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t seems that everyone is talkrng about advanced biofuels,p especially after President Obama announced last year

--#"-that his administration will take the necessary steps tofoster biofuels research and commercialization. In addition,the U.S. Dept. of Energy (DOE) announced the release of5786.5 million in stimulus funds under the American Recov-ery and Reinvestment Act of 2009 to accelerate advancedbioftiels research, development, and deployment.

The term advanced biofuels refers tp fuels other thancom-based ethanol that are derived from renewable biomassand whose greenhouse gas (GHG) emissions are no morethan half those of the fuei they replace. One of the mostcommon lypes of advanced biofuels is celluiosic ethanol,which is derived from the cellulose, hemicellulose, or ligninthat make up plant cell walls.

There are still many unknowns when it comes to cellu-losic biofuels, but gne sure thing is that bioiogical engineersare working together toward biofuei commercialization.This months'SBE Supplement focuses on various aspects ofproducing lignocellulosic biofu els.

In the first feature article, Tim Eggeman and Can'ie Ati-yeh provide an inhoduction to the U.S. biofuels industry andthe strides it has made toward displacing petroleum-basedgasoline with renewable alternatives. Ciirrently, the cellulosicand advanced biofuels industry is at a critical stage of devel-opment and deployment; the federal govemment will play animportant role in facilitating their entry into the market.

In the second article, Brad Cort, Thomas Pschom,and Bertil Shomberg point out that even though uncer-tainties regarding process technology optimization exist,commerciai-scale equipment already being used in the pulpand paper industry can minimize scale-up risks in biofuelproduction from lignocellulosic feedstocks.

In the third article, Blake Simmons, Seema Singh,Bradley Holmes, and Harvey Blanch discuss a new typeof pretreatment for breaking down cellulose. Pretreatmentis required to maximize ethanol yield, and most processesemploy acids, bases, steam, or heat. The new approach,which uses'ionic liquids (salts composed of an anion and acation), reduces the amount ofenzymes needed to achievehigh yields of glucose from lignocellulose.

The foulrth feature.article comes from Michael Ladisch,Nathan Mosier, Youngmi Kim, Eduardo Ximenes. and DavidHogsett. They describe various biological, thermochemical,and chemical technologies being developed to convert ligno-celluiosic feedstock to liquid fuels and discuss some of the

factors that must be considered in developing economic pro-cesses, such as the feedstock, catalyst robustness, tradeoffsbetween selectivify and conversion rate, and cost.

, AIChE's Institute for Sustainability has been explor-ing background data on establishing biofuel metrics. TheRenewable Energy Metrics Committee, under the leadershipof John Carberry, is working on metrics for the assessmentof biofliels development and use, with two primary goals:to develop and adopt a cormon set of technically sor.tnd,economically viable biofuels metrics that can be used by thefederal interagency task force workfng on energ,v metrics;and to identify enabling farming developments andror pro-cessing technologies that would expand the supply, improvethe economics, or improve the sustainability of biofuels. Formore information, visit www.aiche.com/ifs

SBE continues its webinar series in 2010 with presenta-tions from both academic and industry experts on a vanetyof exciting topics. Tune in to Sang Yup Lee's presentationon systems metabologic engineering onApr. 23 and ChaitanKhosla's discussion of modular biocatalysis on June 29.For information about the archived SBE webinars, visitwww. aiche. org/SBElEducation/SBEwebinars. aspx.

Register now for SBE's 2nd International Conferenceon Stem Cell Engineering, May 2-5 in Boston, whichis co-chaired by Peter Zands'tra (Univ. of Toronto) andGeorge Daley (Children's Hospitai Boston, Harvard Medi-cal School). For more info, visit the conference webpage atwww.aiche.orglstemcelleng. SBE is also co-sponsoring the5th International Conference on Bioengineering and Nano-technology in Biopolis, Singapore, Aug. l-4, chaired by Pro-fessor Jackie Ying (A*STAR lnstitute of Bioengineenng andNanotechnology). For details, visit www.icbn20 1 0.com. Savethe date for SBE's 3rd Intemational Conference on Biomo-lecular Engineering, which will take place at the Grand Hyattin San Francisco on Jan. 16-19,2011. This conference isco-chaired by Kurt Deshayes (Genentech) and JeffVamer(Comell Univ.) We hope to see you at one of these meetingsl

SBE continues to strive to become a better society forits members. If you have any ideas or commentq please ietme know at bio@aiche.org. We appreciate your feedbackand value your involvement in making SBE a more dynamicorganization. If you aren't yet a member of SBE, simply goto: http://bio.aiche.org to check us out and join todayl

Miriam Codes-CamineroExecutive Director, SBE

GEP March 2010 www.aiche.org/cep 35

2Kfum ffim&m ffmw ffiffimffmm&ruThe advanced biofuels industry is at a

crlt ical stagg of development and deployment'

nergy drives every part of modem life' In addition topotiAng factories,ofces, and homes, energy fuels'the

24-7 transportation and logistics infrastructure

resources - such as biofueis'Renewable transportation fuels, such as com-derived

ethanol, cellulosic ethanol, and other advanced biofuels' are

the original RFS.Under the RFS, the U.S' Environmental Protection

Agency (EPA) is responsible for implementing regula-tions t ensure that increasing volumes of biofuels for the

36 wwv.aicne.org/cep March2010 CEP

be nearly 13 billion gallons (2)'The RFS2 contmues to support the com ethanol industry'

meet these targets.

