Overview of microbial hydrogen production Carolina Zampol Lazaro Stagiaire postdoctoral – Université de Montréal Supervisor: Prof. Dr. Patrick Hallenbeck Senior Research Associate, National Research Council Department of Biology, US Air Force Academy Reduction of CO2 with Hydrogen Actual hydrogen production: natural gas via steam methane reforming (> 90%) Barrier to overcome: sustainable hydrogen production - electrolysis of water and biomass processing (using a variety of technologies ranging from reforming to fermentation). Biological hydrogen producing microorganisms Great diversity! Metabolic versatility! Source: Chandrasekhar, K., Lee, Y.-J., & Lee, D.-W. (2015). Biohydrogen Production: Strategies to Improve Process Efficiency through Microbial Routes. International Journal of Molecular Sciences, 16(4). Overview of the 50-L horizontal tubular photobioreactor Biophotolysis used for outdoor experiments with C. reinhardtii • Abundant substrate = H2O • Abundant energy source = sun light • Simple products: H2 and O2 • Oxygen sensitive hydrogenase • Low light conversion efficiencies Source: Scoma, A., Giannelli, L., Faraloni, C., & Torzillo, G. (2012). Outdoor H(2) production in a 50-L tubular photobioreactor by means of a sulfur-deprived • Expensive hydrogen impermeable culture of the microalga Chlamydomonas reinhardtii. J Biotechnol, 157(4), 620- photobioreactors required 627. Indirect biophotolysis by Nonheterocystous Cyanobacteria • Separation of the H2 and O2 evolution reactions 1- Production of the biomass (carbohydrates) - open ponds 2. Concentration of biomass – settling pond; 3. Anaerobic dark fermentation (4 H2 /glucose + 2 acetates); 4. Conversion of 2 acetates into Source: Hallenbeck, P. C., & Benemann, J. R. (2002). Biological 8 mol of H (under the light) hydrogen production; fundamentals and limiting processes. 2 International Journal of Hydrogen Energy, 27(11-12), 1185-1193. Indirect biophotolysis by Heterocystous Cyanobacteria • Nitrogen deprivation → cell differentiation • Anaerobiosis permitting nitrogenase to function • Cells where PSII is absent no O2 • Calvin cycle enzymes are absent • Disaccharides imported to Heterocyst Source: P.C. Hallenbeck (ed.), Microbial Technologies in Advanced Biofuels Production, DOI 10.1007/978-1-4614-1208- 3_2, © Springer Science+Business Media, LLC 2012 Photo-fermentation – basic information Diversity of phototsynthetic bacteria: Rhodobacter and Rhodopseudomonas H2 evolved by N2ase (N2 limitation); Energetically demanding → photosynthesis Organic acids, lactate, acetate, and succinate → wastewater Also sugars → SINGLE STAGE Pros and Cons of Photo-fermentation • Complete conversion of • Low light conversion efficiencies organic acid wastes • High energy demand by N2ase • