Rosgaard, Lisa

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Engineering Conferences International

Boosting the cellulytic degradation of steampretreated barley strawLisa Rosgaard Novozymes Inc.Starch R&D•  Babilafuente Bioethanol Project (2003-2007)Babilafuente, Salamanca, Spain• Develop the technology to include lignocellulosefrom barley and wheat husks and straw in a fuel ethanol pilot plant concomitantly utilising barleyand wheat grain• Production capacity is aimed at 200 ML/year of which 195ML will obtained from barley grain and 5ML from lignocellulosic material (barley and wheat husks and straw)• Supported by the EU (5th Framework programme)•  Project partnersProject coordinator•Vehicle testing and marketing of EtOH/gasoline blendPretreatment of wheat straw and SSFPretreatment of barley straw and SSFEvaluate and provide enzymes•  Key research areas at Novozymes• Cellulose conversion• Hemicellulose conversion• Enzyme efficiency• Substrate loading• Lignin-enzyme interaction•  Degradation of celluloseCBHICBHIIEGIEGIIIBGRNRGlucoseCellobioseGlucose oligomerCelloBioHydrolaseEndoGlucanaseStructures modified from NCBI MMDB (Chen J, Anderson JB, DeWeese-Scott C, Fedorova ND, Geer LY, He S, Hurwitz DI, Jackson JD, Jacobs AR, Lanczycki CJ, Liebert CA, Liu C, Madej T, Marchler-Bauer A, Marchler GH, Mazumder R, Nikolskaya AN, Rao BS, Panchenko AR, Shoemaker BA, Simonyan V, Song JS, Thiessen PA, Vasudevan S, Wang Y, Yamashita RA,YinJJ,BryantSH,"MMDB: Entrez's 3D-structure database", Nucleic Acids Res. 2003 Jan; 31(1): 474-7)•  Hypothetical structure of lignocellulosecore ligninnon-core ligninhydrogen bondscellulose lignin hemi-cellulose hemi-cellulosecross linkJ. Bidlack, M. Malone and R. Benson. Okla. Acad. Sci., 1992, Vol. 72.if only it was thatsimple…•  Testing fermentation broths on lignocelluloseSmall scale testing (2g scale at 2% dry matter)6 hours incubationBenchmark: Celluclast:NS188 = 2.3: 1mg enzyme per g reaction ∼8FPU: 13CBU per g solids0.035mg broth protein per g reactionCorresponding to approx. 10% of the total protein loadpH 3.5 50°CpH 5 35°CpH 5 65°CpH 5 50°CpH 4 40°CpH 6 40°CpH 6 60°CpH 6.5 50°CpH 4 60°CL. Rosgaard, S. Pedersen, J. R. Cherry, P. Harris, A. S. Meyer. Biotechnology Progress•  024681012pH6, 60°C pH4, 60°C pH6, 40°C pH5, 65°C pH5, 35°C pH3.5, 50°C pH4, 40°C pH6.5, 50°C pH5, 50°CGlucose g • L-1020406080100120% Cellulose conversion1×2Benchmark2×BenchmarkTesting fermentation broths on lignocellulose-2•  Chaetomium thermophilum -Thielavia terrestris +Thermoascus aurantia +Corynascus thermophilus +Myceliophthora thermophila +Penicillum funiculosum ++ = all optimum at pH 4.8-5, 48-50°C and compare to Celluclast/NS188 on lignocelluloseCellulose conversionpH 4.8, 48°CConclusions• The tested fermentation broth samples significantly boosted the cellulose conversion when benchmarked against Celluclast® + Novozym 188.• The data also demonstrated that spiking, rather than substitution, of Celluclast® + Novozym 188, may be a feasible way forward in industrial, large scale cellulose conversionTesting fermentation broths on lignocellulose- 3•  Increasing substrate loading1101001000100001000001 10 100Shear rate sec-1Viscosity mPa·sec-16 hours24 hours48 hours72 hours• The viscosity of the slurry decrease rapidly within the first hours of hydrolysis. This gives the opportunity to load additional substrateL. Rosgaard, P. Andric, K. D. Johansen, S. Pedersen, A. Meyer. In prep. for Applied Biochemistry and Biotechnology15% DM 10% DM24hours22% DM•  Increasing substrate loading-2• Reaction conditions: whole slurry of dilute acid hydrolysed barleystraw. pH5, 50°C. Full enzyme loading at the start of reaction. time Glucose concentration 6 hours 24 hours 5%10%15%10+55+5+5% Working hypothesisCellulose conversion•  Increasing substrate loading-3050010001500200025000 10 20 30 40 50 60 70Reaction time (Hours)Viscosity (mPas·sec-1)5%10%15%5% +10%+Viscosity (mPas·sec-1)• Viscosity decrease is rapid but does not decrease when additional substrate is added•  Increasing substrate loading 4• Additional substrate loaded does not appear to be efficiently hydrolysedConclusion• There was no net benefit of gradually increasing the substrate loadingwith respect to cellulose conversion or in relation to viscosity. Howeverit is speculated that mixing of substrate and enzyme as well as timing ofadding additional substrate is crucial.010203040506070800 10 20 30 40 50 60 70 80HoursActual conversion %5%10%15%5%+5%+5%10%+5%Cellulose conversion %•  Outlook- Novozymes Biotechnology. Davis, CA.• Enzyme discovery• 2001: US Department of Energy (DOE) and National RenewableEnergy Lab (NREL) collaboration to bring down the cost of enzymesfor ’biomass to bioethanol’ production.• 2005: Target was met and surpassed by a 30-fold reduction in coststo 0.1-0.18$ per gallon.• Extensive research and engineering of cellulases has led to a new generation cellulase products for bioethanol production.

Boosting the Cellulytic Degradation of Steam Pretreated Barley Straw

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Boosting the Cellulytic Degradation of Steam Pretreated Barley Straw

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