantgenome,importantintermedi-
atemoleculesandbiomarkerswillberevealed.Techniquesforisolatingand
preparingrelevantbiomassstandardswillbetransferredtotheprivatesec-
torandmadeavailableforbiomassresearchandcommercialbioreneries.
Whilemuchexistingroboticinstrumentationanddata-reductiontechnolo-
giesareavailableforconstructingHTPmethodologies,severalareasmustbe
addressedtoachieveprojectgoals.Newtechniquesforhomogenizingand
reducingtheparticlesizeofbiomasssampleswithoutdegradationwillbe
neededtopreparebiomasssamplesforautomatedandroboticsystems.New
modules,equipment,andaccessoriesforexistinganalyticaltechniquessuch
ascolumnsforliquidchromatographywillhavetobeadaptedspecically
forbiomasssamples.Rapidbiomass-analysismethodswithlargedynamic
rangeswilldetectmultiplecompoundswithhighprecision,acceptableaccu-
racy,andshortacquisitiontimes.Becausethesemethodsareoftenspecicto
feedstocksandprocesses,manynewonesmayberequired.Integrateddata
fromdierentanalyticalmethodsintoasinglecomprehensiveMVAcalibra-
tionsetwillneedtestingandvalidation.
Data-reductionandstoragemethodswithpredictiveplatformsforproduc-
tionandprocessingpipelinesarenecessaryforretrievingandintegrating
datafrombreedingoreldstudies.Newhands-obioinformaticmeth-
odswillcombine,analyze,andcorrelatedata(includingmetabolomicand
genomicinformation)frommultiplesystems.Multivariateanalysistoolswill
beappliedwherepertinenttoinformationextractionfromcomplexsystems.
Appropriatebiomassanalysisisanecessaryrststepinapplyingsystems
biologytechniquestobioenergyproduction.Inmanycases,analytical
methodsforbiomasssubstratesthatdemonstratetherequiredprecision,
accuracy,andspeedarenotavailable.Analysisusingexistingmethodsistoo
slowandexpensiveforlarge-scalescreeningoutlinedinmanybasictechnol-
ogyresearchprograms.Beforeyieldsofadesiredproductcanbemaximized
orundesirablesidereactionsminimized,analyticalmethodsmustaccu-
ratelymonitorchangesinthechemicalconstituents.Tesemethodsalso
mustbevalidatedinthecomplexmatrixofbiomass-conversionstreams.In
manycases,biomassmethodsdirectthecourseofresearchbyrevealingthe
presenceofimportantproductsorthenatureofyield-reducingsidereac-
tions.Biomass-conversionmethodswillleadusonestepclosertothosefor
accuratelymonitoringcomplexdegradationreactionsinnaturalsystems.
112 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Breakthrough,High-PayofOpportunity:Simplifying
theBioconversionProcessbyUnderstandingCell-Wall
DeconstructionEnzymesExpressedinPlants
ScientifcChallengesandOpportunities
Complexityisoneofthemajorchallengestoeconomicalbiomasscon-
versionforlarge-scaleproductionofliquidtransportationbiofuels.Te
processcurrentlyrequiresmanysteps,includingpretreatment,detoxica-
tion,solidandliquidseparation,cellulaseproduction,cellulosehydrolysis,
biomasssugarfermentation,andproductrecoverysupportedbysuch
ancillarysystemsaswastewatertreatmentandutilities.Tiscomplexity
resultsinaddedoperatingandcapitalcostsaswellasissuesofrobustness,
andsimplerprocedureswouldbehighlydesirable.Althoughengineer-
ingpotentiallycouldimprovetheprocessincrementally,systemsbiol-
ogyapproacheshavethepotentialtorevolutionizeit.Variousstrategies
envisionedtoconsolidateprocessingstepswillgeneticallyincorporate
capabilitieswithinorganisms.Onepotentialstrategywouldinclude
incorporatinggenesthatencodelignocellulose-degradativeenzymesinto
feedstockplants.
Enzymescouldtargetwallpolysaccharides(backbones,sidechains,
andsmallsubstituentsthatcanhavebigeectsonactivityofother
enzymes),simplephenoliccross-linksbetweenpolysaccharidessuchas
thosethattietogetherxylansincerealwalls.
Promoterswouldberequiredthatareactivatedfromsenescence-related
orharvestinsulttriggeredgenesorbyasignalprovidedexogenouslyat
atimedeemedappropriate.
Genesencodingenzymesorotherdisassemblyproteinswouldbe
required,perhapsthoseofmicrobialoriginwhosediscoverywouldbe
amajorengineeringgoalofthelargerprojectandwhoseexpression
wouldbedrivenbythepromotersabove.
ResearchGoals
Whiletargetingspecicobjectivesusingtheexistingstateoftechnologyas
abenchmarkisimportant,researchmustbeopentonewideasandcon-
cepts.Specicareasthatneedtobeaddressedinfundamentalandapplied
areasincludethefollowing.
FundamentalScience
Whatnewlearningwouldenableexpressionofstructuralpolysac-
charide-degradingenzymesinplanttissues?Weneednewpromoters,
genes,transformationtools,andknowledgeaboutthesequestrationand
actionofsuchenzymeswhenexpressedinlivingplants.
Whatistherelativeeectivenessofcellulase-enzymesystemsactingin
cellulose-enzyme-microbecomplexesascomparedtocellulose-enzymecom-
plexes,andwhatisthemechanisticbasisforsuchenzyme-microbesynergy?
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 113

DECONSTRUCTING FEEDSTOCKS
AppliedScience
Canhemicelluloseandcellulosehydrolysisbeaccomplishedwithoutthe
acidicoralkalinehigh-temperatureand-pressurepretreatmentstep?
Tisisparticularlyimportantforadvantageousexpressionofhemicel-
lulasesandcellulaseenzymesinplants.
Canbiocatalystsbedevelopedwhoseperformanceisnotimpacted
signicantlybyinhibitorycompoundsformedasby-productsofpre-
treatment,hydrolysis,orfermentation?Ifso,theyhavethepotentialto
eliminatethecostlyandwaste-intensivedetoxicationstep.
Cansuchupstreamprocessesasstorage(ensilage)orbiopretreatment
bedevelopedthatmakecellulosemicrobrilsmoreamenabletoenzy-
maticcellulosehydrolysiswithouttheneedforrelativelyharshthermo-
chemicalpretreatment?
114 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

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CommodityProductsfromCellulosicBiomass,Curr.Opin.Biotechnol.
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Lynd,L.R.1996.OverviewandEvaluationofFuelEthanolfromCel-
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Lynd,L.R.,R.T.Elander,andC.E.Wyman.1996.LikelyFeaturesand
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Moniruzzaman,M.,etal.1997.FermentationofCornFibreSugarsbyan
EngineeredXylose-UtilizingSaccharomycesYeastStrain,WorldJ.Microbiol.
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Mosier,N.,C.M.Ladisch,andM.R.Ladisch.2002.Characterizationof
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Sarikaya,A.,andM.R.Ladisch.1997.AnUnstructuredMathematical
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BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 117

DECONSTRUCTING FEEDSTOCKS
118 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

SugarFermentationtoEthanol
F
ermentationofsugarsbymicrobesisthemostcommon
methodforconvertingsugarsinherentwithinbiomass
feedstocksintoliquidfuelssuchasethanol.Bioconversion
orbiocatalysisistheuseofmicrobesorenzymestotransformone
materialintoanother.Teprocessiswellestablishedforsome
sugars,suchasglucosefromcornstarch,nowamatureindustry.
Productionoffuelethanolfromthemixtureofsugarspresentin
lignocellulosicbiomass,however,remainschallengingwithmany
opportunitiesforimprovement.Morerobustmicroorganismsare
neededwithhigherratesofconversionandyieldtoallowprocess
simplicationthroughconsolidatingprocesssteps.Tisdevelop-
mentwouldreducebothcapitalandoperatingcosts,whichremain
highbycomparisonwiththoseofcorn.
TegrowingU.S.industrythatproducesfuelethanolfromcorn-
starchhasopportunitiesforincrementalimprovementandexpan-
sion.Processesforthebioconversionoflignocellulosicbiomass
mustbedevelopedtomatchthesuccessinstarchconversion(see
sidebar,Starch:ARecentHistoryofBioconversionSuccess, this
page).Technologiesforconvertingcellulosicbiomassintofuel
ethanolalreadyhavebeendemonstratedatsmallscaleandcan
bedeployedimmediatelyinpilotanddemonstrationplants.Te
challenge,withlimitingfactorsofprocesscomplexity,natureof
thefeedstock,andlimitationsofcurrentbiocatalysts,remainsthe
highercost(seeFig.1.TeGoalofBiomassConversion,p.120).
Tediscussioninthischapterwillfocusonprocessimprovements
thatwillreducerisk,capitalinvestment,andoperatingcosts.Tis
emphasisisdrivenbythegoaltointegrateandmutuallyenhance
theprogramsinDOEsOceoftheBiomassProgram(OBP)
andOceofBiologicalandEnvironmentalResearch(OBER)
relatedtoachievingthepresidentsgoalofaviablecellulosic
ethanolindustry.
Bioconversionmustbuildonitshistoricpotentialstrengthsofhighyield
andspecicitywhilecarryingoutmultistepreactionsatscalescomparableto
thoseofchemicalconversions.Biologycanbemanipulatedtoproducemany
possiblestoichiometricandthermodynamicallyfavorableproducts(seeFig.2.
ExamplesofPossiblePathwaystoConvertBiomasstoBiofuels,p.121),
butbioconversionmustovercomethelimitationsofdiluteproducts,slow
reactions,andoften-limitedreactionconditions.Forcommodityproducts
suchasfuels,biologicallymediatedconversionrepresentsalargefraction
Starch:ARecentHistoryof
BioconversionSuccess
B
iotechnologyhasatrackrecord
ofdisplacingthermochemical
processinginthebiomassstarch
industry.Inthe1960s,virtuallyallstarch
(asugarpolymeringranules)waspro-
cessedbyacidandhightemperatures.
Inhibitoryby-productsandlowercon-
versionratesresultedinasolublestarch
solutionthatwaslowerinqualityand
yieldwhenfurtherfermentedtoethanol.
Developmentofspecicthermostable
high-productivityenzymes(e.g.,alpha-
amylaseandglucoamylase)produceda
higher-qualitysolublestarch,completely
displacingtheacidprocessby1980.Tis
newprocesshasallowedtechnologiesfor
producingethanolfromstarchtocon-
tinuouslyimprovetothehighyieldand
ratelevelsseentodayinwetanddrycorn
mills.Otherstarch-conversionenzymes
(e.g.,glucoseisomerase)havemade
possibleanothercommodityproduct,
high-fructosecornsyrup,whichisused
invirtuallyalldomesticsweetenedbever-
agesandmanyotherproducts(www.
genencor.com/wt/gcor/grain).
References:p.154
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 119

SUGAR FERMENTATION
ofcostsandsellingprices(unlikethepharmaceuti-
calindustry,wherebioconversioncostsaresmall)
(Lynd,Wyman,andGerngross1999).Ultimately,
goalsinthisroadmapseektodeneandovercome
thebiologicallimitationsforkeyconversionparam-
etersofmetabolicuxandproduct,thermal,andpH
tolerancestodeveloparobustbioconversionprocess.
Severalchaptersarticulatepracticaladvantagesand
somechallengesofbiocatalysisandbiomassconver-
sion.Whilemostbiologicalresearchhasfocused
onsystemsrelevanttobasicknowledgeormedical
applications,ithasprovidedawidebaseoftoolsand
knowledgeforapplicationtothebioconversionof
biobasedfeedstocks.
Tisdiscussionfocusesondeningandprioritizing
requirementsforscienceandtechnologypathways
thatreachthemaximalpotentialofbiomassbiocon-
version.Resultsbuildonapproachesdevelopedin
priorworkshops(ScoutenandPetersen1999;Road-
mapforBiomass2002).Tischapterexpandsthat
focusinlightofnewbiologicalresearchtoolsand
understanding.Tenewbiologywillusesuchemerg-
ingtechnologiesasproteomics,genomics,metabo-
lomics,protein-complexcharacterization,imaging,
modelingandsimulation,andbioinformatics.Tis
jointeortwillfurtherguidethedevelopmentof
newhigh-throughput(HTP)biologicaltools(e.g.,
screening,functionalassays,andresequencing).
Somecommonthemesaroseduringtheworkshop.
(1)Atpresent,werearmrecalcitranceoflignocellulosicbiomassasacore
issue,butportionsofboththescienceandtheconversionsolutionclearly
arewithinthemicrobialworld.(2)Understandingmicroorganismswill
enableustomanipulatethemsotheycanreachtheirmaximalpotentialin
human-designedprocesses.(3)Terstthrustistodevelopbiocatalysts
thatwillallowdesignanddeploymentofconversionprocessesthatare
lesscostlyinoperationandcapitalthancurrentlignocellulose-to-ethanol
conversionprocesses.(4)Anothermajorthrustistoeliminateorcombine
separateprocessingstepsbydevelopingamultitalentedrobustmicroor-
ganism.Researchanddevelopmentareaddressingbothstrategies.(5)Even
withmolecularbiologyapproaches,scientistscreatealterations(usually
asinglechange)andobservetheresult.Whileexperimentalvalidation
alwaysisneeded,newglobalgenomicsmethodsoerthepotentialfor
intelligentlypredictingtheimpactofmultiplesimultaneouschanges.
Tenewomictoolsenableadeeperandmorecompleteunderstandingof
themicrobialstateanditsphysiologyinitsenvironmentenablingthe
probingofdynamics,regulation,ux,andfunction.Combiningthisunder-
standingwiththegoalofimprovingmicrobialtraitsbymanipulationwill
Fig.1.TeGoalofBiomassConversion.Securingcost-
eectivebiofuelsfrombiomassfeedstocksrequiresmoving
biologicaltechnologyfromthelaboratory(a.Microbial
CulturesatOakRidgeNationalLaboratory)throughthe
pilotplant(b.NationalRenewableEnergyLaboratorys
ProcessDevelopmentUnit)tothefullindustrialbiorenery
[c.IndustrialBioreneryinYorkCounty,Nebraska(Aben-
goaBioenergyCorporation)].
a
b c
120 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.2.ExamplesofPossiblePathwaystoConvertBiomasstoBiofuels.Tedottedlinesshowexamplesoffactorsthis
roadmapcanaccelerate;solidlinesindicateexistingpaths.[Source:B.Davison,OakRidgeNationalLaboratory]
allowregulationofthemicrobetoachievedesiredoutcomes.Manytraits
orphenotypes,suchasoverallglycolysisrateorethanoltolerance,will
bemultigenic.Toidentifyfurtherpotentialimprovements,weespecially
needrapidmethodstoassessthestateofmicroorganismsthathavebeen
engineeredwithnewpropertieseithernewprocesstraitsorindustrial
robustness.Arststepistoanalyzeofhowcurrentindustrialmicrobes
haveevolvedthroughhumanselectionfromtheirprogenitorstobebetter
adaptedtotheirprocessenvironments.
Asstated,weneedtoachieverapidanalysis,modication,andunderstand-
ingofthebiocatalyticsystemtoaccelerateimplementationoforganismsfor
ecientbioconversionofsugarsintoethanol.Anarrayofbasicmicrobial
requirementsincludesfullmicrobialsystemregulationandcontrol,toolsfor
rapidmanipulationofnovelmicrobes,andnewmicrobialplatforms.More
practicalrequirementsforbiocatalystsincludeutilizationofallsugarsanda
robustmicroorganism.Terstmayrequiredeepermetabolicandregula-
toryunderstanding.Tesecondrequiresanunderstandingofstressresponse
andinhibition.Itcanbeimplementedbyinsertingallcapabilitiesintoone
hostorbyusingmultiplemicrobialspecieswithunique,complementary
capabilitiesinacontrolled,stablemixedculture.Toenablethisresearch
anddevelopment,certainmicrobial-specicenablingtoolsarediscussed.
Troughadeeperunderstandingofthemicrobialsystem,newbiocatalysts
canbedevelopedtoreduceprocesscostandriskindevelopingatrulysus-
tainableindustry.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 121

SUGAR FERMENTATION
Treecorebiologicalbarriershavebeenidentiedashigh-priorityresearch
areasforimprovingcurrentbioconversionprocesses:Optimizingmicrobial
strainsforethanolproduction,developingadvancedmicroorganismsfor
processsimplication,andcreatingtoolsandtechnologiestoenhancethe
analysis,understanding,anduseofmicrobialsystems.Wealsoconsider
severalspeculative,breakthroughopportunitiesoeringnovelapproaches
tobiofuelproductionthatcouldfurtherreducecostandriskinthemore-
distantfuture.Tesebreakthrough,high-payoopportunitiesinclude
useofmicrobialcommunitiesratherthanpureculturesforrobustenergy
production,model-drivendesignofcellularbiocatalyticsystems,direct
productionofmoreenergyrichfuelssuchasalkanesorlong-chainalco-
hols,microbialproductionofupto40%ethanolfrombiomass,andmicro-
bialconversionofbiomass-derivedsyngastoethanolandotherproducts.
Althoughsuchideasasapureinvitromultienzymaticsystemwerecon-
sidered,theyseemedunlikelytocompetewithadvantagesmicrobesoer
inproducing,regulating,andusingcomplexmultistepcarbonandenergy
metabolicpathwaysascommoditiesinthenext20years.
OptimizingMicrobialStrainsforEthanolProduction:
PushingtheLimitsofBiology
Amajorbarrierintheecientuseofbiomass-derivedsugarsisthelackof
microbialbiocatalyststhatcangrowandfunctionoptimallyinchallenging
environmentscreatedbybothbiomasshydrolysisandcellularmetabolism.
Tenewtoolsofbiologywillfacilitatethedevelopmentoftheseadvanced
biocatalysts.Problemsincludeinhibitionbydeleteriousproductsformed
duringbiomasshydrolysis,yieldslimitedbyaccumulationofalternative
products,unnecessarymicrobialgrowth,andsuboptimalspecicproductiv-
ityresultingfromvariouslimitationsintheethanolbiosyntheticpathway
andamismatchinconditionswiththehydrolysisenzymes.Anotherchal-
lengeisthatinhibitionbythemainfermentationproduct(ethanol)results
inlowalcoholconcentration(titer).Teseproblemscontributetothecost
oflignocellulosicethanolbyincreasingcapitalexpenditure,reducingprod-
uctyields,andincreasingwatervolumesthatmustbehandledaspartof
relativelydiluteproductstreams.Teresearchobjectiveistomitigatethese
limitationsthroughconcertedapplicationofemergingtoolsforsystems
biology,workingwithprinciplesfrommetabolicengineeringandsynthetic
biology,andusingevolutionaryapproachescombinedwithquantitative
evaluationofcandidatehigh-producingstrains.
Tofosteranindustrybasedonbiomasssugars,processparametersmust
becomparabletothoseofthecornstarchethanolindustry.Ultimately,
theoverallcellulosicprocesscancompetewithpetroleum,whereas
cornstarchprocessesalonecannotachievetheneededquantities.Cur-
renttechnologyisbasedoncornstarchconversiontoethanolutilizing
yeast.Tisprocessusesglucoseasthecarbonsourceandconvertsitat
highyields(90%),hightiters(10to14wt%),andreasonablerates(1.5
to2.5g/L/h).Recombinantethanologenicorganisms(i.e.,yeast,E.coli,
andZ.mobilis)havebeencreatedtofermentbothglucoseandxylose,
122 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.3.ChangesinMetabolismBroughtAboutbyGeneticEngineering.TeE. coli Bstrain,anorganicacidpro-
ducer,wasalteredtotheE. coli strainKO11,anethanolproducer(ethanologen).TealteredKO11yielded0.50getha-
nolpergxylose(10%xylose,pH6.5,35C).Inthegraphs,biomassreferstothecellmassofE. coli.[Source:L.Ingram,
UniversityofFlorida.BasedondatareportedinH.Taoetal.,EngineeringaHomo-EthanolPathwayinEscherichia
coli:IncreasedGlycolyticFluxandLevelsofExpressionofGlycolyticGenesDuringXyloseFermentation,J. Bacteriol.
183,297988(2001).]
buttheycurrentlyproducelowerethanoltiters(5to6wt%ethanol).
Improvementsinethanolyieldsandtoleranceareneededtoincrease
ratesofproduction(>1.0g/L/h)fromallsugarconstituentsoflignocel-
lulosicbiomass.Onesuccessfulstrategyforutilizationofbothhexoseand
pentosesugarstakesknownethanologenslikeyeastandaddsabilitiesto
utilizepentosesugars.Anotherstrategytakesmixed-sugarconsumerslike
E.coliandreplacesnativefermentationpathwayswiththoseforethanol
production.Figure3.ChangesinMetabolismBroughtaboutbyGenetic
Engineering,thispage,showsanexampleofhowtheoutputofamicrobe
canbechanged.Astitersareincreased,ratesslowdownandeventually
ceaseat~6wt%ethanol,theupperlimitforwild-typeE.coli.Bycom-
parison,wild-typeyeastandZ.mobiliscanreachtitersof>15%ethanol
fromcornstarchglucosebuthavefailedtoachievetheselevelsonpentose
sugars(seesection,OptimalStrains:FermentativeProductionof40%
EthanolfromBiomassSugars,p.149).
Mostmethodsofbiomasspretreatmenttoproducehydrolysatesalsopro-
ducesideproducts(e.g.,acetate,furfural,andlignin)thatareinhibitoryto
microorganisms.Teseinhibitorysideproductsoftensignicantlyreduce
thegrowthofbiocatalysts,ratesofsugarmetabolism,andnalethanol
titers.Inallcases,theimpactofhydrolysatesonxylosemetabolismismuch
greaterthanthatofglucose.Researchdescribedhereoersthepotentialto
increasetherobustnessofethanologenicbiocatalyststhatutilizeallsugars
(hexosesandpentoses)producedfrombiomasssaccharicationatratesand
titersthatmatchorexceedcurrentglucosefermentationswithyeast.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 123

SUGAR FERMENTATION
Fromtheaboveanalysisofpresentandtargetstatesregardinguseofbio-
masshydrolysatesforbiofuelproduction,criticalparametersneededfora
cost-competitiveprocessareclearlyevident:
1. Highyieldwithcompletesugarutilization,minimalby-productforma-
tion,andminimallossofcarbonintocellmass.
2. Highernalethanoltiter.
3. Higheroverallvolumetricproductivity,especiallyunderhigh-solid
conditions.
4. Tolerancetoinhibitorspresentinhydrolysates.
Specically,thefollowingguresofmeritaresuggestedforabiomass-to-
ethanolprocessthatwillbecost-competitiverelativetocurrentcornstarch
ethanoloperations:
Useofbothhexosesandpentosestoproduceethanolatayieldgreater
than95%oftheoreticalyield.
Finalethanoltitersintherangeof10to15wt%.
Overallvolumetricproductivityof2to5gramsofethanolperliterper
hour.
Abilitytogrowandmetabolizeeectivelyinminimalmediaoron
actualhydrolysates(withonlymineralsasaddednutrients).
Toachievetheabovetargets,wemustimproveourabilitytogroworgan-
ismsinaninhibitoryenvironmentofhighconcentrationsofsugarsand
othercompounds,includingethanol.Inaddition,signicantincreasesin
uxthroughthesugar-to-ethanolmetabolicpathwayareneeded.Wepres-
entaroadmapbelowformeetingtheseobjectives.
ScienceChallengesandStrategy
Keyquestionsinclude:
Whataretheimplicationsofsimultaneousvssequentialconsumption
of5-carbonand6-carbonsugarsoncellularmetabolism,ux,and
regulation,especiallywhenxylosemetabolismhasbeenengineered
intoethanologens?
Whatcanallowmorerapidandcontrolledalterationofmicrobes,
especiallyregulatorycontrolsandadaptationtonovelinsertedgenes
ordeletedgenes?Tisconsiderationappliesalsotoknownindustrial
microbes.
Whatmechanismscontrolglycolyticux,andwhataretheirimpli-
cationsforcellularmetabolism?Forexample,couldtheglycolytic
pathwayecientlyhandleanexcessofcarbonuxinanorganism
engineeredtorapidlyconsumeamixtureof5- and6-Csugars(see
Fig.4.RecombinantYeast,S.cerevisiae,withXyloseMetabolismGenes
Added,p.125)? Asystemsbiologyapproachwillallowinsightsinto
themolecularbasisfortheseprocessesanddevelopmentofpredictive
modelstorenetheirdesign.
124 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Whatmolecularmechanismsareused
bycellstocopewithsuchenvironmental
challengesashighconcentrationsofsugars
andethanolandthepresenceofinhibitors
frombiomasshydrolysis?
Whatgeneticandphysiologicalcharac-
teristicsmediateevolutionofwild-type
organismsintorobustlaboratoryorindus-
trialstrains,andwhichonescontroltheir
functionalstateintheprocessenviron-
ment(seesidebar,ProteomicandGenomic
StudiesofIndustrialYeastStrainsandTeir
Ethanol-ProcessTraits,p.126)?
Utilizingacombinationofmetabolicengi-
neeringandsystemsbiologytechniques,two
broadmethodsfordevelopingmorecapable
andmoretolerantmicrobesandmicrobial
communitiesaretherecombinantindustrial
andnativeapproaches.
Recombinantindustrialhostapproach:
Insertkeynovelgenesintoknownrobust
industrialhostswithestablishedrecombi-
nanttools.
Nativehostapproach:Manipulatenew
microbeswithsomecomplexdesirable
capabilitiestodeveloptraitsneededfora
robustindustrialorganismandtoeliminate
unneededpathways.
Tesemethodsrequiregeneticunderstanding
ofthetraitwewishtobeaddedorpreserved
androbusttoolsforgeneticmanipulation.Te
subsetofbiochemicalpathwayspotentially
involvedinglycolysisiscomplex(Fig.5.Some
MetabolicPathwaysthatImpactGlucose
FermentationtoEthanol,p.128).Ourgoalis
toparethisdowntojustwhatisessentialfor
xyloseandglucoseuse(Fig.6.DesiredMeta-
bolicPathwaysforaGlucose-XyloseFer-
mentingEthanologen,p.129).Bothmethods
canhavevalue;forexample,eithereliminate
uneccessarypathwaysinE.coli,whichhasyieldedstrainsthateciently
metabolizebothxyloseandglucose(andallothersugarconstituentsof
biomass)toethanol,oraddxylose-fermentingpathways(andothers)to
ethanol-producingyeast.Anumberofmethodsandapproachessupport
thetwobroadstrategies.Teseandothergoalswillrequirecertainenabling
microbiologicaltools(seesection,EnablingMicrobiologicalToolsand
TechnologiesTatMustbeDeveloped,p.138).
Fig.4.RecombinantYeast,S. cerevisiae,withXyloseMetabolism
GenesAdded.Followingrapidconsumptionofglucose(within
10h),xyloseismetabolizedmoreslowlyandlesscompletely.Ide-
ally,xyloseshouldbeusedsimultaneouslywithglucoseandatthe
samerate,butthexyloseisnottotallyconsumedevenafter30h.
Alsonotethatyieldisnotoptimal.Althoughethanolisthemost
abundantproductfromglucoseandxylosemetabolism,small
amountsofthemetabolicby-productsglycerolandxylitolalsoare
produced.[Source:M.Sedlak,H.J.Edenberg,andN.Ho, DNA
MicroarrayAnalysisoftheExpressionoftheGenesEncodingthe
MajorEnzymesinEthanolProductionDuringGlucoseandXylose
CofermentationbyMetabolicallyEngineeredSaccharomyces Yeast,
Enzyme Microb. Technol. 33,1928(2003).Reprintedwithpermis-
sionfromElsevier.]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 125

SUGAR FERMENTATION
ProteomicandGenomicStudiesofIndustrialYeastStrainsandTheirEthanol-
ProcessTraits:RapidlyFindingtheGeneticandFunctionalBases
C
urrentindustrialyeaststrainshavebeenisolatedfromwildyeastpopulationsformanydecades,selected
fortheircapacitytoproduceethanolunderindustrialsettings.Understandinghowtheseselected
genotypesandphenotypesdierfromundomesticatedstrainsofyeastwouldhelpustounderstandthe
typeofchangesneededtodeveloparobustethanolproducer.Understandinghowcellscopewithhigh-ethanol
mediaconcentrationsisessentialtoimprovefermentationyieldandtiter.Similarstudieswouldbebenecialfor
otherindustrialorganisms,suchasEscherichiacoli.Gaininginsightaboutanorganismsprocessofadaptationto
anindustrialbioreneryenvironmentcanhelpusintentionallyreplicatethesechanges(seeFig.A.Importance
ofAdaptationforRobustInitialStrains,below).Testrategyforstudyingindustrialstrainsfollows.
Compareproteomicandgenomicsequencesofthemostcommonyeaststrainsmanufacturedandsoldfor
ethanolproductionwiththoseoftheirancestralparentstrains.
Compareproteomicandgenomicsequencesofevolvedstrainsproducedthroughmetabolicengineering
andmetabolicevolutionwiththoseoftheirparentalstrains.Proteomicstudieswillbeperformedonsam-
plestakenfromindustrialfermentations.Genomicstudiesofstrainsfromthesameprocessesshouldreveal
dierencesbetweenindustrialandlaboratorystrainsthatwillprovidefundamentalinformationregarding
multigenictraitsessentialforhighmetabolicactivity,producttolerance,andadjustmentstoengineered
changesinmetabolism.
Studyinggenomesofindustrialyeaststrainswillhelpusunderstandcommontraitsofeectiveethanol-pro-
ducingstrains.Proteomicstudieswillrevealproteinsgeneratedunderactualindustrialproductionconditions.
Completemappingandreconstructionofthestrainsnetworkswillbeneededforpropercomparisons.Avail-
ablemodelingtoolsarebeingimprovedcontinuously.Proteomicanalysisofmembraneproteinsisstillachal-
lengeandneedstobedevelopedfurthertoguaranteeamorecompleteandmeaningfulanalysisofsamples.
Datageneratedthroughthiseortwillrequirefulluseofalltoolsavailableforsystemsbiologyandwillstimu-
latehypothesisgenerationandtestingbytheacademiccommunity.Teeectwillbesimilartothosefrom
metagenomicstudiesandcommunityproteomics, inwhichhugeamountsofdataweremadeavailableandare
beinganalyzedbymanydierentgroupsaroundtheworld.
Fig.A.ImportanceofAdaptationforRobustIndustrialStrains.[Source:H.J.StrobelandB.Lynn. 2004.Proteomic
AnalysisofEthanolSensitivityinClostridium thermocellum,presentedingeneralmeeting,AmericanSocietyforMicro-
biology,NewOrleans,La.,May2327,2004.]
126 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

MetabolicEngineering
Yieldandproductivityenhancementwillbeaccomplishedbyapplying
metabolicengineeringconceptsandmethods.Yieldmaximizationistan-
tamounttoby-productminimization,whichisachievedbyeliminating
branchesofcompetingpathwaysthatleadtounwantedproducts.Tis
usuallyisdonebydeletinggenesencodingenzymesthatcatalyzecompet-
ingreactionpathways.Ifsuchpathwaysareresponsibleforsynthesisof
metabolitesessentialforcellgrowthandfunction,downregulationofthese
genesmaybepreferabletocompletegeneknockout.Inallcases,optimal
balancingofenzymaticactivitiesiscriticalforsatisfactoryfunctionofthe
resultingengineeredstrain.Currentmolecularbiologicalmethodscanbe
deployedsuccessfullytothisend,includingspecicgeneknockout,gene
amplicationthroughpromoterlibrariesorregulated(induced)promoters,
andothermethodscombininggeneknockoutordownregulationandgene
amplication.Teabilitytomeasuredetailedcellbehaviorsanddevelop
predictivemodelstorenetheirdesignwillbecriticaltospeedupand
enhancetheseengineeringeorts.
Arelatedpartofthisworkisanalysisandregulationofcellularenergetics.
Carefulalterationofgrowth,energy,andredoxoftenisneeded.Frequently,
decouplinggrowthfromproductionwillincreaseyield.
Productivitymaximizationhasbeendemonstratedinmanyapplicationsof
metabolicengineeringwithE.coliandyeaststrains.Examplesinclude1,3
propanediol,aminoacidssuchaslysineandthreonine,biopolymerbiosyn-
thesis,precursorsofpharmaceuticalcompounds,ethanol,andmanyothers
(seeFig.7.A3GTiterfromGlucose,p.130).Teseexamplesillustrate
thefeasibilityofsignicantspecicproductivityenhancementsbyapply-
inggeneticcontrols,sometimesincombinationwithbioreactorcontrols.
Improvementssuggestthatprojectedenhancementsinspeciccellproduc-
tivityareentirelyfeasibleandthatthenewtechnologiesofsystemsbiology
candramaticallyincreaseandaccelerateresults.
Terstgenerationofspecicproductivityimprovementwilltargetenzymes
importantforthesugar-to-ethanolpathway.Stableisotopeswillbeused
astracerstomapthemetabolicuxesofethanol,includingrelatedpath-
waysproducingorconsumingenergyorredoxmetabolites(e.g.,ATPor
NADPH),andotherkeyprecursorsforethanolbiosynthesis.Fluxmaps,
togetherwithtranscriptionalproles,willbegeneratedforcontroland
mutantstrainstoidentifyenzymescontrollingoverallpathwayux.Gene
amplicationofrate-limitingstepswillbeusedtoovercomeuxlimita-
tions. Tisisanticipatedtobeaniterativeprocess, asnewlimitationsare
likelytoariseassoonasoneisremovedbygenemodulation. Tegoalwill
betoamplifyuxoftheentirepathwaywithoutadverseregulatoryeectson
theorganismsgrowthorphysiology.Again,balancingenzymaticactivities,
removinglimitingsteps,andpruningunwantedreactionsallsupportedby
comprehensiveanalysisandmodelingwillbedeployedforthispurpose.
Inadditiontospecicpathwaysteps,remotegeneswithregulatoryand
other(often-unknown)functionsimpactpathwayux.Modulationof
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 127

SUGAR FERMENTATION
suchgeneshasbeenfoundtoinuencesignicantlythebiosynthesisrate
ofmanyproducts.Suchgeneswillbefoundthroughinversemetabolic
engineering,wherebylibrariesofendogenousandexogenousgenesare
expressedinthehoststrainandrecombinantsareselectedonthebasisof
drasticimprovementsinthedesirablephenotype(e.g.,ethanolproduction
andtolerance).Genesconferringthesephenotypescanbesequencedand
identiedforexpressionincleangeneticbackgrounds.
RecombinantApproach
Tolerancetoinhibitorsisamultigenicproperty.Intheexamplesystems
givenabove,thistraitisfoundedprimarilyonmembraneuidityand
othermembranepropertiesandfunctions.Ingeneral,eortstoimprove
microorganismtolerancebyrecombinantgenemanipulationhavebeen
confoundedbythelimitedabilitytointroducemultiplegenechanges
simultaneouslyinanorganism.Developmentanduseofasystemsapproach
thatallowsmultiple-geneorwhole-pathwaycelltransformationareimpor-
tantmilestones.
EvolutionaryEngineering
Astrategyforincreasingethanoltoleranceorothertraitscoulduseevolu-
tionaryengineeringconceptsandmethods.Tisstrategywouldallowthe
microbialprocesstoevolveundertheproperselectivepressure(inthiscase,
higherethanolconcentrations)toincreasinglyhigherethanoltolerances.
Fig.5.SomeMetabolicPathwaysthatImpactGlucoseFermentationtoEthanol.
Tispathwaymapdemonstratesthecomplexityofevenasimple,widelyutilized,and
relativelywell-understoodprocesssuchasglucosefermentationtoethanol.Glucose
128 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Evolutionaryengineer-
ingcanbeappliedto
theethanol-producing
organismasawholeor
tospecicproteins,in
particularthosewith
regulatoryfunctions.In
thelattercase,evolu-
tionaryengineering
emulatesthemethodsof
directedevolution,which
hasprovenverysuc-
cessfulinengineering
proteinmutantswith
specicdesirablephar-
maceutical,regulatory,or
kineticproperties.
Accuratecharacteriza-
tionofcellandprotein
mutantswillbeneeded
toallowanunderstand-
ingofprinciplesforimprovingandrationallycarryingoutthedesigns.
Tistaskwillrequiresequencing,large-scalebindingexperiments,
andethanolareidentied.[E.Gasteigeretal.,Expasy:TeProteomicsServerfor
In-DepthProteinKnowledgeandAnalysis,Nucleic Acids Res. 31,378488(2003).
Screenshotsource:http://ca.expasy.org/cgi-bin/show_thumbnails.pl.]
Fig.6.DesiredMetabolic
PathwaysforaGlucose-Xylose
FermentingEthanologen.Te
goalistogeneticallyengineer
anindustrialorganismthatcan
metabolizebothsugars.Pathways
indicatethemanyinvolvedgenes
thatwouldhavetobefunctionalin
suchanorganism.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 129

