us road strategic bioethanol program

8/13/2019 US Road Strategic Bioethanol Program

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Chapter1TheRoadto Bioethanol: Strategic Perspective

oftheU.S.Department ofEnergy'sNationalEthanol ProgramJohnSheehan

BiotechnologyCenterfor FuelsandChemicals,NationalRenewableEnergyLaboratory,Golden,C O80401

As the Bioethanol Program at the Department of Energy (DOE)nears the end of twodecadesof research, it is time to take ahardlook at where we have been and where we are going. Thispapersummarizes thestatusof bioethanol technology today and what wesee as the future directions for research and development. All ofthis is placed in the perspective of strategic nationalissuesthatrepresent the drivers for our programthe environment, theeconomy, energy security and sustainability. The key technologypathways include the use of newtoolsfor protein engineering anddirected evolution of enzymes and organisms, as well as newapproaches to physical/chemical pretreatmentofbiomass.

Ethanolis used today as an alternative fuel, a fuel extender, anoxygenateand anoctane enhancer.Fromjust over 10 million gallons ofproductionin 1979, the U.S.fuelethanol industry has grown to more than 1.8 billion gallons ofannualproductioncapacity (/). Almost all of this capacity is based on technology that converts thestarch contained incornto sugars, which are then fermented to ethanol.

From its first days, this industry has been looking for ways to expand theavailable resource base to include many other forms of biomass. The U.S.Department of Energy has, throughout this period, invested in research anddevelopment on technology that will allow the fuel ethanolindustryto achieve its goalofexpandedproductionusing a diversified supplyofbiomassfeedstocks.

We refer to ethanol made fromthese as-yet untapped biomass resources asbioethanol. This paper provides a strategic perspective on this new bioethanoltechnology.

2 2001 AmericanChemicalSociety

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In Glycosyl Hydrolases for Biomass Conversion; Himmel, M., et al.;ACS Symposium Series; American Chemical Society: Washington, DC, 2000.


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3StrategicIssuesThereare several major strategic issuesthat motivate and influence DOE'sresearchprogramfor bioethanol. These include:national security,

the environment, andthe marketplace.

Thougheach ofthese issueshas shifted in importance over the years, all three remainconsistentdriversforourplans. Letme touch on eachofthese issuesbriefly.NationalSecurity.

O il Supply. A recentScience article summarized the strategic situation withregardto oil supply this way:

Naturetook half a billion years to create the world's oil, butobservers agree thathumankindwill consume it all in a 2-centurybingeofprofligate energyuse. (2)

Our dependence has been growing at an alarming rate since the early1980s,ironicallya time when public concern about petroleum has been very low. DOE'sEnergyInformationAdministrationpaints a dismal picture ofourgrowing dependenceonforeign oil (5). Considerthesebasic points:1. Petroleum demand is increasing, especially due to new demand from

Asianmarkets2. New oil will comeprimarilyfromthe PersianGulf3. As long as prices for petroleum remain low, we canexpectour imports

toexceed60% ten yearsfromnow4. U.S. domestic supplies will likewise remain low as long as prices for

petroleumremainlowNot everyone shares this viewofthe future, orseesit as a reason forconcern.The

AmericanPetroleumInstitutedoesnot see foreign imports as a matter of nationalsecurity 4). Others have argued that the prediction of increasing Mideast oildependence worldwide is wrong (5). Nevertheless, the International Energy Agency(IEA)recently announced that itseesannualpetroleum supplies reachingapeaksometimebetween2010 and2020. The IEA is one more voice in a growing chorus ofconcern about the imminent danger ofshrinkingoil supplies (2). Whilesomedisagreewith this pessimistic prediction, concern about our foreign oil addiction is widely heldby abroadrangeofpoliticalandcommercialperspectives 6).

Whilethere may be uncertainty andevencontention over when and if there is anational security issue, there is one more piece to the puzzle that influences ourperspective on thisissue. Put quite simply, 98% of the energy consumed in the U.S.transportation sectorcomesfrompetroleum (mostly in theformof gasoline and dieselfuel). The implication of this indisputable observation is thatevenminor hiccups inthe supply of oil could have cripplingeffects on our nation. This lends special

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4significance to the BioethanolProgramas a means of diversifying the fuelbasein ourtransportation sector.

EnergyDiversity. An important corollary to the notion of increasing energysecurity is the concept of energy diversity. Today,in theU.S.,natural gas, propane,andbiodiesel are establishing a place in the transportation fuel market. Bioethanol isyet another option in the fuel mix that we seek to provide. J.S. Jennings, theChairmanofRoyalDutch Shell, a company recognized as a leading strategic thinkerinthe energy industry, hasstatedthat ...theonly prudent energy policy is one ofdiversityandflexibility (7).

EconomicSecurity. Ourview ofnationalsecurity today must include questionsabout the healthandrobustness ofoureconomy. Energytoday plays an essential roleinour economy. Petroleum imports represent 20% ofourgrowing trade deficit. Thiscannot help but have an impact on our economy. A diverse portfolio of fuels,includingbioethanol, wouldbringmoneyandjobs back into the U.S. economy builton this new renewable energy technology. The associated development of energycrops will likewise provide a neededboostto ouragriculturalsector, a mainstay oftheU.S.economy.TheEnvironment.