Redrucing green*'l*use gas emissionsIn addition to establishing clear production targets' the

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Ec,:*rni* c:mslSerati*s'sDomestic economic development is a significant advan'

tage of the biofuels industr,v,especially in rural areas. In'itsmost recent amual update of the economic contributions ofthe ethanol industry to the U.S. economy, consulting firmLECG projects that the RFS2 ethanol targets will create nearly1 .2 million jobs across all sectors of the economy and wiligenerate more than $630 billion in expenditures to expand theindustry (4). As advanced biofuels technologies capable of uti-lizing a diverse vanefy ofnon-food feedstocks are deployed,direct benefits to local economies will be realizeC in areas ofthe country where biofuels are not currently produced

Cellulosic ethanol technology defloyment is slowlymaking progress rn the early years and is projected to rapidlyincrease over the next decade (following the proverbialhockey-stick growth curve). The federal government iscommitted to working with industry to meet the MS2'saggressive, yet achievable, goals.

The White House report "GrowingAmerica's Fuel,"released in February 2010, is a comprehensive roadmap toadvanced biofuels depioyment l5). This report defines theresponsibilities ofvarious federal agencies for each segmentof the industry, including feedstock development, researchand development of biofuels technology, piiot- and dem-onstration-scale bioreflnery projects, and commercial-scalebiorefineries. The plan is outcome-driven and focused on thefeasibility of the industry at each cntlcal step, with consider-ation of technical, management, economic, market, financial,and environmental issues. The report offers assurance to theadvanced biofuels industry and all of its partners, particulariyin the finance sector, that biofuels are a national priority and aviable industry.

Economic feasibility of the em.erging advanced biofuelsindustry is a critical factor in meeting the targets set forthin the RFS2. Government has an important role to play infacilitating the entry of advanced biofuels into the marketand their ability to compete on an equal economic basis withtraditional fossil-fu el resources.

The finance sector uses the term "valley ofdeath" todescribe the period of project funding between proven early-stage development financed through venture capital andcommercial development using traditional project financing.The advanced biofuels industry cunently sits in the valley ofdeath - milestones have been met in early development butcommercial production has ngt yet been realized. Tax credits,grants, and other useful financial provisions will providenec-essary near-trm ncentives to achieve long-term goals.

However, in orderfor any business to be successful, it- cannot rely indefinitely cn govemment subsidies nd incen-

tives. Eventually, to be competitive and economically viable,

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Community

A Figure 1. Biomass from hybrid poplar trees will supply a 250,000-gal/yrdemonstration-scale biorefinery. Source: Zeachem Inc.

the industry must be able to compete without subsidies againstthe marginal production cost of crude oil. At today's condi-tions, this is approximately $75ibb1. Govemment support atthis stage will help the industry meet this economic goal.

fleedstoc!r. avaitabl{ityFeedstock availability is another cntical issue for the

advanced bioftiels industry, since roughiy 5O-15% of a bio-refinery's operating costs are tied to feedstock. The biofuelsindustry is no different than the oil industry when it comes tothe importance of feedstock supply and cost: It is necessary tosecure reliable, low-cost, and long-term-viable supplies.

In 2005, the U.S. Dept. of Energy (DOE) and U.S. Dept.of Agriculture flJSDA) issued a report known as the "BiliionTon Study," which assesses the ftasibility of displacrng one-third of the nation's petroleum consumption with biomassresources (6).Thereport concludes that meeting such a goalwould require 1 billion tons of biomass annually - and thatsuch supply is indeed feasible.

The administration's recent biofuels report (5) alsoaddresses feedstock feasibility. It points out that the lowest-nsk and highest-potential feedstocks are dedicated (i'e.,specifically grolvn) crops, such as trees, energy grasses, andcane. Establishing contracts between growers and biofuelsproducers wiil reduce risks as the indus@ scales up - byensuring that growers have a market for their product and thatbiofuels producers have the feedstock necessary to meet theirproduction needs. In addition to dedicated crops, the reportie"o--ed. that biorefieris seek to use local residuals(agricultural and forest), thereby making use of extra feed-stocks in close proximity and providing additional income tothe local community. .

This feedstock model provides a useful context for thearticles in this special supplement on cellulosic biofuels. It isthe strategy used by companies like ZeaCfiem Inc., a cel-lulosic biofuels and bio-based chemicals producer. ZeaChem

CEP March2OlO wvwvaiche.org/cep 37

T93E Spscrll 5uppLufl,rEflT: Broru l-s

has contracted with GreenWood Resources, the nation's lead-ing grower of hybrid poplar hees, to supply dedicated woodybiomass (Figure 1) for its 250,000 gaVyr demonshation-scalebiorefinery in Boardman, OR. Dedicated poplar tree feedstockis also available for a commercialellulosic ethanol facilrty.Local agricultural and forest residuals will be used to supple-ment the primary dedicated poplar tree feedstock supply.

Analysis has shown that the use of short-rotation hybridpoplar trees for feedstock initially offers the lowest cost perbone dry ton (BDT)/acrelyear. The feedstock inventory issrored on the stump until it is needed for conversion intoethanol or a chemical product, eliminating storage concerns.Short-rotation hybrid poplar trees can be harvested as often asevery three years, and require replanting only bnce every fiveharvests. Maintarning this high-density energy crop approachminimizes the transportation costs associated with delivery toihe piant and has a low carbon lifecycle.

It is also worth noting some general best practices infbedstock operations. First, longterm supply agreementswith feedstock suppliers will ensure the availability ofsustainable, economical, and plentiful feedstock. Second, theuse of supplemental local'residuals will provide additionalcommunity investment, primarily in rural areas where suchoperations will be located. Finally, a "grow where you go"approach, in which biorefiners co-locate commercial facilitresin the markets they serve with dedicated energy crops, willminimize transportation and logistics costs.

i-ooking aleadThis SBE supplement examines technical breakthroughs

in biofuels production and will assist the advanced-biofuelsindustry in realizing its long-term production goais.