SUGAR FERMENTATION
Fig.7.A3GTiterfromGlucose.Tegraphshows
DuPont-GenencorsuccessinalteringE. coli to
maximizeyieldandtiterof3G(1,3,propanediol).
Projectssuchasthiscouldgreatlybenetfromthe
deepersystemsbiologyunderstandingthatGTL
seeks.[Source:AdaptedfromC.E.Nakamuraand
P.Soucaille,EngineeringE. coli fortheProduction
of1,3-Propanediol,presentedatMetabolicEngi-
neeringIV,Tuscany,Italy,October2002.]
transcriptionalstudies,proteomics,metabolomics,and
physiologicalfunctionalevaluation,allwellsuitedforGTL
capabilitiesdescribedinmoredetailbelow.
Evidenceisgrowingthatmethodsofevolutionaryengineer-
inganddirectedevolutionofregulatoryproteinshavethe
potentialtoachievethetargetsoftolerancetoethanoland
otherinhibitorycompounds.RecentstudieswithE.colihave
increasedthatorganismsethanoltolerancebymorethan
50%whilecomparableincreasesalsohavebeenobtained
foryeastathighethanol(6%)andglucose(100g/L)con-
centrations.ApplyingsystemsbiologymethodsandHTP
technologiesandcomputingwillacceleratetheprocessby
revealingthegenetic,molecular,andmechanisticimpactsof
evolutionarymethods.
Tesemethodsalsowillbeusedtoisolatefast-growing
organisms.Highgrowthratesandnalbiomassconcentra-
tionsareimperativeforachievinghigh-volumetricpro-
ductivities,sincethelatterdependonfermentorbiomass
concentrations.Moredetailedinvestigationisneededon
theeectsofvariousbiomass-hydrolysatecompoundsoncellgrowth,espe-
ciallyfactorsresponsibleforgradualreductioninspecicethanolproduc-
tivityduringfermentationassugarsaredepletedandproducts,particularly
ethanolandotherinhibitors,accumulate.
TechnicalMilestones
Within5years
Mesophilicmicrobesdemonstratedatscalethatarecapableoffulluti-
lizationofalllignocellulosicsugarsforreducedcommercializationrisk.
Tisrequiresoptimizationofdevelopedandpartiallydevelopedstrains.
Increasedstraintolerancetoinhibitoryhydrolysatesandethanol,with
theabilitytouseallsugars,includingmesophileandthermophilestrains.
Understandingofmultigeniccausesofindustrialrobustness.
Candidatemicrobessuchasthermophilicethanologenscompatiblewith
desiredcellulaseenzymeoptima.Tisallowsprocesssimplicationto
single-vesselfermentationwithecientuseofallbiomass-derived
sugars(seesection,AdvancedMicroorganismsforProcessSimplica-
tion,p.132).
Developmentofcoproducts.
Within10years
Rapidtooladaptationandregulationofgeneticallyengineeredstrains,
includinguseofminimalmedia.
Abilitytoengineerethanoltoleranceandrobustnessintonewstrains
suchasthermophiles.
Higher-yieldmicrobesviacontrolofgrowthandenergetics.
130 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Increasedproducttitertosimplifyproductrecoveryandreducewateruse.
Fullpredictivemetabolicpathwaysystemsmodelforcommonindustrial
microbes,includingregulationandidenticationofunknowngenes(see
section,Model-DrivenDesignofCellularBiocatalyticSystemsUsing
SystemBiology,p.142).
Within15years
Termophillicmicrobesdemonstratedatscaletoenablesimultaneous
saccharicationandfermentation.
Furtherrenementofbiofuelprocessandoperation.
TheRoleofGTLCapabilities
Asdiscussed,achievingtheseobjectiveswillrequiretheuseofrational-
combinatorialandevolutionaryapproachestoimprovetheproperties
ofindividualenzymesandorganisms.Toinform,enhance,andacceler-
atemanipulationofnewmicrobes,systemsbiologyanalyses(e.g.,omic
measurements,knockouts,taggingofproteinsandcomplexes,visualiza-
tion,andabioinformaticcorestructurefordata)willbeapplied.Once
thenovelties(e.g.,pathways,proteins,products,traits,andcomplexes)
areidentied,additionalgenetictoolswillmovedesiredgenesandtraits
intoaknownindustrialhostorfurthermanipulatenovelmicrobesinto
anindustrialorganismbyaddinggenetraits.Terearenoconsistentand
rapidtoolsforthesemanipulationsatpresent.
Tecapabilitieslistedbelowwillplayanimportantroleinbothcases.
ProteinProduction
Awiderangeofproteins(regulatory,catalytic,andstructural)willbe
producedandcharacterized,andappropriateanityreagentswillbe
generated.Modiedproteinsalsowillbeusedtounderstandfunctional
principlesandforredesign.Examplesincludeglycolyticproteinsand
alcoholdehydrogenasesfromotherorganismsorthoseevolvedinthelab,
structuralproteinsfromhigh-toleranceorganisms,orregulatoryproteins
withalteredproperties.
MolecularMachines
HTPmethodstoidentifybindingsitesofglobalregulatoryproteinsand
otheraspectsofmembranesandmembraneformationwillberequired.
Specicproteincomplexesofinterestaresugartransporters,solvent
pumps,orotherporins.Tesemeasurementswillinformourunderstand-
ingof,forexample,theinteractionorassociationofenzymesalongthe
glycolyticpathway.Temembranecouldbestudiedasamachinetocon-
trolinhibitorystress.
Proteomics
Althoughrationalandevolutionaryapproachesareenvisioned,acom-
moncomponentofbothistheuseoftoolsthatallowquantitativecellular
characterizationatthesystemslevel,includingexistingtoolsforglobal
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 131

SUGAR FERMENTATION
transcript,protein,andmetaboliteproling.AdditionalHTPtoolsnot
currentlyavailablewillberequiredtomonitorkeyplayersthatdenethe
redoxandcellenergystate[e.g.,ATP,GTP,NAD(P)H,NAD(P)].Capa-
bilitiescouldincludemetabolicuxmapping,amajoractivityinunder-
standingandmanipulatingcellularmetabolism.Temostecientwayto
estimateinvivometabolicuxesisthroughlabelingexperiments.Specic
needsincludeappropriatenuclearmagneticresonance(NMR)andmass
spectroscopy(MS)instrumentationandstableisotopesforvisualizingpen-
toses,hexoses,andcellulose.Intensivemathemathicalandcomputational
powerisrequiredtoachievethenalgoalofuxestimation.HTPtech-
nologiesforglobalidenticationofgenesthatimpactethanolbiosynthetic
pathwaysarerequiredtoselectcellscapableofhighethanolproduction
andotherdesiredfunctions.
CellularSystems
Teabilitytotrackkeymolecularspeciesastheycarryouttheirfunctions
andcreatepredictivemodelsforsystemsprocesseswillbecriticalfordevel-
opingorenhancingcellproperties.
DOEJointGenomeInstitute
Sequencingandscreeningofmetagenomiclibrariesfornovelgenesand
processesandanalyzingnovelorganismswillbecarriedoutatDOEJGI.
Exploitingmicrobialdiversitybyminingfornovelpathwaysororganisms
thatmakeastepchangeinethanolproductioncouldspurtheproduction
ofotherchemicalsthroughfermentation.
AdvancedMicroorganismsforProcessSimplifcation
Methodsandtechnologiesdiscussedabovewillbeappliedtoconsolidating
processsteps,whichiswidelyrecognizedasasignaturefeatureofmature
technologiesandhaswell-documentedpotentialtoprovideleap-forward
advancesinlow-costprocessingtechnology.Inlightofthecomplexity
ofunderlyingcellularprocessesuponwhichsuchconsolidationdepends,
fundamentallyorientedworkwillbeahighlyvaluablecomplementto
mission-focusedstudiesandcanbeexpectedtoacceleratesubstantiallythe
achievementofappliedobjectives.
Realizingthebenetsoftargetedconsolidationopportunitiesrequires
understandingandmanipulatingmanycellulartraits,anapproachmuch
morefruitfulatasystemslevelthanattheindividualgenelevel.As
discussedpreviously,examplesofsuchtraitsincludetransporters,control
mechanisms,andpathwaysrelevanttouseofnon-nativesubstrates(e.g.,5-
Csugarsandcellulose),microbialinhibition(e.g.,bypretreatment-gener-
atedinhibitorsorethanol),andtheabilitytofunctionwellinsimpleand
inexpensivegrowthmedia.Investigationofthesetraitsprovidesanimpor-
tantwaytoapplyandextendnewsystemsbiologytoolstononconven-
tionalhostorganismssuchasthermophiles.
132 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Tecurrentprocesshasundergonemanyimprovementsinthelastdecade.
InFig.4oftheIntroduction,p.14,theprocesscartoonillustratespre-
treatment(probablydiluteacidhydrolysis),followedbyadetoxication
andneutralizationstep,thenseparatefermentationofthesolublepentose
sugars.Somebiomasssolidsareusedtomakethecellulases,whichthen
areaddedtothebiomasssolidstoconvertcellulosetoglucose,followedby
aseparateglucosefermentation.Tissectiondiscussesrecentandongoing
developmentstomakeasinglemicrobeforcofermentationofhexoseand
pentosesugars(e.g.,glucoseandxylose).
Eliminatingprocessstepsmayreducecapitalandoperatingcostsandallow
othersynergisticbenets.Someofthesesimplicationstepsareunder
limitedactiveresearch.Wefocushereonthreeimmediateconsolidation
opportunities:
1. Eliminationofadedicatedsteptodetoxifypretreatmenthydrolysates
beforefermentation.Teseinhibitorscanbeby-productsofthehydro-
lysisprocessandincludeacetate,furfurals,andotherundetermined
substances.Figure8.RecombinantYeastCofermentationofGlucose
andXylosefromCornStoverHydrolysateWithoutDetoxication(this
page)showstheimpactoftheseinhibitors.Inprocesscongurations
underconsideration(e.g.,acidhydrolysis),suchdetoxicationrequires
equipment(e.g.,solid-liquidseparationandtanks),addedmaterials
(e.g.,baseforoverlimingfollowedbyacidforneutralizationbefore
fermentation),andaddedcomplexity.Obvioussavingscanberealized
bydevelopingimprovedbiocatalystsnotrequiringthedetoxication
step.Fordetoxicationelimination,researchwillsupportdevelopment
oforganismshavingahightolerancetopretreatment-generatedinhibi-
torsorthosethat
detoxifythese
Fig.8.RecombinantYeastCofermenta-
inhibitors(e.g.,by
tionofGlucoseandXylose fromCorn
consumingthem)
whilepreserv-
StoverHydrolysateWithoutDetoxica-
tion.Noteslowerxyloseuseandlower
ingotherdesired
ethanoltiterandyieldthaninFig.4. fermentation
Cornstoverhydrolysatewaspreparedby properties.Some
inhibitorshave aqueouspretreatmentfollowedbyenzyme
beenidentied, hydrolysis.[Source:M.SedlakandN.Ho,
suchasfurfurals
ProductionofEthanolfromCellulosic
andacetate,butnot
BiomassHydrolysatesUsingGenetically
allareknown.
EngineeredSaccharomyces YeastCapable
ofCo-FermentingGlucoseandXylose,
2. Simultaneous
Appl. Biochem. and Biotechnol. 114,40316
saccharication
(2004)(alsoseeMosieretal.(2005)]. andcofermenta-
tion(SSCF),in
whichhydrolysisis
integratedwithfer-
mentationofboth
hexoseandpentose
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 133

SUGAR FERMENTATION
sugarsbutwithcellulaseproducedinaseparatestep.Forexample,devel-
opmentofthermophilicethanol-producingorganismsforuseinSSCF
couldallowtheconsolidatedprocesstorunathighertemperatures,thus
realizingsignicantsavingsbyreducingcellulaserequirements.Previous
analyses(Svensonetal.2001)haveshownthatamidtermstrategyto
produceethanolfrombiomasswouldbetodevelopnewstrainscapable
ofyieldingethanolat50CpH6.0,theoptimalconditionsforsacchari-
cationenzymesgeneratedtodaybytheindustry.
3. Combiningcellulaseproduction,cellulosehydrolysis,andcofermen-
tationofC-5andC-6sugarsinasinglesteptermedconsolidated
bioprocessing(CBP).Widelyconsideredtheultimatelow-costcon-
gurationforcellulosehydrolysisandfermentation,CBPhasbeen
showntooerlargecostbenetsrelativetootherprocesscongurations
inbothnear-term(Lynd,Elander,andWyman1996)andfuturistic
contexts(Lyndetal.2005).
TegoalisaprocessmorelikeFig.5oftheIntroduction,p.15.Further
simplicationscanbeenvisionedbeyondtheseexamples.Uniquechallenges
aretheexpressionofmultipleenzymesforcelluloseandhemicellulose
hydrolysisortheengineeringofnativecellulose-hydrolyzingorganisms
toproduceethanol.Selectingoptimalenzymetargetsforexpressionwill
requireextensivescreeningandcharacterizationofheterologousgenes.
Developingauniqueenzymesuitecapableofcompletecelluloseandhemi-
cellulosehydrolysiswillrequireinsightsintotheplantcell-wallassembly
andstructureaswellasnewtoolsforcell-wallinvestigations.
Researchmustdeterminewhicharomatichydrocarbondegradativepath-
wayscansolubilizeligninandhowtheycanbeintegratedintoaproductive
hostforadditionalethanolproduction.Fortunately,aromatichydrocar-
bon-biodegradationpathwayshavebeenstudiedextensivelyoverthepast
twodecades,andmanyareknown.Integratingnecessarycomponentsinto
singlehostsandchannelingcarbontoethanolwillbemajorchallenges.
Itwillbeachallenginggoaltooptimallyachieveallthetraitsatone
time.Expression,regulation,tolerance,growth,andmetabolismmustbe
designedandsynchronizedtofunctionintheprocess. Atpresentfor
thisapproach,weappeartobelimitedtoanaerobicbacteriaandnotthe
aerobicfungiusedtomakecurrentcellulases.Wehavelimitedknowl-
edgeandlessabilitytomanipulatemostofthesebacteria.Tecellulolytic
bacteriaalsohavesomeinterestingdierences,suchasthecellulosome
discussedinthebiomassdeconstructionchapter.
Foralltheseconsolidationopportunities,nativeorrecombinantindustrial
strategieswillbeemployed:
1. Terecombinantindustrialstrategy,engineeringindustrialorganisms
withhighproductyieldandtitersocelluloseorpentosesugarsareused
byvirtueofheterologousenzymeexpression.
2. Tenativehoststrategy,engineeringorganismswiththenativeability
tousecelluloseorpentosesugarstoimproveproduct-relatedproperties
134 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

(e.g.,yield,titer)andprocess-relatedproperties(e.g.,resistancetotoxic
compounds).
However,athirdcombinedstrategyispossible.
3. Mixedcultureconversionstrategytoseparatelymodifymicrobestowork
ondierentpartsofthesubstratesorpathways. Tishasbeensuggested
butnotwelltestedforcofermentationofpentosesandhexoses(seesec-
tion,MicrobialCommunitiesforRobustEnergyProduction,p.140).
Asdescribedbefore,thekeydierenceishowchallengingthecomplex
traitis.Somecasesliketheeliminationoflacticacidasaby-product
mightinvolvethedeletionofasinglegene;inothers,theproductionof
acomplexextracellularcellulaseorcellulosomemayappearimpossibleat
present.Delineatingthegeneticchangesneededtoconferresistanceto
toxiccompoundsgeneratedinbiomassprocessesisevenmorechalleng-
ing.Researchersmustbalancetheeaseofmanipulationagainstthetraits
complexityinpursuingimprovementsofbiocatalystsforindustrialethanol
fromlignocellulose.
ForbothSSCFandCBP,causesofcellulosic-biomassrecalcitranceneed
tobeunderstoodnotonlywithrespecttoenzymatichydrolysis,inwhich
enzymesactindependentlyofcells(cellulose-enzymecomplexes),but
alsotomicrobialhydrolysis,inwhichhydrolysisismediatedbycellulose-
enzyme-microbecomplexes.Growingevidenceshowsthatfree-enzymatic
andmicrobialhydrolyzesdierinsubstantialways.Studiesofrecalcitrance
inmicrobialcellulosehydrolysiswillbuildonandcomplement,butnot
duplicate,investigationofenzymatichydrolysis.
Furtherprocesssimplicationscanbeconsidered.Forexample,develop-
mentofrobust,intrinsicallystablepureormixedmicrobialculturescould
eliminatetheneedforcostlysterilization.Alternativeroutesforprocess
simplicationalsoshouldbeconsideredsuchasgasifactionoftheentire
biomassfollowedbycatalyticorbiologicalconversionintofuelslikeetha-
nol(seesection,AnAlternativeRouteforBiomasstoEthanol,p.152).
ScienceChallengesandStrategiesforProcessSimplifcation
Tephysiologyormicrobialstateofmodiedorganismswithinthecon-
versionprocessneedstobeunderstoodtohelpdeterminewhensimplica-
tionishelpfulandwhatconditionsmustbeachievedtomakeiteective.
Partofthisisregulationofnativeandmodiedpathwaysandtraits,many
ofwhichappeartobemultigenic,complex,poorlyunderstood,anddicult
tocontrol.Forallthreeconsolidationopportunities,understandingthe
sensitivityoforganismperformancetogrowth-mediumformulationwould
benetfromuseofsystemsbiologytools.Althoughseparateprocessesare
morecost-eectiveattimes,simplicationtendstowinhistorically.
A.EliminationofDetoxifcation
Fundamentalmechanismsoftoxicityandresistance.
Evaluationoftoleranceamongadiversityofspeciesandstrainswith
andwithoutopportunityforadaptationandevolution.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 135

SUGAR FERMENTATION
Characterizationandevaluationofdetoxicationmechanisms.
B.SimultaneousSaccharifcationandCofermentation
Fundamentalsoffermentationinthepresenceofhighsolidconcentra-
tion.Temicrobe-enzyme-solidinterfaceshouldbeanalyzed.
Understandingandreconcilingfactorsresponsiblefordierencesin
optimumconditionsforcellulasefunctionandfermentationofsugars
toethanol.
C.ConsolidatedBioprocessing
Akeyquestionis,Howdomicroorganismsbreakdowncellulose?How
isbreakdowninmicrobiallyattachedcellulosomecomplexesdierent
fromenzymatichydrolysiswithaddedfungalenzymes?Asignicant
numberoffundamentalissuesneedingtobeaddressedareoverand
abovequestionsimplicitinseekingtounderstandhowenzymeshydro-
lyzecellulose.Teyinclude:
Bioenergetics,substrateuptake,andmetaboliccontrol(including
regulatorycircuits)relatedtocellulosehydrolysis.
Relativeeectivenessofcellulose-enzyme-microbecomplexesas
comparedtocellulose-enzymecomplexesandthemechanisticbasis
forsuchdierencesandpossiblesynergies.
Extenttowhichproductsofmicrobialcellulosehydrolysisequili-
brateordonotequilibratewiththebulksolutionandthefractionof
hydrolysisproductsthatproceedfromthecellulosesurfacedirectly
toadherentcells.
Featuresofcellulolyticmicroorganismsfavoredbynaturalselection
andhowselectioncanbeharnessedforbiotechnology(especiallyfor
therecombinantstrategy).
Documentationandunderstandingofthediversityofcellulose-uti-
lizingorganismsandstrategiespresentinnature.
Howdomicroorganismsrespondtocellularmanipulationsundertaken
inthecourseofdevelopingCBP-enablingmicroorganisms?Specic
issuesinclude:
Forthenativestrategy,howcellsrespondtochangesinend-product
prolesintermsofthecellsstate(transcriptome,proteome,and
metaboliteproles)aswellaskeypropertiesofindustrialinterest
(producttoleranceandgrowthrate).
Fortherecombinantstrategy,understandinggainedfromrecom-
binantcellulolyticmicroorganismsdevelopedonefeatureatatime,
includingthoseinadditiontohydrolyticenzymes(e.g.,forsubstrate
adhesion,substrateuptake,andmetabolism).Suchstep-wiseorgan-
ismdevelopmentprovidesanoutstandingopportunitytoadvance
appliedgoalsandgainfundamentalinsightssimultaneously.
136 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

D.OtherSimplifcationOpportunities
Tedevelopmentofintrinsicallystableculturescouldinvolvecontami-
nation-resistantthermophilesoracidophilesortechniquestocontrol
mixedmicrobialcultures(seesection,MicrobialCommunitiesfor
RobustEnergyProduction,p.140).
Tedevelopmentofmicrobialgrowth-independentprocesseswill
reducewaste-treatmentvolumesofbiosolidsandallowbetterreturnof
nutrientstothelandassustainablefertilizers.
TechnicalMilestones
Ofthetargetedconsolidationopportunities,CBPisthemostambitiousand
probablywillrequirethelargesteorttoachieve. Tus, wemaywellseesub-
stantialprogresstowardSSCFanddetoxicationeliminationbeforeCBP.
Keymilestonesassociatedwithtargetedconsolidationopportunitiescanbe
pursuedbeneciallybycomplementarymission-orientedandfundamentals-
focusedresearchactivities. Tesemilestonesinclude:
Within5years
Improvehydrolysate-tolerantmicrobes.
AchieveSSCFunderdesirableconditions(highrates,yield,andtiter;
solidsconcentrationandindustrialmedia).
Functionallyexpressheterologouscellulasesinindustrialhosts,includ-
ingsecretionathighlevelsandinvestigationofcell-surfaceexpression.
ConductlabtestsofmodiedinitialCBPmicrobes.
Within10years
Eliminatethedetoxicationstepbydevelopingorganismshighlytoler-
anttoinhibitors.
Havethesameresponsewithundenedhydrolysatesaswithdened
hydrolysates.
MovetopilotdemonstrationofCBP.
Within15years
Developintrinsicallystableculturesthatdonotrequiresterilization.
AchieveCBPunderdesirableconditions(highrates,yield,andtiter;
solidsconcentrationandindustrialmedia),rstoneasilyhydrolyzed
modelcellulosicsubstrates,thenonpretreatedcellulose.
Developmethodstouseorrecycleallprocessstreamssuchasinorganic
nutrients,protein,biosolids,orcoproductcarbondioxide(seesidebar,
UtilizationoftheFermentationBy-ProductCO ,p.138).
2
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 137

SUGAR FERMENTATION
TheRoleofGTLCapabilities
ProteinProduction
Proteinproductionresourcescouldbeveryusefulinsynthesizingenzymes
andmixturesofenzymesascontrolsinexperimentscomparingenzymatic
andmicrobialhydrolysis.Tesecontrolshavethepotentialtobequite
complexandthusdemandingintermsofproteinsynthesiscapability.
MolecularMachineAnalysis
Teseresourcescanprovideadvancedanalyticalandcomputationalscience
tostudycellandcelluloseinteractionandparticularlytogaininsightsinto
whatisgoingoninthegapbetweenanadheredcellandcellulosesurface.
Proteomics
Proteomiccapabilitiescanassistresearchersseekingtounderstandsystem-
levelresponsestometabolicmanipulationinthecourseofdeveloping
microorganismstoachieveallthreetargetedconsolidationopportunitiesas
wellasdiagnosisandalleviationofmetabolicbottlenecksanduxanalysis.
CellularSystems
Tesecapabilitiesalsocanassistresearchersinunderstandingsystem-level
responses,removingbottlenecks,andconductinguxanalysis(e.g.,via
metaboliteanalysis).
DOEJointGenomeInstitute
DOEJGIcanplayakeyroleinsequencinggenomesofnewmicroorgan-
ismswithrelevantfeatures(e.g.,abilitytouseC-5sugars,resistanceto
pretreatment-generatedinhibitors,andcelluloseutiliza-
tion),thusenablingvirtuallyalllinesofinquirydescribed
UtilizationoftheFermentation
inthischapterandinCrosscutting21
st
CenturyScience,
TechnologyandInfrastructureforaNewGenerationof
By-ProductCO
C
2
BiofuelResearch,p.155.
arbondioxideisamajorby-product
ofalcoholicfermentationbyboth
EnablingMicrobiologicalToolsand
yeastandbacteria.Tisrelativelypure
gaseousstreamrequiresnoprimaryseparation
TechnologiesthatMustbeDeveloped
orenrichmentsteptoconcentratetheCO
2
, Cellulosicbiofuelresearchwilluseabroadrangeofpow-
whichcanbesequesteredaspartofthenational erfulomictoolstargetedforfullerdevelopmentofthe
climate-protectionprogramorprocessedby plant,enzyme,andmicrobialarena.However,somespecic
biologicalorothermeanstousefulcoproducts.
microbiologicaltoolswillneedtobecreatedtofurther
Technologiescouldbedevelopedtoproduce
understandandexploitmicroorganisms.Tesetoolsinclude
value-addedcompoundsthatmightprovide
analyticaltechnologiesandcomputationalapproachesand
incomefornancingethanolbioreneries.
technologiesforrevealingthestateofamicrobialsystem,
permittingassessmentofperturbationeectsonthesystem,
Tefundamentalchallengeishowtosupply
andprovidingtheinformationneededtoconstructuseful
chemicalenergytouseandreduceCO
2
,pro-
modelstoguideengineeringeorts.
duceusefulcompounds,andelucidatefactors
governingecientuseofCO
2
.
138 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Techapter,Crosscutting21
st
CenturyScience,TechnologyandInfrastruc-
tureforaNewGenerationofBiofuelResearch,p.155,discussesindetail
barriersin(1)gene-transfermethodsandexpressionofgenesinnoncon-
ventionalhostorganisms;(2)toolsforrapidanalysisandmodelingofcel-
lularcompositionandphysiologicalstate;and(3)HTPscreeningmethods
fornovelandevolvedgenes,enzymes,cells,andcommunities.Additional
requiredtoolsincludethefollowing:
Devicesandrequirementsforpreparingwell-controlledmicrobial
samplesforomicanalysis.
Integratingbiologicalstudiesintoawhole-systemsunderstandingis
beingmadepossiblebynewanalyticaltechniques.Systemsbiology
needstobedrivenbyanorganismsbiologicalcontextanditsphysi-
ologicalstate,whichislinkedtightlytoitscompletehistory,includ-
ingthatofculture.Forexperimentationinallaspectsofworkwith
omictools,high-qualityreproduciblesamplesareparamountfor
subsequentanalysisorpurication.
Controlledcultivationisthemethodtoprovidethesesamples.
Cultivationalsoisthatpartoftheexperimentwhereknowledgeof
thebiologyiscritical,andthequalityofsubsequentunderstanding
isdrivenbythedesignofmicroorganismcultivation.Teemphatic
consensusofworkshopattendeeswasthatchemostatorcontinu-
ous,stirred-tankreactorswillprovidethehighest-qualitybiological
samplesformeasuringmultipleproperties(omics)becausethey
maintainenvironmentalconditionsatasteadystate.Forsomeomic
techniques,batchoperationwillbechosenbecauseoflimitationsin
currentcultivationtechnologyandbecauseofsample-numberand
amountrequirements.Investigatorsmustrealize,however,thatthe
increasedamountornumbercomesatsomecosttoquality.
Apparatusisneededtocharacterizemixedmicrobialpopulations.
Celluloseisdegradedinnaturebymixedpopulationsneedingchar-
acterizationbeyondidenticationofitsmembers.Newtoolsand
approachesarerequiredtounderstandeachpopulationscontribution
tocellulosedegradation.
Developmentofnoveltechniquesandapproachesisneededtocarryout
evolutionarybiotechnology,especiallyformultigenictraits.Newand
moreecientmethodstogenerategeneticandphenotypicvariationin
microbesareneededtoincreasecapabilitiesforobtainingnewpheno-
typesthatrequiremultiplesimultaneouschanges.
Techniquesandapproachesarerequiredforstudyinginteractions
betweencellulolyticmicrobesandtheirsubstrates.Akeystepin
cellulosedegradationinnatureisadhesionofmicrobestothesub-
strate.Tisdynamicprocessneedstobecharacterizedwithnewand
morequantitativeandspatially,temporally,andchemicallysensitive
approachesandtechniques.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 139

SUGAR FERMENTATION
Realizationoftargetedconsolidationopportunitiesandadvancementof
relevantfundamentalswillbeservedbyavarietyofcrosscuttingtechnolo-
giesandcapabilities,includingthefollowing.

Bioreactorsnovelcongurations,insomecasestoevaluateperfor-
manceforconsolidationopportunitiesandtotestseveralkeyhypotheses.
Evolutionarybiotechnologytodevelopneededstrains,includingthose
withnewcapabilities.Applicationofthesetechniqueswillbeadvanced
byminiaturereactorsandautomatedorcontrolledsystems(continuous,
semicontinuous,orserialculture)tomaximizeevolutionrates.Some
specialconsiderationprobablywillberequiredtoadaptevolutionary
biotechnologytoinsolublesubstrates.
Improvedgene-transferand-expressiontechnologiesforunconven-
tionalhostorganismsandparticularlyforGram-positiveorganisms,
whichhavepotentialtobeprofoundlyenablingwithrespecttoallthree
consolidationtargets.
HTPscreeningforfunctionalabilitiesandtraits.Tisisneededforselec-
tionofthemostimprovedstrains,especiallyfornongrowth-associated
functions.Italsoisneededtoidentifythefunctionofunknownorhypo-
theticalgenestoallowbettermodelsandmetabolicengineering.
Toolstounderstandmicrobialmixedculturesinanindustrialcontext.
Scanningandothermicroscopictechniques,aswellasexperimentaland
computationalapproachesdrawnfrombiolmresearch,tocharacterize
adheredcells.