A ir Pollution. A life cycle study conducted by D O E in 1993 evaluated theoverallimpactofbioethanol on several key regulated pollutants targeted by theCleanA irAct Amendments of 1990 (1990 C A A A ) 8). Thisstudy found that, comparedwith reformulated gasoline (RFG),a 95% ethanol/5% gasoline blend (E95) reducedsulfuroxideemissionsby 60 to 80%. Volatile organicemissionsfrom E95-fueledvehicles are 13 to 15% lower. Net (life cycle) emissions of NOx and carbonmonoxide are essentially the same.

These results are encouraging, but of greater importance is the impact thatbioethanol has directly on tailpipeemissions(as opposed to net pollutantlevelsacrossthe life cycle of the fuel). Low blends of ethanol have somepeculiar emissionproblems that go away at higher blendrates(mostly due to Reidvapor pressureincreases that occurbetween10% and 20% volume blends). A surveyofthe availableemissionsdata for high blends of ethanol reveals that, while there is a fair amount ofdata,it is often not consistently obtained. Still,the survey found the following broadtrends for ethanol usedinhigh blendlevelswith gasoline: (9)

CO levels maydecrease as much as 20%, probablybecauseof theoxygen content ofethanolSimilardecreasesinNOxcanbe anticipated as well.Highblends of ethanol cutend-use emissionsof volatile organic carbon(VOCs)by30%.Aldehydeemissionsfrom ethanol combustion in spark-ignitedenginesare,however, substantially higher for ethanol.

Thefirst round of comprehensive emissionstests for flexible fueled ethanolvehicles used in federal fleets was completed in 1996. These tests included a

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5comparisonof21ethanol-fueledChevroletLuminaswith an equalnumberofstandardgasoline modelLuminas 10). The results ofthe extensivestudy of exhaust emissionsconfirmthe trendsseenacross the literature (seeFigure1).

Figure1: EmissionReductionsforE85-fueledFederalFleetVehicles. Thetwosetsofdatarepresentanalyticalresultsfromtwoindependentlaboratories. NMHC-non-methane hydrocarbons)Sustainable Development. Public concern about the quality ofourenvironment hasgrown steadily over the past decade 11). Vice President Al Gore posits anenvironmental crisis that has been brought on by an exploding world population, atechnology revolution that has led to over-exploitation ofournaturalresourcesandanapparentdisregardfor the future. Hecitesthe 1992 EarthSummit in Rio de Janeiroas amajorturningpoint inour thinkingabout the environment.World-renownednaturalistEdwardO .Wilsonechoes thesesentiments in hiscallfor technology development that moves us awayfromfossil fuels and reduces theenergy intensity ofoureconomy. Wilsondescribes very eloquently his notion of anethic of sustainability:

Thecommonaimmustbeto expand resourcesandimprove qualityoflife for as many people asheedlesspopulation growth forces uponEarth,anddo it withrninimalprosthetic dependence. That, inessence,is the ethicofsustainable development. 12)

Bioethanoltechnology represents just oneapproachto moving our economy to amore sustainable basis. We, like many others touting technological solutions, shouldheed his remonstration of over-dependence on what he calls environmentalprostheses thatwillextend the capacity ofourplanet, butwillnot eliminate therisk

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6of environmental catastrophe. Environmentalists and technologists must worktogether toprovidebalanceandreasoninourapproach.

The biggest impediment to sustainable development is our economic system,whichplaces no value on the environment or on the future. The hardtruth, writesA l Gore, is that our economic system is partiallyblind 13). The blindness of themarketplace to environmentalissuesmakes deployment of bioethanol technologyproblematic,but not impossible. It forces a discipline on our development efforts inwhichweseekout opportunities for bioethanol thatmeetmultiple needs. Still,it isclear that something must change in our economic calculus if renewable andsustainable technologiesareto takeholdbefore a crisis forces the issue.ClimateChange. Climatechange is aparticularexample ofthekindofrisksthat areinvolved in ignoring the ethic of sustainable development. Political and publicconcernabout climate change varies with the time ofdayandday oftheweek. A yearwithEl Nino certainly promotes the cause. One reason for the seeminglyarbitrarynatureofourviewson climate change is that it involves a discussion ofrelativerisks,rather than explicit cause-and-effect problems. The reason for this is simple:understandingthe climatic implications ofglobalwarming isnotsimple. Some haveevensuggestedthat we can never understand the complex interaction of variablesinvolvedinunderstandingour climate 14). The salvos continue to go backandforthamongthe scientific experts as to the degree ofwarmingthat has occurred and itsimpact(75,16). Forexample, many critics of climate changeclaimthat satellite dataon global temperature contradict claims of increased temperature over the pastdecade. Researchers have recently demonstrated that decreasing temperature trendsseenin satellite data are actually due to errors caused by not accounting for changingaltitude ofthesatellite. Whencorrected for this change, the satellitedatais consistentwith other surface temperature measurements showing an increase in averagetemperature 17).

Whatthepolicymakersandthepublicneed to do is to make somerationalchoicesabout risk. The research reported in 1957 that confirmed C02accumulation in theatmosphere couched the question of climate change in exactlytheseterms 18), andthere is still no better way to look at theproblem. Giventhe catastrophic natureoftheimplicationsrelated to climate change, howmuchrisks toomuch? Thepotentialriskassociated with climate change has gotten the attention of the insurance industry, agroupall toofamiliarwith the damageandexpensethatcouldbe involved 19). E.O.Wilson'stake on thekindofriskassociated with our environment is along similarlines:

In ecology, as in medicine, a false positive diagnosis is aninconvenience,but afalse negative diagnosiscanbecatastrophic.Thatis whyecologistsand doctors don't like to gamble at all, and if theymust, it is always on the side ofcaution. It is a mistake to dismiss aworriedecologistoraworrieddoctorasan alarmist. 12)

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Inother words, can we afford a false negative diagnosis regarding climate change?Technologies like bioethanol are insurance. Prudence dictates that we takesomeforwardmovement in encouraging the use of such sustainable technologies.