The first article, by Cort e/ a/., discusses biomass handlingsystems, pretreatment reactors, and solid-liquid separationsequipment needed at the front end of a cellulosic biorefinery.The authors present their views liom the perspective of a lead-ing equipment supplier to the pulp and paper industry. Manyof the lessons leamed in that mature industry are directlyapplicable to the emerging cellulosic biofuels industry.

The second article, by Simmons et al., discusses a newionic liquid pretreatrnent process for making lignocellulosicbiomass amenable to enzymatic hydrolysis. This is an impor-tant issue as the industry searches for better, lower-cost pre-treatment systems. Ionic liquids have unique properties thatmake them promising altematives for biomass pretreatment.

The third article, by Ladisch et al., provides an overviewof biochemical and thermochemical technologies for con-verting lignocellulosic biomass int ethanol. The technical

' hurdles for these two production pathways are explored as ameans ofnavigating the path forward.

3A www aiche,org/cep lvlarch 2O1O CEP

The biofuels industry in the U.S. has made great stridesto date in displacing petroieum-based gasoline with renew-able alternatives. Now, the cellulosic and advanced biofuelsindustry is at a critical stage of development and deploy-ment. With the RFS2 rn place and a clear roadmap guidingthe federal governmeirt and the bioftiels industry 36 billiongallons of bioftrel can be achiev ed in2022. ffire

Lrtsntrun ClroU.S, Dept. ofEnergy, "Annual Energy Review 2008,"wwweia,doe. gov/aeripdflaer.pdf, DOE, Energy lnformatiorrAdministration, Washington, DC (June 2009).R.enewable Fuels Association, '12009 Ethanol lndustry Outloolg"www. ethanolfa. orglobjectsipdfloutlook/RFA_Outlook_2009 pdf,RFA, Washington, DC (2009),U.S. Environmental Protection Agenc "EPA Finalizes NewRegulations for the National Renewable Fuel Standard Programfor 2010 and Beyond," www.epa.gov/OMS/renewablefuelsi420fl0007.htrn, EPA, Washington, DC (Feb. 2010).Urbanchuk, J. M., "Contribution of the Ethanol Industry to theEconomy of the United States," www.etlanolrfa.org/objects/documents/2 I 8712008 ethanol-economic_contribution.pdf,prepared by LECG LLC for the Renewable Fuels Association,Washington, DC (Feb. 2009)."Growing America's Fuel: An Innovation Approach to Achiev-ing the President's Biofuels Target," www-whitehouse.gov/sites/defaulVfi les/rss viewer/growin g_americas_fu els.PDF(Feb.20l0).U.S. Dept of Energy and U.S. Dept. of Agriculture, "Bio-mass as Feedstock for a Bioenergy and Bioproducts Industry:The Technical Feasiblity ofa Billion-Ton Aual Supply,"http ://feedStoclaeview.oml. gov/pdf/bi11ion_ton_vision.pdf,DOE and IJSDA (Apr: 2005).

TIM EGGEMAN, P.E., is chieftechnotogy officer ofZeaChem Inc. (165 5. LJnionBtvd., Suite 38o, Lakewood, CO 8ozz8-2257; Phone: Go3) 279-7045;Fax: Go3) 279-9537i E-mail: time@zeachem.com) and a co-inventor ofthe ZeaChem process. Prior to founding ZeaChem, he was an indepen-dent consultant servng clients in the biofuels, syngas and Fischer-Tropsch areas. Previously, he was the process development manager atChronopol, where he supervised a group that devetoped manufacturngtechnology to produce biodegradable ptastics based on po{ytactic acid,and a process design engineer with ihe C. W. Nofsinger Co., where heworked on proiects in the pharmaceutical, speciatty chemical, corn wetand dry mtting, and petroteum industries. He is an expert n chemicalprocess modeting and has numerous patents. He hotds a PhD and MSfrom the Unv. of Kansas and a BS from the UniV. of lll inois, all in chemi'cal engineering, and is a licensed professionat engineer.

CABRIE ATIYEH is director of pubtic affairs at Zeachem Inc. (Phone: $o3)248-7778i E-mail: catiyeh@zeachem.com). She has a decade of experi-ence developing energy and environmental initiatives for [ocat andfederal governments, nongovernmentat organizations, and publc andprivate corporatons. Prior to ioining ZeaChem in 2oo8, she was Green-house Gas Program Administrator for the city and county of Denver,where she planned, managed, and evaluated coJporate and residentialprograms to reduce greenhouse gas emissions. She has also worked atEvergreen Energy, Environmentat Defense, the Federal Tiade Commis-sion, and the Dutko Group. She graduated with honors from Hobart andWitliam Smith Cotleges with a bachetor's degree in political'science.