Systemsbiologytools(e.g.,transcriptome,proteome,metabolome)to
characterizeintracellulareventsassociatedwithtargetedconsolidation
opportunitiesinbothnaturallyoccurringandengineeredcells.Tis
includesomicanalysisforcharacterizationofexistingindustrialmicrobes
underproductionconditionstoinformdevelopmentofnewbiocatalysts.
Quantitativemodelingatthecellularleveltotestfundamentalunder-
standingandprovideguidanceforexperimentalworkrelevanttoall
threeconsolidationopportunities.
Mesoscalemolecularmodelingtounderstandcriticaleventsoccurring
inthegapbetweencelluloseandanadheredcellanditsaccompanying
enzymes(seesidebar,TeCellulosome,p.102).
Modelstoconrmthatconsolidationandprocesssimplicationwillbe
morecost-eectivethanseparateoptimizedsteps(seechapter,Biopro-
cessSystemsEngineeringandEconomicAnalysis,p.181).
Breakthrough,High-PayofOpportunities
MicrobialCommunitiesforRobustEnergyProduction
Mostindustrialbioconversionsrelyonpurecultures.Allenvironmental
bioconversionsarebasedonmixedculturesorcommunities,withspecial-
istsworkingtogetherinanapparentlystablefashion.Examplesofmixed
140 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

communitiescapableofcellulolyticconversionareruminantculturesand
termite-gutcultures.Arethereintrinsicbiologicalreasonswhycommunities
couldnotbeusedforbiofuelproduction?Tefundamentalquestionis,Are
therestableself-regulatingmultiplexsolutionsforbiofuels?
Microbialcommunitiesoerexibilitynotpresentinmonocultures,
becausethecollectivemultiplemetabolicpathwaysofmicroorganisms
areactivatedasconditionsdemand.Forexample,microbialcommuni-
tiespotentiallycouldproducemultipleformsofcellulaseenzymesforuse
inindustrialproductionofethanol.Infact,multiplecellulaseshavebeen
showntobemoreeectivethanasinglecellulaseatprocessingcomplex
andvariablefeedstocks.Mixedculturestendalsotobemorerobust,achar-
acteristicneededforindustrial-scaleuse.
ResearchDirections
Tisgoalwouldrequiretheabilitytomanipulateandusemicrobialcom-
munitiestoachieveindustrialgoalsnotjustthenaturalmicrobialgoals
ofreproduction,survival,andnetenergyutilization.Currentapplications
ofmixedmicrobialculturesprimarilyareforwastetreatment(i.e.,anaero-
bicdigestionorbioltration).However,thesetechnologiesarepoorly
understoodandexploitnaturalselectionforsurvival.Terearelimited
examplesofproductsfrommixedculturesinthefoodindustry,butmod-
ernbiotechnologyhasusedonlypureculturesforpharmaceuticalsorfor
bioproductssuchasethanol.Terststepsinapplyingmicrobialcom-
munitiestobiofuelsare(1)characterizeandunderstandexistingcellulo-
lyticmicrobialcommunitiesofmicrobes(e.g.,ruminant,termite,andsoil)
and(2)developtechniquestounderstandandstabilizeintentionalmixed
cultures.Researchcanelucidatedetailedpopulationinteractions(e.g.,both
trophicandsignaling)thatstabilizethecommunity.Supportalsoisneeded
toevaluaterobustnessandpopulationdriftovertime,sincemanymixed-
cultureoperationswillbecontinuous.Gainingadeeperunderstanding
aboutcommunityevolutionwillallowtheuseofselectivepressuremeth-
odstoevolveconsortiawithincreasedcellulose-processingeciency.As
anadditionalbenet,knowledgeofmixed-culturedynamicsmayallow
developmentofnewmethodstomakepureculturesresistanttobiological
contamination.
ScientifcChallengesandOpportunities
Temostbasicrequirementsareformixed-cultureidenticationand
enumeration.Majorsciencechallengesareanalysisandmeasurementof
themixed-culturestate.Mostcurrentomicanalysesarepredicatedon
knowledgeofthegenesequence.Mixedculturesincreasethechallenge
forsequence,transcriptomics,andproteomics.Techallengeincreases
geometricallyforlower-number(<1%)representativesofthecommu-
nity.Singlespeciesexistingasapopulationofclonalvariantsalsoadds
complexity.Terefore,newtechniquesneedtobedevelopedandtested
forsequence-basedmetaomicanalysis.Alsopossiblearenonsequence-
based,metaomictechniques(e.g.,transcriptfunctionbasedmicroarrays)
(Zhouetal.2004).Temetabolomeistheonlyanalysisthatwillnotbe
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 141

SUGAR FERMENTATION
signicantlymoredicultcomparedwiththatanalysisinpureculture.
Onesubsetofmixedculturesisthemanyindustrialstrainsthatliveas
agroupofclonalvariants(asingle-speciespopulation).Analysisofhow
theseactualstrainshaveadaptedtotheirworkingenvironmentcouldbe
helpful.
Afterbasicanalysis,enumeration,andquantitativemeta-omics,thegoal
istounderstandcommunitystructure.Signalingmoleculesareknownto
beimportantinmanycommunities,soweneedtoidentifyandconrm
thesemoleculesanddeterminetheirimportance.Tenwecanconsider
howtomodifythesesignalstocontrolthecommunity.Regulatoryand
metabolicmodelingofindividualmembers,aswellasthecommunity,will
beessentialtodecipheringtheregulatorystructure.Understandingthe
physicalstructurewillrequireimagingtechnologiestoacquiredetailed
visualizationandspeciclabelingofindividualspecies.Tismightrequire
individual-speciesmodicationtoexpresstaggedmarkerproteinsand
thentoreassemblethemixedculture.Computationalmodelscombining
omicsandbiochemicalandspatialvariableswillbecriticaltoaccomplish-
ingthisgoal.
Reproduciblesamplesandimprovedcultivationtechniquesinhighly
instrumentedchemostats,forexample,alsowillberequired,especially
whenlignocellulosicsolidsareintroduced.Inthiscase,reproducible
samplesareespeciallyneeded.Mixedculturesmaynotbedeterministic.
Someevidenceshowsthatthenalstateishighlyvariable.Previouswork
hasshownthatparallelenrichmentsfromthesamenaturalsourceeachled
todierentpopulationsaftermultipleserialtransfers.
GTLFacilitiesandCapabilities
Amajorpriority,asdescribedintheGTLRoadmap,istounderstand
microbialcommunities.CapabilitiesbeingdevelopedintheGTLprogram
areideallysuitedfordevelopingindustrialuseofmicrobialcommuni-
ties.GTLwillrelyheavilyontheDOEJointGenomeInstitute(JGI)for
sequencingandresultingannotation.Integratedproteomiccapabilities
willbeusefulforawiderangeofomicanalyses.Signalingmoleculesand
taggedproteinsandcloneswouldbeprovidedfromproteinproduction
resources.Foradvancedcommunitymetabolicmodels,cellularsystems
capabilitiesareneeded.Whenstudyingcommunityinteractionswitha
lignocellulosicmedium,theNationalRenewableEnergyLaboratorys
BiomassSurfaceCharacterizationLaboratorywillbevaluable.
Model-DrivenDesignofCellularBiocatalyticSystems
UsingSystemsBiology
Systems-levelmodelingandsimulationisthemoderncomplementtothe
classicalmetabolicengineeringapproachutilizingalltheGTLtechnolo-
giesandcomputing.Microbialorganismscontainthousandsofgenes
withintheirgenomes.Tesegenescodeforallproteinandenzymecompo-
nentsthatoperateandinteractwithinthecell,butnotallproteinsareused
underallconditions;rather,anestimated25%areactiveunderanygiven
142 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

conditioninalivingcell.Wecurrentlyunderstandverylittleabouthow
alltheseexpressedproteinsinteractandrespondtoeachothertocreate
cellularphenotypesthatwecanmeasureortrytoestablish.Withimproved
understandingofsuchcomplexcellularsystems,weshouldgaintheabil-
itytodesigntheminanintelligentmanner.Figure9.MicrobialModels
forProvidingNewInsights,thispage,indicatessomeofthispowerand
complexity.Twogeneralandcomplementarymethodsaretakenintocon-
sideration:Tesyntheticroute,whichembracesdenovocreationofgenes,
proteins,andpathwaysandthenature-basedroute,whichusesexisting
suitesofmicroorganismsandseekstoimprovetheirpropertiesviarational
design.Tisisaqualitativestepbeyondrecombinantandnativestrategies
discussedintheMetabolicEngineeringsection,p.127.
Techallengestartswiththeabilitytocharacterizecellularnetworks
andthenmovestowardestablishingcomputermodelsthatcanbeused
todesignthem.Tesemodelswouldcaptureallaspectsofamicrobes
metabolicmachineryfromprimarypathwaystotheirregulationand
usetoachieveacellsgrowth.Figure5illustratespartofthispathway
complexity.Troughthesecomputationalmodels,wecoulddesignand
optimizeexistingorganismsor,ultimately,createnovelsyntheticorgan-
isms.Specictobiomass-to-biofuelsobjectives,organismsengineered
usingthesetechnologiespotentiallywillconsolidatetheoverallprocess
andreduceunitoperations(seeFig.9.MicrobialModelsforProviding
NewInsights,thispage).
Fig.9.MicrobialModelsfor
ProvidingNewInsights.Infor-
mationgainedinGTLsystems
biologyresearchwillenable
metabolicengineeringand
modelingtoenhancemicrobial
characteristics.Usingdened
experimentalparameters,the
biologycanbechangedtoper-
formdesirednewtasks.Tiswill
allownewbiologicalsystemout-
puts,increaseknowledge,and,
ultimately,improvepredictive
models.[Source:M.Himmel,
NationalRenewableEnergy
Laboratory]
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 143

SUGAR FERMENTATION
ResearchDirections
Tisresearchwouldprovidetheabilitytocontrolandoptimizeamicrobial
transformationfromcarbohydratestoethanolandrelatedvalue-added
coproductsinaselectedmicrobe.Gainingcompletecontrolovercellular
networksimpliesacapabilityforengineeringandconsistentlyperforming
transformationwiththebestavailableyields,rate,andtiters.Italsoincludes
theabilitytodesignthemicrobeintelligentlybyusingcomputationaltech-
nologiesfordeterminingconsequencesandoptimalapproachestointervene
andengineerwithincellularnetworks.Furthermore,thismodelwould
enableustoassessthelimitsofabioprocesssmicrobialbiotransformation
(e.g.themaximalproductivityandratesachievable)andpotentiallytoengi-
neerentirelynovelbiotransformationpathwaysandsystems.
Specicresearchdirectionswouldincludethefollowing.
1.EnumerationofCellularComponents,Interactions,andRelated
Phenotypes.Beforeanypredictivecomputationalmodelscanbebuilt,we
needtogeneratetheunderlyingdatasetsforrelevantprocessconditions:
Identicationofallproteinsandenzymesparticipatinginthemetabolic
pathwaysrelevanttocarbohydratemetabolismforcellgrowth,ethanol
synthesis,andrelatedby-products.Tisalsowouldincludecharac-
terizationofkeyenzymecomplexesrelevanttocellulosedegradation,
carbohydratetransport,andrespiratorymechanisms.Experimental
technologiesthatmaybeusefulincludeproteintagging,proteomics,and
invivoactivitymeasurements.
Characterizationofnovelproteinandgenefunction.About30%of
geneshavenounderstoodfunction,yetsomeoftheseunknowngenes
arethoughttobeinvolvedinthemicrobialmetabolicsystemsandstress
responsesunderprocessconditions.
Identicationandquanticationofallmetabolitespresentwithinthe
cell.ExperimentalapproachescouldinvolveNMRorMS.
Characterizationofthecellsenergeticsundervariousrelevantconditions.
Tesewouldincludemeasurementstocharacterizethestoichiometryof
energy-transducingcomplexes,parameterssuchastheP/OandP/H+
ratios,andacellsmaintenanceenergyassociatedwithcellularfunctions.
Characterizationoftransportmechanisms.Inparticular,thiswould
focusondeterminingthecomponentsassociatedwithtransportof
nutrientsintoandoutofthecellaswellasthoseofthemitochondria
ineukaryoticorganisms.Determiningthestoichiometryoftransporters
andtransportprocesses,aswellastheirkineticsanddierentialregula-
tion,isenvisioned.
Characterizationofmembranecompositionforprocesstolerance
(i.e.,alcoholandtoxintolerance)andenvironmentalandcommunity
interactions.
Elucidationofregulatorynetworksenabledbydevelopmentofexperi-
mentalapproachestoidentifyprotein-proteinandprotein-DNA
interactions.
144 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Localizationdataindicatingwhereproteinsareoperatingwithinthe
cellularandcommunityspace.
2.KnowledgebasetoDevelopDynamicsandKineticsModelingTech-
niques.Oneofthemostattractivefeaturesofamodel-driven,rational
approachisitspredictivecapacity,requiringtheinclusionofregulatory
eventsatthegeneticandmetaboliclevels.Creationofsuchmodelsrequires
datasets,aswellasdevelopmentofHTPtoolswithcapabilitiesbeyond
thosecurrentlyavailable.
Acquisitionofhigh-qualityanddynamicomicdata.
DevelopmentofHTPmethodstoidentifybindingsitesofglobalregu-
latoryproteinsandotherinteractions.
DevelopmentofHTPtoolstomonitorkeyplayersthatdenethecells
redoxandenergystates[e.g.,ATP,GTP,NAD(P)H,andNAD(P)].
HTPquanticationofinvivoenzyme-activitymetabolicuxes.
3.NetworkReconstruction.Fromdatasetsgenerated,wecandevelopthe
completemappingandreconstructionofmicrobialnetworksandphysiol-
ogyrelatedtotheconversionofsugarstoethanol.
Automatedtechniquestointegratedatasetsandrapidlycreaterecon-
structednetworks.
Integratedrepresentationofmetabolism,regulation,andenergetics.
Approachestoaccountfortheimpactofspatiallocalizationofproteins
andenzymecomplexeswithinintegratedmodels.
4.DevelopmentofInSilicoAnalysisTools.Methodsareneededto
interrogateandsimulatethefunctioningofconstructednetworksto
addresskeyquestionsaboutmicrobialphysiology.Anymethodshould
developtestablehypothesesthatcanbeintegratedwithexperimental
studies.Methodsshoulddothefollowing:
Assistinnetworkreconstruction,particularlyinmetabolicpathwaysand
regulatorynetworks.Tesemethodsmayinvolvenewapproachesthat
usearticialintelligence.
Interrogatemechanismsassociatedwithtoxicresponsesandtoleranceto
productandintermediatelevels.
Assessphysicochemicallimitationsofcellularsystemsandenzyme
componentstodeterminemaximumachievablerates(e.g.,identifyrate-
limitingsteps,kineticaswellasdiusionlimited).
Generateprospectivedesignsofcellularnetworksbymodifyingand
testingexistingcellularsystems.
Designsystemsdenovofromcellularcomponents.
5.DesignofCellularSystems.Designingengineeredandsynthetic
organismstoconvertcarbohydratestoethanolthroughtheuseofcompu-
tationalmodelsandmethodswouldincludethefollowing:
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 145

SUGAR FERMENTATION
Dedicatedtransformingmicrobeswithfocusedabilitiestoperformbio-
transformationandnecessarysupportingoperationsfortheconversion
ofcarbohydratestoethanol.
Self-replicatingsyntheticmicrobestosupportbiofuelproductionunder
optimalconditions.
Novelpathwaysforproducingbiofuelsandvalue-addedcoproducts
frombiomasscouldinvolvethegenerationofnewenzymesandorgan-
ismsthroughtheuseofevolutionarydesignconcepts(e.g.,directed
evolutionandadaptiveevolution).
ScientifcChallengesandOpportunities
Toaddressthismodel-drivendesigngoal,anumberofbroadscienticand
conceptualchallengeswillneedtobeovercome,includingtheabilityto
makehigh-qualitymeasurementsofcellularcomponentsandstatestosimu-
latephysiologyanddesignnetworkswithmodelsgeneratedfromthesedata.
TheRoleofGTLCapabilities
ManyGTLcapabilities,eithercentralizedordistributed,canbeleveraged
toaidinaccomplishingthesegoals.Particularonesarenotedbelow.
Protein Production
GTLcapabilitieswillbeusedtocharacterizeproteinsbyrapidisolation,
production,andbiochemicalcharacterizationinanHTPmanner.
Molecular Machines
Molecularmachineanalysiswillenablecharacterizationoflargecomplexes
containingmanyactivecomponentsofbiotransformationnetworks.
Proteomics
HTPanalysisofallproteinspresentinthecell,theirrelativeabundance,spa-
tialdistribution,andinteractionswillbeimportanttomodeldevelopment.
Cellular Systems
Ultimately,cellularsystemsanalysisisaboutdevelopingcomputational
modelsofsystemsthatcanbeusedreliablytoengineermicrobes.Resources
dedicatedtotheanalysisandmodelingofcellularsystemscanbeused
reliablybytechnologiststoengineermicrobesforbiofuelproductiononan
industrialscale.
OutcomesandImpacts
TeGTLRoadmapdescribesscienticgoalsandmilestonesandthe
technologyandcomputingneededtomeettheseresearchdirections.Tese
resourcescanbefocusedontheproblemofengineeringexistingorsyn-
theticorganismsforbiofuelproductionfrombiomass.Tistypeofrational
designandorganismengineeringhasthepotentialtotransformvarious
stagesofbiomassconversiontobiofuelsconsistentwithgoalsofconsolidat-
ingtheoverallprocessandreducingunitoperations.Tepracticalimpact
146 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

isinreducingthetimerequiredtomodifyamicroorganismtoperformas
desiredinanindustrialsetting.
Althoughthedirectappliedbenetwillbeinbiomass-to-biofuelprocesses,
technologiesandmethodsderivedfromtheabilitytoreliablyengineer
biologicalsystemswillhavefar-reachingimpactsonbasicandapplied
researchacrossmanysectorsofbiotechnology.
DirectBioproductionofEnergy-RichFuels
Tisbreakthrough,high-payoopportunityfocusesonmicrobesfordirect
productionofhydrophobicalternativefuels(i.e.,alkanes,longer-chain
alcohols,andfattyacids).Tiswouldovercomeonelimitationofnearlyall
bioconversionstheyresultindiluteaqueousmixtures.Typicalindustrial
productconcentrationsare100to150g/Lforethanolandothersuch
productsasorganicacids.Tislimitationimposesseparationrequirements
thatincreaseprocessandenergycosts.Newfermentationsystemswould
behighlydesirabletoallowsignicantincreasesinproductconcentration,
newtypesofproducts,andnewprocessesforproductrecovery.Strong
increasesineciencyalsocouldbeachievedbydevelopingcontinuous
processes.
ResearchDirections
Microorganismsproduceawidevarietyofpotentiallyusefulcompounds
butinrelativelylowamounts.Recently,becauseofexpandedknowledge
abouttheidentityofgenesforimportantpathwaysandmechanismsof
pathwayregulation,increasingtheuxofmicrobiallyproducedchemicals
byuptosixordersofmagnitude(Martinetal.2003)hasbeenpossible
(fromtracelevelsofprimaryproducts). Anewopportunityisnowoered
toexplorewhetherornotsimilarmethodscanbeappliedtodeveloping
modiedmicroorganismsthatsecretenontoxicmoleculespossiblyuseful
forfuels.Examplesmayincludealkanes,longer-chainalcohols,fattyacids
(VoelkerandDavies1994),esters,andothertypesofmoleculeswithlow
aqueoussolubilitythatfacilitatecontinuousproductremovalduring
fermentation.Advancesinunderstandinghowhydrophobicmoleculesare
secretedbyspecializedcelltypes(Zaslavskaiaetal.2001)mayfacilitate
thedevelopmentofradicallynewproductionsystems.Techallenges
describedhereforfermentationintohydrophobicfuelsalsowouldapply
topotentialphotosyntheticsystems.
Additionally,advancesinsystemsbiologyandproteinengineeringmay
facilitatenewapproachestotheoverallprocessoffermentation.For
instance,developingchemicalregulatorsofcellularprocessessuchascell
divisionmaybepossibletoallowculturestobeheldinhighlyecient
steadystatesforprolongedperiods.Suchprocesscontrolsmaybesyner-
gisticwiththedevelopmentofnovelproducttypesnotnormallypro-
ducedinhighconcentrationsbymicroorganisms.Forexample,cocultures
maypossiblybeusedfordirectlycombiningalcoholsandorganicsinto
etheroresterproduction.Tiswouldbeanadvantageoususeofacetate
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 147

SUGAR FERMENTATION
releasedfrombiomasshydrolysistakingitfromaharmfulby-product
toafuelcosubstrate.
ScientifcChallengesandOpportunities
TeexplosionofsequenceinformationresultingfromGTLandother
genomesequencingprogramshasgreatlyfacilitatedidenticationofgenes
forawidevarietyofprocesses.Tisinformationexpansionalsohasallowed
thedevelopmentofsystemstoolssuchaswhole-genomeDNAchipsfor
measuringgeneexpression.Anext-phasechallengeistobringthatinfor-
mationandassociatedtoolstobearonidentifyingentirepathwaysand
cellularprocessesofrelevancetobiofuelproduction.Additionally,under-
standinghowsuchpathwaysandprocessesareregulatedisessential.New
protein-productionandproteomictoolsenvisionedforGTLwillgreatly
facilitatetheelucidationofpathwaysandtheirregulation.Importantchal-
lengesaretounderstandhowthepermeabilitypropertiesofmembranes
arecontrolledbycompositionandhowthestructureofmembraneproteins
suchastransportersrelatestofunction.Progresshasbeenslowinelucidat-
ingmembraneproteinstructurebyconventionalmethods,requiringnew
approachesthatmaybeaddressedbyGTL.Identicationofmicroorgan-
ismswithhighlevelsofresistancetobiofuelcompounds(butnotneces-
sarilytoanyproductioncapabilities)couldprovideusefulinsightsinto
strategiesforimprovingfermentationeciency.
TheRoleofGTLCapabilities
TefullsuiteofGTLresourcesforgenomicsandsystemstoolswillbe
essentialinclarifyingtheunderlyingmechanismsassociatedwiththese
andrelatedproblems.Examplesofthetypesofcontributionsenvisioned
arelistedbelow.
Protein Production
Proteinproductioncapabilitieswillenableelucidationofenzymefunctionin
novelpathwaysforbiofuelproduction;optimizationofenzymesandtrans-
portersbyproteinengineeringandevolution;andrevelationofcomponents
forinvitropathways. Tiscouldleadtodevelopmentofnovelchemical
regulatorsofmicrobialcellularprocessesforuseinindustrialfermentation.
Molecular Machines
Teseresourceswillallownanoscaleinterrogationofmembraneinter-
actionswithbiofuelcompounds(e.g.,usingpatternedmembranes);
identicationofproteincomplexes;andmechanisticunderstandingof
transportersinvolvedinbiofuelsecretion.Developmentofnanoscalemate-
rialswillfacilitateproductseparations.
Proteomics
Teproteomicapproachinvolvesbiological-stateomicsformicrobesunder
inhibitorystress;characterizationofpost-translationalmodicationsof
proteinsthatregulateenzymesorpathwaysforbiofuelproduction;and
analysisofbiofuelexposureeectsonmicrobialgeneexpression.
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Cellular Systems
Cellularsystemcapabilitiesincludemodelingofcellularcarbonuxfrom
uptakeofbiomass-derivedsugarstosecretionofnishedbiofuelcom-
pounds,systemsengineeringofbatchandcontinuousfermentationfor
biofuelproduction,andmodelingofproteinstructuresinaqueousand
nonaqueousenvironments.
DOE Joint Genome Institute
DOEJGIwillcharacterizeorganismswithsuchusefulpropertiesashigh
productivityoforresistancetoprospectivebiofuelcompoundsandwill
developgene-expressioninterrogationsystems.
Other Needs
Otherneeds(e.g.,screeningfornewpathwaysandfunctions)include
assessmentofmaximalredoxbalances(reducedfuelproductsyieldmore
CO infermentation).
2
OutcomesandImpacts
Ifalternatefuelsweremadewithhigherfuelvalue(i.e.,diesel,alkanes,
lipids),bothseparationsandlife-cyclecostswouldbealteredbecausethese
hydrophobicfuelswouldseparatespontaneouslyfromwater.Fuel-density
issuesofethanolalsowouldbereduced.Additionally,transportationcosts
mightbeloweredbecausecompoundssuchasalkaneswouldbesigni-
cantlylesscorrosivethanethanol.Tesebiofuelscouldbeusedmoreeasily
inthenationscurrenttransportationinfrastructure.Ifcontinuousfermen-
tationwithproductremovalwereimplemented,higherthroughputwould
resultinlowercapitalexpendituresaswellascostsassociatedwithproduct
dehydration,asinethanolproduction.
TranslationtoApplications
DOEEEREwouldleadinpilot-scaletestsofstrainsthatproducenovel
biofuelsandindevelopingfermentationprocessesbasedonnewstrains,
products,andproduct-recoveryprocesses.EEREwouldanalyzethepoten-
tialmarketandcostimpactsfornewandexistingbiofuelsandthentake
theleadinseparationtechnologiesandinintegrativeseparations.Addi-
tionally,EEREwouldcarryouttestingandpossibleengine-designmodi-
cationsfornewtypesofbiofuels.
OptimalStrains:FermentativeProductionof40%Ethanolfrom
BiomassSugars
Currentcorn-to-ethanolprocessingplantstypicallyproducetitersof10to
14%w/w.Becauseoflimitsonbiomasssugarconcentrations,theseethanol
levelsareatleastthreefoldhigherthanthoseproducedfromlignocellulose
usingcurrenttechnologyandbiocatalysts.Tthediluteproductstreamfor
lignocellulosicethanolimposesatwo-tothreefoldincreaseinfermenta-
tionvolumeperannualgallonofethanol,withcorrespondingincreases
inpumps,nutrients,andmanagement.Useofmodernmoleculartoolsto
harvestthecatalyticdiversityofnatureshouldfacilitateconstructionof
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SUGAR FERMENTATION
revolutionarybiocatalyststhatcouldincreaseethanoltitersupto40%and
lowerneededinvestmentsincapitalandoperatingcostsforbiomass-to-
ethanolplants.Tischallengeispresentedasanexampleofhowsystems
biologymayallowapplicationstoexceedcurrentbiologicallimits.Strat-
egiesforobtainingorganismsthatproduceandtoleratehigh-ethanol
concentrationsinclude:
Engineeringcurrentethanolproducerstoretainhighmetabolicactivity.
Engineeringnaturallyethanol-tolerantorganismstoproduceethanol.
Exploringnativediversityofmicroorganismstoidentifythosethat
retainglycolyticandfermentativeactivityinthepresenceofhigh-
ethanoltiters.
ScientifcChallengesandOpportunities
Ifachieved,productionoffermentationbrothcontaining40%ethanolfrom
biomassorstarchsugarswouldrevolutionizeprocessdesigns.Incremental
progresstowardthisgoalwouldreducedramaticallythesizeoffermentation
plantsbydecreasingfermentorcapacityandassociatedpumps,nutrientcost,
waterusage,andwaste-watertreatmentandrecovery.Forinstance,doubling
ethanoltitersfrom5to10%wouldreduceprocesswatervolumeby63%for
equivalentethanolproduction.Furtherdoublingthetiterfrom10to20%
wouldreducethewaterneededbyanadditional55%.Forfermentation
brothwitha40%titer,waterusagewouldbeonlyone-tenththeamountcur-
rentlyneededforthebiomass-to-ethanoltechnologythatproduces5%titer.
Atethanoltitersabove40%,viablealternativestodistillationcouldreduce
energycostsassociatedwithpurication.However,distillationisamature
technologyanditsenergycostsdonotsubstantiallylimitthecurrentprocess
becausewasteheatisreused.
Improvingthedistillationprocessalsowouldrequiredevelopingbetter
upstreamprocessesorconversiontechnologiestoprovidehighlyeective
sugarconcentrations.A30%biomassslurrycanyieldonlya15to20%
sugarstreamthatresultsinjust7to10%ethanol.However,reectingon
thischallengeillustrateshowtofurtherpushbiologybeyondcurrentlimits.
Grainethanolplantsproducefrom10to15%ethanol(Lynd, Wyman,
andGerngross1999),reachingupto20%ethanolwhenprovidedwith
veryhighsubstratelevels(ScoutenandPetersen1999).Evenhigherlevels
ofethanol(25%)areproducedveryslowlybysakeyeastsatalowtem-
perature.Temostethanol-tolerantmicroorganismsknown,Lactobacillus
homohiochiiandL.heterohiochii,wereisolatedasspoilagebacteriainsake
(RoadmapforBiomass2002;Svensonetal.2001;Lyndetal.2005).Te
membranelipidsinthesebacteriacontainunusuallylongfattychains
thatsupposedlyareanadaptationtogrowthinhighethanol.However,no
systematicsearchforaclassofextremophileswithresistancetoethanolhas
beenreported.
Cellsneednotbeviabletometabolizesugarstoethanol.Atabiochemi-
callevel,ethanolproductionfromsugarsisastronglyexergonicreaction
whencoupledtoATPhydrolysis.Individualglycolyticenzymesinyeast
150 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

andZymomonasmobilishavebeenshowntofunctionwellinthepresence
of20%ethanol(Zhouetal.2004)andareprogressivelymoreinhibitedat
increasinglyhigherethanolconcentrations.Invitrodisruptedcellprepara-
tionsofbothorganismshavebeenshowntoremainactiveandcontinueto
produceethanoleveninthepresenceof20%ethanol.Althoughorganisms
continuingtogrowatethanolconcentrationsabove30%areunlikelyto
befound,developingmicroorganismsthatremaincatalyticallyactiveand
metabolizesugarstoachieveveryhighlevelsofethanolisquiteplausible.
Forinstance,overhalftheethanolincommercialyeastfermentationsis
producedaftergrowthhasbeeninhibitedbyaccumulatedby-products.
Formulatingnewbiocatalystsforbiomasspresentsachallengeandopportu-
nityforengineeringimprovementstoprovideconcentratedsugarfeedstocks
(Zhouetal.2004).
Describedbelowareseveralapproachesformovingtowardthegoalof
producingfermentationbrothcontaining40%ethanol,includingtheisola-
tionofnativenovelmicroorganismscapableofgrowingorsurvivinginthe
presenceofhighlevelsofethanol.Suchorganismscanbeusedasaplatform
fromwhichtoengineerethanolproduction,ifneeded,andasasourceof
genesandenzymestoimprovealcoholproductionincurrentethanolo-
genicbiocatalysts. TisworkisexpectedtorelyheavilyonGTLresources
forsequencingandtranscriptomeandproteomeinvestigations,whichwill
identifymolecularrequirementsforethanoltoleranceduringgrowthandfor
maintenanceofactivemetabolisminthepresenceofhighlevelsofethanol.
Retainingthetraitsofcurrentbiocatalyststhatdonotdisturbthemetabo-
lismsofallcarbohydrateconstituentsoflignocellulose(hexoses,pentoses,
anduronicacids)willbeimportant.Additionalgenesforusingvarious
biomasscarbohydratesandothercomponentsmaybeneededtoprovide
high-substratelevelsforhighethanoltiters.Tesegenesperhapsinclude
hydrolasesforcelluloseandhemicelluloseaswellasuptakesystemsfor
solubilizedproducts.
Otherbiomasscomponentsoerfurtheropportunitiestoincreaseyield.
Acetatelevelsequivalentto10%ofhemicelluloseweightrepresentapoten-
tialsourceofoxidizedsubstrate.Equivalentlevelsofsolubilizedlignins
representasourceofreducedsubstrate,apotentialelectrondonortoconvert
portionsofbothsubstratesintoadditionalethanol.Cometabolizingthese
substratesbyengineeringknowngenesandpathwaysfromsoilorganisms
couldincreaseethanolyieldupto5%.Acid-stableproductsrepresented
by4-O-methyl-glucuronoxyloseand4-O-methyl-glucuronoxylobiosein
acidhydrolysatesofhemicellulosecurrentlyarenotmetabolizedbyany
ethanologenicbiocatalysts.Teserecalcitrantproductstypicallyarenot
measuredbyhigh-performanceliquidchromatography analysisandcan
representupto10%oftotalcarbohydrate.Discoveryofnewgenesand
organismstometabolizethesesaccharidesandincorporatethemintobio-
catalystscouldprovideafurtherincrementalincreaseinethanolyieldwith
no increaseincapitalor operatingcosts. Together, themorecompleteuse of
allsolubilizedcomponentsfromlignocelluloseandtheincreaseinethanol
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 151

SUGAR FERMENTATION
titerswouldreducedramaticallythesizeoffutureethanolplantsandthe
amountofwateruse.
ResearchDirections
TeburgeoningamountofsequenceinformationandGTLscurrentability
torapidlydeterminegenomesequencesfornewalcohol-resistantorgan-
ismsfromnatureprovideanexcellentopportunity.Wecanidentifythe
genesandfunctionsrequiredforgrowth,survival,andcontinuedmetabo-
lisminthepresenceofhighlevelsofethanol.Transcriptomeandpro-
teomicanalysesresourceswillgreatlyassistthesestudies.
Characterizingmicrobialmembraneandwallstructures,includinglipids,
proteins,andcarbohydrates,representsadicultanalyticalproblemanda
necessarychallengeforfutureGTLcapabilities.Envelopestructureispre-
sumedtorepresentamajordeterminantforcontinuedmetaboliccellactivity
inhigh-ethanolandotherextremeenvironments.Newtoolsareneededto
facilitatedesignandmodicationofbiocatalystsformanyfutureprocesses.
Otheryet-to-be-discoveredopportunitiesincludepotentialmetabolic
pumpsforsolventsandproducts,possiblyevolvedbycellstomaintainlow
intracellularproductconcentrations.Newanalyticaltoolswillbeneededto
investigatepropertiesandfunctionsofthesebiomachines.
Processimprovementsalsowillbeneededtomakeavailablethehigh
amountsofsugarsneededtoachievetheseethanoltiters,leadingtosolid-
statefermentationinthefuture.
AnAlternativeRouteforBiomasstoEthanol:MicrobialConversion
ofSyngas
Biomasscanbegasiedtoproducesyngas(mostlyamixtureofCOand
H ).Perhapssurprisingly,syngashasbeenshowntobeconvertedbycer-
2
tainmicrobesintoproductsincludingethanol(Klassonetal.1990;Gaddy
2000).Tesemicrobesarenotwellunderstood,buttheprocesshasbeen
takentosmallpilotscale.Teattractionofthisalternativeapproachto
bioethanolisthatthetheoreticalyieldisquitehighsinceallthebiomass
potentiallyisavailableassyngasforanaerobicfermentation.Tisgives
theoreticalyieldsgreaterthan130galperdrytonofbiomass.
Background
Gasicationisacombinationofpyrolysisandcombustionreactionsfor
convertingasolidmaterial,suchasbiomass,toagasiedproduct(syngas).
Gasicationisarobustandtraditionaltechnology,yetnotextensively
implemented.
Biopowercanusethissyngasasafuelforpowerproduction.Oncesulfur
compoundshavebeenremoved,thisgascanbeconvertedtootherproducts
throughcatalyticFisher-Tropschreactionsathightemperaturesandpres-
sures.However,theseprecious-metalcatalystsforgas-to-liquidconversion
havebeenexploredforover50yearswithincrementalimprovements.Bio-
catalystsforsomeconversionmethodsarerelativelyunstudied,operatingin
152 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

aqueousmediawiththesyngasbubbledpastatambienttemperatureand
pressuresandrepresentingastrongalternativetotraditionalcatalysis.
Challenges
Howdothesebiocatalystscarryouttransformationsthatotherwisework
onlywithpreciousmetalsathightemperatureandpressure?Which
enzymesandmolecularmachinesallowthesetransformations?Can
increasedunderstandingoftheseproteinstructure-functionrelationships
aiddevelopmentofeitherbetterbiocatalystsorinsightstoimprovedinor-
ganicmetalcatalysts?
Trialanderrorexperimentationhasshownthatprocessconditionsand
reactordesignwillshiftthemicroorganismstohigherproductyields.Tis
isthefundamentalandunexploredbiologicalquestion:Howdoesthe
regulationofthefermentationpathwayallowtheseenvironmentalshifts
(e.g.,pH,andmediumcomposition)toinducehigheryields?
SyngasStatusinIndustry
BioengineeringResources,Inc.isasmallcompanydevelopingandsoon
tobedemonstratingitspilotsyngasbioethanolprocess(EERE2005;BRI
Energy2006).TeUniversityofOklahomahasassembledanintegrated
gasicationandbiologyprogram;however,ratesremainslowandarelim-
itedbythefundamentalbiologyandmasstransfer(Klassonetal.1990).
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 153

SUGAR FERMENTATION
CitedReferences
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process01.html).
EERE.2005.SynthesisGasFermentation(www.eere.energy.gov/biomass/
synthesis_gas_fermentation.html).
Gaddy,J.L.2000. BiologicalProductionofEthanolfromWasteGases
withClostridiumljungdahlii,UnitedStatesPatent6,136,577.
Klasson,K.T.,etal.1990.BiologicalProductionofLiquidandGaseous
FuelsfromSynthesisGas,Appl.Biotech.Biochem.2425,85773.
Lynd,L.R.,etal.2005.ConsolidatedBioprocessingofCellulosicBio-
mass:AnUpdate,Curr.Opin.Biotechnol.16,57783.
Lynd,L.R.,C.E.Wyman,andT.U.Gerngross.1999.Biocommodity
Engineering,Biotechnol.Prog.15,77793.
Lynd,L.R.,R.T.Elander,andC.E.Wyman.1996.LikelyFeaturesof
MatureBiomassEthanolTechnology,Appl.Biochem.Biotechnol.5758,
74161.
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richiacoliforProductionofTerpenoids,Nat.Biotechnol.21,796802.
Mosier,N.,etal.2005.OptimizationofpH-ControlledLiquidHotWater
PretreatmentofCornStover,Bioresour.Technol.96,198693.
RoadmapforBiomassTechnologiesintheUnitedStates.2002.Biomass
ResearchandDevelopmentTechnicalAdvisoryCommittee(www.biomass.
govtools.us/pdfs/FinalBiomassRoadmap.pdf ).
Scouten,W.,andG.Petersen.1999.NewBiocatalysts:EssentialToolsfora
Sustainable21stCenturyChemicalIndustry,CouncilforChemicalResearch
(www.eere.energy.gov/biomass/pdfs/biocatalysis_roadmap.pdf).
Svenson,C.J.,etal.2001.StrategiesforDevelopmentofTermophiles
forEthanolProductionfromLignocellulosics,presentedat23rdSympo-
siumonBiotechnologyforFuelsandChemicals,Breckenridge,Colo.,May
2001.
Voelker,T.A.,andH.M.Davies.1994.AlterationoftheSpecicityand
RegulationofFattyAcidSynthesisofEscherichiacolibyExpressionofa
PlantMedium-ChainAcyl-AcylCarrierProteinTioesterase,J.Bacteriol.
176,732027.
Zaslavskaia,L.A.,etal.2001.TrophicObligateConversionofaPho-
toautotrophicOrganismTroughMetabolicEngineering,Science292,
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&Sons,Inc.
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Crosscutting21stCenturyScience,
Technology,andInfrastructurefor
aNewGenerationofBiofuelResearch
OpportunitiesandChallenges
E
cientlyandinexpensivelyproducingethanoloralternativeprod-
uctssuchasalkanes,fattyacids,andlonger-chainalcoholsfrom
biomasswillrequiresignicantadvancesinourunderstandingand
capabilitiesinthreemajorareasexploredatthisworkshop:Feedstocksfor
Biofuels,DeconstructingFeedstockstoSugars,andSugarFermentation
toEthanol.Asystems-levelapproachtounderstandingandmanipulating
plantsandmicroorganismscentraltoprocessingbiomassintoliquidfuels
dependsonobtainingandusingdetailedchemicalandbiochemicalinfor-
mationonorganismstatesandstructurestobuildfunctionalmodelsthat
guiderationaldesignandengineering.Asystems-levelunderstandingof
modelplantswillfacilitaterationalimprovementofplantcell-wallcom-
positionincropsdedicatedtoconversionintobiofuels.Newapproaches
andtoolswillbenecessarytocharacterizedenitivelythedetailedorga-
nizationalstructuresofprincipaltypesofplantcelluloseandtheirrelative
energiesandinterrelationshipswithsuchotherstructuralcomponentsas
ligninsandnoncellulosicpolysaccharides.
Similarly,systems-levelexplorationsareneededtodeterminegenetic
makeupandfunctionalcapabilitiesofsuchmicrobialcommunitiesas
thoseinvolvedinbiomassdecompositionandsugarfermentation.Emerg-
ingtoolsofsystemsbiologytogetherwithprinciplesandapproaches
frommetabolicengineering,syntheticbiology,directedevolution,and
evolutionaryengineeringwillhelptoovercomecurrentobstaclesto
bioprocessingcellulosicfeedstockstoethanol.Increasedemphasismust
beplacedonachievingapredictiveunderstandingofplantandmicro-
bialbiologyincludingdynamics,regulation,ux,andfunction,withthe
ultimategoalofrationaldesigntoimprovetraitsofbioprocessingmicro-
organismsandplantfeedstocks.Forexample,amajorbarrierinecient
useofbiomass-derivedsugarsisthelackofmicroorganismsthatcangrow
andfunctionoptimallyinthechallengingenvironmentcreatedthrough
biomasspretreatment,hydrolysis,andcellularmetabolism.Amilestonein
surmountingthisbarrieristoidentifyandunderstandmolecularmecha-
nismsusedbycellstocopewithsuchenvironmentalchallengesashigh
sugarandethanolconcentrationsandthepresenceofinhibitorsfrombio-
masspretreatmentandhydrolysis.Becauseresistancemechanismstypi-
callyinvolvecomplexsubsystemsandmultiplegenes,systemsbiologyis
neededforrationallyengineeringmicrobestoovercometheselimitations.
Ultimately,thesystemsbiologyfocusoftheGenomics:GTLprogram
References:p.180
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 155