Thecurrentpolitical setting for discussing climate change frames the question asanall or nothingproposition. Eitherclimate change is arealproblemorit is not. Ifitisreal,then we should treat it as a crisis ;otherwise, we are wasting our time. TheKyoto agreement signed by representatives of countriesfromaround the world isdoomed tofailif we continue to view theissuein this ill-conceivedframework.Agroup of prominent energy and environmental leaders recently met at the highlyrespected Aspen Institute to address theissueof climate change. In a letter to theWhiteHouse, they urged theClintonadministrationnot to send the Kyoto agreementto Congress, where itwilltooreadilybe dismissed 20). Instead, theysuggestthat theU.S. take a leadership role in establishing a long-term strategy for dealing withclimate change. Climatechange, they wrote, is a longtermproblem, andthe focusshouldbe on achieving sustainablelevelsof greenhouse gas concentrations at theleastcost,not only on near-term emission reductions. Thisapproach recognizes climatechange as a question ofriskrather than a black and whiteproblemthat must be dealtwith usingDraconianmeasures. In the end, renewable energy options like bioethanolbenefitfromthis type of longer-term strategy. Reasonable and sustained support iswhat is neededifbioethanol is to play apartinourenergy future.TheMarket. The bottom line for bioethanol is what, if any, market opportunitiesexistfor this fuel. It can be used as a fuel additive or extender in blends ofaround10%, or it can be used as a fuel substitute. Intoday'sU.S.fuelmarket,ethanol can beused in flexible fuel vehicles that can operate using blends of85%ethanol (and 15%gasoline).

AlternativeFuelsMarket. Fora long time, thegreatestimpediment to ethanol'suse as an alternative fuel was the lack of ethanol-compatible vehiclesintheU.S. Thishas changed dramatically. Today, bothFord andChrysleroffer standard modelsdesigned torunon either 85% ethanol (E85) or gasoline. Theyare offering this fuelflexibility at no additionalcostto the consumer 21, 22). Whilethe availability ofvehicles is no longer anissue, there is still a paucity of fuel stationsand fueldistributioninfrastructure for E85. Today,45 publicly available E85stationsareavailable in theU.S. Thirtymore limitedaccessstationsare available 23). The lackof basic infrastructure and the higher price of ethanol versus gasoline are majorconstraints on this market.

FuelAdditiveMarket. Use of ethanol as an additive in gasoline has become amajormarket. Startingfromliterally nothing a little over 20 years ago, ethanol as afuel additive has become a billion gallon per year market. It has value as anoxygenate in C Ononattainment markets, and as a fuel extender and octane booster.Thevalue of ethanol in the oxygenate and octane booster market is around 70 to 80centsper gallon.

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8EthanolSelling Price and Tax Incentives. Passageof1998'soverhaul of the

highwaybillbrought with it an extension of the ethanol tax incentiveprogram. Thisprogramadds about 50centsper gallon to the value of ethanol soldinthe fuelmarket.Whenadded on top of the market value for ethanol as anoxygenateand an octanebooster, this tax incentive allows ethanol to sell on the market for around $1.20 to$1.40 per gallon. The ethanol tax incentive willremain in place through2007.Withoutcontinued authorizationfromCongress, this incentivewillgo away. A majorstrategy of the BioethanolProgram is to take advantage of this tax incentive bydeveloping near term technology that can compete in the current ethanol market. Inthe meantime, our research is geared toward achieving cost reductions that willeliminate the need forfurtherextensionsofthe tax incentive.

TheTechnologyTodayOurworkingdefinitionofbiomass is plant matterproducedvia photosynthetic uptakeof carbonfromatmospheric C02 . It is important to understand this definition. Thephotosynthetic uptake of carbon imparts many of thebenefits of biomass-derivedfuels, such as sustainability and greenhouse gas reductions 24). The BioethanolProgramis, more specifically, concerned with the conversion of carbon present assugarsinbiomass to fuel ethanol.

Attheriskof oversimplifying the Bioethanol story, we prefer to view ethanoltechnology in terms of only four basicsteps(see Figure2). Production of biomassresults in thefixingof atmosphericcarbondioxide into organiccarbon. Conversionofthis biomass to auseablefermentation feedstock (typicallysomeformofsugar)canbeachieved using a variety of different process technologies. Theseprocessesfor sugarproduction constitute the critical differences among all of the ethanol technologyoptions. Using biocatalysts (microorganismsincludingyeastandbacteria) to fermentthe sugars releasedfrombiomass to produce ethanol in a relatively dilute aqueoussolution isprobablytheoldestformof biotechnology developed byhumankind. Thisdilute solution can be processed to yield ethanol thatmeetsfuel-grade specifications.Finally,the economics of biomass utilization demands that any unfermented residualmaterialleftover after ethanolproductionmust be used, as well.