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in biorefi.neries is a key parameterin achieving signif cant economical'oroduction of alternative liquid bio-fuels. Because of the low densttYof biomass, transPorlation costsare high, such that 40-50 miles tsthe mximum distance consideredeconomically feasible for biomasstransport. Figure 3 plots the number

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EthanolCoproducts

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Bi*chemicatandthernrochernica{pr0ce55technoiogies belng developed to convertwood anC other t ignocel lulosic feedstocks

to {iquicl fueis ri l l drive the transitiottf rcm corn-based ethanoL to advanced biofuels '

he recent National Academies report "America's

Bt.tgy ettute" concluded that alternative liquid

"tr-uu. the potential to reduce dependence onimported oi l, enhance energy securitv'. and n"t", itl1l11.T:::"r'rvur Leu ""' -'*^--

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consumPtion levels (1)'Biochemicalandthermochemicalprocessesthatffans-

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Figure 1. Bioprocessing of ligocellulose to ethanol involves pretieatment' hydrolysis' fefmenttion and

seoaraton. Source: ( / //'

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The radeoff is selectivity vs' conversion rate - selectivity

is high for bioprocesses and low for thermal' whereas con-

u"rri"on rates are low for bioprocesses and high for thermal.

Feedstock, cataiyst robustness, and costs are key factors that

mustbeaddressedtoachieveeconomicalplocessesforbothbiochemical and thermochemical technolcgies'

Bioprocesses for making ethanol from celiulose have a

iong history Q)-Thepotentiai to produce ethanol from cel-lulo"se usin! min"ral acid catalysts was demonstrated

prior to

Wbrld War iI' Thermochemical and acid routes for obtain-

ing fermentable sugars were mature technologies more than

Tdyears ago, whereas en4'rne biocatalysts that perform

similar functrons were identifled more recently (?' 3/ Theseenzylnes have been punfied and characterized' and

the genes

thaiencode them have been sequenced' Current produc-

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microorganisms to produce cellulolytic enzymes that are

used in the food and consumer products industries' as well

as in the emerging biofuels industry'

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,* Figure 3. Number of siies in the U.S. vs. biomass availability withina 40-mile radius 0f the site. Note that lT sites can provide more fian7,000 dry tons of biomass per day, which is equivalent to more than2.5 million toniyr. Source: lil.

sufficient through the combustion'or gasification of residuallignocellulose. If only corn grain is processed, the remainingsolids that are high in protein are recovered as coprodttctsand sold as animal feed, which has a higher value than boilerfuel. Carbon dioxide is recycled into plant matter throughcrop-production agriculture.

Efcient conversion of cellulose to biofuels requiresthat the cellulosic feedstock, whether wood, corn stalks,sugarcane bagasse; grasses, or other non-food crops, firstbe pretreated. Pretreatments include processes that cook thematerial in liquid hot water, or use steam, acid or base, toopen-the plant cell wall structure in a way that exposes the

Biomassof sites in the U.S. that could supplyvarions amounts of biomass within a40-mi radius of potential biorefinerylocations. For exampie, i7 sites can pro-vide more than 7,500 dry ton/d. Becauseof the wide variation in potential supply,the size of biorefineries is likely to varyconsiderably (1).

Cel lulosic biolnass consists pt' intar-ily of cellulose and hemicellulose, whichupon pretreatment and hydrolysis areconverted to monosaccharides, princi-pally glLrcose (a hexose) and xylose (apentose). Based on the compositions ofthe various sources of biomass (Table1), it is possible to caicr-riate the potentialquantify of sugars that would be obtainedl'rom complete hydrolysis. and from Lhis.the maximum yield of ethanol can beestimated. Although actual yields willbe lower, since some of the sugars aredirected to cell biornass and energy forcarrying out various metabolic functionsof the microorganisms, the data in Table

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Figure 2. A typical thermochemical route to biofuel involves gasification of biomass t0 syngasfollowed by catalytic Fischer-Tropsch (FT) conversion t0 biodiesel Source: (7/).

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1 provide reasonable estimates of achievable converston' Asa rule of thumb, maximurrr ethanol yieids are on the order of100 galiton for both thermal and biochemical processing'

Pretreatment is required to maximize ethanoi yield' andvarious pretreatments (e.g, acids, bases, water, steam,and/or heat in sotne combination) have similar effects (4' 5)'Without pretreanent, yields of fermentable sugars from cel-lulosic biomass by enzymatc hydrolysis are 3-20oA of theo-retical. After appropnate pretreatment, yields exceeding 90%oof theoretical can be achieved. The remaining organic solidmaterial is lignin, which has a cmbustion value sufficient tosupply the energy for the rest of the plant when bumed in acogeneration faciiity.

f i i*qh'*rnieai p-r oces*mgThe processing of cellulosic biomass requires five steps,

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enzymes. Other than feedstock'enzymes account for the major costof converting cellulosic rr'ateriais to

ethanol. The combination of pretreat-

ment with CBP produces a lignin thatis suitaUte for use as a boiler fuel and

contins enough energy to run theconversion Process'

The selection ofProcess andprocess conditions dePends on the

fre of feedstock' For examPle' wood

ot .o* stalks may lend themselves topretreatment followed by fermenta-iion,leaving a solid lignin coprod-uct that can be used as a chemicalfeedstock or boiler fuel' Gasificationfollowed by catalyic reforming maybe more aPProPnate for municiPalsolid wastes' which maY be a mxflffe

of plastic, cellulose and other types

ofrganic components that are notreadiiy broken down bY enzymes ano

microbial fermentatron'

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a Figure 4. The basic unit opefatons in a biorefinefy -afe-feedstock

pfeprJigl:lf119^ltjlent' hydrolysis' Yeast is most commonly used infermeniation, and distiilarcn. ril is iecovereo u"-*ptooutt no'gasifieo

or combusted' making the industrial ethanol fermentations to

;ilffi;r'sr;rit;-iutririrnt. dource:Adapted from Ref' 22' produce both fuels and beverages'

cellulosetoenzymesthathydrolyzethesemacromolecules.T:.::thedifferenttypesofyeasls,Saccharomycescer.(5, 6). Hydrorysis produces bothfive-carbon and six-carbon "r*ro"'l*'il"f"r.d

b";r. oiit, ab'ity to produce ethanol

sugars in a ratio of about 5:3. at high .ol".nt utions and to perform reliably in commer-