CROSSCUTTING RESEARCH
(GTL)hasthepotentialtoenableconsolidationoftheoverallbioconver-
sionprocessandreducethenumberofbioreneryoperations.
Powerfulnewtoolsandmethods,includinghigh-throughputanalytical
andimagingtechnologiesanddata-handlinginfrastructure,alsowillbe
neededtoobtain,manage,andintegrateinformationintomodels.Related
tasksinvolvesequencingmodelcrop-plantgenomes,understandingthe
structureandfunctionofbiomassdeconstructionenzymes,andmodeling
metabolicandregulatorynetworksofmicrobialsystemsinvolvedinbio-
conversion.Someofthesetechnologies,whicheitherexistnoworareenvi-
sionedintheGTLRoadmap,areplannedcomponentsoftheproposed
GTLcapabilityinvestments;otherswillneedtobeenhancedordeveloped
forspecicresearchoutlinedinthisreport.
AnalyticalToolstoMeettheChallenges
ofBiofuelResearch
Systemsforeectivelyconvertingbiomasstoliquidfuelswillrequirea
widearrayofinnovativeanalyticalcapabilitiestofacilitatefundamentaland
appliedscience.Tesecapabilitiesencompassnewmethodsforrapidand
sensitiveanalysisofbiomasspolymersandsubunitsandhigh-throughput
characterizationofplantcellwallsandmicrobialpopulationscatalyzing
bioconversionreactions.Analyticalmethodsshouldprovidedetailedinfor-
mationonchemicalmoieties,chemicalbonds,andconformationofplant
cell-wallpolymers;theyshouldrapidlyassessthestateofmicrobeshaving
newpropertiesorgrowingundervariousdenedenvironmentalconditions.
Asdiscussedinprecedingsections,acapabilityrequirementisanalysisof
membranecomponentsincludinglipids,proteins,andcarbohydrates.Tese
structurespossiblyrepresentmajordeterminantsforcontinuedmicro-
bialactivityinhigh-ethanolandotherextremeenvironments.Membrane
analyticsareachallengingproblembutanessentialcapability.Newtools
areneededtofacilitatebiocatalystdesignandmodicationformanyfuture
processes,andtechnologies,methods,andcomputationalandinformational
toolswillbeestablishedtosupportcoresystemsbiology.Suchtoolsinclude
genomics,transcriptomics,proteomics,glycomicsandlignomics,and
uxomicsasdescribedbelow;imagingtechnologiesatvariouslengthsand
timescales;structure-characterizationtechniques;biomasscharacterization;
cultivation;andaccompanyingmodelingandsimulationcapabilities.
Genomics
Capitalizingonpotentialbiofuelproductionfromcellulosicbiomass
requiresthecontinuedcommitmentofDOEsJointGenomeInstituteto
sequencevariousorganismsthatcontributetotheprocess.Teseorganisms
includecropplants,industrialyeaststrains,andmicrobialcommunities
involvedinbiomassdecompositionandsoilproductivity.Geneticblue-
printsprovidedbyDNAsequenceswillallowtheuseofsystemsbiology
forrationaldesignandprocessconsolidationtoincreaseproductionof
biomasscropsandoptimizebiofuelconversionprocesses.Supportwillbe
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requiredforsequenceassembly,opticalmapping,andothertechniquesto
placeassembledcontigsfromaplant,microbe,orcommunityonaphysical
map.Comparativegenomeanalyseswillinfersimilaritiesordierencesin
regardtootherorganisms,andexpressedsequencetagandotherlibraries
willfacilitategeneticandmolecularanalysisoffeedstockplants.
Genomicinformationwillenableidenticationandmolecularcharac-
terizationofthediversityandactivityofrelevantbiomassdeconstruc-
tionenzymesfromsuchnewsourcesasfungi,bacteria,anduncultured
microbes.Genomicanalysiswillopenthedoortouseofgeneticresources
presentinmicrobialcommunitiesspecializinginlignocellulosedegrada-
tion.Genomicprojectsareenvisionedinthefollowinggeneralareas.
FeedstockPlants
Tisreportoutlinestheimportanceofnewandimprovedsourcesoffeed-
stockbiomassforconversiontoliquidfuels.Consideringprojectscale,soil
andclimatedierencesacrosstheUnitedStates,andthedesiretomaintain
biologicaldiversityonagriculturallands,investigatorsrecognizethatavari-
etyofbiomasscropsmustbeused.Grassespotentiallyareaprimesource
ofbiomass,sodeterminingtheDNAsequenceofoneormoregrassesisa
highpriority.Tegeneticblueprintwillassistinshort-termdevelopment
ofgrass-breedingstrategiesandtheuseoflonger-termsystemsbiology
methodstorealizetheplantspotentialasasourceofbiomass.Someplant
systemstobestudiedincludeBrachypodium(slatedforsequencingatDOE
JGI)andPopulus(alreadysequenced).
SoilMicrobialCommunities
Conversionintoahigh-valuecommodityislikelytoaltertheamount
andcompositionofpostharvestplantmaterialandresiduesreturnedto
thesoil.Sustainabilityofbiofueltechnologiesrequiresascienticassess-
mentofproductionpracticesandtheirinuenceonsoilquality,including
impactsonmicrobialcommunitiesandtheprocessestheycatalyze.Given
thatthetechnologyinvolvescropstailoredtothriveinindividualregions,
anumberofsitesrepresentingvariousecosystemsmustbechosenforsoil
metagenomicanalysis.Intheshortterm,suchanalyseswillcontributeto
anunderstandingofeectsonsoilsustainability.Tedataalsowillbecriti-
calinpreventingunwantedlong-termeectsonsoilsustainabilityandfor
assessingmore-directeectsofsoilmicrobesonplantgrowth.
FermentationandBiomassDecayCommunities
Biomassconversiontosugarsandbiofuelsrequiresoptimizingmicrobial
breakdownofstructuralsugarsandfermentationofcomplexsugarmixtures.
Anumberofmicrobialcommunitieshaveevolvedovermillionsofyearsto
maximizeandcoordinatethesecapabilities,withseveralofthebetter-studied
onesassociatedwithruminantsandthehindgutoftermites. Tus, areason-
ablenumberofmodelfermentativecommunitiesthatcandegradelignocel-
lulosearecriticaltargetsformetagenomicanalysis.Intheshortterm,new
insightsintoactivitiespresentorcoordinatedamongmembersofthesewell-
establishedcommunitieswillresult. Tisinformationpotentiallycanbeused
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CROSSCUTTING RESEARCH
todesignsecond-generationbiofuelsystemscomposedofmicrobialcommu-
nitiesorconsortiaconsideredmorerobustanddiverseinregardtoenviron-
mentalconditionstheycanwithstandandtypesofsubstratestheycanuse.
IncreasedProductionSystems
Adaptiveevolutionofmicroorganismsusingselectivepressureinfermen-
torsorchemostatshasenormouspotentialtoselectfortraitsthatbenet
biofuelproduction.Tounderstandmutationsthatgiverisetodesiredtraits,
determiningthemolecularbasisofsuchchangesiscritical;weneedto
sequenceindustrializedstrainsandcomparethemtotheirprogenitors.In
thelongterm,thisinformationisvitalindesigningnewmicrobialsystems
thatincreasebioconversioneciencyandlowerbiofuelcost(seesidebar,
ProteomicandGenomicStudiesofIndustrialYeastStrainsandTeir
Ethanol-ProcessTraits,p.126).
Transcriptomics:High-ThroughputExpressionAnalyses
MeasuringRNAexpression(transcriptomics)providesinsightintogenes
expressedunderspecicconditionsandhelpstodenethefullsetofcell
processesinitiatedforcoordinatedmolecularresponse.Tisinformationcan
beusedtoelucidategeneregulatorynetworksandevaluatemodelsofcellu-
larmetabolism.Currenttechnologiesincludemicroarray-basedapproaches
applicabletothestudyofplantsandhomogeneousmicrobialpopulations
inwell-mixedsystems.Custommicroarraystargetedtonewplantvarieties
ormicroorganismsarecriticallyneeded.Inheterogeneoussystemssuchas
thoseinmixedmicrobialpopulations,currentmicroarray-basedapproaches
arelessusefulbecausetheyprovideonlyanaveragegene-expressionprole
acrossanentirepopulation.Terefore,single-cellgene-expressionmethods
applicabletodiversecelltypesarerequired.
Proteomics
Althoughcarbohydratesarebiomassconversionsprimarysubstrate,pro-
teinsareworkhorsecatalystsresponsibleforconstructingbiomass-forming
polymers,depolymerizingbiomassbeforefermentation,andconverting
sugarstodesiredendproductssuchasethanol.Tointerrogatethesevarious
processes,identifyingandquantifyingproteinsandproteincomplexesfor
variousplantandmicrobialsystemsandsubsystemsarevital.Tegenomes
informationcontentisrelativelystatic,butproteinsproducedandmolecu-
larmachinesassembledforspecicpurposesaredynamic,intricate,and
adaptive.Allproteinsencodedinthegenomearecollectivelytermedthe
proteome. Tecell,however,doesnotgeneratealltheseproteinsatonce;
rather,theparticularsetproducedinresponsetoaspecicconditionis
preciselyregulated,bothspatiallyandtemporally,tocarryoutaprocessor
phaseofcellulardevelopment.
Proteomicscanbeusedtoexploreamicrobesprotein-expressionprole
undervariousenvironmentalconditionsasthebasisforidentifyingprotein
functionandunderstandingthecomplexnetworkofprocessesfacilitated
bymultiproteinmolecularmachines.Identifyingthesuiteofproteins
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involvedinconstructionandbreakdownofcell-wallpolymersisimportant
inunderstandingbiomassconversiontobiofuels.Inadditiontoanaly-
sisofintracellularproteins,alldegradationenzymesandotherproteins
secretedbybiomass-degradingmicrobesneedtobecharacterizedbecause
theyprobablyarekeytothedepolymerizationprocess.Membraneprotein
systemsareparticularlyimportantandneedtobeimprovedbecausethey
controlcelluloseproduction,cellulolyticenzymeexcretion,andferment-
ablesugartransport.
Tofacilitatemassspectroscopy(MS)detectionandquantitationofpro-
teinsincomplexmixtures,improvementsininstrumentationandchem-
istryareneededtoenhanceproteinandpeptideionization,increase
sensitivityandmassresolution,quantifyproteinlevelsincomplexmixtures,
andwidenthedynamicrangeoftheirdetection.Newinformationandbio-
informatictoolsarerequiredtoanalyzeproteomicdata.
Metabolomics
Analysisofthecellsmetabolitecontenthaslaggedbehindtranscriptand
proteinprolingbutisanequallyimportantindicatorofcellularphysiol-
ogy.Temetaboliteprolemaybeabetterindicatorofcellphysiology
becausemetaboliteconcentrations(anduxes)occursoonerinresponse
tochangesintheextracellularenvironmentthandogeneexpressionand
proteinproduction.Also,metabolitesareprecursorstothecellstranscripts
andproteinsandallothercellmacromolecules,andregulationofcellular
processesmaynotalwaysbereectedintranscriptorproteinproles.
Tedicultyinprolingmetabolitesderivesfromtheirstructuralhet-
erogeneityandshortlifetimesinsidethecell.ProteinsandRNAareeach
composedofaconstrainedorlimitedsetofprecursors(aminoacidsin
proteins,nucleicacidsinRNA).Dierenceslieinaminoacidornucleic
acidsequences,enablingasingleseparationmethodtoanalyzetheentire
transcriptorproteinproleatonce.UnlikeRNAsandproteins,metabo-
liteheterogeneitymakesanalysisnearlyimpossiblewithasinglesepara-
tiontechnique.Althoughsuchtechniques(e.g.,high-performanceliquid
chromatography,thinlayerchromatography,gaschromatography,and
capillaryelectrophoresis)canseparatemetabolitegroupshavingcommon
structures,veryfewreportsonseparationandanalysisofacomprehensive
setofcellularmetaboliteshavebeenissued.Furthermore,metabolitestend
tohavemuchshorter(secondsorless)half-livesthandoproteins(hours)
orRNAs(minutes).Shorterhalf-livesmakerapidmetabolismsampling
andquenchingevenmoreimportantthananalysisofRNAsandproteins.
Methodssuchasnuclearmagneticresonance(NMR)thatcaneectively
integrateandstoreinformationaboutuxthroughparticularpathwaysand
potentiallycanbeappliedtolivingsystems.Localizedmetaboliteconcen-
trationssuchasthosewithinanorganellealsoneedtobemeasured.
Anothercomplicationisthatametabolitesintracellularconcentration
sayslittleaboutitsimportanceincellularphysiology.Indeed,someofthe
mostpotentcellular-signalingmoleculesfoundrecentlyexistatrelatively
lowconcentrationsinsidethecell,andsomemetabolitesproducedand
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 159

CROSSCUTTING RESEARCH
consumedatthehighestrateshaverelativelylowintracellularconcentra-
tions.Intheformercase,extremelysensitivemethodsarenecessaryto
measurethesemetabolites.Inthelattercase,themetabolicuxprole
ismoreimportantthanabsolutemetaboliteconcentrations(seesection,
Fluxomics,p.161).
Metabolomicresearchwilladdressfuelproduction,includingmethodsto
isolate,extract,andanalyzelabilemetabolitessuchasthoseinvolvedincel-
lularenergymetabolism(e.g.,ATP,GTP,NADP,andNADPH).Multidi-
mensionalseparationmethodsarevitaltoanalyzingplantormicrobial-cell
metabolites.Newanalyticaltechniquesarerequiredforcontinuousmetab-
olitemeasurements,monitoringprocesses,andinvivometaboliteanalyses.
GlycomicsandLignomics
Asparallelconceptstotranscriptomics,metabolomics,andproteomics
describedintheGTLRoadmap,biomass-to-biofuelsresearchwilluse
similarhigh-throughputandhigh-contentanalysiswithcompoundshav-
inglowmolecularweightandinvolvementinsynthesisanddegradationof
plantcell-wallpolymers.Glycomics(prolingmaterialsrelatedtostruc-
turalpolysaccharides)andlignomics(prolingmaterialsrelatedtolignin)
areessentialcapabilities.Newpowerfulanalyticaltoolswillprovideinfor-
mationforsystems-levelunderstandingofbiomassstructureandchemistry
anditsroleinbiomassconversiontofuelsandvaluablechemicals.
Informationfromtheseanalyseswillhelpusunderstandnativeand
modiedpathwaysforsynthesisofcell-wallpolymersbytrackingprecur-
sorconsumption,generatingandutilizingintermediatestructures,and
exploringtheirconnectiontoplantcell-wallchemicalcompositionand
physical-chemicalstructure.Atoolboxofoptionsforplantbreedersand
cropscientistseventuallywillbeavailabletoimprovefeedstocksubstrates
specicallyforfuelproduction.Newglycomicandlignomictoolsalsowill
beapplicabledirectlytootherplant-developmentneeds.
Informationobtainedfromlignomicsandglycomicswillelucidatesub-
stratemodicationbytrackingstructuralchangesandconcentrationuxes
insaccharicationproductsthatmaybelinkedtoharshpretreatments.
Saccharication-productprolingalsowillguideselectionofappropriate
enzymaticcocktailsbyidentifyingspecicchemicalbondsandfunctional
groupsinsolidsandlargeroligomericproducts.Teadditionofenzymes
tailoredtoknownrecalcitrantstructurescouldsignicantlyenhance
productyields.Informationonthenatureofrecalcitrantstructuresalso
canbeusedtoguideplantbreedingandpretreatmentconditions.Tese
approacheswillprovideinsightintorate-limitingstepsthatariseinsub-
strate-andenzyme-limitedsystems.
Accurateandrobustglycomicandlignomicanalyticaltoolswillplayan
importantroleinfermentationresearch.Manylignin-andcarbohydrate-
derivedcompoundsarefermentationinhibitors,andothermaterialsmay
beconvertedtoundesirableside-products. Trackingthesematerialsfrom
160 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

feedstockthroughproductswillprovidevaluableinsightsintopossible
improvementsinallstagesofbiomass-to-biofuelsconversion.
Severalspecicchallengespersistintheapplicationofhigh-sensitivity,
high-throughputtoolsforligninandcarbohydrateanalysis.Manyrobotic
andautomatedtoolsusedinconventionalmetabolomicsresearchcannot
accommodatethelargersamplesizesrequiredforrepresentativesampling
ofbiomasssubstrates.Multivariateanalysistoolswillneedtobedeveloped
andvalidatedforaccuratequanticationofcomplexmixturesofsubstrates,
pretreatmentcatalysts,conversionenzymes,andmicrobes.Manyanalyti-
calmethodsforsupportingasystemsapproachtobiomassconversionwill
requirebiomassstandardsnotreadilyavailablefromcommercialsources.
Obtainingsmall-moleculestandardswillrequireprep-scalesynthesisand
isolationofmoleculesofinterestandtheabilitytomodify(e.g.,byisotopi-
callylabeling)isolatedmaterialsusingadvancedtechniquesofcarbohy-
drateandnatural-productorganicsynthesis.
Fluxomics
Useofmicroorganismsforliquid-fuelproductionfromlignocellulosic
biomassdependsonuxesofdiversesubstratesthroughcomplexnetworks
ofmetabolicpathways.Quantifyingmetabolicuxesinmicroorganisms
allowsidenticationofrate-limitingstepsinabiosyntheticpathwaythat
couldbeimprovedbygeneticmanipulationorbyalterationsincultiva-
tionconditions.Substrateturnoverbyanenzymeinametabolicpathway
canbedeterminedbymeasuringchangesinisotopicallylabeledsubstrate
levelsoveraspecicperiodoftime.Usingisotopomeranalysis,quantities
oflabeledsubstratesandmetabolicproductsderivedfromthemcanbe
measuredandcompared.Similarapproachescanbeusedtomapmetabolic
sugaruxestoethanolandrelatedpathways.
Simultaneouslymeasuringtheturnoverofallintracellularmetabolites
oftenisdicult,ifnotimpossible.Onceuxforcertainenzymaticreac-
tionsismeasuredinthelaboratory,uxthroughothercellularpathways
canbecalculatedusingmathematicalmodelsoftheentiremetabolicnet-
work.Bymodelingagivenorganismsmetabolism,scientistscanquantify
theeectsofgeneticmanipulationsorchangesingrowthconditionson
thecellsentiremetabolicnetwork.Inputstoux-basedmodelsaretheset
ofpotentiallyactivemetabolicreactionsandmeasurementsofthesteady-
stateproductionratesofsuchmetabolitesasDNA,RNA,carbohydrates,
fattyacids,andproteins.Improvedestimatesofcelluxescanbeobtained
byfeedingalabeledcarbonsourceandusingmeasuredtransformeduxes
asinputtothemodel.
Arangeoftechniquesandmethodswillbeneededtodetermineuxesin
metabolicpathwaysofmicrobialcellsemployedforbiomassfermentations.
Tesetechniquesincludestableandradioactiveisotopelabelingandassoci-
atedmethodsforestimatingunmeasureduxesfromisotopedistribution
inmetabolitesandmacromolecules.MS- andNMR-basedmethodswill
determineisotopedistributionsincellularmetabolitesandmacromolecules.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 161

CROSSCUTTING RESEARCH
TheSuperImager
T
hepotentialistocreatecompound,
multifunctionalinstrumentsthat
individuallyincludemanyofthe
followingcapabilities:
Mappingofmolecularspeciessuchas
RNA,proteins,machines,andmetabo-
litesthroughtheuseofuorescenttagsof
variouskinds
Multipleexcitationanddetectionwave-
lengthsincludingbothuorescentand
infraredabsorptionmethods
High-speed3Dimaging
Nonlinearcontrastimagingincluding
second-andthird-harmonicgeneration
andcoherentRamanscattering
Lifetimemappingassensitiveprobesof
localenvironments
Rotationalcorrelationmappingforin
situanalysisofproteinstructureand
function
Magneticresonanceimagingwith
10-micronscaleanalysesofmetabolite
concentrationsandprovidingdataondif-
fusionpropertiesandlocaltemperatures
Acousticalimagingatmicron-scalereso-
lutionofthesystemsphysicalparameters
Atomicforcemicroscopy(AFM)
mappingofstructureswithadded
informationprovidedbythecontrolled
interactionoflightandsharpmetallic
AFMtipstoobtainopticalresolutionsof
~20nm,one-tenththediractionlimit
Highspatialresolution(nanometerscale)
usingX-rayandelectronmicroscopies,
includingtheuseofspecialDOEfacili-
tiesorperhapsthedevelopmentoflabo-
ratory-basedX-raysourcesforimaging
[Sources:Report on the Imaging Workshop
for the Genomes to Life Program, Oceof
Science,U.S.DepartmentofEnergy,2002
(www.doegenomestolife.org/technology/
imaging/workshop2002/);GTLRoadmap,
pp.18287]
Newanalyticaltechniquesareneededtoquantifyextracellular
metabolitesandquicklyandeasilydeterminebiomasscomposition.
EnzymeStructureandFunction
Developinganddeployingimprovedenzymesandmultienzyme
complexesforbiomassdeconstructionandconversionwillrequire
moreunderstandingandtheproductionofsuitablesubstratesas
wellasenzymesandtheirappropriatecomplexes.Teywillbe
usedtoachieveamechanisticunderstandingofcelluloseandcell-
wallinteractionswithdegradingenzymes.
DefningandProducingSubstrates
Despitenearly100yearsofpretreatmentresearch,detailedunder-
standingofcellulosicbiomasssfundamentalphysicalandchemi-
calfeaturesisstilllacking.Teplantcellwallthesubstratefor
degradativeenzymesystemsiscomplex,containingvariousforms
andquantitiesofcellulose,hemicellulose,andlignin.Chemical
andphysicalpretreatmentmayalterbiomasstomakeenzymatic
feedstockdigestionmoredicult,somergingpretreatmentand
deconstructionstepswouldsimplifyprocessing.Criticaltounder-
standinganyenzymaticreactionishavingbothareliableassay
andadenedsubstrate,thusallowingactivitiestobemeasured
accurately.Constructingstandardizedsubstratessuitableforhigh-
throughputassaysisrequiredtooptimizetheseenzymes.Synthesis
andcharacterizationofmodelsubstratesrequiremultipletechnolo-
gies,rsttodenethesubstratesbyemployingmultipleanalytical
andcomputationalmethodsandthentosynthesizethesubstrates.
High-throughputmethodsareneededforcompositionalanalysis
andcharacterizationofbiomasssubstrates,onceafamilyofbasic
referencestructureshasbeenidentied.
IdentifyingEnzymesandDegradativeSystems
Multipleclassesofenzymesarerequiredforbiomassconversion
toachievemaximumsugaryieldsincludinghemicellulasesand
ligninases.Terate-limitingstepismakingcelluloseaccessiblefor
subsequentsaccharicationsteps.Surveyingandidentifyingsuit-
ableenzymesrequiremanytechnologieslistedintheGTLRoad-
map,includingmetagenomicanalysesofmodelbiomass-degrading
communities,high-throughputproteinexpression,generation
ofanitytags,reassemblyofcomplexes,activitymeasurements,
andbiochemicalandbiophysicalcharacterizationsofenzymes
andcomplexes.Degradativesystemsfallintotwogeneralclasses:
Individualenzymesworkingsynergistically;andthecellulosome,
alarge,mega-Dacomplexnormallyattacheddirectlytocellsand
foundtodateonlyinanaerobicbacteria.Tecellulosomeisa
LEGO-likesystemwithmanyinterchangeablestructuralanddeg-
radativeproteincomponentsdeployedwhenspeciedforparticular
substrates.Tissystemprovidesthebasisforengineeringtocreate
162 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

cellulosomesoptimizedforparticularsubstrates.Tecellulo-
somecanbedetachedfromcellsandfunctions,evenunder
aerobicconditions.Withappropriatemodications,thecellulo-
somehaspotentialforenhancedstabilityandactivity.Individual
enzymesandthoseincellulosomesmightbenetfromimmobi-
lizationtechnologiesdesignedtoincreaseactivityandstability.
Howmembraneproteinssuchassugartransportersinterface
withcellulosomesattachedtocellwallsisunknown(seesidebar,
TeCellulosome,p.102).
ImagingTechnologies
Toughtisimpossiblewithoutanimage. Aristotle,325B.C.
Imagingtechnologieswithwideapplicationswillbecriticalto
manyoftheresearchchallengesidentiedinthisreport.
Biomassdeconstructionisacrucialstepinconversion,yet
relativelylittleisknownaboutthedetailedmolecularstructure
ofplantcellwallsandhowtheyareconstructedfromvarious
components.Toaddressthislimitation,newandimproved
methodsareneededtoanalyzeplantcell-wallcompositionand
structureatthenanometerscale.Organizationofpolymer
componentsinbiomassstructuresshouldbeanalyzedinthree
dimensionsusingnoninvasivetools.Suchnewcapabilitiesare
anticipatedtorevealkeymolecularprocessesoccurringinreal
timeduringthefulllifecycleofcell-wallformationand
maturationandduringexperimentstotransformcropspecies
intofeedstockssuitableforbioconversionbyoptimizingcell-
wallmakeup.Manyrequirementsgenerallyhavebeenantici-
patedbyGTLandbycapabilitydevelopmentandplanningas
documentedonpp.18287intheGTLRoadmap(U.S.DOE
2005)andImagingReport(U.S.DOE2002).
Imagingneedsforeachspecicresearchareaaresummarized
below.Teyincludeadvancesinawiderangeofimagingtech-
nologiesusingNMR,optical,X-ray,andelectron-basedmeth-
odsaswellasatomicforceandscanningtunnelingmicroscopies.
Givendierencesinresolution,sensitivity,andselectivity,the
fullimpactofthesemethodswillberealizedbestwhenused
incombinations,eitherwithinasingleinstrument(seesidebar,
TeSuperImager,p.162)orparallelapplicationsassembledand
correlatedthroughadvancedimage-managementsoftware.
ImagingNeedsforFeedstockResearch
Within5years,methodswillbedevelopedanddeployedfor
chemical-specicimagingoverawiderangeofspatialscales
(0.5nmto50m),withcontrastmethodsandtagsenabling
manymolecularcomponentstobedistinguishedeasily.Imag-
ingofliving(ornever-dried)materialsisperhapsjustas
SomeImagingTechnologies
RelevanttoFeedstock
Characterization
AtomicForceMicroscopy(AFM)
InAFM,ascanning-probetechnique
allowsthedirectstudyofsurfaceusing
tappingprobes,thetipsofwhichproject
lessthanamicron.Tedynamicbehavior
ofsurfacesandmoleculesoftencanbe
observed.Agreatadvantageofscanning
forcemicroscopyovermostotherhigh-
resolutiontechniquesisitsabilitytooperate
inaliquidenvironment.High-resolution
AFMimagesofacellulosesurfacerecently
havebeenreported.UsingAFMtosupport
interpretationofpretreatmentandenzyme
actiononbiomasssurfacesrepresentsa
tremendousopportunity.
ScanningElectronMicroscopy(SEM)
SEMreallyisthebackboneoftraditional
biologicalsurfaceanalysis.Newdevelop-
mentsinthedesignofSEMsample
chambersandopticspermittheanalysisof
samplescontainingsomenaturalmoisture,
whichiscriticalforbiomassfractions.
Also,newstrategiesforcreatingreplicas
ofbiologicalsamplesprovidemoreversa-
tilityinanalyzingproteinsandmicrobial-
cellsurfaces.
TransmissionElectronMicroscopy(TEM)
Cryoelectronmicroscopyistheleading
techniqueforhigh-resolutionmolecular
machinestructural-biologyresearch,and
TEMisthemostcommonplatformfor
thismethod.Thetechniquemakesthe
followingspecialdemandsonelectron
microscopy:Ultrahighandcleanvacuum
forcontamination-freeobservation,
stablelow-driftcryotemperaturehold-
ers,specialfunctionstoprovidelow-dose
imagingconditions,andcooledslow-
scanchargecoupleddisplaycamerasfor
low-dosedigitalimagerecording.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 163











































CROSSCUTTING RESEARCH
criticalasimagingharvestedmaterialsthathavebeendried,eitherfor
storageinaprocessingfacilityorforstructureexamination.Teseimag-
ingcapabilitiesshouldenableobservationofcell-walldeconstructionand
construction.Newopticaltechnologiesandadvancesatsynchrotronlight
sourceswillallowsomeimagingmethodstofunctionashigh-throughput
devices,producinghundredstothousandsofimagesperminutewitha
singleinstrument.Importantpossibleconsequencesaredramaticreduc-
tionofcostandprocessingtimepersampleandgreatercommunity
accesstothesetechnologiesaspartofauserfacility.Second,enhanced
imagingspeedenablesreal-timeobservationofbiologicalprocesses.
Availabilityofreal-timedataisvitaltobuildingandvalidatingmodels
thatallowasystemsbiologyapproach.
Temporalimagingofdynamicsubcellularsmallmolecules,including
metabolites,isamongadvancesexpectedtotakelongerdevelopment
times.Improvedmolecule-specicimagingtagsandameansforintro-
ducingthemintocellswithoutdisruptingfunctionarecriticalneeds.
Tescaleofvariousbiomassconversionswillrequireenhancedimaging
forobtaininghigh-resolution,large-volumetomographicimagesaswell
asreal-timeimagingoflivingsystemsathighspatialresolutionandfor
useintheeldatlowerspatialresolution.Tecriticalaspectofenzyme
engagementwithbiomasscomponents,suchascellulaseinterfacingwith
cellulose,requiresnewcapabilitiesforcharacterizingenzymebindingsites
byatomicforcemicroscopy,scanningelectronmicroscopy,transmission
electronmicroscopy,andelectronspectroscopyforchemicalanalysis[see
sidebar,SomeImagingTechnologiesRelevanttoFeedstockCharacteriza-
tion,p.163,andImageAnalysisofBioenergyPlantCellSurfacesatthe
OBPBiomassSurfaceCharacterizationLab(BSCL),p.40].Similarly,
capabilitiesareneededtocharacterizeinteractionsbetweenmicrobialcells
andtheirsolid-phasesubstrates.
ImagingNeedsforMicrobialCommunitiesinDeconstructionand
ConversionofBiomasstoEthanol
Meetingimagingrequirementsforprocessingbiomasstoethanolwilldraw
ontheabovecapabilitiesbutwillfocusmoreonmicrobial-celland-com-
munityimagingtoaidindelineatingsub- andextracellularorganizationof
proteins,proteincomplexes,transporters,andmetabolites.Appropriatetags
andmethodologieswillberequiredforinvestigatingmulticellularinterac-
tionsinmixedmicrobialpopulations(e.g.,thoseobservedinfermentation)
andcommunities(e.g.,thoseobservedincolonizationofdecayingbiomass)
withsolidsubstratessuchasplantcellwalls.Understandingtheseinterac-
tionswillrequireinformationonamounts,types,andlocationsofsecretion
products,reactionstheycatalyze,andhowthesereactionsandproducts
impactcellviability.Imaging-basedapproachesareessentialtoachieve
temporalandspatialresolutionsucientforunderstandingtheseprocesses
(seeFig.1.ProbingMicrobialCommunities,p.165).
164 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.1.ProbingMicrobialCommunities. Microbialcommunitiesandecosystemsmustbeprobedatthe
environmental,community,cellular,subcellular,andmolecularlevels.Teenvironmentalstructureofa
communitywillbeexaminedtodenemembersandtheirlocations,communitydynamics,andstructure-
functionlinks.Cellswillbeexploredtodetectandtrackbothextra- andintercellularstatesandto
determinethedynamicsofmoleculesinvolvedinintercellularcommunications.Probingmustbedoneat
thesubcellularleveltodetect,localize,andtrackindividualmolecules.Preferably,measurementswillbe
madeinlivingsystemsoverextendedtimescalesandatthehighestresolution.Anumberoftechniques
areemergingtoaddressthesedemandingrequirements;abrieflistingisontherightsideofthegure.
[Figuresource:GTLRoadmap,p.176(http://doegenomestolife.org/roadmap/).]
MicrobialCultivation
Tebioreneryenvironmentiscomplex,andcultivationtechnologiesmust
becapableofreproducingcriticalaspectsofindustrialsystems.Ecient
conversionofbiomasstoliquidfuelswillrequirenewandinnovative
approachesforcontrollingcultivationandsimultaneouslymonitoringcell
physiologicalstatesandmetabolicprocessesunderarangeofconditions.
Suchcapabilitiesareneededtoidentifygeneregulatoryandmetabolic
networksandtodevelopandevaluatemodesofcellmetabolism.Obtain-
ingasystems-levelunderstandingoffermentationorganismswillrequire,
insomecases,thousandsofsamplesfromsingle-andmultiple-species
cultures;andtechnologiesforcontinuouslymonitoringandcontrolling
cultureconditionsandinterrogatingthephysiologicalstateofmicrobial
cells.Relativelyhomogeneousandcomplexmicrobialpopulationswill
betestedinphysicallyandchemicallyheterogeneousenvironmentssuch
asthoseassociatedwithsolids(plantbiomass).Necessaryinfrastructure
willsupportcultivationatscalessucienttoobtainadequateamountsof
sampleforanalysisandtogrowmicrobialcellsinmonoculturesandin
nonstandardconditions(e.g.,inassociationwithsolidsorbiolms).Tese
cultivationsystemswillbeenhancedbyadvancedcomputationalcapabilities
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 165

CROSSCUTTING RESEARCH
LaboratoryCultivationTechniquestoSimulateNaturalCommunityStructure
T
oidentifythefunctionofgenespreferentiallyexpressedbyspecicpopulationsinastructuredmicro-
bialcommunity,suchasthosedeconstructingbiomassinsoilsorinabioreactor,newcultivation
techniquesarebeingdevised.Duringthepastdecade,researchershavedevelopedreactorsinwhich
biolmscanbeimagedusingconfocalscanninglasermicroscopy(CSLM)and
otherlight-microscopictechniques(Wolfaardtetal.1994).Whencombinedwith
uorescentinsituhybridizationtodistinguishpopulationsofcellsinmultipopu-
lationbiolmsanduorescentreporters(greenuorescentprotein)offunctional
geneexpression,CSLMhasbeenusedtodemonstratehowgeneexpressionby
onepopulationaectsgeneexpressioninanotherproximallylocatedpopulation
(Molleretal.1998).
Temobilepilot-plantfermentorshownherehasa90-Lcapacityandcurrentlyis
usedtogeneratelargevolumesofcellsandcellproductssuchasouter-membrane
vesiclesunderhighlycontrolledconditions.Futuregenerationsoffermentorswill
bemorehighlyinstrumentedwithsophisticatedimagingandotheranalytical
devicestoanalyzeinteractionsamongcellsinmicrobialcommunitiesunderan
arrayofconditions.
P
a
c
i