Figure2.GeneralschemeforconvertingbiomasstoethanolTheNature Of Sugars In Biomass. Thedegreeof complexity and feasibility ofbiomass conversion technology depends on the nature ofthefeedstockfromwhichwe

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9start. Theleastcomplicatedapproachto fuel ethanolproductionis to use biomass thatcontains monomeric sugars, which can be fermented directly to ethanol. Sugarcaneand sugar beets are examples of biomass that contain substantial amounts ofmonomeric sugars. Upuntilthe1930s,industrial grade ethanol was produced in theUnitedStatesvia fermentation ofmolassesderivedfromsuch sugar crops(25). Thehighcostof sugarfromthesecrops has madethesesourcesprohibitivelyexpensive intheUnitedStates(26,27).

Sugars are more commonly found in the form of biopolymers that must bechemically processed to yield simple sugars. IntheUnitedStates,today's fuel ethanolis derived almost entirelyfromthe starch (abiopolymerofglucose)contained incorn.Starchconsistsof glucose molecules strung together by -glycosidiclinkages. Theselinkages occur in chains of a-1,4 linkages with branches formed as a result of a-1,6linkages (seeFigure3).Theterms and are used to describe different stereoisomers ofglucose. A not-so-obvious consequence of the linkages in starch is that this polymer is highlyamorphous,making it more readily attacked by human and animal enzymesystems.The ability to commercially produce sugarsfromstarch is the result of one of theearliestexamplesofmodernindustrialenzyme technologytheproductionanduse of -amylases, glucoamylases and glucose isomerase in starch processing (28).Researchers have long hoped to emulate thesuccessofthisindustryin the conversionofcellulosic biomass to sugar(29).

Figure3. Thepolymeric structureofglucoseinstarchtendsto beamorphousCellulose,the most commonformofcarbonin biomass, is also a biopolymer of

glucose. In this case, the glucose moieties are strung together by -glycosidiclinkages. The -linkages in cellulose formlinear chains that are highly stable andmuch more resistant to chemical attack because of the high degree of hydrogenbondingthat canoccurbetweenchains of cellulose (seeFigure4). Hydrogenbondingbetweencellulose chains makes the polymers morerigid,inhibitingthe flexing of themolecules that mustoccurinthehydrolytic breakingofthe glycosidic linkages.

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Figure4. Linearchainsofglucoselinkedby glycosidic bondscomprisecellulose

Yet a third form of sugar polymers found in biomass is hemicellulose.Hemicelluloseconsistsof short, highly branched, chains of sugars. It contains fivecarbonsugars (usually D-xylose andL-arabinose)and sixcarbonsugars (D-galactose,D-glucose and D-mannose) and uronic acid. The sugars are highly substituted withacetic acid. Its branched nature renders hemicellulose amorphous and relativelyeasyto hydrolyze to its constituent sugars. When hydrolyzed, the hemicellulose fromhardwoodsreleasesproducts high inxylose(afive-carbonsugar). The hemicellulosecontained insoftwoods,by contrast, yields more sixcarbonsugars(30).

The four forms of sugar in biomass represent a range of accessibility that isreflected in the history ofethanolproduction. Simple sugars are theoldestandeasiestto use feedstock for fermentation to ethanol. Nextcomesstarch, now the preferredchoice of feedstock for fuel ethanol. Starch-containing grain crops, like sugar crops,have higher value for food and feed applications. Because many animals (includinghumans) candigeststarch, but not cellulose, starch will likely continue toserveaunique and important role in agriculture(31). The remaining two formscelluloseandhemicelluloseare the most prevalent forms ofcarbonin nature,andyet they arealsothe most difficult to utilize. Cellulose's crystalline structure renders it highlyinsoluble and resistant to attack, while hemicellulose containssomesugars that havenot, until recently, beenreadilyfermentable to alcohol.ThreeTechnology Platforms. As indicated earlier, the technology pathways pursuedin the Bioethanol Program differprimarilyin the approach used to produce sugarsfrom biomass(step2 in Figure 2). Regarding sugar recovery, releasing the sugarsfrom the biopolymers in plant matter involves hydrolysis of the linkagesbetweenthesugar moieties. Hydrolysisis a simple chemical reaction in which a water molecule isadded across the glycosidic linkages in order to break the bonds. The discovery ofsugar production by acid hydrolysis of cellulose datesback to 1819 (32, 33). By1898, a German researcher had already attempted to use this chemistry in acommercialprocess for producing sugars from wood. Thisearly process includedfermentation of the sugars to ethanol (34). In the one hundred years since then,researchers have continued to pursue different approaches to achieving high yields offermentable sugars from the acid hydrolysis of biomass. It is easyto lose thishistoricalperspective onacidhydrolysis technologies.

The Bioethanol Program supports development of threetechnologiesbased ondifferent approaches toproducingsugars:

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11LowTemperature, ConcentratedAcidHydrolysisHighTemperature,DiluteAcidHydrolysisEnzymatic Hydrolysis.

The two acid hydrolysis technology platforms have the longest history ofdevelopment, while the use of enzymes to produce sugarsfrombiomass is, in theschemeofthings, a relatively recent concept.

Concentrated Acid Hydrolysis Process. The concentrated acid process forproducingsugars and ethanolfromlignocellulosic biomass has a long history. Theabilityto dissolveandhydrolyze native cellulose in cotton using concentrated sulfuricacidfollowed by dilution with water was reported in the literature as early as 1883(55). The concentrated acid disrupts the hydrogenbondingbetweencellulose chains,converting it to a completely amorphous state. Once the cellulose has beendecrystallized, it is extremely susceptible to hydrolysis at this point. Thus,dilutionwith water at modest temperatures provides completeandrapidhydrolysis to glucose,with little degradation.