Modem biotechnorogy (7) emproysgenetic modiflcation "iat star"',-to-ethanol facilities' s' cerevisiae metabolizes '

of.yeast and bacteria to enable them to 1"r*:1g.;* nr*or", glucose into ethanol under anaerobic conditions

through the

and pentoses derived fro ceilulosic substrates Ji" "rrr*"r *u"n-tvr"yerhof pathway, making con as a byproduct'

or orher biotuel molecules (Step 4 in Figure ? i;;; il;- . Th;;;i*it'o'g*itm ro' ttre conversion of celluiosic

fast, consolidated bioprocessine (CBP) *r**t'tt"p' bi"-;';;;oiy'ut"'loto ethanol should be tolerant of

and 4 by integrating cellulase production *d ,";i;;;"- lfrfilors and pioducts' consum a wide range of substrates

tation i one microorganism d carrying "", n;"fii, ""d lborh h"*or" and pentose sugars), and have high productiv-

fermentation in a singre vesser (Figure 5). ity to result in hig-h yield. s. cerevisiaemeeti most of these

CBp is made possibie by the genetic engineering of yeast

"iit"",;"'iifuJt' ttt"uUility to consume a wide range of

58 wwwaiche org/cep March 2010 CEP

34.60/o 332%43.8o/o 41 .Oo/oCellulose 21 .o%14.9% 15.Oo/o 18.3o/oXylan 3.2%5.6% 0.0% 2.5%Arabinan, Mannan,

Galactan^ '70/^

NotAvailable

2.5o/oAcetyl 3,6%

10.8% 10.2%Extractives 3.o/o

3.0%Not

Available5.7o/o

Protein NotAvailable

NotAvailable

17.70/o 17.9%Lgnin 29.1%

29.1%1.0o/o 10.2% 3.70/o

Ash 1.1o/o93'8o/o 94.1o/o 97.4o/o

Total 101.7% 95 99Estimated Maximum Ethanol

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substrates - it is unable to pro-cess five-carbon (pentose) sugars.

The rnajor focus for the pastf5 years has been on introducinggenes that allow microorganisms,suclr as S. cerevisiae. Zvmomo-nas ntobilis, Pichia stipitis andEscherichia coli. to fermentxylose to ethanol. Metabolic engi-neering has created a variety ofmicrobial strains, both prokary-otic bacteria and eukaryotic yeast,that are able to ferment five- andsix-carbon sugars from cellulosicbiomass.

Both metaboiic engineer-ing and traditional selectiveculturing approaches have beenapplied to various microorgan-isms to enhance biofuel produc-tion performance. This requiresfinding new micioorganisms orimproving the e.xisting industrialmicrooreanisms to increase therange of sugars that can be metabolized, to improvetolerance to ethanol or other fuel molecules, and toimprove tolerance to other inhibitors present in cel-iulosic materiai processing streams (e.g., acetic acidand sugar-degradation products such as furfural and5 -hydroxymethylturfu ral).

Metabolic engineering of S. cerevisiae to enableit to fement pentose sugars commonly found in cel-lulosic biomass. such as D-xylose and L-arabinose,has been a area of active development for more thana decade (8). Figure 6 illustrates the rnetabolic path-ways for D-xylose and L-arabinose to enter centralmetabolism. Pathways found in bacteria (blue) do notinvolve the production ofsugar alcohols orreliance onNAD(P)H and NAD(P)+. which is typical of tungalpathways (green).

Because xylan is the largest pentose constituent ofcellulosic biomass and D-xylose metabolism is conceptuallysimpler than that of L-arabinose, engineering of D-iyloseutilization has been the focus of significant effort. Two mainapproaches have been pursued, relying on either fungalor bacterial genes to convert D-xyloseto D-xyhilse-5-phosphate, which can subsequently be converted to ethanolby genes native to S. cerevisiae. Ofthe two approaches. theuse of bacterial genes has shown somewhat greater promise,since the bacterial pathway does not require balancing the

A Figure 6. D-xylose and L-arabinose can enter central metabolism via bacterial(blue) or fungal (green) metabolic pathways.

enzymatic cofactors N'AD(P)+ and NAD(P)H. The bacterialpathway for L-arabinose utilization has a similar advantage.In addition to introducing genes for pentose metabolism,improvements in utilization of pentose have been realizedby introducing additional modifications related to pathway

. regulation, cofactor utilization, and sugar transport.Organisms for CBP need to produce hydrolytic enzymes

(e.g., cellulases and hemicellulases) that will depolpenzeand debranch the polysaccharides in pretreated biomass toform sugars that it can feiment. The number of hydrolytic

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most organisms that arenaturally capable of breaking down plant biomass expressm'ore than 50 such enzymes.

Significant progrcss has been made in engineering strainsof S. cerevisiae that are capable ofbreaking down bothxylan and cellulose (9). Expression ofas few as two to fiveenzymes appears to be sufficient to achieve some degree ofsugar release from xylan and cellulose' However' there issubstantial room for improvement in both extent and rate ofconversion of cellulosic biomass to ethanol.

As additional and improved hydrolytic enzymes are suc-cessfully expressed in S. cerevisiae, eSpecially when coupledwith improvements in pentose sugar utilization and toleranceto inhibitors, the cost for CBP will fall.

