A PowerPoint presentation given as part of the 2005 Hydrogen Program Review, May 23-26, 2005, in Washington, D.C

Czernik, S.

Maness, P. C.

Smolinski, S.

UNT Digital Library

Fermentative Approaches to Hydrogen Production Pin-Ching Maness, Stefan Czernik and Sharon SmolinskiNational Renewable Energy LaboratoryDOE HFC&IT Program ReviewMay 24, 2005Project ID # PD18This presentation does not contain any proprietary or confidential informationNREL/PR-560-38184Overview• Project start date: FY 05• Project end date: on going• Percent complete: NA• Production Barriers addressed– Barrier AI: H2 Molar Yield– Barrier AK: Feedstock Cost• Total project funding– $200K (DOE share)• Funding received in FY04: $0.00• Funding for FY05: $200KTimelineBudgetBarriers• Interactions/collaborationsDr. Bruce Logan, Dr. Jay Regan, Penn State UniversityDr. Lee Lynd, Dartmouth CollegeDr. David Levin, Univ. of Victoria (Canada)PartnersObjectives• The long-term goal is to assist DOE in developing direct fermentation technologies to convert renewable biomass resources to H2• The objectives in FY05 are to: – Screen and identify cellulolytic microbes which can produce H2 directly from cellulose and hemicellulose, major constituents of biomass– Identify up to 3 suitable strains of fermentative microbes to select one from for pathway engineering to improve H2 molar yield in FY06 and beyond (FY05 Milestone)Approach to Address Feedstock Barrier (AK)• Problem: Near 75 to 90% of lignocellulosic biomass is composed of sugars, ideal substrates for H2 production. NREL’s Biomass Program is developing technologies to lower the cost of glucose from biomass to 8 cents per pound by 2015• Approach: Bio-prospect cellulolytic microbes that can convert cellulose and hemicellulose (xylose) directly, in lieu of glucose, to H2 as an alternative and valid strategy to lower the feedstock cost barrierComponent % Dry WeightCellulose 40-60%Hemicellulose 20-40%Lignin 10-25%Approach to Address H2 Molar Yield (AI)• Problem: Molar Yield of H2 (mol H2 /mol sugar) is too low (2 to 2.5) due to the simultaneous production of other fermentation waste byproductsGlucose + 6H2 O             6CO2 + 12H2Glucose + 2H2 O             2Acetate + 2CO2 + 4H2Glucose                          Butyrate + 2CO2 + 2H2• Approach (FY2006 and beyond): Select a suitable cellulolytic microbe of known genome sequence for metabolic pathway engineering – Block competing pathways has been demonstrated in literature in improving H2 molar yieldTechnical Accomplishment/Progress – Screening 9 Strains of Clostridium thermocellum• Avicel® is the most recalcitrant cellulose• Fermentation was carried out at 55 °C• H2 production resumes when the headspace H2 was displaced with an inert gas 02000400060008000100000 50 100 150 200HoursHydrogen, nmol/ml gas phase7.7.107.8.37.10.47.12.1YSAvicel celluloseTechnical Accomplishment/Progress – Screening 9 Strains of C. thermocellum02000400060008000100000 20 40 60 80Hoursnmol H2/ml gas1.1.1ATCC• Cellulose utilization is noted by a change in colorStrains were kindly provided to us by Profs. Lee Lynd (Dartmouth College) and Ed Bayer (Weizmann Institute of Science, Israel)celluloseTechnical Accomplishment/Progress – Identified the Suitable H2 ProducerStrains Rate of H2 Production*ATCC 10181.1.1 595YS 4777.10.4 4477.12.1 4077.7.10 357.8.3 Traces6.3.2 Traces7.9.4 Traces• Screened 9 strains of cellulolytic microbes• ATCC strain has the highest rate. Work is ongoing to optimize its growth conditions to eliminate lag phase  • Strain 1.1.1 was selected for scale-up experiment due to its fast growth rate in cellulose• Screening effort is ongoing• Using cellulose in lieu of glucose will meet the technical target of lowering the feedstock cost* nmol H2 /hr/ml culture gas phaseBioreactor Configurations for Cellulose Fermentation• pH and temperature