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o
r
t
h
w
e
s
t

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a
t
i
o
n
a
l

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a
b
o
r
a
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y

thatallowsimulationofcultivationscenariosandidenticationofcritical
experimentalparameters.
Mostindustrialbioconversionsrelyonpurecultureswhile,incontrast,
environmentalbioconversionsaremorecommonlycatalyzedbymixed
populationsorcommunitieswithspecialistsworkingtogetherinan
apparentlyintegratedandstablefashion.Afundamentalquestionis,Are
therestable,self-regulatingmultiplexsolutionsforbiofuels?Inthisregard,
newtechniquesareneededtounderstandandreproducecompositionand
functioninstabilizedmixedcultures(seesidebar,LaboratoryCultivation
TechniquestoSimulateNaturalCommunityStructure,thispage).
Biologicalsystemsareinherentlyinhomogeneous;measurementsofthe
organismsaveragemolecular-expressionproleforacellpopulationcan-
notberelatedwithcertaintytotheexpressionproleofanyparticularcell.
Forexample,moleculesfoundinsmallamountsinsampleswithmixed
microbialspeciesmaybeexpressedeitheratlowlevelsinmostcellsorat
higherlevelsinonlyasmallfractionofcells.Consequently,asarene-
ment,newanalyticalandimagingtechniqueswillbenecessarytointer-
rogateindividualcellphysiologicalstatesinheterogeneousculturesystems.
Forexperimentationinallaspectsofworkwithomictools,productionof
high-qualityreproduciblesamplesisparamount.
DataInfrastructure
Progresstowardecientandeconomicprocessesforconvertingbiomass
tobiofuelswillbenetfromaresearch-datanetworkabletomanage,
preserve,query,andecientlydisseminatelargeamountsofexperimen-
talandanalyticaldata,asoutlinedintheGTLRoadmap.Becausemany
researchactivitiesaredataintensiveandwillengagealargenumberof
investigatorsfromnationallaboratories, academia, andtheprivatesec-
tor,theseactivitieswillbenetfromadistributeddataenvironment.
166 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.2. GTLIntegratedComputationalEnvironmentforBiology:UsingandExperimentallyAnnotating
GTLsDynamicKnowledgebase.Attheheartofthisinfrastructureisadynamic,comprehensiveknowledge-
basewithDNAsequencecodeasitsfoundation.Oeringscientistsaccesstoanarrayofresources,the
knowledgebasewillassimilateavastrangeofmicrobialandplantdataandknowledgeastheyareproduced.
[Figuresource:GTLRoadmap,p.83(http://doegenomestolife.org/roadmap/).]
Consistingoffederateddatabasesandrepositories,thedataenvironment
willcomprisedescriptive,quantitative,andvisualinformation,method
libraries,queryanddata-miningtools,andacommunicationnetwork.
Tisenvironmentprovidescontrolledsharingandmanagementandcom-
municationofbiochemical,genetic,andothertypesofbiologicaldata
andinformation.Whenavailablethroughasingleportal,suchacyberin-
frastucturecanhaveadramaticimpactinecientuseofnewknowledge.
Additionalbenetsfromecientdatasharingincludefosteringcollabo-
rationamongprojects,facilitatingstandardsdevelopment,andaccelerat-
ingimplementationofnewenablingtechnologies.GTLalsowillneeda
centralizedresearchdatanetworkformodelplantsandspecicbiomass
cropplants.
Data-infrastructurerequirementsofbiomass-to-biofuelsresearchalign
extensivelywiththeIntegratedComputationalEnvironmentforBiology
describedintheGTLRoadmap(seeFig.2.GTLIntegratedCompu-
tationalEnvironmentforBiology,thispage),reproducedasaprogram-
maticsubsetoftheGTLinformationinfrastructure.Biomass-to-biofuels
researchwillrequiremultipledatabases,imagingarchives,andLIMSin
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 167

CROSSCUTTING RESEARCH
eachofitsmajorresearchareas:FeedstockforBiofuels,Deconstructing
FeedstockstoSugars,andSugarFermentationtoEthanol.
Forexample,researchondeconstructingfeedstockswillnecessitate(1)
LIMSfortrackinganddocumentationofsamplesforenzymaticpretreat-
mentexperimentsandhigh-throughputcompositionalanalysisofcell-wall
material;enzymedatabasesforcharacterizingsuchkeyenzymesasglyco-
hydrolases,esterases,andligninases;metagenomicsequencedatabasesfor
characterizinglignocellulolyticandothermicrobialcommunities;imaging
repositoriesforavastarrayofmicroscopiesincludingoptical,Xray,elec-
tron,andatomicforce;andarchivesformoleculardynamicsimulationsof
themolecularmachinerythatbreaksdownplantcell-wallcomponents.
Feedstockandfermentationresearchgoalshavecomparabledata-infrastruc-
tureneeds,withdescriptiveandquantitativedatatypesdieringmainlyin
experimentalandanalyticalmethodsemployed.Proteomic,metabolomic,
transcriptomic,andplantandmicrobialgenomicdatawillrequirecorre-
spondingdatabasesandintegrationwithintheGTLKnowledgebase.
Avarietyofcomputationaltoolswillbeneededtosupportbiomass-
to-biofuelsresearchandsuchenablingtechnologiesasproteomicsand
imaging.Forexample,computationaltoolsarenecessaryforstreamlining
theconversionofisotopomerux-tracingdataintouxdistributionsfor
subsequentanalysis;theprocesscurrentlyislimitedbythecomputational
sophisticationofrequiredmethodsratherthanbytheabilitytogener-
aterawdata.High-throughput,automatedimageacquisition,storage,
processing,andanalysiswillberequiredforexaminingdataonmolecular
machinesinvivo.Tesetoolsalsowillaidinlocalizingandvalidating
complexes,dynamics,docking,intercellularcommunication,extracellular
matrix,andmetabolitedistribution.
ComputationalModeling
Computationaltechnologieswillbecriticaltotheoverallplatformthatwill
enablesuccessindevelopingeectivetechnologiesforconvertingbiomass
tobiofuels.Signicantamountsofdatageneratedatanumberofinvesti-
gativelevelsmustbemadeaccessibletoinvestigatorsatdierentinstitu-
tions.Datasetswillneedtobeanalyzedandusedtodevelopandevaluate
computermodelsthatwillcontributetoguidingoveralldecisionmaking
andprogramdesign.Tesedecisionscouldinvolvethebestlignocellulosic
compositionofbiomassforagivenprocessandhowtocreateitaswellas
designingenzymesorengineeringmicrobesforoptimalethanolproduction.
Computationalmodelingtechnologiesandtoolswillbeneededtoaddress
thesechallenges(seeFig.3.FromGenomeDatatoFullCellSimulation,
p.169).Somekeyneedsandexamplesarediscussedbelow.
Modeling:GenomeSequenceAnalysis
Awiderangeoforganismsacrossmanydierentkingdomsoflife
includingplants,fungi,andbacteriaarerelevanttobiomassconversionto
biofuels.Withthepowerofmoderngenomesequencingandcapabilitiesat
168 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Fig.3.FromGenomeDatatoFullCellSimulation.Tisconceptdiagramschematicallyillustratesapathfrom
basicgenomedatatoamoredetailedunderstandingofcomplexmolecularandcellularsystems.Newcomputational
analysis,modeling,andsimulationcapabilitiesareneededtomeetthisgoal.Tepointsontheplotareveryapproxi-
mate,dependingonthespecicsofproblemabstractionandcomputationalrepresentation.Researchisunderway
tocreatemathematics,algorithms,andcomputerarchitecturesforunderstandingeachlevelofbiologicalcomplexity.
[Figuresource:GTLRoadmap,p.89(www.doegenomestolife.org/roadmap/).]
DOEsJointGenomeInstitute,obtaininggenomesequencesforessentially
anyorganismispossible.Researchwillusesequence-analysistoolssurpass-
ingtraditionalgenomeannotation.Tecomplexityofbiomassconversion
tobiofuelswillnecessitatetheevaluationandintegrationofsequencedata
frommultipleorganisms.Teneedtoestablishfunctionalrolesforamulti-
tudeofproteinsinvolvedinmediatingplantcell-wallbiologyandden-
ingmetabolicandregulatorypathwayswillrequirerobustbioinformatic
capabilities.Withineachgenomesequenceisawealthofinformationthat
canbeminedandaccessedtoimproveourknowledgeabouttheseorgan-
isms.Insomecases,traditionalbioinformaticapproachesmaybesucient.
Manyareas,however,stillneedresearchadvances.
Sequence-basedanalysismethodsandtoolsarevitalinmanyareas.Cre-
ationofanimproved-qualitystandardizedpipelineforgenome-sequence
annotation,includingdenitionofopenreadingframesandfunctional
assignmentofgenes,isacriticalneedforessentiallyallsequencedorgan-
ismsthataresubjectsofGTLmission-directedresearch.Teseannotations
needtobeupdatedcontinuouslybecauseadvancementscanberapidin
manyareasandshouldbeappliedtosingleorganisms,includingplants,
andmetagenomesofsoilecosystemsandfermentativeconsortia.Specic
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 169

CROSSCUTTING RESEARCH
tobiomass-to-biofuelsresearcharecomputationalmethodsforimproving
analysisofmetagenomicdatatoidentifynewenzymesrelatedtolignocel-
lulosedegradationandfermentation.Directedevolutionhasthepotential
torapidlyevolvenewandusefulmicrobialstrainsforfermentationreac-
tions.Comparativegenomicsofevolvedmicrobesfollowingresequenc-
ingwillidentifyspecicchangesinthegenome.Comparativeanalysesof
multipleplantgenomeswillgeneratefamilyclustersofgenecomponents
mediatingcell-wallbiosynthesis(i.e.,enzymes,regulatoryfactors,and
processingproteins).Amoregenericrequirementisenhancedannota-
tionrelatedtorecognitionofinteractingproteins,regulatorysignals,and
structures.
Modeling:Molecular
Anumberofcriticalbiochemicalprocesses,occurringatthemolecular
scale,governtheoverallconversionofbiomasstobiofuels.Teseprocesses
include,forexample,mobilizationofbuildingblocksforplantcell-wall
synthesis,machineryforcell-wallformationinplantsandtheirbiophysi-
calcharacteristics,degradationoflignocellulosicmaterialsbyenzymes,and
transportofsugarsintomicrobesforfurthermetabolismandconversionto
desiredendproducts.Incombinationwithstructuralinformation,mecha-
nisticmodelsofplant-derivedpolymericsubstratesandtheirenzymatic
conversionareneededtoidentifybottlenecksinbioconversionofligno-
cellulosicbiomass.Gainingadetailedunderstandingofthesemechanisms
willhelpindesigningsuperiortechnologiestoaddresskeybarriers.Asa
componentofthisresearch,newcomputerprogramsandcodesareneeded
forultralargebiologicalmodelsofmorethanamillionatomstoenablethe
analysisofcomplexmolecularmachinessuchasthecellulosome.
Molecular-scalemodelsarecriticaltoanumberofbiomass-to-biofuels
aspects.Forexample,biophysicalmodelsofplantcell-wallcomposition
areneededtodelineatepathwaysforsynthesisandtransportofkeycell-
wallcomponents.Ourknowledgeoffundamentalpolysaccharide-water
interactionswithinlignincarbohydratecomplexeswillbenetgreatly
frommoleculardynamicssimulations,aswillourunderstandingofwater
andchemicaltransportalongorthroughtheplantcellwall.Dynamic
molecularmodelswillaidindeterminingcell-wallenzymaticdegradation
andinteractionsamongtheseenzymesandtheirsubstrates.Specically,
molecularmodelsofsuchcell-walldegradingenzymesashemicellulases
andligninaseswillhelptoestablishstructure-functionrelationshipsfor
substrateinteractionsandguidethedevelopmentofnovelenzymes.Simi-
larly,dynamicmodelingoflargemolecularmachinesandtheirsubstrates
onnano-tomillisecondmeasureswillprovideinsightsintothestructure
andfunctionofkeyproteincomplexessuchasthecellulosome.Molecu-
larmodelingatascaleexceedingthatforindividualenzymeswillenable
understandingofcriticaleventsoccurringattheinterfacebetweencellulose
andanadheredcell.Onthemicrobialside,modelsareneededtopredict
membranecompositionandchangesinresponsetostressinducedbyhigh
concentrationsoffermentationendproducts.
170 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy













































Modeling:PathwaysandNetworks
Molecularmachinesthatcarryoutindividualtransformationsandproc-
essesimportantinbiomass-to-biofuelsconversionsoftenoperatewithin
muchlargersystemsofinteractingproteinsandmetabolicpathwaysincells.
Understanding,controlling,andmanipulatingtheoverallphenotypicalstate
ofthesesystemsineitherplantormicrobialcellswillbenecessarytoover-
comeidentiedtechnicalbarriers.Computationalmodelsofthesecellular
systemsandpathwayswillfacilitateintegrativeanalysisofexperimentalomic
datasetswhilealsoprovidingthebasisforpredictivesimulationsthatcan
generatetestablehypotheses. Toincreasetheeciencyoffermentationreac-
tions,improvedmetabolicandregulatorymodelsareneededtounderstand
mechanismsthatcontrolglycolyticuxanditsimpactoncellularmetabo-
lism.Becausecarefulalterationsofgrowth,energy,andredoxconditions
oftenarerequiredtooptimizefermentationreactions,robustmodelsfocused
onanalysisandregulationofcellularenergeticsarenecessary.Regulatory
andmetabolicmodelingatcommunityandindividuallevelsisessentialfor
identifyingandunderstandinggeneregulatorynetworks.
Anumberofspecicbiomass-to-biofuelstopicswillrequiresystems-level
modelsandsimulationmethods.Genome-scalemodelsofkeyindustrial
microbesareneededtounderstandmetabolismdetailsandallowrational
designforimprovedbiofuelproduction,includingwaystoidentifynovel
biotransformationroutes.Dynamicpathwaymodelsincorporatingkey
enzymekineticsforethanol-producingpathwaysandotherrelevantcel-
lularsubsystemsareaspecicneed.Methodsalsoarerequiredtoassess
physicochemicallimitationsofcellularsystemsandenzymecomponents
todeterminemaximum-achievablemetabolismrates(e.g.,identifyrate-
limitingsteps,bothkineticanddiusionlimited).Fromtheplantper-
spective,detailedpathwaymodelsofcell-wallbiosynthesisinthecontext
ofoverallplantmetabolismareneededtogeneratetestablehypothesesfor
controllingcell-wallcomposition.Tesemodelsshouldincorporateinfor-
mationaboutcatalyticactivity,geneexpression,mechanismsofcontrol,
andinterspeciesvariations.
Genericmodelingcapabilities,manyofwhicharerequiredinthebroader
GTLprogram,includeintegratedmodelingofmetabolicpathwaysand
regulatorynetworksandsimulationoftheirfunctionalcapabilities.Insup-
port,automatedtechniquesareneededtointegratedatasetsandrapidly
reconstructmetabolicpathwaysandgeneregulatorynetworks.Methods
alsowillberequiredtoincorporatethenextlevelofcomplexityintosys-
tems-levelcellmodelsbyaddinginformationfromcellular-component
imaging.Othermethodswillaccountfortheimpactofproteinandenzyme
spatiallocalizationwithinintegratedmodelsofcellularsystems.
Modeling:BiorefneryProcess
Telong-termvisionforbiomass-to-biofuelsresearchinvolvesallcellular-
andmolecular-basedprocedureswithinanintegratedbiorenery.To
determinedesignrequirementsandalternatives,theoverallprocessshould
bemodeledcomputationally.Modelingisimportant,asisobtaining
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 171

CROSSCUTTING RESEARCH
experimentalvalidationoflarge-scaledesignsthatwillenableimple-
mentationofgenomicandfundamentalscienceresearch.Inaddition,
physicalpropertiesandmaterialhandlingincludingfeed,mass,anduidic
transportparametersneedtobedetermined.Withouttheseparameters,
designingbioprocessingfacilitiesincludingbioreactors,heatexchange,
ltration,bioseparations,centrifugation,pumps,valves,andotherchemical-
engineeringunitoperationsisdicultandnotcomprehensive.Tiscapa-
bilityencompasseseconomicmodelsandthosethatassessthenetcarbon
balanceimpactofdierentprocessingschemesandtheadvantagesand
disadvantagesofvariouscoproductprocesses.Modelsdoexistforsome
oftheseprocesses,buttheyarenotreadilyavailabletothecommunity.
Toallowresearcherstoassessthefeasibilityofdierentdesignconcepts,
resultsandmethodologiesshouldbereadilyaccessibleandsubjecttocon-
tinuousfeedback-basedimprovements.
Processmodelingwillbeveryusefulinevaluatingthefeasibilityof
consolidatedbioprocessing.Consideringthegoaltoprovidethescience
underpinningoneormorecommercialprocessesforproducingliquid
fuelsfrombiomass,aproperevaluationofmost-probablefuturesce-
nariosisneeded.Forexample,onescenarioenvisionedformoreeciency
andeconomyissimultaneoussaccharicationandfermentation(SSF).
Saccharicationenzymemixescurrentlyareoptimalat50Candata
pHbetween4and6,soasindicatedbyotherstudygroups,noneofthe
currentethanol-producingmicroorganismsissuitable.Processmodeling
willevaluatetheimportanceandsensitivityofSSFvsatwo-stepprocess.
Modelingisneededtodemonstraterationaldesignofintegratedbiomass
processingbypredictingandthenverifyingoverallhydrolysisyieldsfor
nativeandmodiedbiomassspeciesusingdierentpretreatmentchemis-
tries,temperatures,andspecicenzymes.
Insummary,researchisneededtodevelopprocessmodelsforassessing
massbalancesandeconomicmodelsforbioprocessengineeringandto
disseminatethemforcommunitybenet(seechapter,BioprocessSystems
EngineeringandEconomicAnalysis,p.181).
CapabilitySuitesforBioenergyResearch
andFacilityInfrastructure
Toaddressmissionscienceneedsinenergyandenvironment,theDOE
OceofSciencehasproposedestablishingverticallyintegratedresearch
centersthatwilldrawupontherangeofadvanced,high-throughputtech-
nologiesandinformation-managementcomputingdescribedintheGTL
Roadmap.Terstofthesecenterswillfocusonbioenergy.Tissection
providesanoverviewofexistinganddevelopingGTLcapabilitiesthatwill
berequiredtoacceleratebiomass-to-biofuelsresearch.TeGTLprogram
hasestablishedpilotsofmostkeytechnologiesrequiredtoperformsys-
temsbiologythatmightbeincorporatedintoresearchcenters.Biological
capabilitiesneedtobeinvestigatedatmanyscales(seeFig.9.Understand-
ingBiologicalCapabilitiesatAllScalesNeededtoSupportSystems
172 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

BiologyInvestigationsofCellulosicBiomass,p.21).Overthepastdecade,
genomicsequencinghasbeenestablishedasahighlyecientproduction
componentattheDOEJointGenomeInstitute.Suchcapabilitiesprovide
thegenomicfoundationforresearchdescribedinthisreport.
DOEJointGenomeInstitute
DOEJGIsmissionistoprovideintegratedhigh-throughputsequenc-
ingandcomputationalanalysestoenablegenomic-scale,systems-based
scienticapproachestoDOEchallengesinenergyandenvironment.JGI
capabilitieswillhaveanimmediateimpactonbiomass-to-biofuelsscience
byprovidinghigh-qualitygenomesequencesofrelevantcropplantsand
microorganisms,includingnaturallyoccurringandengineeredmicroorgan-
ismsusedforbiomassconversionandsoilmicrobialcommunitiesasthey
relatetosoilqualityandsustainability.
Identicationofgenesthatcontrolcell-wallcompositioninbiomassspe-
ciesanddevelopmentoftoolssuchasgenechipsdependontheavailability
ofgenomesequencesforbiomasscropspecies.Sincemicro-RNAsare
expectedtoplayaroleincontrollingexpressioninmanyrelevantgenes,
DNAsequencingmustbecomprehensiveenoughtoidentifyallmicro-
RNAsinthesespecies.
ImplementatingeectivebiomassconversionwillrequireJGItorapidly
andcost-eectivelydeterminethesequencesofanumberoforganisms,
consortia,andmetagenomes.Inadditiontobiomasscropplants,JGIcould
providegenomesequencefornewspeciesofwhiterotfungiandbrownrot
fungi,actinomycetes,andsuchotherbiofuel-relevantorganismsasnatu-
ralconsortiaandcommunitiesinvolvedinbiomassdecomposition.Full
metagenomicsequencingwillelucidatehemicelluloseandligninbreak-
downobservedinmixedmicrobialpopulationsandwillallowharnessing
oftheircollectiveprocesses.
ProductionandCharacterizationofProteinsandMolecularTags
FunctionalCapabilities
Tesecapabilitieswouldenablethehigh-throughputexpressionofpro-
teinsmediatingcell-wallbiosynthesis(laccases,peroxidases,andglycosyl
transferases)inArabidopsisandothermodelandfeedstockplants.Most
enzymesofinterestincell-wallbiosynthesis,suchasglycosyltransferases,
arethoughttobemembraneassociatedand,therefore,diculttopurify
andcharacterizebyconventionalmethods.Heterologouslyexpressed
proteinswouldbecharacterizedbiochemicallyandusedasaresourcefor
rst-passidenticationoffunctionandthegenerationofantibodiesand
tags.Toimproveenzymesforlignocellulosedeconstruction,theprotein
productioncapabilitywouldbearesourcefornativeandrecombinant
formsofenzymesandmodiedenzymesfromdirectedevolutionand
fromrational-design,site-directedmutagenesisapproaches.Forpilotand
validationstudies,signicantquantitiesofproteinwouldbeproduced.
Proteins,naturalandmodied,couldbeusedtorapidlyreconstitute
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 173

CROSSCUTTING RESEARCH
machinessuchascellulasesandcellulosomesforimprovedperformance.
Antibodyresourceswouldhavemultipleapplications,includingpinpoint-
ingcellularlocalizationoftargetproteins;proteincomplexidentication;
quanticationofnaturalfunctionaldiversity;andquantitationofmolecular
outcomesfrombreedingexperiments.Inaddition,thisresourcewould
generatetagsandprobesforimagingcell-wallpolymersandprogressof
enzymesthatdegradethesepolymers.
Assaysformanycell-wallsynthesisanddeconstructionenzymeswill
requirenewapproachestoscreenforactivity.Inparticular,theywillinvolve
accesstodierentpolysaccharidesubstratesandcapabilities.Incombi-
nationwithproteomiccapabilities,theresourcewouldprovidevaluable
informationaboutproteinsandmechanismsthroughwhichbiomassis
convertedtobiofuels.Tisknowledgewillenableengineered-organism
combinationstooptimizebiofuelproduction.
Tesecapabilitieswillbecriticalforgeneratingandcharacterizingnew
proteinsandbiomarkersforimaging.Forexample,acollectionofuo-
rescentlylabeledproteinsforsuchspecicmodelplantsasArabidopsisor
Brachypodiumwouldbeparticularlyvaluable.
InstrumentationandMethods
Fromgenomesequences,proteinproductionandcharacterizationmethods
willexpressproteinsandgeneratereagentsforinterrogatingcellfunction.
Specically,thegoalwillbetocreatecapabilitiestoproduceondemandall
proteinsencodedinanygenome;createmoleculartagsthatalloweachpro-
teintobeidentied,located,andmanipulatedinlivingcells;gaininsights
intofunction;andperformbiophysicalandbiochemicalproteincharacter-
izations.Usinghigh-throughputinvitroandinvivotechniques(i.e.,cellular
andcellfree)willlowerprotein-productioncoststolevelsthatwillallow
comprehensiveanalysisofcellularproteins.Tesemethodologieswillbe
appliedtotheproductionandcharacterizationofmodiedandnativepro-
teins.Productsandanalysiscapabilitieswillbemadeavailabletoscientists
andtechnologists.
Anityreagentswillbegeneratedinparallelwithproteinproduction,
usingmanyofthesametechnologies.Taggedproteinsornucleicacids
willpermitdetectionandtrackingofindividualproteinsinlivingsystems,
includingcomplexmolecularassemblages;intracellularpositionofallpro-
teinsandtheirspatialdynamics;ifsecreted,extracellularlocalizationand
interactionwithothercommunitymembers;andtechniquesformanipu-
latingproteinactivityintheenvironment.
Potentialcoreinstrumentation:
Gene-synthesisandmanipulationtechniques.
High-throughputmicrotechnologiesforprotein-productionscreening.
Roboticsystemsforproteinandanity-reagentproductionandcharac-
terization.
174 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Computingfordatacaptureandmanagement,genomiccomparative
analyses,controlofhigh-throughputsystemsandrobotics,and
production-strategydetermination.
CharacterizationandImagingofMolecularMachines
FunctionalCapabilities
Tesecapabilitieswillenableidenticationandcharacterizationofprotein
complexesresponsibleforessentialfunctionsofenergy-relevantpheno-
types,includingcell-wallbiosynthesis.Understandingthefunctionofthese
cell-wallsynthesizingcomplexesandtheirinteractionswithmetabolic
pathwaysthatproducesugarnucleotidesinthecytosolwillbeimportant
forunderstandingpolysaccharidebiosynthesis.Identifyingkeyprotein-
proteininteractions(interactomics)involv-
ingsuchbiomass-deconstructionenzymesas
polysaccharidases,hemicellulases,ligninases,
andesterases,alongwithinteractionswiththeir
substrates,alsoisapriority.Becausepolysac-
charidesprobablyaresynthesizedbyamulti-
enzymecomplexintheGolgi,thatalsowillbe
identiedandanalyzed.
Advancedcapabilitiesareneededtounderstand
thestructureandfunctionofcellulaseandcellulo-
somemolecularmachinesandimprovedesigner
cellulosomesbylinkingessentialenzymestothe
desiredsubstrateandeliminatingsuperuous
proteinsandreactions. Teseadvancedcapabili-
tiesincludeanalysisandcomputationforprobing
interactionsamongcells,theirassociatedsecreted
enzymecomplexes,andcellulose;andparticularly
forunderstandingthebiologyoccurringatthe
cellsurfacecelluloseinterface.
InstrumentationandMethods
Capabilitieswillidentifyandcharacterize
Fig.4.VisualizingInteractionNetworks.Graphicalmaps
molecularassembliesandinteractionnetworks
displayproteininteractiondatainanaccessibleform.Tese
(seeFig.4.VisualizingInteractionNetworks,
visualizationssummarizedatafrommultipleexperimentsand
thispage).Resourceswillisolateandanalyze
alsoallowquickdeterminationsofproteinsthatmightbecore
molecularmachinesfrommicrobialcellsand
constituentsofaparticularproteincomplexandthosethatmight
plants;imageandlocalizemolecularmachinesin
playrolesinbridginginteractionsamongdierentcomplexes.
cells;andgeneratedynamicmodelsandsimula-
Tegureabove,generatedusingCytoscape,summarizesprotein
tionsofthestructure,function,assembly,and
interactionsincomplexesisolatedbyanityapproachesfrom
disassemblyofthesecomplexes.High-through-
Shewanella oneidensis.Nodes(yelloworredcircles)represent
putimagingandcharacterizationtechnologies
proteins.[Source:GTLCenterforMolecularandCellularSys-
willidentifymolecularmachinecomponents,
temsatOakRidgeNationalLaboratoryandPacicNorthwest
characterizetheirinteractions,validatetheir
NationalLaboratory;previouslypublishedinGTLRoadmap,
occurrence,determinetheirlocationswithinthe
p.68(doegenomestolife.org/roadmap/).]
cell,andallowresearcherstoanalyzethousands
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 175

CROSSCUTTING RESEARCH
ofmolecularmachinesthatperformessentialfunctionsinsideacell.Te
capabilityforcompletelyunderstandingindividualmolecularmachineswill
bekeyindetermininghowcellularmolecularprocessesworkonawhole-
systemsbasis,howeachmachineisassembledin3D,andhowitisposi-
tionedinthecellwithrespecttoothercellularcomponents.
Potentialcoreinstrumentation:
Roboticculturingtechnologiestoinducetargetmolecularmachinesin
microbialsystemsandsupportrobotictechniquesformolecular-complex
isolation.
State-of-the-artmassspectroscopyandothertechniquesspeciallycon-
guredforidenticationandcharacterizationofproteincomplexes.
Variousadvancedmicroscopiesforintracompleximagingandstructure
determination.
Imagingtechniquesforbothspatialandtemporalintra-andintercellu-
larlocalizationofmolecularcomplexes.
Computingandinformationsystemsformodelingandsimulationof
molecularinteractionsthatleadtocomplexstructureandfunction.
AnalysisofGenomeExpression:TheOmics
FunctionalCapabilities
Anorganismselectivelyproducesportionsofitsproteomeinresponseto
specicenvironmentalorintracellularcues.Studyingitsconstantlychang-
ingproteinexpressionthusleadsto
betterunderstandingofhowandwhy
anorganismturnsgenomeportions
onando.Identifying,quan-
tifying,andmeasuringchangesin
globalcollectionsofproteins,RNA,
metabolites,andotherbiologically
signicantmoleculesmediatedby
proteinsincludinglipids,carbohy-
drates,andenzymecofactorsare
importanttothisunderstanding. Te
abilitytomeasuretimedependence
ofRNA,protein,andmetabolite
concentrationswillrevealthecausal
linkbetweengenomesequenceand
cellularfunction(seeFig.5.Gene-
Protein-MetaboliteTimeRelation-
ships,thispage).High-throughput
omictools(transcriptomics,prote-
omics,interactomics,metabolomics,
glycomics,lignomics,anduxomics)
willbecriticalforsystems-level
investigationofplantcell-wall
Fig. 5.Gene-Protein-MetaboliteTimeRelationships.Toaccuratelyestab-
lishcausalityamongmeasuredgene,protein,andmetaboliteevents,sampling
strategiesmustcoverthefullcharacteristictimescalesofallthreevariables.
Littleisknownaboutthetimescaleofgene,protein,andmetaboliteresponses
tospecicbiologicalstimuliorhowresponsedurationsvaryingenesandspe-
cies.[FigureadaptedfromJ.Nicholsonet al.,Metabonomics:APlatformfor
StudyingDrugToxicityandGeneFunction,Nat. Rev. 1,153-61(2002);pre-
viouslypublishedinGTLRoadmap,p.158(doegenomestolife.org/roadmap).]
176 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

constructionandprocesseswherebythecellwallisdeconstructedand
convertedtoethanol.Multipleanalyticalcapabilitieswillbeseamlessly
integratedwithmodelingtoachievetheneededlevelofunderstanding.
Useofproteomicswillallowcomprehensiveanalysisofplantproteincom-
plexes,factorscontrollingtheircreationandfunction,andensuingpro-
cesses,beginningwiththemodelplantArabidopsis.Tethousandormore
proteinsinvolvedincell-wallsynthesisormodicationarehighlyinteract-
ingorlocatedincomplexes.Knowingwhichproteinsareincomplexes
beforeattemptingtodevelopsurrogateexpressionsystemsforenzyme
characterizationwillbeessential.Manysuchenzymesaremembrane
associated,soinnovativemethodsforcharacterizingmembranecomplexes
mustbedeveloped.Tesecapabilitiesarevitaltodocumentingthemolecu-
larmakeupoflivingcelltypesinvascularandothertissues,includingray
parenchymaandphloem.
MSandNMRwouldbeusedtodevelopverysensitive,high-throughput
assaysforglycosyltransferaseactivity.Synthesis,purication,andcharacter-
izationofglycansneededasacceptorswouldbeimportant.
Aglycochiporrelatedmethodforassayingenzymesinvolvedinpolysac-
charidesynthesisandmodicationalsowouldbeneeded.High-through-
putMScapabilitieswouldbewellsuitedfordevelopingtheanalytical
aspectsofaglycochip.Teseresourcesalsowouldbenetanalysisofmicro-
bialsystemsusedforplantcell-walldeconstructionandsugarfermenta-
tion.Forexample,suchcapabilitiescouldquantifyresponseofwhiterot
fungitochangingcultureconditionsandprovideasystems-levelunder-
standingforimprovingtheenergeticsandcarbon-allocationeciencyfor
cellulaseandcellulosomeproduction.
Optimizingmicrobialculturesforbioconversionwillrequiretoolsthat
allowquantitativecellularcharacterizationatthesystemslevel,including
thoseforglobaltranscript,protein,andmetaboliteprolinginconjunc-
tionwithmetabolicandregulatorymodeling.HTPmethodstoidentify
bindingsitesofglobalregulatoryproteinswillberequiredformodelsof
globalgeneregulation.AdditionalHTPtoolswillmonitorkeymetabolites
thatdenecellredoxandenergystates[e.g.,ATP,GTP,NAD(P)H,and
NAD(P)]forfermentationoptimization.
InstrumentationandMethods
Consolidatedomicswillbecapableofgaininginsightintomicrobialfunc-
tionsbyexaminingsamplestoidentify(1)allproteinsandothermolecules
createdbyaplantorindividualorcommunityofmicrobesundercon-
trolledconditionsand(2)keypathwaysandotherprocesses.Integrating
thesediverseglobaldatasets,computationalmodelswillpredictmicrobial
functionsandresponsesandinferthenatureandmakeupofmetabolicand
regulatoryprocessesandstructures.
Potentialcoreinstrumentation:
Largefarmsofchemostatstopreparesamplesfromhighlymonitored
andcontrolledmicrobialsystemsunderawidevarietyofconditions.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 177

CROSSCUTTING RESEARCH
NumerousspecializedmassandNMRspectrometersandotherinstru-
mentscapableofanalyzingthemolecularmakeupofensemblesamples
fromthousandsofdiversemolecularspecies.
High-performancecomputingandinformationcapabilitiesformodel-
ingandsimulatingplantormicrobial-systemfunctionsunderdierent
scenarios.Tesestudieswillinformthedesignofsystems-levelexperi-
mentsandhelptoinfermolecularprocessesfromensuingdata.
AnalysisandModelingofCellularSystems
FunctionalCapabilities
Biomasscomplexity,coupledwithintricateprocessesinvolvedinconver-
siontoethanol,requiresexperimentalcapabilitiesandmodelsforanalyzing
thebiomass-enzyme-microbesystem,specicallycell-wallsynthesisand
microbialdecompositionofbiomass.Tesecapabilitiesalsocancontribute
todeterminingtheresponseofcomplexsoilmicrobialcommunitiesto
variouscroppingregimes.
Onthebasisofsimilaritywithgenesofknownfunction,advancedcom-
putationwillbeusedforcomprehensiveanalysisofgenesfrommodeland
biomasscropplantsimplicatedinpolysaccharidesynthesisormodica-
tion.AcquisitionandanalysisofHTPgene-expressiondatafrommodel
andcropplantswouldbepowerfulinassigningprobablefunctiontogenes
implicatedincell-wallsynthesis.Asenzymefunctionemerges,asystems-
levelplant-cellmodelwouldincorporatebiophysicalandstructuralproper-
tiesandknowledgeaboutpertinentproteins.Modelsofthistypewould
greatlyfacilitatetherationaldevelopmentoffeedstockspeciesbasedon
designprinciples,inwhichwallchemicalcompositionisoptimizedas
feedstockwhileplantproductivityissimultaneouslymaximized.
Teseresourcesshouldincludecapabilitiesforgeneratingmodelsthat
enableimprovedcellulaseproductionfromnear-termhostssuchasTricho-
dermareeseiandproceedtobacterialandyeastsystemsthatwillrequire
longertimesforresearchanddevelopment.Othergoalsaretoimprove
productionofspecicpreferredcomponentsandfosteramoreglobal
understandingofhyperproductionatindustrialscalesinindustrialstrains.
Anintegratedmodeloffermentativemicroorganismsandtheabilityto
testitexperimentallywillsupportnewconceptstoproduceliquidfuelsand
understandtheproducersunderlyingmetabolismandresponsestostressin
producingbiofuelssuchasethanol.Modelsandnewroundsofmodica-
tionandexperimentationthenwillbeusedtoalleviatestressresponsesand
increasecarbohydratedegradation,minimizecellgrowthandsideproducts
ofsynthesis,andmaximizefuelproduction.
Metabolicuxanalysis(asdescribedearlier)isessentialinimproving
understandingandmanipulationofcellularmetabolism.Possibleanalytical
needsincludeappropriateNMRandMSinstrumentation;stableisotopic
labelingforkeymolecularmoietiesinvolvedinmetabolicprocesses;and
178 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

synthesisofprocessintermediates.Intensivemathematicalandcomputa-
tionalpowerisrequiredtoachievethenalgoalofuxestimation.
InstrumentationandMethods
Technologiesandmethodologiesforanalyzingcellularsystemswillbethe
capstoneforultimateanalyticalcapabilitiesandknowledgesynthesisto
enableapredictiveunderstandingofcell,organism,tissue,andcommu-
nityfunctioncriticalforsystemsbiology.Imagingmethodswillmonitor
proteins,machines,andothermoleculesspatiallyandtemporallyasthey
performtheircriticalfunctionsinlivingcellsandcommunities.Within
theirstructures,microbialcommunitiescontainnumerousmicroniches
thatelicituniquephenotypicalandphysiologicalresponsesfromindividual
microbialspecies.Teabilitytoanalyzethesenichesandthemicrobial
inhabitantswithiniscrucialtoourultimategoal.Tisgrandbiological
challengemustbeaddressedbeforescientistscanpredictthebehaviorof
microbesandtakeadvantageoftheirfunctionalcapabilities.Modeling
resourceswillenabledescriptionofbiologicalinteractionswiththephysi-
cochemicalenvironmentandpredicthowthesystemwillevolvestructur-
allyandfunctionally.
Potentialcoreinstrumentation:
Highlyinstrumentedcultivationtechnologiestopreparestructured
microbialcommunitiesforsimulatingnaturalconditionsinahighly
controlledenvironment.
Instrumentsintegratingnumerousanalytical-imagingtechniquesto
spatiallyandtemporallydetermine,inanondestructiveway,relevant
molecularmakeupanddynamicsofthecommunityenvironment,com-
munity,andmicrobesthatcompriseit.
Computingandinformationcapabilitiestomodelandsimulatecomplex
microbialsystems,designexperiments,andincorporatedata.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 179

CROSSCUTTING RESEARCH
CitedReferences
Moller,G.M.,etal.1998.InSituGeneExpressioninMixed-Culture
Biolms:EvidenceofMetabolicInteractionsBetweenCommunity
Members,Appl.Environ.Microbiol.64,72132.
U.S.DOE.2005.Genomics:GTLRoadmap:SystemsBiologyforEnergyand
Environment,U.S.DepartmentofEnergyOceofScience
(doegenomestolife.org/roadmap/).
U.S.DOE.2002.ReportontheImagingWorkshopfortheGenomestoLifePro-
gram,April1618,2002,OceofScience,U.S.DOE(doegenomestolife.
org/technology/imaging/workshop2002).
Wolfaardt,G.M.,etal.1994.MulticellularOrganizationinaDegradative
BiolmCommunity,Appl.Environ.Microbiol.60,43446.
180 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

BioprocessSystemsEngineering
andEconomicAnalysis
C
ompleteprocessmodelingshouldbeinitiatedtoguidethesci-
enticworkdescribedinthisRoadmap,includingsystemsengi-
neeringandeconomicanalyses,toevaluatethemost-probable
scenarios;tocoordinateadvancesandneededresearchacrossthefeedstock,
deconstruction,andfermentationdomains;andtoreduceriskasthe
developmentcycleproceeds.Resultsandmethodologiesshouldbemade
availabletothecommunitythroughthewebandshouldbesubjecttocon-
tinuousimprovementsbasedoncommunityfeedback.
Biomass-conversionliteraturehasmanyexamplesofpretreatment,hydro-
lysis,andfermentationsystemsthataretechnicallyeectivebuthaveno
realchancetobecompetitiveeconomically.Acommontendencyamong
biotechnologistsistoattemptprocessesthatwillachieve,forexample,
thehighestyields,rates,andtiters.Processmodeling,however,veryoften
revealsthatdierentcombinationsoftheseparametersareneededtopro-
duceacommerciallyviableprocess.Disciplinedsystemsengineeringand
economicanalysisusingmassbalancesandstandardanalyticalmethods
caneliminateineectiveapproachesratherquickly,narrowingthefocusto
themost-promisingoptions.Currentadvancedsaccharicationenzyme
systemshavedemonstratedwhataconcerted,focusedprogramofenzyme
developmentcanachieve.Tesesystems,however,weredesignedforaspe-
cicacid-basedpretreatmentandthebiomassrawmaterialofcornstover.
Othercombinationsofpretreatmentsandbiomassmaterialswillneedto
beanalyzedandsubjectedtoprocessengineeringandeconomicanalysesas
theymature.
ResearchGoals
Tisworkseekstointegrateimprovementsinthemolecularbiologyof
plantmaterialsandthoseinpretreatment,enzymehydrolysis,andfermen-
tationwhileconductingrigorousbioprocesssystemsengineering(incor-
poratingnewprocessesintoamodelrenery)andeconomicanalysis.Te
approachintegratesallelementsofenhancedandcost-eectivemeansto
convertbiomassintoethanolandotherbioproducts.
Practicaloptionsforlignocelluloseconversiontoliquidfuelsaresharplycon-
strainedbytheneedtoproducefermentablesugarcostingafewcentsper
pound,preferablywithminimalsugarlossanddegradation.Furthermore,
inabiorenery,alldownstreamprocesseswillbeaectedbypretreatment
choice.Processintegration,therefore,iscrucial.Finally,noevidencesuggests
thatbiologicalmethodsalone(i.e.,withoutathermochemicalpretreatment
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 181

SYSTEMS ENGINEERING
step)willbeabletoobtainthehigh-yield,low-costconversionsofcellu-
loseandhemicelluloserequiredforeconomicviability.Researchelements
wouldinclude(1)correlationofcell-wallchemistryandstructurewiththe
eciencyofcellulasepolysaccharidedepolymerization,withandwithout
pretreatment;(2)multidisciplinarystudiesofspecictargetedlignocellu-
losicmaterialswithpromisingpretreatmentsandadvancedenzymes,using
standardanalyticalmethodsanddetailedmassbalances;(3)examination
of theeectsofindividualandcombinedenzymaticactivitiesonpretreated
substrates,withtheprimarytechnical-evaluationcriterionofmaximum
sugaryieldatminimumenzymeloadings;and(4)processengineeringand
economicmodelingoftheseintegratedpretreatmentandhydrolysisstudies
toidentifypromisingcombinationsandeliminateless-attractiveoptions.
Tisworkmustbecoordinatedwithalldomainsoftheresearchportfolioto
havethedesiredeectoptimalprocessdevelopment.
Consideringthegoaltodeveloponeormorecommercialprocessesfor
ethanolproductionfrombiomass,aproperevaluationofmost-probable
futurescenariosisneeded.Forexample,severalgroupsenvisionsimultane-
oussaccharicationandfermentation(SSF)foramoreviableprocess.Since
saccharicationenzymemixesworkbetterat50

CandapHbetween4
and6,thenasindicatedbyotherstudygroups,noneofthecurrentethanol
producersmicroorganismsissuitable.Underthisscenariotheparameters
ofsaccharicationmustbeincludedindesigningnewproductionmicro-
organisms.Processmodelingisneededtoevaluatetheimportanceand
sensitivityofSSFvsatwo-stepprocess.Similarly,ethanolfermentations
willbeevaluatedbythreemainparameters: Yieldfromcarbohydrates,rate
ofproduction,andnaltiter.
Modelingwillbeveryusefulinevaluatingthefeasibilityofconsolidated
bioprocessing.Asanexample,currentspecic-activityvaluesofthethree
enzymesusedtodaytodegradecellulose(i.e.,exoglucanases,endogluca-
nases,andbeta-glucosidases)maypossiblyallowpredictionoftheprotein
amountthataconsolidatedstrainwillhavetoproduceperunitoftimeto
degradecelluloseandliberatesugarsquicklyenoughtosustainfastratesof
ethanolproduction.
Milestones
Within5yearsorless
Initialresearchshouldaddresscorrelationofchangesinplantcell-wall
structurewitheaseofhydrolysis.Tiscouldbedonethroughchemical
compositionornanoscaleandmolecular-scaleimaging(suchaswouldbe
availablefromNationalRenewableEnergyLaboratory).Terelationship
betweenchangesinplantcell-wallstructure(e.g.,lignincomposition)
wouldenablebetterformulationofmore-eectiveandmore-productive
advancedenzymesintheirinteractionswithasolidsubstrate.Emphasis
wouldbeoncouplingchangesinplantcell-wallstructure,changesinpre-
treatmentconditions,anddecreasesinenzymeusagetoachieveconversion
extentandratecurrentlyattainedwithmuch-higherenzymeloadings.
182 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Within10years
Oncescientistsdeterminetheimpactofchangesinplantcell-wallstruc-
tureonhydrolysisextentandtheeasewithwhichaplantcellwallcanbe
deconstructed,furthermechanisticstudiesforimprovingtheeciencyof
enzymehydrolysiswillbecarriedout.Certaintypesofplantcellwallsmay
havelignincontenthigherthanordierentfromcurrentbiomassmateri-
als;utilizationofenzymesincludinglignases,hemicellulases,andcellulases
couldbeconsidered.Amodelwouldbehelpful,forexample,inwhicha
lignin-richmaterialisconvertedecientlyintoitsfractionsoflignin,cel-
loligosaccharides,andxylans.Lignincouldbeusedasasourceofenergy
foracellulose-to-ethanolprocessingplant.Tepropercompositionbalance
inlignintopolysaccharidescouldmakesuchafacilityself-sucientwith
respecttoenergyusage.Systemsintegrationofthesevariousstepswillbe
carriedoutinalonger-termproject.Scientistsexpectinitialimprovements
intheeciencyofcellulose-to-ethanolconversioninanexistingplant
(suchasadrymill)tobepossiblewithin10years.
Within15years
Engineering,molecularevolutionofnewenzymes,andgeneticmodica-
tionofagronomictraitsshouldbecombinedtoyieldaverticallyinte-
gratedtechnologyforconvertingbiomassmaterialstoethanolandother
bioproducts.Combiningmolecularbiologywithprocessengineeringmay
yieldplantsthatarerobustintheeldbuteasilyaccessibleforconversion
usingspecializedenzymesaddedafterharvest.Transportationofbiomass
materialsisknowntobeakeycostfactor,soenzymesintegratedintoplant
materialcouldmakeitmorecompressibleafterharvest.Lignin-degrading
enzymesmaymakepossiblehigh-ligninplants(includingcertaintypesof
grasses)thatareeasilydeconstructedonceexposedtoproperconditionsin
aprocessingplant,yieldinganadequateamountforpoweringthefacility.
TheRoleofGTLandOBPFacilitiesandCapabilities
Techallengeinusingadvancedanalyticalandmodelingcapabilitiesisto
integrateresearchverticallyacrossseveraldomainsintoaviable,techni-
callyandeconomicallysoundbioreneryconcept.Researchresultsmustbe
rapidlyassimilatedintoanoverallsystemsmodelthatwillprovideinsight
intotheimpactofnewphenomenaorprocesses;concomitanttechnolo-
giesmustbeincorporatedintothesystemtooptimizetheseeects.Such
analysiswillguideaverticallyintegratedresearchportfoliothatsupports
allaspectsofbioprocessengineering.Asummaryfollows.
ProteinProduction
Proteinproductioncapabilitieswillbecriticalingeneratingandcharacter-
izingnewproteinsandbiomarkersforimagingandforidentifyingkey
protein-proteininteractions,includingenzymesforlignin,polysaccharide,
andhemicelluloseesterdegradation.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 183




SYSTEMS ENGINEERING
Proteomics
Proteomicmeasurementscanprovidevaluableinformationaboutproteins
andmechanismsbywhichmonosaccharideconversiontoethanoland
otherproductsisachieved.Combinationsofnativeandgeneticallymodi-
edorganismscanachievebetterconversioninexistingethanolfacilities.
CellularSystems
Cellularsystemsanalysisandmodelingmaybeemployedtogenerateand
monitormicrobialcellsandcommunitiesthatwouldenableexpressionof
newtypesofenzymesorproteinssosucientquantitiesareavailablefor
research.
DOEJointGenomeInstitute
DOEJGIwillbeimportantindiscoveringnewmicroorganismsand
enzymesthatmaybeusefulinconvertingmore-recalcitrantformsofcellu-
losicmaterialstomonosaccharides.
184 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

APPENDICES
AppendixA.ProvisionsforBiofuelsandBiobasedProductsintheEnergyPolicyActof2005.......................186
AppendixB.WorkshopParticipants..........................................................................................................188
AppendixC.WorkshopParticipantBiosketches.........................................................................................192
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 185

APPENDICES
AppendixA.ProvisionsforBiofuelsandBiobased
ProductsintheEnergyPolicyActof2005
OnJuly29,2005,CongresspassedtheEnergyPolicy
Section941.AmendmentstotheBiomassResearchand
Actof2005,
1
andPresidentGeorgeBushsigneditinto
DevelopmentActof2000.
lawonAugust8,2005.Te$14billionnationalenergy
Tissectionpresentsseveralchangesinthewordingof
planincludesprovisionsthatpromoteenergyeciency
theBiomassResearchandDevelopmentActof2000.
andconservation,modernizethedomesticenergyin-
Someimportantchangesincluderedeningtheobjec-
frastructure,andprovideincentivesforbothtraditional
tivesoftheBiomassResearchandDevelopmentInitia-
energysourcesandrenewablealternatives.Tefollow-
tivetostatethattheinitiativewilldevelop(1)tech-
ingsectionsoftheEnergyPolicyActrelatetobiofuels
nologiesandprocessesnecessaryforabundantcommer-
andbiobasedproducts.
cialproductionofbiobasedfuelsatpricescompetitive
withfossilfuels;(2)high-valuebiobasedproductsto
TitleIXResearchandDevelopment
enhancetheeconomicviabilityofbiobasedfuelsand
Section932.BioenergyProgram. powerand[serve]assubstitutesforpetroleum-based
Research,development,demonstration,andcom-
feedstocksandproducts;and(3)adiversityofsustain-
mercialapplicationactivitiesunderthisprogramwill
abledomesticsourcesofbiomassforconversiontobio-
addressbiopower,biofuels,bioproducts,integrated
basedfuelsandbiobasedproducts.Anotherimportant
bioreneries,crosscuttingresearchanddevelopment
amendmentintroducesfourtechnicalareasthatthe
infeedstocks;andeconomicanalysis.GoalsforDOE
initiativewilladdressinitsresearchanddevelopment
biofuelandbioproductprogramsincludepartnering
activities:(1)developcropsandsystemsthatimprove
withindustrialandacademicinstitutionstodevelop:
feedstockproductionandprocessing,(2)convertrecal-
(1)advancedbiochemicalandthermochemicalcon-
citrantcellulosicbiomassintointermediatesthatcan
versiontechnologiescapableofmakingfuelsfromlig-
beusedtoproducebiobasedfuelsandproducts,(3)de-
nocellulosicfeedstocksthatareprice-competitivewith
veloptechnologiesthatyieldawiderangeofbiobased
gasolineordieselineitherinternalcombustionengines
productsthatincreasethefeasibilityoffuelproduction
orfuelcell-poweredvehicles;(2)advancedbiotech-
inabiorenery,and(4)analyzebiomasstechnologies
nologyprocessescapableofmakingbiofuelsand
fortheirimpactonsustainabilityandenvironmental
bioproducts,withemphasisondevelopmentofbio-
quality,security,andruraleconomicdevelopment.
renerytechnologiesusingenzyme-basedprocessing
Section942.ProductionIncentivesforCellulosicBiofuels.
systems;(3)advancedbiotechnologyprocessescapable
ofincreasingenergyproductionfromlignocellulosic
Tissectioncallsfortheestablishmentofaprogram
feedstocks,withemphasisonreducingthedependence
thatprovidesproductionincentivesonaper-gallon
ofindustryonfossilfuelsinmanufacturingfacilities;
basisforcellulosicbiofuels.Tepurposeoftheincentive
and(4)otheradvancedprocessesthatwillenablethe
programistoensurethat1billiongallonsinannualcel-
developmentofcost-eectivebioproducts,including
lulosicbiofuelproductionareachievedby2015;cellulos-
biofuels. TroughIntegratedBioreneryDemonstra-
icbiofuelsarecost-competitivewithgasolineanddiesel;
tionProjects,DOEwilldemonstratethecommercial
andsmallfeedstockproducersandruralsmallbusinesses
applicationofintegratedbioreneriesthatuseawide
arefullparticipantsinthecellulosicbiofuelindustry.
varietyoflignocellulosicfeedstockstoproduceliquid
transportationfuels,high-valuebiobasedchemicals,
Section977.SystemsBiologyProgram.
electricity,substitutesforpetroleum-basedfeedstocks, Tissectionprovidesfortheestablishmentofaresearch
andusefulheat. programinmicrobialandplantsystemsbiology,protein
science,andcomputationalbiologytosupportDOE
energy,nationalsecurity,andenvironmentalmissions.
Fundswillbeavailableforprojectstoplan,construct,
1
U.S.Congress.2005.EnergyPolicyActof2005,Pub.L.109-
oroperatespecialinstrumentationorfacilitiesfor
58(www.epa.gov/OUST/fedlaws/publ_109-058.pdf).
186 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix A. Energy Policy Act

researchersinsystemsbiologyandproteomicsandas-
sociatedbiologicaldisciplines.Biomedicalresearchon
humancellsorhumansubjectsisprohibited.
TitleXVEthanolandMotorFuels
Section1501.RenewableContentofGasoline(Renewable
FuelsStandard).
TissectionamendsSection211oftheCleanAir
Act.Animportantamendmentistheestablishmentof
therenewablefuelprogram.Inthisprogram,gasoline
soldintheUnitedStatesisrequiredtobemixedwith
increasingamountsofrenewablefuel(usuallyethanol)
onanannualaveragebasis.In2006,4billiongallonsof
renewablefuelsaretobemixedwithgasoline,andthis
requirementincreasesannuallyto7.5billiongallons
ofrenewablefuelby2012.For2013andbeyond,the
requiredvolumeofrenewablefuelwillincludeamini-
mumof250milliongallonsofcellulosicethanol.
Section1505.PublicHealthandEnvironmentalImpactsof
FuelsandFuelAdditives.
TissectionamendsSection211(b)oftheCleanAir
Actbyrequiringstudyoftheimpactsofincreaseduse
offueladditives(e.g.,ethanolandotherchemicals)on
publichealth,airquality,andwaterresources.
Section1506.AnalysisofMotorVehicleFuelChanges.
TissectionamendsSection211oftheCleanAirAct
byrequiringstudyoftheeectsofethanol-gasoline
mixesonpermeationoffuelmoleculesintoplasticand
rubbercomponentsoffuelsystemsandtheevaporative
emissionsresultingfromthispermeation.
Section1511.RenewableFuel.
TissectionamendstheCleanAirActbyaddinga
sectionthatallowsfundstocoverloanguaranteesfor
nomorethanfourprojectscommerciallydemonstrat-
ingthefeasibilityofproducingcellulosicethanol.Each
projectisexpectedtoproduce30milliongallonsof
cellulosicethanolannually.Fundsalsohavebeenautho-
rizedforaresourcecenterthatfurtherdevelopsbiocon-
versiontechnologiesforethanolproduction.
Section1512.ConversionAssistanceforCellulosicBiomass,
Waste-DerivedEthanol,ApprovedRenewableFuels.
TissectionamendsSection211oftheCleanAirAct
byauthorizingDOEtoprovidegrantsforbuilding
productionfacilitiestoproducersofcellulosicethanol
andotherapprovedrenewablefuels.
Section1514.AdvancedBiofuelTechnologiesProgram.
TissectionauthorizestheEPAAdministratorto
establishaprogramtodemonstrateadvancedtechnolo-
giesfortheproductionofalternativetransportation
fuels.Priorityisgiventoprojectsthatincreasethegeo-
graphicdistributionofalternativefuelproductionand
makeuseofunderutilizedbiomassfeedstocks.
Section1516.SugarEthanolLoan-GuaranteeProgram.
TissectionauthorizesDOEtoissueloanguarantees
toprojectsthatdemonstratethefeasibilityofproducing
ethanolfromsugarcaneorsugarcaneby-products.
TitleIIRenewableEnergy
Section208.SugarCaneEthanolProgram.
TeEnvironmentalProtectionAgencyistoestablisha
$36millionprogramtostudysugarcane,productionof
ethanolfromsugarcane,andsugarcaneby-products.
Teprojectwillbelimitedtosugarandethanolpro-
ducersinFlorida,Louisiana,Texas,andHawaii.Infor-
mationwillbegatheredonhowtoscaleupproduction
oncethesugarcaneindustryisreadytolocatesitesfor
andconstructethanol-productionfacilities.
Section210.GrantstoImprovetheCommercialValue
ofForestBiomassforElectricEnergy,UsefulHeat,
TransportationFuels,andOtherCommercialPurposes.
TeSecretaryofAgricultureortheSecretaryofthe
Interiormayprovidegrantstoanypersoninapreferred
community(e.g.,Indiantribe,townorunitoflocal
governmentwithfewerthan50,000individuals,orone
thathasbeendeterminedtoposeare,disease,orin-
sect-infestationhazardtofederalorIndianland).Own-
ersoffacilitiesthatusebiomasstoproduceelectricity,
usefulheat,ortransportationfuelsmayreceivegrantsof
upto$20pergreentonofbiomassdeliveredtooset
thecostofrawmaterial.Grantsofupto$500,000may
beawardedtoosetthecostsofdevelopingresearch
opportunitiesthatimprovetheuseoforaddvalueto
biomass.Foreachscalyearfrom2006through2016,
$50millionisauthorizedtobeappropriatedtocarry
outthissection.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 187

APPENDICES
AppendixB.WorkshopParticipants
Ackerman,Eric
PacicNorthwestNationalLaboratory
eric.ackerman@pnl.gov
Adams,Justin
BPPLC
justin.adams@uk.bp.com
Armstrong,Katherine
DowAgrosciencesLLC
karmstrong@dow.com
Atalla,Rajai
33/314ForestProductsLaboratory
rhatalla@wisc.edu
Baldwin,Sam
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
sam.baldwin@ee.doe.gov
Bayer,Ed
DepartmentofBiologicalChemistry
WeizmannInstituteofScience
ed.bayer@weizmann.ac.il
Bownas,Jennifer
GenomeManagementInformationSystem
OakRidgeNationalLaboratory
bownasjl@ornl.gov
Brady,John
DepartmentofFoodScience
CornellUniversity
jwb7@cornell.edu
Bull,Stan
NationalRenewableEnergyLaboratory
stan_bull@nrel.gov
Cameron,Doug
Cargill,Incorporated
doug_cameron@cargill.com
Casey,Denise
GenomeManagementInformationSystem
OakRidgeNationalLaboratory
caseydk@ornl.gov
Cavalieri,Ralph
WashingtonStateUniversity
cavalieri@wsu.edu
Chapple,Clint
DepartmentofBiochemistry
PurdueUniversity
chapple@purdue.edu
Chum,Helena
NationalRenewableEnergyLaboratory
helena_chum@nrel.gov
Cleary,Michael
SanDiegoSupercomputerCenter
UniversityofCalifornia,SanDiego
mcleary@sdsc.edu
Collart,Frank
ArgonneNationalLaboratory
fcollart@anl.gov
Colson,Steven
FundamentalScienceDirectorate
PacicNorthwestNationalLaboratory
steven.colson@pnl.gov
Cotta,Mike
NationalCenterforAgriculturalUtilizationResearch
AgriculturalResearchService
U.S.DepartmentofAgriculture
cottama@ncaur.usda.gov
Dale,Bruce
DepartmentofChemicalEngineeringandMaterialsScience
MichiganStateUniversity
bdale@egr.msu.edu
Davison,Brian
LifeSciencesDivision
OakRidgeNationalLaboratory
davisonbh@ornl.gov
Dean,William
DaniscoGenencorInternational
bdean@danisco.com
Donohue,Tim
BacteriologyDepartment
UniversityofWisconsin,Madison
tdonohue@bact.wisc.edu
Drell,Daniel
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
daniel.drell@science.doe.gov
Ferrell,John
BiomassProgram
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
john.ferrell@hq.doe.gov
Foust,Tom
NationalRenewableEnergyLaboratory
thomas_foust@nrel.gov
Fredrickson,Jim
PacicNorthwestNationalLaboratory
jim.fredrickson@pnl.gov
Gonzalez,Ramon
DepartmentofChemicalandBiomolecularEngineering
RiceUniversity
ramon.gonzalez@rice.edu
188 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix B. Workshop Participants

Greene,Rich
OceofInternationalResearchPrograms
AgriculturalResearchService
U.S.DepartmentofAgriculture
richard.greene@nps.ars.usda.gov
Hames,Bonnie
NationalRenewableEnergyLaboratory
bonnie_hames@nrel.gov
Harrison,Maria
BoyceTompsonInstituteforPlantResearch
mjh78@cornell.edu
Heineken,Fred
NationalScienceFoundation
fheineke@nsf.gov
Hennessey,Susan
DuPontCentralResearchandDevelopment
susan.m.hennessey@usa.dupont.com
Himmel,Mike
NationalBioenergyCenter
NationalRenewableEnergyLaboratory
mike_himmel@nrel.gov
Hladik,Maurice
IogenCorporation
mauriceh@iogen.ca
Houghton,John
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
john.houghton@science.doe.gov
Ingram,Lonnie
FloridaCenterforRenewableChemicalsandFuels
DepartmentofMicrobiologyandCellScience
UniversityofFlorida
ingram@u.edu
Jacobs-Young,Chavonda
NationalResearchInitiative
CooperativeStateResearch,Education,andExtensionService
U.S.DepartmentofAgriculture
cjacobs@csrees.usda.gov
Jofuku-Okamura,Diane
DivisionofBiologicalInfrastructure
NationalScienceFoundation
dbipqr@nsf.gov
Kaempf,Doug
BiomassProgram
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
douglas.kaempf@ee.doe.gov
Kahn,Michael
BasicEnergySciences
OceofScience
U.S.DepartmentofEnergy
michael.kahn@science.doe.gov
Kaleikau,Ed
NationalResearchInitiative
CooperativeStateResearch,Education,andExtensionService
U.S.DepartmentofAgriculture
ekaleikau@csrees.usda.gov
Keasling,Jay
PhysicalBiosciencesDivision
LawrenceBerkeleyNationalLaboratory
jdkeasling@lbl.gov
Keegstra,Ken
MSU-DOEPlantResearchLaboratory
MichiganStateUniversity
keegstra@msu.edu
Klembara,Melissa
BiomassProgram
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
melissa.klembara@hq.doe.gov
Knotek,Mike
m.knotek@verizon.net
Ladisch,Mike
LaboratoryofRenewableResourcesEnergy
PurdueUniversity
ladisch@purdue.edu
Lohman,Kent
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
kenton.lohman@science.doe.gov
Lynd,Lee
TayerSchoolofEngineering
DartmouthCollege
lee.r.lynd@dartmouth.edu
Manseld,Betty
GenomeManagementInformationSystem
OakRidgeNationalLaboratory
manseldbk@ornl.gov
Matteri,Bob
AgriculturalResearchService
U.S.DepartmentofAgriculture
rmatteri@pw.ars.usda.gov
McLean,Gail
NationalResearchInitiative
CooperativeStateResearch,Education,andExtensionService
U.S.DepartmentofAgriculture
gmclean@csrees.usda.gov
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 189

APPENDICES
Michaels,George
BioinformaticsandComputationalBiology
PacicNorthwestNationalLaboratory
george.michaels@pnl.gov
Miranda,Amy
BiomassProgram
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
amy.miranda@hq.doe.gov
Mitchinson,Colin
DaniscoGenencorIntl.Inc.
cmitchinson@danisco.com
Moorer,Richard
OceofEnergyEciencyandRenewableEnergy
U.S.DepartmentofEnergy
richard.moorer@hq.doe.gov
Morrison,Mark
DepartmentofMicrobiology
OhioStateUniversity
morrison.234@osu.edu
Palmisano,Anna
CooperativeStateResearch,Education,andExtensionService
U.S.DepartmentofAgriculture
anna.palmisano@usda.gov
Patrinos,Ari
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
ari.patrinos@science.doe.gov
Ragauskas,Art
InstituteofPaperScienceandTechnology
GeorgiaInstituteofTechnology
art.ragauskas@ipst.gatech.edu
Ralph,John
U.S.DairyForageResearchCenter
CooperativeStateResearch,Education,andExtensionService
U.S.DepartmentofAgriculture
DepartmentofForestry
UniversityofWisconsin,Madison
jralph@wisc.edu
Remington,Karin
J.CraigVenterInstitute
Sarkanen,Simo
KaufertLaboratory
DepartmentofBio-BasedProducts
UniversityofMinnesota,TwinCities
sarka001@umn.edu
Schilling,Christophe
Genomatica,Inc.
cschilling@genomatica.com
Shanklin,John
BiologyDepartment
BrookhavenNationalLaboratory
shanklin@bnl.gov
Shoemaker,Sharon
DepartmentofFoodScienceandTechnology
UniversityofCalifornia
spshoemaker@ucdavis.edu
Shoham,Yuval
DepartmentofBiotechnologyandFoodEngineering
Technion-IsraelInstituteofTechnology
yshoham@tx.technion.ac.il
Smith,Lloyd
DepartmentofChemistry
UniversityofWisconsin,Madison
smith@chem.wisc.edu
Somerville,Chris
DepartmentofPlantBiology
DepartmentofBiologicalSciences
CarnegieInstitutionofWashington
StanfordUniversity
crs@stanford.edu
Stephanopoulos,Greg
DepartmentofChemicalEngineering
MassachusettsInstituteofTechnology
gregstep@mit.edu
Stevens,Walt
ChemicalSciences,Geosciences,andBiosciencesDivision
BasicEnergySciences
OceofScience
U.S.DepartmentofEnergy
walter.stevens@science.doe.gov
Stone,Bruce
DepartmentofBiochemistry
LaTrobeUniversity
b.stone@latrobe.edu.au
Stults,Ray
NationalRenewableEnergyLaboratory
ray_stults@nrel.gov
Tabita,Bob
DepartmentofMicrobiology
OhioStateUniversity
tabita.1@osu.edu
Tomas,Steve
Ceres,Inc.
sthomas@ceres-inc.com
Tomassen,David
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
david.thomassen@science.doe.gov
190 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix B. Workshop Participants

Tuskan,Gerald
EnvironmentalSciencesDivision
OakRidgeNationalLaboratory
tuskanga@ornl.gov
Uberbacher,Ed
LifeSciencesDivision
OakRidgeNationalLaboratory
ube@ornl.gov
Valle,Fernando
DaniscoGenencor,Inc.
fvalle@Danisco.com
Vogel,John
WesternRegionalResearchCenter
jvogel@pw.usda.gov
Vogel,Kenneth
AgriculturalResearchService
U.S.DepartmentofAgriculture
DepartmentofAgronomyandHorticulture
UniversityofNebraska,Lincoln
kpv@unlserve.unl.edu
Weatherwax,Sharlene
BiologicalandEnvironmentalResearch
OceofScience
U.S.DepartmentofEnergy
sharlene.weatherwax@science.doe.gov
Wheeler,Nick
MolecularTreeBreedingServices,LLC
nickwheeler@scattercreek.com
Wilson,David
DepartmentofMolecularBiologyandGenetics
CornellUniversity
dbw3@cornell.edu
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 191

APPENDICES
AppendixC.WorkshopParticipantBiosketches
EricAckerman
EricAckermanisamolecularbiologistatPacicNorthwest
NationalLaboratory,havingearnedadoctorateinbiophys-
icsfromtheUniversityofChicagoin1979.HewasaHelen
HayWhitneyFellowattheLaboratoryofMolecularBiology
inCambridge,England,for3yearsandthenastascientistat
NIHNationalInstituteofDiabetesandDigestiveandKidney
Diseasesuntil1996.Hehasbeeninvolvedindevelopmental
biologyprojectswithXenopuslaevis,themethodsbywhich
toxinskillcells,mechanismsofnucleases,biochemistryof
nucleotideexcisionDNArepair,andanextremelysensitiveand
quantitativeassaytomeasureradiationeectsonmultiplekinds
ofDNArepairusingasfewas3000cellsand0.1-ngDNA.
MostrelevantfortheBiomasstoBiofuelsworkshophavebeen
Ackermansstudiesofnovelmechanismsforimmobilizing
enzymesinfunctionalizednanoporousmaterialsforenhanced
activityandstability.Recently,hebeganimplementinghigh-
throughput,cell-freeproductionofproteinsandtheircharac-
terizationsinhopesthatthisapproachmightyieldoptimized,
stableproteincomplexesthatcouldcontributetoenergygenera-
tion.HealsowasinvolvedinstrategicplanningforGenomics:
GTLproductionfacilities,particularlyfortheFacilityforPro-
ductionandCharacterizationofProteinsandAnityReagents.
JustinAdams
JustinAdamsjoinedBPPLCin2003andcurrentlyisdirector
oflong-termtechnologystrategyinBPsOceoftheChief
Scientist.Inthisrolehehelpsbuildandshapethestrategic
agenda,coordinatesalllong-termactivitiesacrossthecompany
toensurealignmentandbalance,andoverseesspecicprograms
managedbythecentraltechnologyfunction.
BeforejoiningBP,AdamswasfounderandCEOofHigh
PowerLithium,aSwisscompanydevelopingnext-generation
batterymaterialsforhybridelectricvehiclesincollaboration
withToyota.HealsowasanadvisortoKonarkaTechnologies,a
Massachusetts-basedstartupdevelopingnext-generationsolar
cellsusingconductingpolymersandnanostructuredmaterials.
HepreviouslyworkedasaconsultantwithArthurD.Little,
ultimatelyleadingitsAdvancedEnergySystemspracticein
Europe.ArthurD.Littledeliversstrategicandtechnoeconomic
consultingonemergingenergytechnologiestomanyofthe
worldsleadingenergymajors.
Adamsholdsjointhonorsinmanagementandtechnologyfrom
theUniversityofBath(England)andtheUniversityofRich-
mond(Virginia).
KatherineArmstrong
KatherineArmstrongisgloballeaderfortraitgeneticsand
technologiesR&DatDowAgroSciencesinIndianapolis,
Indiana.SheearnedherbachelorsdegreefromtheUniversityof
Virginiaandmastersinmolecularandpopulationgeneticsfrom
theUniversityofGeorgia.ShehasbeenanR&Dscientistwith
Dowfor25yearsandhasstudiedplantgeneexpressionatthe
molecularandcellularlevels.Currentlysheoverseesthedevel-
opmentofcorntraitsthroughproductlaunch.Sheholdsseven
U.S.patentsintheareaofplantgeneexpressionandhaswritten
numerouspublications.Relevantareasofresearchincludeopti-
mizationofcorngeneticsforethanolextractionfrombothgrain
andcellulosicfeedstocks.
RajaiH.Atalla
RajaiAtallareceivedhisbachelorsdegreefromRensselaerPoly-
technicInstitutein1955andmastersanddoctorateinchemi-
calengineeringandphysicsfromtheUniversityofDelaware
by1960;hisworkfocusedonspectroscopicstudiesofames.
During8yearsatHerculesResearchCenter,hestudiedphase
transitionsinsemicrystallinepolymersandevaluatedanoma-
lousspectraofmanycompounds.Hewasrsttorecognizethat
anomalousprotonnuclearmagneticresonance(NMR)spectra
of(alkylphosphito)hydridesofcobaltandironindicatedthe
occurrenceofuxionalmolecules.Healsodevelopedtherst
theoreticalmodelforphotodegradationofinorganicpigment.
AsprofessorofchemicalphysicsandengineeringattheInstitute
ofPaperChemistry,AtallapioneeredtheapplicationofRaman
spectroscopytostudiesofcelluloses.Findingacceptedcrystal
structuresinconsistentwithRamanspectra,heinvestigatedthe
13
Csolid-stateNMRspectraofnativecelluloseswithDavid
VanderHartoftheNationalInstituteofStandardsandTechnol-
ogy.Teydeterminedthatallnativecellulosesarecomposites
oftwoformsI andI (1984).WithUmeshAgarwal,usinga