Ferrnertation irhl bitorsThe major inhibitors present in biomass hydrolysates

- weak acids, furan derivatives, and phenolics - exertinhibitory effects by different mechanisms. Furan derivatives(i. e., furfural and'5-hydroxymethylfurfiral) resuit from thedegradation of the sugars found in the hemicellulose andcellulose fractions during processing. Phenolic compoundsare formed by the degradation of the lignin fraction. Portionsofhemiceliulose are acetylated, and acetylated groups arereleased as acetic acid during the pretreatment process.

These inhibitors negatively affect product yield, volu-metric productivity (grams of product per liter per hour),and microorganism growth rate. For exampie, furfuraland 5-hydroxymethyl furfural strongly inhibit cell growththrough direct inhibition of key dehydrogenase enzymesin the metabolism of sugars.-Fuel molecules produced byfermentation (i. e., ethanol, butanol, isobutanol) becometoxic to the microorganism as their concentrations increaseduring the course of the fermentation (7' l0' 11)' From aprocess economics standpoint, concentrations of biofuelproduct in excess of 5% (w/v) alcohol in aqueous fermen-tation medium is desired. Higher concentrations reduceproduct recovery costs.

Direct genetic manipulation of eukaryotic organisms toimprove tolerance to inhibitors is difficult because cellularresponse to stress involves multiple genes, cell signaling,and various transcription ftors. Selective cultunn'g ofmetabolically engineered microorganismsralready able toferment the sugars from cellulosic biomass is the methodthat has generally been more successful for imprvingtolerance.

Selective culturing involves.applying Darwinian seiec-. tive pressures to favor microorganisms with improved

fermentation performance in the presence of inhibitors.Through spontaneous or induced mutations, a population

.

of microorganisms is grown through multiple generationsin media containingth inhibitors toward which improvedresistance is desired. More-tolerant organisms grow morequickly and eventually dominate the population. Two-, five-,and even ten-fold improvernents in volumetric productivityare not uncommon after 100-200 generations.

Hn:yn':e inh:bitorsEnzymes constitute a major cost in the bioconversion

of cellulose to ethanol (12). Factots that reduce enrpeactivity inciude: nonproductive adsorption of enzyme ontolignocellulosic substrates prior to reaction; intermediate andend-product inhibition; mass-transfer limitations affectingthe transport of e enzyineto and from insoluble substrates;the distribution of lignin in the ceU wall; the presence ofhemicellulose, phenolic compounds, proteins and fats;lignocellulose particle size; and crystallinity and degree ofpolymerization of the cellulose substrate.

Enzyme hydrolysis of pretreated cellulosic materialsslows as the concentation of solid biomass increases, evenif the ratio of enzyme to cellulose is kept constant (l 3, 14)'This form of inhibition is distinct from substrate and productinhibition, and has been observed in lignocellulosic materi-als such as wood, com stover, switchgrass, and com wetcake at solids concentrations above 10 g/L.

Identifying enzyme inhibitors and moderating thetreffects is very important. Achieving favorable ethanol-production economics requires at least 200 glL of cellulosicsubstrates to produce monosaccharide concentrations of100 gA and, in turn, ethanol titers (concentrations) of50 g/L'Most inhibitors that reduce hydrolysis activity of cellulaseenzymes are released as the cellulosic biomass is brokendown in the pretreatment and hydrolysis steps. These com-pounds include vanillin, syringaldehyde, ffans-cinnamic acid,hydroxybenzoic acid, soluble xylans (xylo-oligosaccharides)and xylose, and the products ofcellulase action (i'e., cello-biose and glucose) (14). Allsignificantly inhibit hydrolysis ofcellulose and must be removed to maiimize enzyme activity,either by hydrolysis or other means.

Yherrnochern ical eonversio nUnlike bioconversion processes, thermal processing

requires high temperatures and a source of heat to initiateand propagate reactions that convert the chemical backboneof plant matter (r.e., cellulose, hemicellulose, lignin andorganic extractives) to CO, H' and water. In biochemi-cal processes, the pretreatment step that precedes ceilulosehydrolysis may constitute a significant expense, whereasin thermal processing routes, gasification and gas clean-upare significant expenses. In pretreatment, plant biomass is

6Gl' www.aiche.org/cep March 2010 CEP

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processed in a soiid or aqueous form before it is incubatedwith enzymes to achieve further hydrolysis. In thermalprocessing, the solid feedstock is converted directly to asynthesis gas by thermal or auto-catalytic gastfication (i.e.,partial combustion) of the organic material in the presence ofconholled amounts of oxygen. The costs of enz)rmes, in thecase ofbioconversion, or inorganic catalysts, in the case ofthermal processing, add significant additional expense, andhence, motivate research in biocatalysis/catalysis researchaimed at reducing these costs. Overall, thermochemicalprocesses are more complex than cellulose bioconversionprocesses (compare Figures 1 and2), but the technologiesIre more mature.

Cellulose may be broken down through thermal process-ing that gasifies lignocellulosic materials, including the ligninfraction, to a mixture of CO, tlr, *d CO, athigh tempera-tures. When th gases are cleaned up and then passed oversolid catalysts, alcohols higher than ethanol or other fuelmolecules are formed. Since the reactions are carried out bythe Fischer-Tropsch process, the resulting fuels are some-times termed FT tiquids. An alternative process in which thegases are fermented by bacteria that are capable ofconvertingCO and H, to ethanol has also been proposed (23).