controlled• Operate two reactors simultaneously• On-line continuous sampling of reactor gas phase via gas chromatography• H2 /CO2 is vented continuously,no pressure buildup Technical Accomplishment/Progress: H2 from Cellulose in Bioreactor• 0.5% (w/v) Avicel was consumed completely• Fermentation waste byproducts are ethanol, acetic, and butyric with traces of lactic and formic acids• Carbon mass balance approaching 95%• H2 molar yield near 2• First demonstration of H2 molar yield data from cellulose0204060801001200 25 50 75 100 125 150 175HoursH2 or CO2, mmolH2CO2C. thermocellum 1.1.1Corn Stover Pretreatment: Steam ExplosionCorn StoverSteam Explosion (acid or neutral) Aqueous Hemicellulose  (5- and 6-carbon sugar oligomers)Solid Lignocellulose(cellulose & lignin)Liu et al. 2002. Biotech Bioengineering 77: 121-130.Technical Accomplishment/Progress: H2 from Corn Stover Lignocellulose Solids020004000600080000 20 40 60 80Hoursnmol H2/ml gas phaseNeutralAcidC. thermocellum 1.1.1Solids before and after fermentationNeutral: 220 °C, 3 min Acid: 190 °C, 1 min¾ Near 98% and 90% of cellulose and hemicellulose were consumed, respectively.Responses to Previous Year Reviewers’ Comments• This new project started Oct 1, 2004 and has not been reviewed previously Future Work• Remainder of FY2005:– Screen additional cellulolytic microbes such as Clostridium cellulovorans, C. cellulolyticum, etc.– Further optimize fermentation parameters in scale-up bioreactor– Determine carbon balance and H2 molar yield– Identify the best microbe of known genome sequence for metabolic engineering in FY2006 (FY2005 Milestone)• FY2006:– With the selected model microbe, conduct metabolic profiling to determine the most effective strategy to re- direct biochemical pathways (FY2006 Milestone)– Begin genetic engineering to block competing pathways to improve molar yield of H2Publications and Presentations• Publications– Datar, R., J. Huang, P. C. Maness, A. Mohagheghi, S. Czernik, and E. Chornet. Hydrogen production from the fermentation of corn stover biomass pretreated with a steam explosion process. Submitted to Environ. Sci. Technol.– Lee, J. Z., D. M. Klaus, P. C. Maness, and J. R. Spear. Characterization of the effect of butyrate on hydrogen production in photofermentation for use in Martian Resource Recovery. Submitted to Intl. J. Hydrogen Energy• Presentations– The 10th Annual Meeting of Institute of Biological Engineering, Athens, GA. March 2005– Graduate Student Seminar Series, Dept. of Civil & Environ. Engineering, Penn State University, PA. April 2005Hydrogen Safety• The most significant hydrogen hazard associated with this project is the use of H2 - containing anaerobic glovebox for sample preparations under anaerobic environment– Anaerobic glovebox routinely contains 2-3% H2 (in N2 ), provided via a 10% H2 gas cylinder (in N2 )– Inside glovebox are small electrical devices and power cords needed for sample preparationsHydrogen Safety• Our approaches to deal with this hazard are:– Install H2 /O2 gas monitor inside the glovebox, with alarms set at 10% H2 and 300ppm O2 (Factory preset)– Maintain H2 level inside the glovebox at 2-3% (in N2 ) – Activate palladium catalyst frequently– The power cord is unplugged from the mains (outside) first prior to any (dis)connection inside the glovebox– Use a flammable gas detector to detect potential H2 leaks out from the glovebox– NREL laboratory ventilation system provides 6 to 10 complete air exchanges per hour in the event of a catastrophic leak– The DOE Hydrogen Safety Review Team visited NREL in 2004 and we have incorporated their suggestions in our AOP.

Fermentative Approaches to Hydrogen Production

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Fermentative Approaches to Hydrogen Production

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