Itseemsas though most of the research on concentrated acidprocesseshas beendone usingagriculturalresidues,particularlycorncobs. In 1918, researchers atUSDAproposedaprocess schemeforproductionofsugarsand other productsfromcorncobsbased on a twostageprocess. These researchers introduced the idea of using diluteacidpretreatment ofthebiomass to remove hemicellulose before decrystallization andhydrolysisof the cellulosefraction36). The ability to isolate hemicellulosic sugarsfromcellulosic sugars was an important improvement to the process, because the fivecarbonsugars were not fermentable.

In1937, theGermansbuiltandoperatedcommercialconcentratedacidhydrolysisplants based on the use and recovery ofhydrochloric acid. Several such facilitieswere successfully operated. DuringWorld WarII, researchers atUSDA'sNorthernRegional ResearchLaboratoryin Peoria, Illinois further refined the concentratedsulfuricacidprocess for corncobs 37). Theyconducted process developmentstudieson a continuous process that produced a15-20%xylose sugar stream and a10-12%glucose sugar stream, with the lignin residue remaining as abyproduct. The glucosewas readily fermented to ethanol at 85-90%of theoretical yield. The Japanesedeveloped a concentrated sulfuricacidprocess that was commercialized in 1948. Theremarkablefeature of their process was the use of membranes to separate the sugarandacid in the product stream. Themembrane separation, a technology that was wayahead of its time, achieved 80% recovery of acid 38). Research and developmentbased on the concentrated sulfuricacidprocess studied byUSDA(and which came tobe known as the PeoriaProcess ) picked up again in the UnitedStatesin the1980s,particularlyat Purdue University 39)and at T V A 40). Amongthe improvementsaddedbytheseresearchers were: 1) recycling of dilute acidfromthe hydrolysisstepforpretreatment, and 2) improved recycling of sulfuricacid. Minimizingthe use ofsulfuricacidandrecyclingthe acidcosteffectively arecriticalfactors in the economicfeasibilityofthe process.

Commercial success in the past was tied to times of national crisis, wheneconomic competitiveness of ethanol production could be ignored. Conventional

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12wisdom in the literaturesuggeststhatthis processcannot be economicalbecauseofthe high volumes ofacid required 41).

Today,despitethat wisdom , two companies in theU.S. are working with D O Eand NREL to commercialize this technology by taking advantage of nicheopportunities involving the use of biomass as ameansofmitigatingwastedisposal orother environmental problems. Arkenol,a company which holds aseriesofpatentsonthe use of concentrated acid to produce ethanol, is currently working with D O E toestablish a commercial facility that will convert rice straw to ethanol. Arkenolplansto take advantage of opportunities for obtaining rice straw in the face of newregulations that would restrict the current practice of open fieldburningof rice straw.The economics ofthisopportunity are driven by the availability ofacheapfeedstockthatposesa disposal problem. Arkenol's technology further improves the economicsof raw straw conversion by allowing for the recoveryandpurification of silica presentinthe straw. The facility would be located in SacramentoCounty 42).

Masada,a company which holds severalpatentsrelated toM S W (municipal solidwaste)-to-ethanol conversion, is working with D O E to contruct a MSW-to-ethanolplant, which will be located in Orange County, NY. The plant willprocessthelignocellulosic fraction ofmunicipalsolidwasteinto ethanol using technology basedonT V A 's concentrated sulfuric acid process. Concentrated acid hydrolysis produceshigh yields ofsugarwith little decomposition. Therobustnessofthis processmakes itwell suited to complex and highly variable feedstockslike municipal solidwaste.Masada's NewYorkprojecttakesadvantage of relatively high tippingfeesavailableinthe area for collection and disposal ofmunicipalsolidwaste. Masadais finalizingengineering andproject financing, andexpectsto break ground on the plant in the year2000.

Dilute SulfuricAcidProcess. Dilute acid hydrolysis of biomass is, by far, theoldesttechnology for converting biomass to ethanol. As indicated earlier, the firstattempt at commercializing aprocessfor ethanol from wood was done inGermanyin1898. It involved the use of dilute acid to hydrolyze thecellulosetoglucose,and wasable to produce 7.6 liters of ethanol per 100 kg of woodwaste(18 gal per ton). TheGermanssoondeveloped an industrialprocess optimized for yields of around 50gallons per ton of biomass. Thisprocess soonfound its way to the UnitedStates,culminating in two commercial plants operating in thesoutheastduringWorldWarI.These plants used what was called the American Process a one stage dilutesulfuric acid hydrolysis. Though the yields werehalf that of the original Germanprocess(25 gallons of ethanol per ton versus 50), the productivity of the Americanprocesswas much higher. A drop in lumber production forced the plants tocloseshortly after the end ofWorldWar I 43). In the meantime, a small, butsteadyamounto fresearch on dilute acid hydrolysis continued at theUSDA'sForest ProductsLaboratory.In 1932, the Germans developed an improved percolation processusing dilutesulfuricacid, known as the Scholler Process. These reactorsweresimplesystemsinwhich a dilute solution of sulfuric acid was pumped through a bed of wood chips.Several years intoWorldW arII, the U.S. found itself facingshortagesof ethanol and

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13sugar crops. The U.S. War ProductionBoardreinvigorated research on wood-to-ethanol as an insurance measure against future worsening shortages, andevenfundedconstruction ofaplant in Springfield, Oregon. The board directed the ForestProducts lab to look at improvements in the Scholler Process 44). Theirworkresulted in the Madison Wood Sugar process, which showed substantialimprovements in productivity and yield over itsGermanpredecessor 45). Problemswith start up of the Oregon plant prompted additional process development work ontheMadisonprocess atT V A ' s Wilson Dam facility. Theirpilot plantstudiesfurtherrefinedthe process by increasing yield and simplifying mechanicalaspectsof theprocess 46). The dilute acid hydrolysis percolation reactor, culminating in the designdeveloped in 1952, is still one of the simplest and mosteffectivemeans ofproducingsugars from biomass. It is a benchmark against which we often compare our newideas. In fact, suchsystemsare still operatinginRussia.In thelate 1970s,a renewed interest in this technology took hold in the U.S.because of the petroleum shortages experienced in that decade. Modeling andexperimentalstudieson dilute hydrolysissystemswerecarriedoutduringthe first halfofthe1980s. DOE andUSDAsponsoredmuchofthis work.