The processes that occtu in a gasifier are drying, pyrolysis,combustion, and gasificatio n ( I 5 , I 6) . Wood and biomassform tars, particularly in updraft gasifiers; consequently,downdraft configurations (in which the cellulosic biomassflows through the gasifier in a downward direction) are morecommon. Air is injected near the bottom of the gasifier, wherecombustion occurs at an oxidation temperature above 1,000'C(the theoretical maximum is 1,450'C). Heat released duringcombustion dries and pyrolyzes the biomass above it. Organicacids are released from the biomass during the drying process,

Neutr"alizing

A Figure 7. Bodiesel can be produced from vegetable oils and anmal fats via chemicalprocessing. Source'. (1 7).

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necessitating a corrosion-resistant construclion (1 5).The combustion reactions are:

c+oz*cqzHz+ 02---2HzOC+CO2

-2COc+H2O*CO+H2CO+Hr

-COz+Hz2Hz; CH+COr+F1"-CO+HzO

(+ 393 MJlkg-mole) (1)(+ 393 MJ/kg-mole) (2)(-64.9MJ,&g-mole) (3)(- 122.6 MJ/kg-mole) (4)(+ 42 MJ/kg-mole) (5)(+ 75 MJ/kg-mole) (6)(- 42.3 MJ/kg-mole) (7)

Endothermic reduction (Reactions 3,4 andT) reducesthe temperature of the reaction mixture to the range of800-1,000.c.

Pyrolysis products (i.e., tars) are also formed duringcombustion. In theory these pyrolysis products should becracked into smaller molecules as they pass through the bedof hot charcoal created bythe partial combustion of the bio-mass at the bottom of the gasifier. In practice, however, thecracking of pyrolysis products in a gasifier is less than 1.00%efficient, so filtration of the hot gases or other separationmethods are needed to remove the tars before the synthesisgas is passed over the catalyst.

The gas must be cleaned up to reduce catalyst foulingand achieve the catalyst life required for economic opera-tion. When the reaction is carried out in the absence ofoxygen and the miterial is heated to about 800'C, an oilymaterial forms. This oil may be processed further (throughcracking) into diesel-like molecules, sometimes referred toas green diesel. The viscosity ofthe oil and its tendency toform a tar-like material are issues that need to be addressedby research ifrobust scale-up is to be achieved (17).

An alternative approach to chemical catalysis forconveding CO and H2 to fuels is a biocata-lytic process. A group ofbacteria knowncollectively as acetogens can convert thesegases into a mixture of ethanol, butanol,acetic acid, butyric acid, and methane.Low ethanol yields, relatively low produc-tivities, and the challenges inherent ingas/liquid mass kansfer must be solved tomake this a commercially viable approach.The recovery oflow concentrations ofthefinal products requires separations that addto the cost. Hence, the cost of pretreatmentand enzyme hydrolysis is replaced by adifferent set of costs that include catalystseparation and recovery.

. The. benefit of simplified front-end-processing in a thermochemical process is

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a Figure L Biomass-to-biofuel conversion technologies are in various stages of development.Source: (17.

higher alkanes is known as a p-series fuel(19, 20). Depending on the composition,P-series fuels can be derived from non-petroleum sources, such as waste paper,agricultural residues, and urbadindustrialwaste. The Biofine process developeC atPacifi c Northwest National Laboratoryproduces MTHF from lelulinic acid in asingle step with high yielde.

The development of industriallyrobust catalysts, optimization of reac-tions to'achieve high yields, and securingcost-effective sources of relatively puresugars are challenges that must stili beaddressed (17, 19,20).ofFset by the increased complexity of post-gasification pro-

cessing and the loss of the sugars'functionality. pretreatmentis preferred when the goal is to obtain sugars for biochemi_cal and biological fermentation routes.

Ch*rnicat {$nversonThrough a series ofcatalytic reactions, lignocellulosic

biomass can be converted into levulinic acid, which can then

octane number as regular gasoline (87), tow Reio vaporpressure (5.7 psi), and a lower heating value (32 MJ/kg) thatis slightly higher than that of ethanol (26.7 MJkg). This setof properties allows it to be mixed with ethanol and naturalgas iiquids to create a transportation fuel.

A mixture of ethanol, MTHF, butane, pentanes, and

eornparing hieefNernieaIand therrllocl'e-r ica { paf f?ways

Starch- and sugar-based ethanol processes are maturecommercial technologies, with com the dominant feedstockin the U.S. and sugarcane in Brazil. Corn (including thecorn stalks) has the potential to provide up to 22 billiongallons ofethanol per year ifboth celluloie and starch areconvefted to ethanol (21).

wastes generate biomethane.Biodiesel has been produced both for use as a chemical

and as a biofuel in the U.S. Vegetable oils are the pnncipalfeedstock for a process (Figure 7) that reacts methanol withliquid vegetable oil using an acid catalyst to form methyl

Sugar/Starch EthanolCellulose EthanolP-SeriesBiodiesel and HDRD-Synthetic Bio. FT DieselMethanolDmerhyt Ether (DME)BiomethaneBiosynthetic Natural GasGreen Pyrolysis Diesel

' HDRD = Hydrogenation-Derived Flenewable Diesel (r'.e..

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esters (1.e., biodiesel). In this case, the preflx blo- refers tothe source of the feedstock - a biologically derived veg-etable oil (rather than the type ofprocess, such as fermenta-tion or digestion).

