8/14/2019 Cell Bacteria

1/4

Advances in Research at theNotional Renewable Energy laboratory

Mini-Manhattan Project for CellulasesNREL Pursues Enzyme DevelopmentCritical to Viability of BiofuelsThe National Renewable EnergyLaboratory (NREL) is working tomake ethanol Americas automotivefuel of the future by domesticallyproducing it from lignocellulosicbiomass, the most abundant renew-able resource on earth. Extensiveproduction of this environmentallycompatible fuel is contingent on theability to convert wood, grasses,agricultural wastes, wastepaper, orother biomass to ethanol at pricescompetitive with gasoline. A keyelement of achieving competitiveproduction costs is the ability toeconomically produce the cellulase

enzymes that convert cellulose intosugar so yeasts can ferment the sugarinto ethanol.Cellulose and hemicellulose, thefibrous and woody material that con-stitutes the bulk of most plants, arecarbohydrates. But unlike starchesand sugars, they are ones thathumans and most animals cannotdigest and most yeasts cannot fer-ment. The common enzymes thatconvert starch to sugar are ineffectiveon the bonds that hold together thechains of sugar molecules in cellu-lose and hemicellulose. In cellulose,

This shelffungus (Ganodermaapplanatum)produces cellulase enzymes that enable itto digest cellulose.NREL scientists are using cellulases from microscopic bacteria andfungi to produce ethanol from lignocellulosic biomass.

there are also hydrogen bond inter-connections between the chains ofsugars. The resulting crystallinestructure is not soluble in water,which makes enzymatic attackdifficult.Carbohydrate chains are brokendown to sugars by a chemical reac-tion called hydrolysis. Dilute acidsare commonly used to hydrolyzehemicellulose, but for cellulosehydrolysis, enzyme catalysts calledcellulases produce the best results.Cellulase enzymes are needed, how-ever, in large amounts to be effective.This requirement is a major obstacleto cellulose hydrolysis because of thehigh cost of the enzymes. That highcost is caused by the slow growthand production rates of the fungigenerally used to make the cellulases.Fungi Versus BacteriaNREL and its subcontractors areworking with bacteria and fungi tofind the most efficient means ofproducing highly effective cellulases.Most cellulose-digesting fungi andbacteria produce sets of at least threedifferent kinds of ccllulase enzymesto effectively attack the complexstructure of cellulose. (They oftenalso produce separate sets of hemi-cellulases, enzymes that attackhemicellulose.)The three general components ofcellulase enzyme systems are endo-glucanases, exoglucanases, and beta-glucosidases. Endoglucanases attackthe cellulose crystalline structure atrandom points, breaking the linearchains of glucose molecules to

8/14/2019 Cell Bacteria

2/4

Cellulase

dons and enzyme preparations,dead ends'' fromthis and subsequentarchived for possible use in futureL research or for transfer to industry.

Because there are so many potential "dead ends," esearchersmust process manycellulases (several of which may come from a single organism) and their genes throughthe pathway shown above.produce shorter chains. Each breakproduces two new chain ends. Exo-glucanases attach to these exposedends of the chains and, workingdown the chains, release cellobioseand some glucose. Cellobiose, whichconsists of two connected glucosemolecules, is then split by beta-glucosidases.In nature, fungi tend to producemore cellulase enzyme than bacteria;however, cellulases produced bybacteria are often more effectivecatalysts. They may also be less in-hibited by the presence of material

that has already been hydrolyzed(feedback inhibition). Researchershave not definitively determinedwhich organisms have greater effec-tiveness.Fungi currently produce most com-mercial (extensively used by the foodprocessing and detergent industries)and laboratory cellulase. But severalfactors suggest that bacteria mayhave greater potential. Bacteria growmore rapidly and easily. Also, somebacterial cellulases tolerate very hightemperatures, a valuable charac-teristic because chemical reactions