Aftera century of research and development, dilute acid hydrolysis has evolvedinto a process in which hydrolysis occurs in two stages to accommodate thedifferencesbetweenhemicellulose and cellulose 47). The firststagecan be operatedundermilder conditions, which maximize yield from the more readily hydrolyzedhemicellulose. The second stageis optimized for hydrolysis of the more resistantcellulose fraction. The liquid hydrolyzates are recovered from each stageandfermented to alcohol. Residual cellulose and ligninleftover in the solids from thehydrolysis reactorsserveas boiler fuel for electricityandsteamproduction.

Whilea variety of reactordesignshave been evaluated, the percolation reactorsoriginallydeveloped at theturnofthe centuryarestill the mostreliable. Thoughmorelimitedin yield than the percolation reactor, continuous cocurrent pulping reactorshave been proven atindustrialscale 48). N R E Lrecently reported results for a diluteacidhydrolysis ofsoftwoodsin which the conditionsofthe reactorswereas follows:

Stage1: 0.7% sulfuric acid, 190C, anda 3 minute residence time Stage2: 0.4% sulfuric acid,21 5C , anda 3 minute residence time

These benchscaletestsconfirmed the potential to achieve yields of89% for mannose,82% forgalactoseand 50% for glucose.FermentationwithSaccharomycescerevisiaeachieved ethanol conversion of90% ofthe theoretical yield 49).

B CInternational(BCI) and theDOE'sOfficeofFuels Development have formeda cost-shared partnership to develop a biomass-to-ethanol plant based on dilute acidtechnology. The facility will initially produce 20 million gallons per year of ethanol.B C Iwill utilize an existing ethanol plant located in Jennings,Louisiana. Dilute acidhydrolysis will be used to recover sugarfrombagasse,thewasteleftover after sugarcane processing. A proprietary, genetically engineered organism will ferment thesugarsfrombagasseto ethanol 50, 51).

Enzymatic Hydrolysis Process. Enzymes are the relative newcomers withrespect to biomass-to-ethanol processing. While the chemistry of sugar production

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14from wood has almost two centuries of research and development history and ahundredyears of process development, enzymesfor biomass hydrolysis can barelyspeak of fifty years of serious effort. The search for biologicalcausesof cellulosehydrolysis did not begin in earnest untilWorldWar II. The U.S.Armymounted abasic researchprogramto understand thecausesof deterioration ofmilitaryclothingandequipment in the jungles of the South Pacifica problem that was wreckinghavoc with cargo shipmentsduringthe war. Thiscampaign resulted in the formationofthe U.S.ArmyNatickLaboratories(52). Out of this effort to screen thousands ofsamples collected from the jungle came the identification of what has become one ofthe most important organisms in the development of cellulase enzymesTrichodermaviride(eventually renamedTrichodermareesei).reeseiis the ancestor of many ofthe most potent cellulase enzyme-producing fungi incommercialuse today.

Ironically,the research oncellulaseswas prompted by a need to prevent theirhydrolyticattack on cellulose. Today,weturntotheseenzymesin hope of increasingtheir hydrolytic power. Thisturning point in the focus of cellulase research did notoccuruntil the early1960s,when sugarsfromcellulosewererecognized as a possiblefood source(53),echoing similar notions expressed by researchers in earlier days onacidhydrolysis research(54). In the mid-1960s, the discovery that extracellularenzyme preparations could be made from the likes of T.reesei (55) acceleratedscientific and commercial interest in cellulases. In 1973, the army was beginning tolook atcellulasesas a means of converting solidwasteinto food and energy products(56). In a keynote address at a major symposium on cellulases, the HonorableNormanR.Augustine, thenUnderSecretary oftheArmy,spoke with vision about thepotential impact thattheseenzymescouldhave on oursociety(52):

Asthe army's developmentof ENIAC provedto be the stimulus forthe worldwide computer industry, I look forward to this emergingtechnology whosebirthstemsfrom a lonely fungus found in NewGuineamanyyearsago, to haveanequivalent worldwide impact onourwayoflife.

By 1979, geneticenhancement ofreeseihad already produced mutant strainswith up to 20 timesthe productivity of the original organisms isolated from NewGuinea(57,58). Forroughly 20 years,cellulasesmadefromsubmerged culture fungalfermentations have been commercially available. In another ironictwist,the mostlucrative market forcellulasestoday is in thetextileindustry, where they have foundvaluable niches such asintheproductionof stone-washed jeans.