Ramanmicroprobe,Atalladevelopedtherstdirectevidenceof
ligninorientationinsecondarywalls(1984).
In1989,asheadofchemistryandpulpingresearchattheU.S.
DepartmentofAgriculture(USDA)ForestServiceandadjunct
professorinchemicalandbiologicalengineeringattheUniver-
sityofWisconsin,Madison,Atallaleddevelopmentofinorganic
analogsofligninperoxidasesforuseinliquid-euentfreepulp-
ingandbleachingsystems.Teprocesseswerefeasibleeconomi-
cally,buttheindustryseconomicconditionledtosuspensionof
theprogram.Freedofadministrativeresponsibilitiesin1999and
elevatedtoseniorandpioneeringresearchscientistin2005,he
returnedtostudiesofmoleculararchitectureinplantcellwalls,
withemphasisonsecondarywallsandnativecelluloses.
Atallahaspublishedmorethan150papers,editedabookon
cellulosestructures,andisafellowoftheInternationalAcademy
ofWoodScienceandoftheTechnicalAssociationofthePulp
andPaperIndustry.HereceivedtheAnselmePayenAwardof
theAmericanChemicalSocietysCelluloseDivisionaswellas
192 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix C. Participant Biosketches

multipleUSDAawards,includingtheForestServiceChiefs
DistinguishedScientistAward.
EdBayer
EdBayerisaprofessorintheDepartmentofBiologicalChem-
istryattheWeizmannInstituteofScience,Rehovot,Israel.He
wasawardedabachelorsdegreeinliberalartsfromtheUni-
versityofMichiganin1969,amastersinbiologyfromWayne
StateUniversityin1971,andadoctorateinbiophysicsfromthe
WeizmannInstituteofSciencein1976.Sincetheearly1970s,
hehasbeeninvolvedindevelopingtheavidin-biotinsystem
asageneraltoolinthebiologicalsciences.Hewasrsttouse
biotinylationproceduresforantibodiesandotherproteinsand
carbohydratesaswellasavidin-conjugationandcomplexation
techniques.Teworkinitiallywaspublishedinthemid-1970s,
andmanyoftheproceduresarestillinroutineusetoday.He
receivedtheSarstedtAwardforhiscontributionstotheavidin-
biotinsystemforbiomedicalanalysis.
TogetherwithRaphaelLamed,Bayerintroducedthecellulo-
someconceptintheearly1980s.In1999,hewasorganizerand
cochairoftherstGordonResearchConferenceonCellulases
andCellulosomes,andheservedaschairmanofthesame
conferencein2001.In1994,heproposedtheuseofdesigner
cellulosomesforbiomassdegradationandwastemanagement
andasageneraltoolinthebiologicalsciences.Sincethen,he
hasworkedsystematicallytowardthecontrolledconstructionof
sucharticialcellulosomesviaself-assemblyandhasproduced
agrowingrepertoireofdivergentcellulosomalcomponentsfor
thispurpose.
Duringhiscareer,hehascollaboratedwithgroupsintheUnited
States,Canada,Holland,Belgium,Germany,GreatBritain,
France,Spain,Finland,Denmark,Guatemala,andtheRepublic
ofGeorgia,andhehasauthoredmorethan250articlesand
reviewsinbothelds.HecoeditedVol.184onavidin-biotin
technologyintheMethodsinEnzymologyseriesandsince1999
hasservedaseditorofthereviewjournalBiotechnologyAdvances.
In2002,hewaselectedafellowoftheAmericanAcademyof
Microbiology.Hecontinueshisworkinboththeavidin-biotin
andcellulosomeelds,andhisinterestsstillfocusonprotein
engineering,nanobiotechnology,andthestructuralandfunc-
tionalconsequencesofprotein-proteinandprotein-ligand
interactions.
JohnBrady
JohnBradyisaprofessorintheDepartmentofFoodScienceat
CornellUniversity.Hereceivedabachelorsdegreeinchemistry
fromtheUniversityofNorthCarolina,ChapelHill,in1975
andadoctorateinchemistryfromtheStateUniversityofNew
YorkatStonyBrookin1980.Duringmuchofhisgraduate
study,hewasavisitingstamemberatLosAlamosNational
LaboratoryinNewMexico.Hereceivedhispostdoctoraltrain-
inginchemistryatHarvardUniversity,workingwithMartin
Karplus.
Hisresearchprimarilyinvolvesbiopolymerdynamicsand
hydrationandtherelationshipsamongstructure,conformation,
andfunctioninbiologicalsystems.Specicexamplesinclude
thesolutionbehaviorofbiopolymers,factorsthatdetermine
secondaryandtertiarystructureinpolymers,enzymaticreaction
mechanisms,rationaldrugdesign,eectsofpointmutations
inproteins,andthepossibilityofengineeringdesirablemodi-
cationsinthefunctionofwild-typeproteins.Hisworkuses
techniquesofcomputationaltheoreticalchemistrytomodel
propertiesofbiopolymersandsolutionsnumerically.Tese
techniques,oftencalledmolecularmechanics,includecomputer
graphicsbasedmoleculardocking,energyminimizationand
conformationalenergycalculations,andmoleculardynamics
simulations.
AprincipalfocusofBradysresearchisoncarbohydratestruc-
ture,dynamics,andhydration.Hehascontributedtoadvancesin
carbohydratemodeling,includingtherstmoleculardynamics
simulationsonasugar,therstrelaxedconformationalenergy
mapforadisaccharide,therstfree-energysimulationsofsugar
energydierencesinsolution,andtherstpotentialofmean-
forceorconformationalfree-energymapforadisaccharide.As
anoutgrowthofhisprimaryinterestsincarbohydrates,heis
studyingcarbohydrateinteractionswithproteins.Inacurrent
project,Bradyisusingmolecularmechanicssimulationstostudy
thecatalyticmechanismandmodeofsubstratebindinginvari-
ouscellulases,includingE2fromTermomonosporafusca,inthe
hopeofdesigningamoreactiveenzymethatcouldbeproduced
bysite-directedmutagenesis.
DougCameron
DougCameronreceivedabachelorsdegreeinbiomedicalengi-
neeringin1979fromDukeUniversityandadoctorateinbio-
chemical engineeringfromMIT in 1986. He servesasdirector
ofbiotechnologyforCargillResearch,withanadjunctprofessor-
shipintheDepartmentofChemicalandBiologicalEngineering
attheUniversityofWisconsin,Madison(UWM).From1986to
1998,CameronwasaprofessorintheDepartmentofChemical
EngineeringandanaliateintheMolecularBiologyProgram
atUWM.In1996hewasaguestprofessorintheInstitutefor
BiotechnologyattheSwissFederalInstituteofTechnologyin
Zurich.From1979to1981,heheldthepositionofbiochemical
engineeratAdvancedHarvestingSystems,aplantbiotechnol-
ogycompanyfundedbyInternationalHarvester.
CameronisafellowoftheAmericanInstituteofMedical
andBiologicalEngineeringandisontheeditorialboardsof
MetabolicEngineeringandBiomacromolecules.Heservesonthe
MinnesotaGovernorsBioscienceCouncilandtheboardof
directorsofMinnesotaBiotechnologyIndustryOrganization.
HeisamemberoftheMITBiologicalEngineeringvisiting
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 193

APPENDICES
committeeandonthemanagingboardofthenewlyformed
SocietyforBiologicalEngineering.Cameronalsoisaconsult-
ingprofessorintheDepartmentofChemicalEngineeringat
StanfordUniversity.
ClintChapple
ClintChapplereceivedhisdoctorateinchemistryfromtheUni-
versityofGuelphin1989.Afterdoingpostdoctoralworkwith
ChrisSomervilleatMichiganStateUniversity,hejoinedthe
facultyofPurdueUniversityintheBiochemistryDepartment.
Chapplesresearchintheareaofligninbiosynthesisandplant
secondarymetabolism,usingArabidopsisasamodelsystem,has
earnedhimthetitleofPurdueUniversityScholarandafellow-
shipintheAmericanAcademyfortheAdvancementofScience.
ResearchbytheChapplegrouphelpedtochangethetraditional
paradigmoftheroleofferulicandsinapicacidsinbuildingplant
cellwalls.Ratherthancontributingtotheproductionoflignin,
thegroupfoundthatthesetwoacidsserveasendproductsinan
essentialbiochemicalpathwayforcell-wallconstruction.
Tephenylpropanoidpathwaygivesrisetoawidearrayofsolu-
blemetabolitesinplants.Tesecompoundsparticipateinmany
plantdefenseresponsesandabsorbpotentiallydamagingUVB
radiation.Tepathwayalsogeneratesthemonomersrequired
forligninbiosynthesisferulicacidandsinapicacid.Ligninis
integratedintotheplantsecondarycellwall,whereitprovides
structuralrigiditytoplanttissuesandenablestrachearyelements
towithstandthetensiongeneratedduringtranspiration.
Chapplereceivedthe2001AgriculturalResearcherAwardfrom
thePurdueSchoolofAgricultureforhispatentedworkinengi-
neeringplantstostoreandstabilizeplasticmonomerprecursors
invacuoles.
HelenaL.Chum
Trainedinphysicalandindustrialchemistry,HelenaChum
hasworkedinbioenergyandrenewableenergysince1979at
theSolarEnergyResearchInstitute,nowNationalRenew-
ableEnergyLaboratory(NREL),andhasledR&Dbranches,
divisions,andcentersatNRELsince1992.Hergeneralresearch
involvestechnologydevelopmentforconversionofbiomassand
avarietyoforganicwastesintobiofuels,chemicals,electricity,
andhigh-valuematerials.Shehascoauthoredabook,85peer-
reviewedpublications,and150meetingpapers.Shehaspresented
100invitedlecturesworldwideandjointlyholds18patents.
Herspecicresearchareasarebiomasschemicalanalyses,
standardsdevelopment,andrapidspectrometricanalysismeth-
odologies;biomassandurbanandplasticresidueconversionto
chemicalsandbiofuels;thermochemicalconversiontomultiple
products;biomassfractionation;electrochemistryappliedto
biomassandderivedcompounds;environmentaltechnologies;
thermallyregenerativeelectrochemicalsystemsandfuelcells;
technologydevelopmentandgovernment-industry-academia
partnershipsinR&D;andanalysesofU.S.governmental
biomassandhydrogenprogramsandtheirimpactoncommer-
cialtoolsandsystemstosupportrecommendationsforfuture
programs.
ChumisafellowoftheAmericanAssociationfortheAdvance-
mentofScienceforintegratingindustry-academia-govern-
mentresearchpartnershipsinbiomassandbiobasedmaterials
andalsooftheInternationalAcademyofWoodSciencefor
demonstratedleadershipofbiomassanalysisandstandards
activitiesworldwide.Shereceivedacerticateofappreciation
fromtheU.S.DOEAssistantSecretaryoftheOceofEnergy
EciencyandRenewableEnergy(EERE)forcontributions
andleadershipindepartmental,presidential,andcongressional
environmentalinitiatives,includingtheNationalEnvironmental
TechnologyStrategy,andfordedicationtoEEREprograms
(1995).
MikeCleary
JosephMichaelClearyisExecutiveDivisionDirectorofSci-
encesR&DattheSanDiegoSupercomputerCenter(SDSC),
UniversityofCalifornia,SanDiego.Hereceivedhisbachelors
degreeinbiologyfromStanfordUniversityin1970andhisdoc-
torateinmolecularbiologyfromtheUniversityofCalifornia,
LosAngeles,in1980.AtSDSC,hedirectsgroupsthatsupport
thecyberinfrastructureneedsofresearchersbyproducingdata
systemsandcomputationaltoolstofacilitatediscoveriesinthe
naturalsciences.Hisresponsibilitiesincludetheinitiationof
interdisciplinaryresearchprojectsforlifescienceprogramswith
biomedicalandbiologyresearchersatuniversity,government,
andindependentresearchinstitutions.
BeforejoiningSDSCin2003,Clearyheldbiotechnology
researchandmanagementpositionsforover20yearsatMerck
andMonsanto,whereheworkedinfermentationandmicrobial-
straindevelopment,withemphasisonbiosyntheticpathwaysfor
producingcommerciallyvaluablebacterialpolysaccharides.He
isanadjunctprofessorofbiologyatSanDiegoStateUniversity
andservesasaconsultanttoseveralbiotechnologybusinesseson
mattersrelatedtoindustrialmicrobiology.
FrankCollart
FrankCollartismanageroftheRoboticMolecularBiol-
ogyFacilityintheBiosciencesDivisionatArgonneNational
LaboratoryandCloningandExpressiongroupleaderofthe
MidwestCenterforStructuralGenomics(MCSG).Hereceived
hisdoctorateinmedicalsciencesfromtheMedicalCollegeof
OhioandmastersdegreeinchemistryfromBowlingGreen
StateUniversity.Hehasusedculturedcellmodelstodelineate
criticalsignaltransductioneventsinvolvedindierentiationof
hematopoietic,melanoma,andbreastcelllineagesandhasover
50scienticpublicationsand4patents.
194 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

























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 oneiden-
sis and Geobacter sulfurreducens. Tese organisms have potential
for degrading organic pollutants and bioremediating metals. Te
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 auto-
mated protocols for high-throughput generation and analysis of
bacterial expression clones.
Steve Colson
Steve Colson received his bachelors 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 Pacic 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 spec-
trometric methods to address fundamental problems in physi-
cal and analytical chemistry. General research interests include
high-sensitivity spectroscopy and microscopy, photochemistry,
photophysics, molecular dynamics, electronic structures of mole-
cules, 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 depart-
ments and industry. Te power and excitement of interdisciplin-
ary, 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.
Michael Cotta
Michael Cotta is research leader for the Fermentation Biotech-
nology 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 bachelors and masters 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 sci-
ence in 1985 and joined USDA ARS as a research microbiologist
in October 1984. His research interests include microbial ecology
of gastrointestinal environments and animal wastehandling
systems, ecophysiology of ruminal microorganisms, and micro-
organism interactions in the bioconversion of complex polysac-
charides.
Bruce E. Dale
Bruce Dale is professor of chemical engineering and former
chair of the Department of Chemical Engineering and Materi-
als Science at Michigan State University (MSU). He earned his
bachelors degree (summa cum laude) in chemical engineering
from the University of Arizona, Tucson, in 1974 and masters
degree from the same university in 1976. He then studied under
George T. Tsao at Purdue University, receiving his doctorate
in 1979. His rst academic position was in the Department of
Agricultural and Chemical Engineering at Colorado State Uni-
versity, where he rose to professor in 1988. In that same year, he
joined Texas A&M University, where he was professor of chemi-
cal 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 appoint-
ment 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. Specically, he is
interested in the environmentally sustainable conversion of plant
matter to industrial productsfuels, 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 Hydro-
carbon Age is winding down during the current century. His
concern with sustainable resources was inuenced by growing
up in the copper-mining town of Ruth in eastern Nevadaa
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 Commercializa-
tion 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.
Biofuels Joint Roadmap, June 2006 Ofce of Science and Ofce of Energy Efciency and Renewable Energy U.S. Department of Energy 195

APPENDICES
BrianH.Davison
BrianH.Davisonischiefscientistforsystemsbiologyand
biotechnologyatOakRidgeNationalLaboratory(ORNL),
wherefor2yearshewasdirectoroftheLifeSciencesDivision.
Hepreviouslywasadistinguishedresearcherandleaderofthe
BiochemicalEngineeringResearchGroup.Inhis20yearsat
ORNL,hehasconductedbiotechnologyresearchinavarietyof
areas,includingbioconversionofrenewableresources(ethanol,
organicacids,andsolvents);nonaqueousbiocatalysis;systems
analysisofmicrobes(cultivationandproteomics);bioltration
ofvolatileorganiccompounds;mixedcultures;immobilization
ofmicrobesandenzymes;metalbiosorption;andextractive
fermentations.Hisresearchhasresultedinmorethan80publi-
cationsand6patents.
Davisonreceivedhisdoctorateinchemicalengineeringfromthe
CaliforniaInstituteofTechnologyandhisbachelorsinchemical
engineeringfromtheUniversityofRochester.Hechairedthe
15
th
to26
th
SymposiaonBiotechnologyforFuelsandChemicals
andservedasproceedingseditorinAppliedBiochemistryand
Biotechnologyfrom1994to2005. Tesymposiumgrewfrom150
to400attendeesduringhis12yearsaschair.Davisonreceived
anR&D100Awardin1977forProductionofChemicals
fromBiologicallyDerivedSuccinicAcid.Healsoisanadjunct
professorofchemicalengineeringattheUniversityofTennes-
see,Knoxville.
BillDean
BillDeanreceivedhisdoctorateinbiochemistryfromSyracuse
University.Heisvicepresidentofdevelopmentandprocess
sciencesatDaniscoGenencorInternational,Inc.;previously,he
heldthepositionsofvicepresidentoftechnologyprogramsand
vicepresidentofmanufacturingdevelopment.Beforejoining
Genencor,hewasintheResearchDivisionofCorningGlass
Works,whereheworkedonenzymeimmobilizationtechniques
andbioreactordesignandwasresponsibleforoverseeingthe
enzymesubcontractwiththeNationalRenewableEnergyLab-
oratorytodevelopcost-eectivebiomasscellulases.Currently,he
isresponsibleforgrain-processingtechnologyatGenencor.
TimDonohue
TimDonohueisprofessorofbacteriologyattheUniversityof
Wisconsin,Madison.HeearnedhisdoctoratefromPennsylva-
niaStateUniversityin1980.Hestudiesphotosyntheticbacteria
thatconvertsolarenergyintoalternativefuels(hydrogen)or
removegreenhousegasesandotherenvironmentalpollutants.
Hehasusedmoleculargenetic,biochemical,andsystemsbiol-
ogytechniquestostudyglobalsignal-transductionpathways,
alternativesigmafactors,andsignalsthatcontrolexpressionof
well-studiedcomponentsoftherespiratoryandphotosynthetic
electron-transportchains.
Donohuesmostrecentworkincludesidenticationofcellular
pathwaysusedbyphotosyntheticmicrobestosensethepres-
enceofsingletoxygenanddefendthemselvesfromthistoxic
substance.Tisknowledgemayleadtotheabilitytonetune
thedesignofmicrobialandplantphotosyntheticsystemsto
minimizetheharmfuleectsofsingletoxygenandtoenhance
energyproduction.Photosynthesisprovides>90%ofnetenergy
inputintothebiosphere.Terefore,light-drivenprocesseswithin
photosyntheticorganismshaveenormouscapacityforthepro-
ductionofsustainable,carbon-neutral,solar-poweredtechnolo-
giesthatreducetheglobaldependencyonfossilfuels.
Telong-rangegoalsofDonohuesprojectsaretoidentify
importantmetabolicandregulatoryactivities;obtainathorough
understandingofenergy-generatingpathwaysofagricultural,
environmentalandmedicalimportance;andgeneratecomputa-
tionalmodelstohelpdesignmicrobialmachineswithincreased
capacitytousesolarenergy,generaterenewablesourcesof
energy,removetoxiccompounds,orsynthesizebiodegradable
polymers.
TomFoust
TomFoustjoinedtheNationalRenewableEnergyLabora-
tory(NREL)in2004asdirectorofbiomassresearch.Hehas
adoctoratefromtheUniversityofIdaho,mastersfromJohns
HopkinsUniversity,andbachelorsfromPennsylvaniaState
University,allinmechanicalengineering.Healsoisalicensed
professionalengineer.
Inhiscurrentrole,heguidesanddirectsNRELsresearcheorts
todevelopbiomassconversiontechnologyviabothbioconver-
sionandthermoconversion.Tisresearchisfocusedondevelop-
ingthenecessaryscienceandtechnologyforconvertingbiomass
tobiofuelsinaneconomicalmannerandcoversthegamutof
fundamentaltoappliedscience.Hisparticularareaofexpertise
isincomplexowandchemical-reactionmodelingasitrelates
tobiomass-conversionprocessesandin-processseparations.
BeforejoiningNREL,Foustspent7yearswiththeIdaho
NationalLaboratorywherehewastheresearchleadforthe
biomassfeedstocksprogram.Hisprimaryareaofresearchwas
incomplexmultiphaseowanalysisasitrelatedtophysical
fractionationofbiomass.Hehasover20yearsofexperience
inresearchandresearchmanagement,specializinginbiomass
feedstocksandconversionresearch.Hehaswrittenmorethan
15peer-reviewedpublicationsrelatedtobiomassfractionation
andtechnology-developmentissues.
JimFredrickson
JimFredricksonobtainedabachelorsinsoilsciencefromthe
UniversityofWisconsin,StevensPoint,andadvanceddegrees
insoilchemistryandsoilmicrobiologyfromWashingtonState
University.HeisachiefscientistwithintheBiologicalSciences
DivisionatPacicNorthwestNationalLaboratory,specializing
196 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix C. Participant Biosketches

inmicrobialecologyandenvironmentalmicrobiology.Withhis
researchfocusedonsubsurfacemicrobiologyandbiogeochem-
istry,hehasbeenresponsibleforlaboratoryandeldresearch
programsinvestigatingthemicrobialecologyandbiogeochem-
istryofgeologicallydiversesubsurfaceenvironmentsandis
recognizednationallyandinternationallyforthesestudies.
Fredricksonalsohasservedassubprogramcoordinatorfor
DOEsSubsurfaceScienceProgramfrom1991topresent.In
thisrole,hecoordinatedthetechnicalaspectsofDOEsDeep
SubsurfaceMicrobiologySubprogramatthenationalleveland
assistedtheprogrammanagerinsettingprogrammaticresearch
directions.Tissubprograminvolvedmorethan15projectsat
universitiesandnationallaboratoriesnationwideandfocusedon
multidisciplinaryeld-scaleresearch.AttherequestofDOE,
hecurrentlyisnationalcoordinatorfortheShewanellaMicro-
bialCellProject,partoftheGenomics:GTLprogram.Hewas
appointedchiefscientistin2005toserveasspokespersontothe
sciencecommunityfortheGTLprogramandfacilities.
RamonGonzalez
RamonGonzalezistheWilliamW.AkersProfessorinthe
DepartmentofChemicalandBiomolecularEngineeringat
RiceUniversity.Heholdsdegreesinchemicalengineering
fromtheUniversityofChileandtheCentralUniversityofLas
Villas,Cuba,andalsoisalicensedprofessionalengineer.His
researchaddressessuchchallengingissuesinmicrobialcataly-
sisasunderstandingandmanipulatingvitaminsandcofactor
biosynthesis,anaerobicfermentationofnontraditionalcarbon
sources,simultaneousmetabolismofsugarsinsugarmixtures,
andunderstandingandmodifyingrespiratoryandfermenta-
tivesystemsforsynthesisofreducedproducts.Specicresearch
areasincludemetabolicengineeringandinverse(metabolic)
engineering,functionalgenomicsandsystemsbiology,microbial
fermentation,molecularmodeling,andhigh-performanceliquid
chromatographyoptimization.
Gonzalezusesawidespectrumofapproachesandstate-of-the-
arttechniquestypicallyviewedundersuchdierentscientic
andengineeringdisciplinesasmolecularbiology,biochemistry,
andchemicalengineering.Hecurrentlyisusingtranscriptomics
andproteomictoolsinconjunctionwithuxomictoolstoeluci-
datebiologicalfunctionofindividualgenesatcellularlevels.He
advocatesthesystemicmethodforitsintegrationofmathemati-
calandcomputationaltoolsandisusingthisglobalandintegra-
tiveapproachtounderstandcomplexmetabolicandregulatory
networksinbacterialsystems,thebasisforunderstanding
similarprocessesinmorecomplexorganisms.Teultimategoal
ofhisresearchisthedesignofspecicgenotypesbasedonthe
desiredphenotype.
BonnieHames
BonnieHamesleadsthebiomasschemicalcharacterization
teamswithintheNationalBioenergyCenterattheNational
RenewableEnergyLaboratory(NREL).Sheearnedabachelors
degreeinchemistryfromRegisUniversityandadoctoratein
organicchemistryfromtheUniversityofDenver. Workingin
thegroupofBernardMonties,shealsocompletedapostdoc-
toralassignmentinligninchemistryattheCentreNationalde
laRechercheScientique,InstituteNationaldelaRecherche
Agronomique,LaboratoiredeChimieBiologique,Tiverval-
Grignon-Paris,France.
Herextensiveexperienceinbiomasschemistryincludesmore
than18yearsofdevelopingstandardwetchemicalmethodsfor
characterizingbiomassfeedstockandbiomass-derivedmateri-
als,preparingstandardreferencematerialsforqualityassurance
andqualitycontrol,andapplyingstandardmethodstofueland
chemicalproductionfrombiomass.Shecurrentlyleadsthe
NRELBiomassProgramtodevelopnew,rapid,andinexpensive
methodsforbiomasscompositionalanalysis.Tesemethods
includeadvancedtoolsforchemicalcharacterizationofbiomass
feedstocksandbiomass-derivedmaterialsbasedoninfrared
spectroscopyandadvancedmultivariateanalysistechniquesrst
developedintheAgenda2020programsponsoredjointlyby
DOEOceofIndustrialTechnologiesandtheforestproducts
industry.In2000,herprogramsreal-timebiomassanalysiswon
anR&D100awardandwashonoredbyDOEasaBestof
Agenda2020Project.
Hamesalsodevelopedpatentedtechniquesforbiomassfrac-
tionationandligninisolation.Shehasextensiveexperiencein
ligninchemistryincludingstructuralcharacterization,synthesis
ofligninmodelcompounds,anddevelopmentofmethodsfor
theselectiveoxidationofligninusingorganmetalliccatalysts
andbiomimeticsystems.Sheholds3U.S.patents,hasauthored
3bookchaptersandmorethan25papersinpeer-reviewed
journals,andhasmademorethan80presentationsattechnical
meetings.ShecurrentlychairstheAmericanSocietyforTesting
andMaterialscommitteeE48onstandardsforbiotechnology
andsubcommitteeE48.05onstandardsofbiomassconversion.
MariaHarrison
MariaHarrisonearnedherbachelorsdegreewithhonorsin
microbiologyfromtheUniversityofNewcastleUponTyne,
England,andherdoctoratein1987fromtheInstituteofSci-
enceandTechnology,UniversityofManchester.Sheconducted
postdoctoralresearchattheSamuelRobertsNobleFoundation
inArdmore,Oklahoma,underthedirectionofR.A.Dixon.She
hasservedasanadjunctprofessoratOklahomaStateUniversity
andTexasA&MUniversity;in2003,shejoinedthestaatthe
BoyceTompsonInstituteforPlantResearch,withanadjunct
appointmentintheDepartmentofPlantPathologyatCornell
University.
Mostvascularoweringplantsareabletoformsymbioticasso-
ciationswitharbuscularmycorrhizal(AM)fungi.Teseassocia-
tionsdevelopintheroots,wherethefunguscolonizescortical
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 197

APPENDICES
cellstoaccesscarbonsuppliedbytheplant.Tefungalcontribu-
tiontosymbiosisincludestransferofmineralnutrients,par-
ticularlyphosphorus,fromthesoiltotheplant.Inmanysoils,
phosphatelevelsarelimitingtoplantgrowth.Consequently,
additionalphosphatesuppliedviaAMfungicanhaveasigni-
cantimpactonplantdevelopment,andthissymbiosisinuences
thestructureofplantcommunitiesinecosystemsworldwide.
Telong-termgoalsofHarrisonsresearcharetounderstand
themechanismsunderlyingdevelopmentofAMsymbiosis
andphosphatetransferamongsymbionts.Sheusesamodel
legumeMedicagotruncatulaandAMfungiGlomusversiforme,G.
intraradices,andGigasporagiganteafortheseanalyses.Currently,
acombinationofmolecular,cellbiology,genetic,andgenomic
approachesisbeingusedtoobtaininsightsintosymbiosisdevel-
opment,communicationamongplantandfungalsymbionts,and
symbioticphosphatetransport.
SusanHennessey
Inalmost20yearsatDuPont,SusanHennesseyhasapplied
herexpertiseasachemicalengineertoanumberofimproved
chemicalproductionprocesses.Herteamofscientistshasused
immobilizedbacterialcellsandotherencapsulationtechniques
toincreaseenzymedurabilityandconcentrationforcontinu-
ousproduction.Herworkontheintroductionoftheherbicide
Milestoneforcropprotectionearnedherteamthe2002Indus-
trialInnovationAwardfromtheMid-AtlanticRegionofthe
AmericanChemicalSociety.Otherworkcombiningthesynergy
ofbiologywithnewchemicalsynthesistechnologiescontinues
toresultinnewhigh-valueproductsusingenvironmentally
preferredprocesses.
MikeHimmel
Duringhis23-yeartermatDOEsNationalRenewableEnergy
Laboratory(NREL,formerlySERI)inGolden,Colorado,Mike
HimmelhasworkedtosupportmanytechnicalaspectsofDOEs
BiomassProgram.Morerecently,hehasbeenresponsiblefor
establishingthefacilitiesandstanecessarytopursueprojectsin
proteinengineering,specicallycellulases.Today,hemanagesthe
award-winningEnzymeTechnologyTeamandthemajorindus-
trialandacademicsubcontractsthatsupportthiswork.Teteam
hasassembledworld-classproteinpuricationandcharacteriza-
tionfacilitiesatNREL,withspecialemphasisonroboticsystems
forscreeninglibrariesderivedfromdirectedevolutiontechnol-
ogy.Himmelhascontributed300peer-reviewedpapersand
meetingabstracts,4books,and16patentstotheliterature.He
alsochairedorcochaired15internationalmeetingsintheeld
ofbiochemistryandbiotechnology,includingthe2003Gordon
ResearchConferenceonCellulasesandCellulosomes.
MauriceHladik
MauriceHladikisdirectorofmarketingforIogenCorporation,
whichspecializesindeveloping,manufacturing,andmarketing
enzymestomodifyandimprovetheprocessingofnaturalbers
withinthetextile,animal-feed,andpulpandpaperindustries.
OneofIogensmajoractivitiesisresearchoncellulosicethanol.
Hladikhasextensiveinternationalbusinessexperience,particu-
larlyintheUnitedStates,Germany,andtheUnitedKingdom
aswellasseveralAsiancountriesincludingChina(alsoHong
Kong),SouthKorea,andTailand.Aparticularstrengthishis
abilitytolocatebusinesscontactsandcommercialintelligencein
aforeignsetting.
BeforejoiningIogen,from1978to1998heservedintheCana-
dianForeignService.HisassignmentsincludedseniorCanadian
tradeocerpostedtoBangkok(3years),HongKong(3years),
Beijing(1year),Seoul(2years),andMunichasconsulgeneral
(3years);anddirectorgeneraloftheGrainMarketingBureauat
AgricultureCanada.Inthisposition,Hladikwaschiefadvisor
totheCanadiangovernmentoninternationalgrainpolicyand
marketingissues.HealsoservedtheCanadianInternational
DevelopmentAgencyasdirectorforAsia,IndustrialCoopera-
tionDivision.
LonnieIngram
LonnieIngramisadistinguishedprofessorofmicrobiology
anddirectoroftheFloridaCenterforRenewableChemicals
andFuelsattheUniversityofFlorida.ElectedtotheNational
AcademyofSciencesin2001,Ingramandhiscoworkerswere
therstintheworldtodevelopgeneticallyengineeredE.coli
bacteriacapableofconvertingallsugartypesfoundinplantcell
wallsintofuelethanol.Ingramsorganismproducesahighyield
ofethanolfromsuchbiomassassugarcaneresidues,ricehulls,
forestryandwoodwastes,andotherorganicmaterials.
Ingramsbreakthroughbioconversiontechnologywasselectedto
becomeLandmarkPatentNo.5,000,000bytheU.S.Department
ofCommerce.Morethan30additionalpatentsarependingor
havebeenissuedforthistechnology,whichisbeingcommercial-
izedwithassistancefromtheU.S.DepartmentofEnergy.BC
InternationalCorp.,basedinDedham,Mass.,holdsexclusive
rightstouseandlicensetheengineeredbacteria,dubbedK011
byIngram.In1993,IngramreceivedaU.S.Departmentof
AgricultureDistinguishedServiceAwardforhisbreakthrough
research.Teagencyshighesthonor,theawardrecognizedhis
outstandingcontributionstoresearchandtheconsumer.
JayKeasling
JayKeaslingearnedabachelorsdegreeinchemistryandbiol-
ogyin1986fromtheUniversityofNebraska,Lincoln,and
adoctorateinchemicalengineeringfromtheUniversityof
Michiganin1991.Heisprofessorofchemicalengineeringand
bioengineeringattheUniversityofCalifornia,Berkeley.Healso
servesasdirectorofthePhysicalBiosciencesDivisionandheads
thenewSyntheticBiologyDepartmentatLawrenceBerkeley
NationalLaboratory.Teideabehindthisdepartmentisto
designandconstructnovelorganismsandbiologicallyinspired
198 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix C. Participant Biosketches

systemstosolveproblemsunsolvablebynaturalbiological culturalandBiologicalEngineeringatPurdueUniversity,with
systemsandalsoprovidenewinformationaboutlivingcells. ajointappointmentinbiomedicalengineeringandacourtesy
KeaslingsmanyhonorsincludeelectionasfellowoftheAmeri- appointmentinfoodscience.In1973heearnedhisbachelors
canInstituteofMedicalandBiologicalEngineeringandrecipi- degreefromDrexelUniversityandin1974and1977,respec-
entoftheAIChEAwardforChemicalEngineeringExcellence tively,hismastersanddoctoratefromPurdueUniversity,allin
inAcademicTeaching.Healsoisfounderoftwocompanies, chemicalengineering.
AmyrisBiotechnologiesandCodonDevices,whichhavegrown
Hehasabroadbackgroundinbioscienceandbioengineer-
fromdiscoveriesathislaboratory.
ingandhasauthoredatextbook,BioseparationsEngineering:
KeaslingandcollaboratorswereawardedaGatesFoundation Principles,PracticeandEconomics(Wiley,2001)and150journal
grantthatwillseektocreateinthelaboratoryaninexpensive andproceedingspapers.Hehas14patents(issuedandapplied
antimalarialdrug,artemisinin.Tisdrugcouldbesoldforone- for)andhaspresentedover100papers.HereceivedtheMarvin
tenthoftodayspriceandcompetewiththeformerlyfront-line J.JohnsonAwardinBiochemicalTechnologyoftheAmerican
antimalarialnowconfrontedbydisease-resistantstrainsaround ChemicalSocietyin2002andtheFood,Pharmaceutical,and
theworld.KeaslingandhisteamatBerkeleyalreadyhave BioengineeringDivisionAwardoftheAmericanInstituteof
workedoutmethodsforextractingthegenesresponsiblefor ChemicalEngineersin2001.HewaselectedtotheNational
makingartemisininandhavetransplantedthemintoaharmless AcademyofEngineeringin1999.
strainofE.coli.
LadischservedasamemberofU.S.delegationsandadvisory
ResearchintheKeaslinglaboratoryfocusesonthemetabolic panelstoRussia,Tailand,China,andJapantoreviewthestatus
engineeringofmicroorganismsfordegradationofenvironmen- ofbiotechnologyprograms.In1991and1992,hechairedthe
talcontaminantsorforenvironmentallyfriendlysynthesis.To NationalResearchCouncilsCommitteeonBioprocessEngi-
thatend,hehasdevelopedanumberofnewgeneticandmath- neering,whichstudiedresearchprioritiesandpolicyissues
ematicaltoolstoallowmorepreciseandreproduciblecontrolof relatedtocommercializationofbiotechnologyandpublished
metabolism.Tesetoolsarebeingusedinsuchapplicationsas thereportPuttingBiotechnologytoWork:BioprocessEngineering
synthesisofterpenedrugsandbiodegradablepolymers,accumu- (NationalAcademyPress,1992).
lationofphosphateandheavymetals,degradationofchlorinated
Hisresearchaddressesfundamentaltopicsinbioprocessengi-
andaromatichydrocarbons,biodesulfurizationoffossilfuels,
neeringastheyapplytobioenergy,bioproducts,biorecovery,and
andcompletemineralizationoforganophosphatenerveagents
bionanotechnology.Teresearchaddressestransformationof
andpesticides.Genomics,proteomics,andmetabolomicsare
renewableresourcesintobioproducts,propertiesofproteinsand
beingemployedtoinvestigateeectsofthesechangesoncellular
livingorganismsatsurfaces,rapidprototypingofmicrouidic
physiologyandtooptimizecellularredesign.
biosensors,andbioseparations.Tisworkhasresultedinnew
KenKeegstra
industrialbioenergyprocessesandsystematicapproachesand
correlationsforscaleupoflaboratorychromatographicpuri-
KenKeegstraisdirectorofthePlantResearchLaboratoryand
cationtechniquestoprocess-scalemanufacturingsystems;it
aUniversityDistinguishedProfessorinthedepartmentsof
alsohasresultedinscaledownofbioseparationsandtherapid
BiochemistryandPlantBiologyatMichiganStateUniversity
prototypingofmicrouidicbiochipsforquickdetectionof
(MSU).HereceivedhisdoctorateinchemistryattheUniver-
pathogenicmicroorganisms.Ladischteachesbioseparations,
sityofColorado,whereheinvestigatedthestructureofplant
bioprocessengineering,andbiotechnologyatboththegraduate
cell-wallcomponentsandtheirinteractionswithinthewall.For
andundergraduatelevels.
morethan20yearshestudiedotherbiologicalproblems,mainly
chloroplastbiogenesisandthetargetingofnuclear-encodedpro-
LeeLynd
teinintochloroplasts.AtthetimeofhismovetoMSUin1993,
LeeRybeckLyndisaprofessorofengineeringandadjunct
hereinitiatedworkonplantcellwalls.Temajorfocusofhis
professorofbiologicalsciencesatDartmouthCollegeanda
currentresearchisthebiosynthesisofplantcell-wallpolysaccha-
professorextraordinaryofmicrobiologyattheUniversityof
ridesproducedintheGolgibeforedeliverytothecellwall.Te
StellenboschinSouthAfrica.Hereceivedabachelorsdegreein
greatestamountofeorthascenteredonxyloglucanbiosynthe-
biologyfromBatesCollege;mastersinbacteriologyfromthe
sis,buthisresearchgroupalsohasinvestigatedthebiosynthesis
UniversityofWisconsin,Madison;andmastersanddoctorate
ofmannans,glucomannans,andarabinoxylansaswellasafew
fromtheTayerSchoolofEngineering.
otherwallcomponents.
Lyndleadsaresearchgroupinbiochemicalengineeringand
MichaelLadisch
appliedbiologyrelevanttoprocessingcellulosicbiomass.His
MichaelLadischisdirectoroftheLaboratoryofRenewable
laboratorysresearchtopicsarechosentoaddressaprimary
ResourcesEngineeringandDistinguishedProfessorofAgri-
technicalimpedimenttorealizingacarbohydrateeconomy:
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 199