Nature's Woody Biom ass Recyc lersAlthough humans and yeasts cannot digest the cellulose and hemicellulose..... ... ........................... ......come to mind cannot d o so inherently Fungi, which derive all their energyfrom dead or living trees or other plant material, do have the ability tobreak cellulosic material down to sugars, a s do the various soil and aquaticbacteria responsible for the decay of plant material. These two classes oforganisms are nature's primary agents for recycling lignocellulosic biomass.Nearly all other consumers of cellulosic material from beavers to grass-eating cows and horses, to wood or paper-eating termites and cockroaches,rely on the bacteria, fungi, or, in the case of termites, protozoa colonizingtheir digestive systems. These symbiotic guests break down bonds in thefibrous material and enable their hosts to live on a diet of biomass. Fungiand bacteria produce enzymes that attach to the chains of molecules in

I. 1 7 , . .I 1 .. I 1 . . . ... . . . . .ceiiuiose ana nemiceiiuiose ana nyaroiyze or D r e a K aown rnis material intodigestible or fermentable sugars. NREL researchers seek ways to produceand employ these enzymes on an efficient industrial scale so they can uselignocellulosic material as a feedstock for fermentation into ethanol.

Several factors suggest thatbacteria m ay have greaterpotential, particularly theease wi th which bacteria canbe genetically engineered.are generally faster and more costeffective at elevated temperatures.High-temperature tolerance oftenalso indicates a greater tolerance ofother physical stresses, such as shearforces. Of greatest potential impor-tance, however, is the ease withwhich bacteria can be geneticallyengineered. This factor is likely tobe invaluable in achieving cellulaseproduction goals.Imp rov ing Technology fo rUse in Pilot PlantFor the near term, NREL is concen-trating on the extant fungal produc-tion system. That system generallyuses the two endoglucanases and thetwo exoglucanases produced by thefilamentous fungus Trichodemareesei, and beta-glucosidase from thefungus Aspergillus n i p - . The immedi-ate objective is to maximize thepotential of the current system bylate 1994.At that time, NREL plansto begin operating a fermentationprocess development unit (I'DU) orpilot plant now under constructionat its Golden, Colorado, site. Thegoal is for the new PDU to employ

8/14/2019 Cell Bacteria

3/4

ategy

y So,.,e]I fsucceed NoI Re-enaineer Icontrhingcomponents1 fgene I

the best cellulase production technol-ogy available.One current project strives to identifythe optimum ratio of the four enzymestaken from T. reesei . Research hasshown that the ratio of endoglucan-ases and exoglucanases is critical.However, that research has generallybeen done on refined feedstockmaterials, and NREL seeks to iden-tify the best enzyme mix for several"real world" biomass feedstocks thatthe PDU will likely use.A second project aims to improveassay techniques for identifying andmeasuring endoglucanase enzymes.This is important for screening andselecting the best enzyme producers.These projects are being conductedboth in house and by subcontract.A third project will focus on selectingalternatives to the A . n i p ungalstrain to more effectively producebeta-glucosidase. Beta-glucosidaseplays a critical role because highconcentrations of cellobiose (con-verted to glucoseby beta-glucosidase)inhibit the action of the T. reeseiexoglucanases.Moving Beyond CurrentTechnologyOne advanced technology projectthat NREL has subcontracted is theinvestigation of cellulase/substratesurface interactions. The binding ofcellulases to cellulose is crucial tothe process, but the chemistry of thisevent is not understood in detail. A

Fe'

Fcould be the ability to select or builda single "super" endoglucanase orexoglucanase.Although genetic engineering iseasier with bacteria, it can also beaccomplished with some fungi. Cel-lulase technology could be advancedby transferring modified fungal

or bacterial cellulase genes withdesirable traits into T. r m e i . Aprimary objective for such engineer-ing would be to obtain enzymes thatare less inhibited by the presence ofcellobiose or glucose than are theenzymes currently used.