In many ways, however, our understanding of cellulases is in its infancycomparedto other enzymes. There aresome good reasons for this. Cellulase-cellulosesystemsinvolve solubleenzymesworking on insoluble substrates. Thejumpincomplexityfromhomogeneousenzyme-substrate systemsis tremendous. It becameclear fairlyquicklythat the enzyme known as cellulase was really a complexsystemofenzymesthat worktogethersynergistically to attack native cellulose. In 1950, thiscomplex was crudely described as asystemin which an enzyme known as C i actstodecrystallize the cellulose, followed by a consortiumof hydrolyticenzymes, known as

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15C x which breaks down the cellulose to sugar(59). Thisearly concept of cellulaseactivity has beenmodified,addedtoandargued about for the past forty years 60,61).

Though many researchers still talk in terms of the original model of anonhydrolyticCx enzyme and a set ofC xhydrolytic enzymes, our current picture ofhowtheseenzymes work together is much more complex. Three majorclassesofcellulase enzymes are recognized today:

Endoglucanases, which act randomly on soluble and insoluble glucosechains

Exoglucanases, which include glucanhydrolases that preferentiallyliberate glucose monomers from the end of the cellulose chain andcellobiohydrolases that preferentially liberate cellobiose (glucosedimers)fromthe endofthe cellulose chain

-glucosidases, which liberate D-glucosefromcellobiose dimers andsoluble cellodextrins.Fora long time, researchers have recognized thatthesethreeclassesof enzymes

work together synergistically in a complex interplay that results in efficientdecrystallization and hydrolysis of native cellulose. In reaching out to non-scientific audiences, promoters of cellulase research often oversimplify the basicdescriptionof howtheseenzymes work together to efficiently attack cellulose(62).The danger in such oversimplifications is that they may mislead many as to theunknowns and the difficulties we still face in developing a new generation ofcosteffective enzymes. While our understanding of cellulase's modes of action hasimproved,we have much more to learn before we can efficiently develop enzymecocktails with increased activity.

Thefirstapplicationof enzymes forhydrolysisofwoodinanethanol process wasobvioussimply replace the acid hydrolysisstepwith an enzyme hydrolysisstep.Thisconfiguration, now often referred to as separate hydrolysis and fermentationSHF) is shown inFigure5(63). Pretreatment ofthebiomass isrequiredto make thecellulose more accessible to the enzymes. Manypretreatment options have beenconsidered,includingboththermalandchemicalsteps.

The most important process improvement made for the enzymatic hydrolysis ofbiomass was theintroductionof simultaneous saccharificationandfermentation(SSF),as patented byGulf OilCompanyand the University ofArkansas(64, 65). Thisnewprocess scheme reduced the number of reactors involved by elirninating the separatehydrolysisreactor and, more importantly, avoiding the problem ofproductinhibitionassociated with enzymes. In the presence of glucose, -glucosidasestopshydrolyzingcellobiose. Thebuildup of cellobiose inturnshutsdown cellulose degradation. IntheSSFprocess scheme, cellulase enzymeandfermenting microbes arecombined.Assugars are produced by the enzymes, the fermentative organisms convert them toethanol. The SSF process has, more recently, been improved to include thecofermentationofmultiple sugar substrates. ThisnewvariantofSSF,known asSSCFfor Simultaneous Saccharification and CoFermentation, is shown schematically inFigure6.

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Figure5.Theenzyme processconfiguredasSeparateHydrolysisandFermentationSHF

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17

Figure6. Theenzyme processconfiguredforSimultaneousSaccharificationandCoFermentation SSCF)Assuggestedearlier, cellulaseenzymesare already commercially available for a

variety of applications. Mostoftheseapplications do not involveextensivehydrolysisofcellulose. For example, thetextileindustry applications forcellulasesrequirelessthan 1% hydrolysis. Ethanol production, by contrast, requires nearly completehydrolysis. In addition, most of the commercial applications for cellulaseenzymesrepresent higher value markets than the fuelmarket. Forthesereasons, there is quite alarge leap from today's cellulase enzyme industry to the fuel ethanol industry. Ourpartners in commercialization ofnear-termethanol technology are choosing to beginwithacidhydrolysistechnologies becauseofthe highcostof cellulase enzymes.

Twocompanies have plans to deploy enzyme technology for ethanolproduction.Petro-Canada, the second largest petroleum refining and marketing company inCanada,signed an agreement withIogenCorporationinNovember of 1997 to co-fundresearch and development on biomass-to-ethanol technology overaperiodof 12 to 18months. Petro-Canada, Iogen and the Canadian government will then fundconstruction of a plant to demonstrate the process, which is based onIogen'sproprietarycellulase enzyme technology 66). .

B CInternational(BCI),mentioned in the previous section, will begin operationoftheir Jennings,Louisianaplant using diluteacidhydrolysis technology. The choiceof dilute acid technology is strategic, in that it allows for the eventual addition ofenzyme hydrolysis when cellulase productionbecomes cost effective. BCI iscurrentlyevaluating options for utilizingenzymes 67). BCIplans to utilize cellulaseenzymesin a projectpartiallyfunded by the Department ofEnergythat will lead to acommercialricestraw to ethanol facility inGridley,C Aby2003.

Technology PathwaysThe PromiseOfBiotechnologyFroma big picture technological perspective, there is every reason to believe

that the progress made over the past fewdecadesingeneticengineering technology

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18

couldbe dwarfed by future advances. Biotechnology isanexplosive field. Newtoolsandbreakthroughs areoccurringat an exponential pace. Knowledge in the biologicalsciences is doubling every five years. In the field of genetics, the amount ofinformationis doubling annually 68).In 1997,BusinessWeekdeclared the 21stcentury to be The BiotechCentury.TheyciteNobelPrizewinning chemistRobertF. Curl,whostatesthat the20thcenturywas the century of physics and chemistry. But it is clear that thenextcentury will be

the century of biology 69). JeremyRifkin,a frequent critic of biotechnology, stillacknowledges theprofoundimpact thatgeneticengineering will have 70):

The marriage of computers andgeneticscience,in just thelasttenyears, is oneofthe seminalevents of ourageandislikelytochange ourworld more radically than any other technological revolution inhistory.