APPENDICES
Overcomingtherecalcitranceofcellulosicmaterialstobiological
conversion.Specictopicsinclude,inorderofincreasingscale,
metabolicengineeringtoimproveproductyieldsinthermophilic
bacteria,microbialphysiologyofanaerobiccellulolyticmicroor-
ganisms,kineticsandreactordesignforenzymaticandmicrobial
hydrolysisofcellulosicmaterials,andconversionofreal-world
cellulosicmaterialssuchaswastesludgeproducedfrompaper
mills.AparticularfocusoftheLyndgroupisconsolidatedbio-
processing,awidelyapplicablepotentialbreakthroughinwhich
productionofcellulaseenzymes,hydrolysisofbiomassber,and
fermentationofresultingsugarsareaccomplishedinoneprocess
stepbyasinglemicrobialcommunity.
LyndisarecipientoftheNationalScienceFoundationsPresi-
dentialYoungInvestigatorAwardandatwo-timerecipientof
theCharlesA.LindberghAwardforhiseortstopromotebal-
ancebetweentechnologicalprogressandpreservationofnatural
andhumanenvironments.Professionalactivitiesincludeservice
asassociateeditorforBiotechnologyandBioengineering,member
ofapresidentialadvisorycommitteeonreducinggreenhouse
gasemissionsfrompersonalvehicles,andorganizingcommittee
memberfortheAnnualSymposiumonBiotechnologyforFuels
andChemicals.Lyndhasauthoredmorethan60peer-reviewed
manuscriptsandholds5patents.
GeorgeMichaels
InMay2004,GeorgeMichaelswasnamedassociatelaboratory
directorofthenewlyformedComputationalandInformation
SciencesDirectorateatPacicNorthwestNationalLaboratory
(PNNL).Tisnewdirectoratedeliversinnovativesolutionsto
addressnationalandglobalproblemsbyenablinglarge-scale
scienticdiscoveriesthroughR&Dinscience-drivencomput-
ing.Heholdsadoctorateinbiochemistryandmolecularbiology
andabachelorsdegree(1974)inmicrobiology,allfromthe
UniversityofFlorida.
HejoinedPNNLinApril2003asdirectorofbioinformatics
fortheBiomolecularSystemsInitiative.Heisaninternationally
recognizedpioneerinbioinformaticsandinthepracticaldevel-
opmentofbiotechnologicalapproachesfordiscovery.During
hiscareerspanningnearly30years,hehasprovidedincreasingly
signicanttechnicalandleadershipcontributionstohiseld.He
holdspatentsinmethodsfordesigningDNA-bindingproteins
andformorphologicalreconstruction.
Mostrecently,MichaelsheldleadershippositionsatMonsanto
inSt.Louis,Missouri,wherehedesignedanintegratedexpres-
sion-prolingprogram.Healsocofoundedandservedasvice-
presidentandchiefscientistofGenomeDynamics,aMaryland
biotechnologystartupcompany.Whileanassociateprofessor
atGeorgeMasonUniversityinFairfax,Virginia,heinitiated
oneofthenationsrstdoctoralprogramsinbioinformaticsand
computationalbiology.Healsohasservedasaspecialexpertto
theoceofthedirectoroftheNationalInstitutesofHealth.
ColinMitchinson
ColinMitchinsonearnedhisbachelorsdegreeinbiochemistry
fromtheUniversityofEdinburghanddoctorateinbiochemistry
fromtheUniversityofNewcastle.Hehasextensiveresearch
experienceinthestudyofstructure-functionrelationshipsin
proteinsrangingfrom(Ca++/Mg+)ATPaseofmusclesarco-
plasmicreticulum,ribonuclease,andsubtilisin.Healsohasper-
formedproteinengineeringonstarch-processingenzymesand
cellulasesandwasprojectleaderandprincipalinvestigatorfora
multidisciplinaryeorttodevelopanewcellulase.Hecurrently
servesasseniorstascientistatDaniscoGenencor,wherehis
researchfocusesondevelopmentofnewcellulaseproductsfor
biomassconversion.Hispublicationsincludereviewsonprotein
folding,substratebinding,andactivesitecharacterizationusing
moleculargenetic,biochemical,andbiophysicaltechniques.A
representativerecentpublicationisM.Sandgren,J.Stahlberg,
andC.Mitchinson,StructuralandBiochemicalStudiesofGH
Family12Cellulases:ImprovedTermalStability,andLigand
Complexes,ProgressinBiophysicsandMolecularBiology89,
24691(2005).
MarkMorrison
MarkMorrisonisaprofessorintheDepartmentofAnimal
ScienceatOhioStateUniversityandalsoholdsanonsalaried
appointmentintheDepartmentofMicrobiology.Hehasalong-
standinginterestingastrointestinalmicrobiologyandbacterial
physiology.Muchofhisresearchhasfocusedontheecophysiol-
ogyofplantbiomassdegradationinherbivoresandthemolecu-
larbiologyunderpinningcellulosedegradationandbacterial
adhesiontoplantstructuralpolysaccharides.Heservesasproject
leaderfortheNorthAmericanConsortiumforGenomicsof
FibrolyticBacteria,involvingscientistsfromTeInstitutefor
GenomicResearch,Cornell,UniversityofIllinois,andUniver-
sityofGuelph.Teconsortiumsactivitiesincludesequencingof
fourrumenbacterialgenomes(Fibrobactersuccinogenes,Prevotella
bryantii,Prevotellaruminicola,andRuminococcusalbus)aswellas
comparativeandfunctionalgenomicstudieswiththesebacteria.
ArtRagauskas
ArtRagauskasreceivedhisdoctorateinchemistryfromtheUni-
versityofWesternOntarioin1986,withsubsequentpostdoc-
toralresearchattheUniversityofAlbertaandColoradoState
University.HeisafellowoftheInternationalAcademyofWood
ScienceandtheTechnicalAssociationofthePulpandPaper
Industry(knownasTAPPI).HisresearchprogramatGeorgia
InstituteofTechnologyisseekingtounderstandandexploit
innovativesustainablelignocellulosicmaterialsanddevelop
newandimprovedapplicationsfornaturespremiererenewable
biopolymersincludingcellulose,hemicellulose,andlignin.
Ragauskassresearchisdirectedtowardinnovativeprocesses
forconvertinglignocellulosicbiomassintoinnovativebioma-
terialsandbiofuels.Achievingthisgoalrequiresresearchin
200 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix C. Participant Biosketches
severaleldsofstudy,includinglignocellulosicberchemistry
andphysicalproperties;carbohydrate,lignin,andextractive
chemistry;nanobiomaterials;biotechnology;andmaterialand
polymerscience.Tesestudiesaresupportedbyexpertisein
advancedspectroscopy,imaging,nanotechnology,chemoenzy-
maticbiotechnology,coldplasma,composites,bleaching,and
pulpingtechnologies.Hisresearchissponsoredbyaconsortium
ofindustrialpartners,DefenseAdvancedResearchProjects
Agency,NationalScienceFoundation,U.S.Departmentof
Agriculture,DepartmentofEnergy,andGeorgiaTraditional
IndustriesProgram(TIP3).
RagauskushasbeenaLuso-AmericanFoundationTeaching
FellowattheUniversidadedaBeiraInterior,Portugal;aninvited
guestteachingprofessoratChalmersUniversityofTechnol-
ogy,SwedenandSouthChinaUniversityofTechnology;and
aninvitedresearchprofessoratRoyalInstituteofTechnology,
Stockholm.Hehaspublished185papers,patents,andconfer-
enceproceedings.HeisanassociateeditorfortheJournalof
PulpandPaperScience,Holzforschung,andtheJournalofChem-
istryandTechnologyandhasservedonseveraladvisoryboards
andreviewpanels.
JohnRalph
JohnRalphreceivedhisbachelorsdegreewithhonorsinchemis-
tryin1976fromtheUniversityofCanterbury, NewZealand, and
hisdoctorateinchemistryandforestryin1985fromtheUniver-
sityofWisconsin,Madison(UWM).Heisaresearchchemist
attheU.S.DairyForageResearchCenterandaprofessorinthe
UWMDepartmentofForestEcologyandManagement.
Anorganicchemistandnuclearmagneticresonance(NMR)
spectroscopistspecializingincell-wallmodelcompound
synthesesandmonolignolpolymerizationreactions,Ralphis
involvedprincipallyinstudiesaimedatdetailingthemecha-
nismsoflignin-polysaccharidecross-linkingandtheireecton
limitingcell-walldegradability.HedevelopedNMRmethods
forcell-wallstructuralanalysis,includinganNMRdatabaseof
modelcompoundsforligninandrelatedwallcomponents,as
wellasmethodsforanalyzingligninstructureandnondegrada-
tivelysolubilizingtheentirecell-wallfactionofnelydivided
plantcellwalls.Hehasadditionalexpertiseinsyntheticorganic
chemistry,specicallyinthesynthesisofcell-wallmodelcom-
pounds,ligninoligomers,enzymeprecursors,andproducts.
SimoSarkanen
SimoSarkanenisaprofessorinligninchemistryandbiochem-
istryattheDepartmentofBiobasedProducts,Universityof
Minnesota.HereceivedhisundergraduatetrainingatKings
College,Cambridge(England),andwasawardedadoctorate
inchemistryfromtheUniversityofWashington,Seattle.His
rstpublicationswereintheoretical(computational)chemis-
try,buthisdoctoraldissertationwasinbioorganicchemistry
(enzymekinetics).AtthepostdoctorallevelintheDepartment
ofChemicalEngineeringattheUniversityofWashington,he
embarkedonajourneyintovariouscontroversialaspectsof
ligninchemistry.Currently,hisresearchinterestsrangefrom
ligninbiosynthesisandbiodegradationtonewformulationsfor
lignin-basedthermoplastics.
Mostligninchemistsandbiochemistshavethoughtthatcon-
gurationsofligninmacromoleculesarerandom(orcombi-
natorial),butSarkanensgroupistryingtodevelopanexplicit
workinghypothesisforreplicatingspecicligninprimary
structuresduringligninbiosynthesis.Terststepinligninbio-
degradationgenerallyhasbeenconsideredunderthecontrolof
ligninperoxidase,manganese-dependentperoxidase,orlaccase-
mediatorsystems.Sakanenandhiscoworkersactivelypromote
theviewthatacompletelydierentkindoflignindepolymerase
mayberesponsibleforcleavingligninmacromoleculesinvivo.
Finally,theyhaveproducedtherstseriesofthermoplasticswith
promisingmechanicalpropertiescomposedpredominantlyor
entirely(85to100%)ofsimpleligninderivatives.Previouswork
intheeldtypicallyhadencounteredincorporationlimitsof25
to40%forligninsinpotentiallyusefulpolymericmaterials.
ChristopheSchilling
ChristopheSchillingisacofounderofGenomatica,Inc.He
receivedhisdoctoraldegreeinbioengineeringunderBernhard
PalssonattheUniversityofCalifornia,SanDiego,wherehewas
aPowellFoundationandWhitakerFoundationFellow.Healso
holdsabachelorsdegreeinbomedicalengineeringfromDuke
University,wherehewasaHowardHughesUndergraduate
ResearchFellow.
AspresidentandchiefscienticocerofGenomatica,hecur-
rentlydirectsallthecompanysbusinessandscienticeorts
towardapplyingadvancedmodelingandsimulationtechnolo-
giestoanumberofmetabolism-drivenproducts.Tisincludes
overseeingdevelopmentofGenomaticasintegratedcomputa-
tionandexperimentalplatformtodrivethemetabolicengineer-
ingofmicrobestosupportnext-generationbioprocessesbeing
developedbythecompanysindustrialpartners.
Heiscoauthorofnumerousscienticarticlesinsystemsbiology
andmetabolicmodeling,andheisaninventoronanumberof
patentlingssurroundingGenomaticascoretechnologies.He
wasfeaturedintheFebruary2001editionofGenomeTechnology
asoneof16upandcomerswhohavethetalentanddriveto
makegreatstridesinscience,technology,andbusiness.In2003he
wasnamedoneofthetop100younginnovatorsunder35whose
innovativeworkintechnologywillhaveaprofoundimpactonthe
world,asselectedbyMITsTechnologyReviewmagazine.
JohnShanklin
JohnShanklinisaseniorbiochemistintheBiologyDepartment
atBrookhavenNationalLaboratoryandanadjunctprofessorin
theBiochemistryDepartmentattheStateUniversityofNew
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 201

APPENDICES
YorkatStonyBrook.Hereceivedhisbachelorsdegreeinphysiol-
ogyfromtheUniversityofLancaster,UnitedKingdom,in1981
andhismastersinforestryfromtheUniversityofWisconsin,
Madison(UWM),in1984.Hewasawardedadoctorateinhorti-
culturefromUMWin1988,workingontheubiquitinsystem.
Shanklinscurrentinterestsfocusonplantlipidbiochemistry
anddevelopingplantoilsasrenewableindustrialfeedstocks.
Specicallyhestudiesstructureandfunctioninlipidmodi-
cationenzymes.Tisclassofenzymesperformshigh-energy
chemistryonawidevarietyofsubstrateswithvariouschemical
outcomes.Inadditiontostudyinghowspecicfattyacidsare
synthesized,healsoconductsmetabolicengineeringexperiments
onhowmodulationsinpathwaycomponentsaectstorageoil
accumulationforbothusualandunusualfattyacids.
Shanklinisthewinnerofseveralawards,includingtheOce
ofEnergyResearchYoungScientistAward,aPresidentialEarly
CareerAward,andtheTerryGalliardMedalforPlantLipid
Biochemistry.HewaschairoftheScienticProgramCommittee
fortheNationalPlantLipidCooperativemeetingsfrom2001to
present.HehasservedonpanelsfortheDepartmentofEnergy
andtheNationalScienceFoundationandisontheScientic
AdvisoryBoardforGeneticEngineering,PrinciplesandMethods
andAdvancesinPlantBiochemistryandMolecularBiology.Hecur-
rentlyisamemberoftheCouncilforEnergyBiosciencesonthe
BasicEnergySciencesCommitteeofVisitors(2005).
SharonShoemaker
SharonShoemakerjoinedtheUniversityofCalifornia,Davis
(UCD),in1991asfounderandexecutivedirectoroftheCali-
forniaInstituteofFoodandAgriculturalResearch(knownas
CIFAR).ShealsoisresearchleaderattheUCForestProducts
LaboratoryandsitedirectoroftheNationalScienceFounda-
tionUCDCenterforAdvancedProcessingandPackaging.
Shoemakerholdsabachelorsdegreeinchemistry,masters
infoodscience,anddoctorateinbiochemistryandnutrition
fromVirginiaTech.Afterpostdoctoraltraininginbiochemical
engineering,shejoinedCetusCorporationinBerkeley,Cali-
fornia,therstU.S.biotechnologycompany.After7years,she
leftCetustojoinGenencor,acompanymorealignedwithher
interestsinapplyingfermentationandenzymologytoproblems
infoodandagriculture.
Shoemakersindustrialexperienceledtopatentsonnovelyeast
strainstoconvertbiomasstoethanolandonnovelbacterial
strainstoproducenewformsofcellulose.Shealsoledteam
eortstoimproveenzymesystemsforconvertingbiomassto
sugarsandsubsequentlyfermentingthemtochemicalsandto
characterizeanddevelopligninasesystemsforuseinthepulp
andpaperindustry.Herresearchinterestsfocusoncellulose
applicationsinbiomassconversion(e.g.,ricestraw,wood,
andmixedwastepaper);integrationofvariousunitopera-
tionsinbiomass-conversionprocesses(membraneltration
andenzymes);anddevelopmentofnewanalyticalmethodsfor
quantifyingspeciccellulaseactivities.Shoemakerisactivein
regional,national,andinternationaltaskforces,reviewpanels,
andprogrammingonnewandemergingbiobasedprocessing
technologies,carbonsequestration,andcellulaseR&D.
YuvalShoham
YuvalShohamhasbeenheadsince2004oftheDepartment
ofBiotechnologyandFoodEngineeringattheIsraelInstitute
ofTechnology(Technion)inHaifa.Hereceivedhisbachelors
degreeinbiologyfromTelAvivUniversityin1980,hismastersin
microbiologyin1982,andhisdoctorateinbiochemicalengineer-
ingfromMITin1987.In1988hejoinedtheTechnion,wherehe
isdirectoroftheOttoMeyerhoMinervaCenterforBiotechnol-
ogyandholdstheErwinandRoslPollakChairinBiotechnology.
HeisafellowoftheAmericanAcademyofMicrobiology.
Shohamsresearchfocusesonthecatalyticmechanismsand
structure-functionrelationshipsofindustrialenzymes,especially
glycosidehydrolases,andongeneregulationofthehemicellu-
lolyticsysteminGeobacillusstearothermophilusandcellulosome-
relatedgenesinClostridiumthermocellum.Hehasbeeninvolvedin
severalindustrialprojects,includingthedevelopmentofalarge-
scaleprocessforbleachingpaperpulpwithalkalinethermostable
xylanasesandanenzymaticprocessformakingcomplexlipids
withnovelSN-2lipases.Hehasauthoredmorethan120articles
andbookchaptersandholdsseveralpatents.
LloydM.Smith
LloydM.SmithisJohnD.MacArthurProfessorofChemistry
anddirectoroftheGenomeCenterattheUniversityofWis-
consin,Madison(UWM),wherehehasbeensince1988.He
receivedabachelorsdegreeinbiochemistryfromtheUniversity
ofCalifornia,Berkeley,in1977andadoctorateinbiophysics
fromStanfordUniversityin1981.In1982hemovedtothe
CaliforniaInstituteofTechnology,wherehedevelopedtherst
uorescence-basedautomatedDNAsequencinginstrument.
SmithhasbeennamedoneofScienceDigestsTop100Inno-
vatorsandhasreceivedthePresidentialYoungInvestigator
Award,EliLillyAnalyticalChemistryAward,Associationof
BiomolecularResourceFacilitiesAwardforthedevelopment
ofautomatedDNAsequencing,andtheAmericanChemical
SocietyAwardinChemicalInstrumentation.Hehasservedon
theNIHNationalHumanGenomeResearchInstituteAdvi-
soryCouncilandtheNIHHumanGenomeStudySection,
hasauthoredmorethan165scienticpapers,andisinventor
on20issuedU.S.patents.Heisacofounderofthebiotechnol-
ogycompany,TirdWaveTechnologies,andisamemberof
theboardofdirectorsofGWCTechnologies,Inc.,andGenTel
Biosurfaces,Inc.,wherehealsoischairoftheScienticAdvisory
Board.Hisprimaryareaofresearchisthedevelopmentofnew
technologiesforanalysisandmanipulationofbiomolecules.
202 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy




























Appendix C. Participant Biosketches
Chris Somerville
Chris Somerville is director of the Carnegie Institution Depart-
ment of Plant Biology and professor in the Department of Bio-
logical Sciences at Stanford University. He has published more
than 170 scientic papers and received patents in plant and
microbial genetics, genomics, biochemistry, and biotechnology.
His current research interests are focused on the characteriza-
tion of proteins such as cellulose synthase, which is implicated
in plant cell-wall synthesis and modication. He is a member
of the senior editorial committee of Science magazine and of the
scientic 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 Soci-
ety of London, and Royal Society of Canada. He has received
numerous scientic 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 bachelors degree from the National
Technical University of Athens, masters from the University
of Florida, and doctorate from the University of Minnesota, all
in chemical engineering. Upon nishing his doctorate in 1978,
he joined the faculty of the California Institute of Technol-
ogy, 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 Technol-
ogy (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
SingaporeMIT Alliance (2000).
Stephanopouloss current research focuses on metabolic engi-
neering 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 genomics-
based technologies are applied to the elucidation of cell physiol-
ogy 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 Meta-
bolic Engineering and serves on the editorial boards of seven sci-
entic 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 rst 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 (199599) and Bioinformatics (2000).
Bruce Stone
Bruce Stone received his bachelors degree from the University
of Melbourne in 1948 after majoring in chemistry and bio-
chemistry. 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 Chemi-
cal 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 ocial 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 Biologi-
cal Sciences at La Trobe (197678, 1987-91), was president
of the Australian Biochemical Society (198890), and was chair-
man of the Royal Australian Chemical Institute, Cereal Chem-
istry Division (197879). He currently is editor-in-chief of the
Journal of Cereal Science and assistant director of the Australian
Academy of Science and Technologys Crawford Fund, an orga-
nization supporting international agricultural research.
His major research interest in the chemistry and biochemistry
of plant polysaccharides arose during his rst appointment
through an investigation of cellulose-breakdown enzymology.
His specic studies on polysaccharide components of the cell
walls of cereals and grasses encompassed their structure, biosyn-
thesis, depolymerization, and their interactions with lignins and
proteins. Teir 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(13)--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 (13)--D-glucan, curdlan.
Biofuels Joint Roadmap, June 2006 Ofce of Science and Ofce of Energy Efciency and Renewable Energy U.S. Department of Energy 203

APPENDICES
BobTabita
BobTabitaisprofessorofmicrobiologyandplantbiology
andanOhioEminentScholaratOhioStateUniversity.His
doctoralworkinthelateDonLundgrenslaboratoryatSyracuse
Universityintroducedhimtothemetabolismandbiochemistry
ofautotrophicbacteria;hispostdoctoralresearchwasinBruce
McFaddensgroupintheChemistryDepartmentatWashing-
tonStateUniversity.Akeyexperiment,inwhichhediscovered
thatgrowthwithareducedelectrondonorupregulatesRubisco
synthesis,hasbeenthefoundationforhisentirecareerwith
photosyntheticbacteria,enablingstudiesontheenzymologyof
Rubiscoandotherpathwayenzymes.Hislaboratorycontinues
thisstronginterestinmolecularregulation,biochemistry,and
enzymologyofcarbondioxideassimilationandthecontrolof
Rubiscosynthesis.
AllorganismsrequireCO
2.
Itisusedinmanyenzyme-catalyzed
reactionsinprocessesasimportantandvariedascarbohydrate
metabolism,lipidbiosynthesis,andproductionofvitalmeta-
bolicintermediatesforthecell.Withtherealizationthatmany
microorganismsuseCO
2
toelicitpathogenesis,CO
2
metabolism
anditscontrolarerecognizedashavinggreathealthrelevance.
Carbondioxidealsomaybeemployedasthesolesourceof
carbonbyalargeanddiversegroupoforganisms.Forthisrea-
son,CO
2
xationisassociatedwithglobalissuesofagricultural
productivity,carboncycling,andindustrialproductivity.Carbon
dioxidealsoisrecognizedasthechiefgreenhousegasandhas
beenimplicatedingeneralwarmingoftheearthsbiosphere.
Forallthesereasons,researchonvariousaspectsofCO
2
xation
control,biochemistry,andecologyhaveattractedwideinter-
est.MicrobialsystemsstudiedintheTabitalaboratoryinclude
R.palustris,C.tepidum,andR.sphaeroides.
SteveThomas
SteveTomassresearchexpertisespansworkoninsecthor-
monesattheUniversityofCalifornia,LosAngeles,toplant
biotechnologyresearchattheARCOPlantCellResearchInsti-
tuteinDublin,California.TroughtheNationalRenewable
EnergyLaboratory,TomaswasmanagerfortheDOEOce
ofFuelDevelopmentprojectentitled,ProductionofCellulases
inTobaccoandPotatoPlantBioreactors.Hehasworkedonthe
SugarProcessingIntegrationTask,surveyingthecompositional
variabilityofgeographicallyandgeneticallydiversecornstover
residuestominimizeriskassociatedwithcommercializationof
biomass-conversiontechnology.HerecentlyjoinedCeres,Inc.,
asaprincipalscientist.
JerryTuskan
GeraldA.Tuskan,adistinguishedscientistinOakRidge
NationalLaboratory(ORNL)EnvironmentalSciencesDivi-
sion,holdsabachelorsdegreeinforestmanagementfrom
NorthernArizonaUniversity;amastersinforestgenetics
fromMississippiStateUniversity;andadoctorateingenetics
fromTexasA&MUniversity.Healsoisaresearchprofessor
intheUniversityofTennesseesdepartmentsofEntomology,
PlantPathology,PlantSciences,andGenomeSciencesand
Technologywhereheadvisesgraduatestudents,interactswith
departmentalfaculty,andprovidesguestlecturesandgraduate
seminars.BeforejoiningORNL,heservedasassociateprofessor
ofhorticultureandforestryatNorthDakotaStateUniversity
andwasaninstructorintheForestScienceDepartmentatTexas
A&MUniversity.
AtORNL,TuskanisresponsibleforcoordinatingtheDOE
eorttosequencethePopulusgenome.Tisincludesprojects
withcarbonallocationandpartitioninginwoodyplantsasa
meanstoenhancebioenergyconversionandcarbonsequestra-
tion,genome-enableddiscoveryofcarbonsequestrationgenesin
poplar,environmentalinuencesonwoodchemistryanddensity
ofPopulusandloblollypine,formationofaninternationalPopu-
lusgenomeconsortium,andcreationofaPopuluspostsequence
scienceplan.Hisresearchhelpsidentifygenesassociatedwith
cell-wallchemistry,geneticmappinginPopulus,particularly
relatedtocarbonallocationandpartitioning,andtheuseof
genomicsinformationtoacceleratedomesticationofPopulus.
EdUberbacher
EdUberbacherisleadscientistforcomputationalbiologyin
theLifeSciencesDivisionatOakRidgeNationalLaboratory.
HereceivedabachelorsdegreefromJohnsHopkinsUniversity
in1974andadoctorateinchemistryfromtheUniversityof
Pennsylvaniain1979.HeisthecodeveloperofGRAIL,the
rstofmanygene-ndingprogramscreatedduringtheHuman
GenomeProject.Next-generationalgorithmssuchasGrail-Exp,
whichcanusebothESTandcompletecDNAdata,provide
anotherleveltotheanalysisofdraftdata.
AsamemberoftheComputationalBiologyInstitute,Uber-
bacherworkswithinacollaborativeenvironmentthatcombines
theexpertiseofbiologists,computerscientists,andmathemati-
cianswithhigh-performancecomputingtocreateandprovide
toolsandinfrastructuretoadvancesystemsandcomputational
biology.HeisCEOofGenomixCorporation,aproviderof
analysissystems,informationresources,andpartneringoppor-
tunitiestoguideR&Dteamsthroughcriticalstepsinpharma-
ceuticaldiscoveryanddevelopment.Healsoservesasafaculty
memberincomputationalbiologyandbioinformaticsatthe
UniversityofTennesseeORNLGraduateSchoolofGenome
ScienceandTechnology.
FernandoValle
FernandoValleisstascientistforprocessscienceatDanisco
GenencorInternational,withexpertiseinmicrobialpathway
engineeringforthebiologicalproductionofenzymes,thera-
peuticproteins,peptides,antibiotics,vitamins,aminoacids,and
organicacids.Hissignicantresearchaccomplishmentsinclude
theoptimizationandpathwayengineeringofanE.colistrain
204 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy

Appendix C. Participant Biosketches

totransformglucoseinto1,3-propanediol,amonomerusedin
producingpolyesterbers.
JohnVogel
JohnVogelsresearchprogramisfocusedonapplyingmolecu-
larbiologytoimproveherbaceousenergycrops.Majorprojects
includeusingdsRNA-mediatedgenesilencingtoreducelignin
contentinswitchgrassandstudyingthemodelgrassBrachypodium
distachyontoidentifygenescontrollingcell-wallcomposition.
Recentaccomplishmentsincludecreationoftransgenicswitch-
grassplantswithsilencedligninbiosyntheticgenes,development
ofanAgrobacterium-mediatedtransformationmethodforBrachy-
podium,andsequencingof>20,000BrachypodiumESTs.
HeisaUSDAAgriculturalResearchServicemolecular
biologistattheWesternRegionalResearchCenterinAlbany,
California.Hisexperienceincludesextensiveresearchinplant
pathologyasbothanassistantprofessorattheUniversityof
California,Riverside,andpostdoctoralfellowattheCarnegie
InstitutionofWashingtoninStanford,California.Hisgraduate
workfocusedoncytokininandethylenesignalinginArabidopsis.
KenVogel
KennethVogelisaresearchgeneticistattheUSDAAgricultural
ResearchServiceWheat,Sorghum,andForageResearchUnit
inLincoln,Nebraska.Hereceivedhisbachelorsandmasters
degreesfromColoradoStateUniversityin1965and1967anda
doctoratefromtheUniversityofNebraskain1974.
Hisresponsibilitiesareinresearchmanagementandperennial
grassbreedingandgenetics.Hisspecicresearchinterestisto
developimprovedperennialgrasses,switchgrassinparticular,for
useonmarginallandsoftheCentralGreatPlainsandmid-
westernstatesalongwithassociatedmanagementpractices.Tis
researchinvolvesgermplasmevaluationandcharacterization
ofnativeandintroducedgrassesandbasicandappliedgenetic
researchinadditiontoappliedbreedingwork.Since1990,he
hasbeenconductingresearchtodevelopswitchgrassintoa
biomassenergycropfortheseareas.
NicholasWheeler
NicholasWheelerreceivedanundergraduatedegreeinforest
sciencesfromtheUniversityofWashingtonin1973,amasters
inforestgeneticsfromMichiganStateUniversityin1974,and
adoctorateinplantbreedingandgeneticsfromWisconsinin
1981.Hehasworkedasatreebreederforthegovernmentof
BritishColumbiafor4yrs,asascientistintheforestproducts
industryfor22yrs,and,morerecently,asaprivateconsultantin
hisownbusiness(MolecularTreeBreedingServices).
Wheelersresearchincludesprojectsinphysiologicalandquanti-
tativegenetics,genecology,andmoleculargenetics.Hecurrently
isanadjunctfacultymemberintheDepartmentofForestryand
EnvironmentalResourcesatNorthCarolinaStateUniversity,
workingonaNationalScienceFoundationgrantfocusedon
associationgeneticsinloblollypine.Healsoisanaliatefaculty
memberatOregonStateUniversityDepartmentofForest
Sciences.Hemostrecentlycompletedaprojectinvestigatinga
novelapproachtobreedinginpoplars.
DavidWilson
DavidWilsonisaprofessorofbiochemistryandmolecularand
cellbiologyatCornellUniversity.Hereceivedhisbachelors
degreefromHarvardin1961andhisdoctorateinbiochemistry
fromStanfordMedicalSchoolin1966.Hedidpostdoctoral
workattheDepartmentofBiophysicsatJohnsHopkinsMedi-
calSchoolfrom1966to1967beforegoingtoCornellasan
assistantprofessorin1967.WilsonisamemberoftheAmerican
SocietyofBiologicalChemists,AmericanSocietyofMicro-
biologists,andAmericanAssociationfortheAdvancementof
Science.HealsoisamemberoftheJohnsHopkinsSocietyof
ScholarsanddirectoroftheCornellInstituteforComparative
andEnvironmentalToxicology.
Wilsonslaboratoryusesacombinationofgenomics,protein
engineering,andmolecularbiologytostudytheenzymology
ofplantcell-walldegradation,withamajorfocusoncellulases.
Enzymesthatdegradeinsolublesubstrateshavesignicant
dierencesfrommostenzymeswhosesubstratesaresmall
solublemolecules.Inaddition,cellulasesareimportantindustrial
enzymesandhavepotentialintheproductionofrenewable,
nonpollutingfuelsandchemicals.Hisgrouphasbeenstudying
thehighG-CgramvariablesoilbacteriumTermobidafusca,a
moderatethermophile,formorethan20years.T.fuscaisamajor
microorganismdegradingplantcellwallsinheatedplantwastes
suchascompostpiles.Healsoisusingagenomicapproach
tocompareaerobicandanaerobicorganismstodiscovernovel
mechanismsfordegradingcellulose.
Researchprojectsinvolvethebiochemistryandchemistryofthe
cellwallsofhigherplants,withspecialreferencetocerealsand
grassesandthestructure-functionrelationshipsofwallpolysac-
charidesandproteins:chemistryoflignin-carbohydrateand
lignin-proteinassociationsinwallsofforageplants,withspecial
referencetotheirimpactonruminantdigestion;enzymologyof
-glucanhydrolasesactivesitechemistryinrelationtospecic-
ityandtothedesignofspecichydrolaseinhibitors;molecular
mechanismsofbiosynthesisof-glucansinplantcellwallsand
bybacteria;anddevelopmentofmonoclonalantibodiesforthe
specicdetectionofcell-wallpolysaccharides.
BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy 205

APPENDICES
206 BiofuelsJointRoadmap,June2006 OfceofScienceandOfceofEnergyEfciencyandRenewableEnergy U.S.DepartmentofEnergy