Biomethane is a common product of some iandf'lls'as well as digestion processes that handle animal wastes

material. Methane is less likely to be usqd as a transportatlonfuel because of its low physical and energy density ancl thelimited infrastn-rcture for its distriblltion (17)'

Figure 8 compales various chemical, therrnochemicaland biogical tehologies for kansforming lignocelluiosicfeedstocks to ethanol and their stages of development'

{ Ii}s,i :1.: i 1.':: i:3 1"1-f,.tEthanol is produced in iarge quantities, and an esti-

mafed 12 biliion gal will be derived from corn in 2010'Since the celluiosic portion ofthe corn kernel is a potentialsource of an advanced biofliel as well, cellulosic ethanol islikely to be the first such fuel on the market' The technolo-gies io process wood and other lignocellulosic feedstocksurrently under development wiil enable the rapid expan-sion of cellulosic ethanoi production from non-food feed-stocks and lead the way for other advanced biofliels oYg,*.=the next ten years. l'"'i{:i

AcxowlroGMENTsThe authors thank Jm Ftatt and Todd Polanowicz of luascoma Corp for therreview ofthis articte and he{pfut suggestions' The material in this work wassupported by DOE contract DE-FC-3'o8Gor8ro3 and Mascoma L0rp'

Lrrsutunr rso1

Biotechnol Bioeng, 16, pp. 141 1-149i (197 4)'NIandels, M.o et al., "lvleasurement of Saccharifting Cellulase""Biotechnol. Bioeng. Symp

' 6, pp- 21--23 (1916)'

Sierra. R., e/ a/., "Producing Fuels and Chemicals from Ligno-cellulosic Biomass," SBE Special Section, Chem' Eng' Progress'104 (8), pp S10-S18 (Aug.2008).Wyman, C.8., et al., "Coordinated Deveiopment of LeadingBiomass Pretreatment Technologies," Boresource Tbch'' 96'pp. t9s9-1966 (2005).Mosier, N. S', e! trL, "Features of Promising Technologies for

Prereent of Lignocellulosic Biomass," Bioresource Tech ' 96'pp. 673-686 (2005)'Mosier, N., and M. Ladisch, "Modem Biotechnology: Connect-ing Innvaiions in Microbiology and Biochemistry to EngineeringFundamentats," Miey, Hoboken, NJ ; p' 433 (2009)'Hahn-Hgerdal ,8., et aL, "Metabolic Engineering for PentoseUtilrzation in S ccharomyces cerevisiae," Adv' B iochem Eng'Biotechnol., 108, pp- 141-177 (2007)'

Kumar, R., and C. E. Wyman, "Effect of Supplementation atModerate Cellulase Loadings on Initial Glucose and Xylose Releasefrom Com Stover Solids Pretreated by Leading Technologies"'Biotechnol Bioeng., 102 (2), pp- 4547 Q009)'

14. Ximenes, 8., et al,, "Inhibition oICellulases by Phenols"' Enz

Microb. Technol., 46 (34), pp. 170-176 (2009)'15. Rajvanshi, A. IC, "Biomass Gasification," Chapter 4 in "Alterna-

tive Energy inAgriculture," Vol. II, Goswami, D Y', ed'' CRCPress, Boca Raton, FL, pp. 83-102 (1986)'

16. Jdrapur, R, andA. K. Rajvanshi, "sugarcane Leaf-Bagasse Gas-ifiers for Inciustrial Heating Appl ications"' Biomass and Bioenergy'i3 (3), pp. 141-146 (199'7)-

17. Schwietzke ,5., et aL, "Analysis and Identificatin of Gaps inResearch for the Production ofsecond Generation Liquid Transpor-tation Fuels," www.ieabioenergy com, T41 (2) (2008)'

18. Pacific Northwest National Laboratoryo "Conversion of BiomassWastes to Levulinic Acid and Derivatives"' www pnl gov'/biobased/completed.stm (1999)"

19. U.S. Dept. of Energy, "P-Series," DOE Energy Efficiency and

Renewalld Energy,lternative Fuels andAdvanced Vehicles DataCenter, www. afcl c.energy' gov/afdc/fu e1s/emergiir grseries'html(19ee).

20. Iluber, G.'W.,.et al-, "Synthesis ofTransportationFuels ftomBiomass: Chemistry, Catalysts, and Engineering," ChemicalReviews, 106 (9), pp. 40444098 (2006)'

21. Schwietzke,S., et al',."Erhaol Production ftom Maize"' tn

"Molecular Genetirc Approaches toMaize Improvement"' Kris'

A. L., and B. A. Larkins, eds., Springer Verlag, Berlin' 63' pp'

347-364 (2009).22. Eggeman, T., and R. T. Elander, "Process and Ecoomic Analysis

of-iretreatment Technologies''l Bjo resource Technolog' 96 (lE)'pp. 2019-202s (200s).

23, Datar, R. P. , et aL, "FermenaJion of Biomass-Generated ProducerGas to Ethanol,' Biotechnol- Bioeng ,86 (5),pp' 587-594 (2004)'

",

1

g. Van Zyl, W. fi'., et aL, "Consolidated Bioprocessing for Bioethanol

Production usrng Saccharomyces cerevisiae," Adv' Biochem' Eng'

Biotechnol.,108,pp.205-235 (2001)' !10, Ladisch, M. R' "Fermentation-Derived Butanol and Scenaris for

its Uses in Energy-RelatedApplications," Enz' Miuob' Technol ' 73'

pp.280-283 (1991).11. Shota, A'' ef /., 'Non-Fermentative Pathl/ays fo;: Synthesis of

Brnched-Chain Higher Alcohols as tsiofueq" Iy'ature' 451 (3)'pp. 86-89 (2008).

12. Ifoughton, J' C., et al., "Breaking the Biologicai Barriers toCellulose Ethanol' A Joint Research Agenda," Publication No'

DOE/SC-0095, U.S. Dept' of Energy, Washington' DC (2006)'

GEP March 2010 www arcne org/cep Gi3