It is in this broadercontextof biotechnology's bright future that we buildaroadmapfor bioethanol technology. We see the path for technology development asone that uses computer technology, biochemistry and molecular biology as theessentialtoolsfor fundamental improvement.Cellulase Enzyme Development. Dr. Ghose, one of the pioneers in cellulaseresearch, spokethesewords almostthirtyyears ago:

Microorganisms have no difficulty digesting cellulose. Theyaccomplish it rapidly and effectively. Why is it then that we cannotutilize theirsystemsto develop a practical conversion of cellulose tosugar? The answer is rather simple; we can-if we pour into thisproblemthe effort itrightlydeserves. 71)

Despite his optimism, we have yet to crack the secretsofmicrobial cellulosehydrolysis. We still share his optimism. Learninghow to use cellulase enzymestoefficientlydigestcellulose to sugar requires aconsistenteffort that simply hasn't beenappliedup to now. Furthermore, we haveaccessto exciting new biotechnologicaltoolsunimagined by Dr. Ghose in 1969. These newtoolswill make it possible toproduce newenzymesspecifically designed for use inindustrialproductionprocesses.

Because of the importance of cellulaseenzymesin the process, D O E andN R E Lsponsored a series of colloquies with experts and stakeholders in industry andacademia to determine what types of improvements in enzyme production andperformance offer thegreatestpotential forsuccessin the short term 72). There wasa clearconsensusinthesediscussions that the prospects for enzyme improvementthroughprotein engineering are very good. We identified the followingtargetsforproteinengineering:

IncreasedThermalStability.Simply by increasing the temperature atwhichtheseenzymescan operate, we can dramatically improve the rateof cellulose hydrolysis. Thegeneticpool available in our labs and in

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20Substantial improvement in biomass conversion can be achieved by making the

following additional improvements in ethanolproducingmicroorganisms: Ethanolproducing microorganisms capable ofproducing5% ethanol at

temperatures greater than or equal to5 0 C ,and Ethanolproducing microorganisms capable of converting cellulose toethanol.

We have recently shown that a doubling of the rate of biomass hydrolysis forevery 2 0 C increase in temperature of saccharification can be expected if T.reesei-likecellulasesare used. The development ofethanologenscapable of fermentation attemperatures greater than50Ccan potentially reduce thecostof cellulase enzyme byone-half. Thisisbecausethecurrentethanologenscan onlymeetdesired performanceat temperatures of3 0 -3 3 C .

The most advanced processing option is one in which all biologically mediatedsteps(e.g.,enzyme production, enzymatic cellulose hydrolysis, and biomass sugarfermentation) occur in a single bioreactor (80). Thisprocess, alsoknown as directmicrobialconversion (DM C ) or Consolidated Bioprocessing(CBP), can becarriedout to various extentsby a number of microorganisms, including fungi, such asFusariumoxysporum and bacteria, such as Clostridiasp. However, known D M Cstrains often exhibit relatively low ethanol yieldandhave not yet been showneffectiveinhandlinghigh concentrations ofbiomass.

Ourprogram plancalls for introducing a high temperature ethanologen by2005.This new organism should be able to operate at 5 0 C , while maintaining thebestcharacteristicsofthecurrentethanologens.Ethanol Cost Savings In The Future. The improvements in enzyme andethanologen performance will impact the process in2005 and 2010. Geneticallyengineered feedstocks with higher carbohydrate content might happen in2015though the timing for thislastitemneedsto be determined more precisely. Figure7showsthe decline in bioethanolpricingbased ontheseresearch targets. The upperand lower bounds on theerrorbars reflect the results of sensitivitystudiestoassesstheeffectof feedstock price. The lower bound is a price projection for $15 per dryU.S. ton($17.50perM T )feedstock andtheupperboundis a price projection for $44per dry U.S. ton ($40 perM T ) feedstock.Conversiontechnology improvementscould providea 35centsper galloncostreductionover thenextten years. Combiningtheseimprovements with geneticallyengineeredfeedstocksbrings the savings to 40centsper gallon.

ConclusionsNew technology for the conversion of biomass to ethanol is on the verge of

commercialsuccess. Overthe course of thenextfew years, we should see new acidhydrolysis-based bioethanol plants come on line, which use niche feedstocksthataddress an environmentalissue,such as solidwastedisposal. As improvements inenzyme technology become available, weexpectto see bioethanolproductioncoming

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21

$1.40

>$0.40$0.20 '

-1995 2000 2005 2010 2015 2020

ear

Figure7. PriceTrajectoryforEnzyme-BasedProcessTechnology

online that provides ethanol at prices that can compete with other fuel additives andblendingcomponents without any subsidy. Thistechnology should be available justas the existing incentives for fuel ethanol are scheduled to end. As concern aboutclimate change, sustainability and other environmental issues increase, theopportunities for bioethanolwillcontinue to grow. Thenextten years should provean interesting time for bioethanol, a time when bioethanoltakesonmuchgreaterimportanceinthe fuelmarket.

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us road strategic bioethanol program

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