8/14/2019 Cell Bacteria

4/4

Mini-Manhattan ProjectCentral to all intermediate and long-range cellulase production efforts isone that NREL researchers thimk ofas the Mini-Manhattan Project.NREL is aggressively seeking to iden-tify and purify various fungal andbacterial cellulases and their genesso the efficacy of these enzymes canbe compared head to head. Morethan 60 fungi and 50 bacteria areknown to produce cellulase, but inthe absence of central coordination,research has been unable to deter-mine which organisms and enzymesare best for industrial use. By stand-ardizing procedures, NREL plans todetermine the effective activity leveland other characteristicsof the vari-ous cellulases. This multiple-taskinitiative is being carried out byin-house and subcontract researchers.Seven subcontracts contribute toenzyme development and supply,and one is for development of astandard testing procedure.

On the basis of these results, NRELwill develop systems for productionof the best-performing enzymes.Ultimately researchers seek oconstruct a multicellulase geneexpression system in an industrialbacterium. This would require iden-tification of the most effective com-bination of cellulase enzymes andthe genetic material for producingthem in the largest quantity. Thatgenetic material would then be trans-ferred into one or two bacterial hoststhat are environmentally acceptableand can be quickly and inexpen-sively grown on an industrial scale.

Critical Cost Factor forEthanol and Much MoreIn both the short and long terms,cellulase improvement is one aspectof the ethanol-from-biomass processwith great potential for cost reduc-tion. The capability to efficientlyproduce cellulase-and therebyfermentable sugars and their

PublicationsGrohmann, K.; Wyman, C.E.; Himmel, M.E. (1991).Potentialfor FuelsfromBiomass and Wastes. Emerging Materials and C hemicalsfr om Biomass. ACSSeries 476, Washington,Dc:American Chemical Society; pp. 354-392.Himmel, M.E.; Adney, W.S.; Baker, J.O.Cellulase Assays: A Review.Energyfro m Biomass and Wastes X U . hicago, I L IGT. (In press).Himmel, M.E.; Hinman, N.D.; Thomas, S.R. (1993). Cellulase Research forthe DOE Biofuels Program. Accepted for the IEASymposium on Biotechnol-ogy for the Conversion of Lignocellulosics, Helsinki, Finland, June 6-9, 1993.Himmel, M.E.; Leatham, G.F., eds. (1991).Enzym es in Biomass Conversion.ACS Series 460, Washington, D.C.: American Chemical Society.Himmel, M.E.; Thomas, S.R. (1993). Cellulase Research for DOE: An Over-view. Proceedings or the 1992 An nu al Auto moti ve Technology DevelopmentContractors Coordination M eeting . Warrendale, PA Society of AutomotiveEngineers, Inc., (P-265); pp. 125-128.Stockton, B.C.; Mitchell, D.J.; Grohmann, K.; Himmel, M.E. (1991).Opti-mum a-D-Glucosidase Supplementation of Cellulase for Efficient Conver-sion of Cellulose to Glucose. Biotechnology LPtters, 13(1);pp. 57-62.Tucker, M.P.; Mohagheghi, A.; Grohmann, K.; Himmel, M.E. (1989). Ultra-Thermostable Cellulases from Acidothermus cellulolyticus: Comparison withPreviously Reported Cellulases. Bio/Technology, 7(8); p. 817-820.

I

-.

P

NREL researcher collects cellulase-producing bacteria fromhotsprings.One of t h e heat-tolerant species collectedshows great promise for cellulaseproduction.derivatives from cellulosic po ly m er salso has tremendous potentialbeyond ethanol. Whole new indus-tries could evolve from the abilityto produce chemicals and materialsfrom renewable biomass instead offossil fuels.

For More lnformatlonGeneral Information and otherNREL Research Briefs:Technical Inquiry Service(303) 275-4099Technical Information on thisNREL Project:Michael Himmel

(303) 384-6299Steven Thomas(303) 384-6187NREL Business Information:Technology Transfer Office(303) 275-3008

Produced by the Communications and MISBranch for the Alternative Fuels DivisionNRELNK-336-5676-Rev. Revised 8/95National Renewable Energy Laboratow,1617 Cole Boulevard. Golden, Colorado80401-3393Operated fo r t h e U.S. Department 01 Energy by the Midwest Research Instituter.2 Printedwith a renewable source ink on paper containing at least 50%wastepaper V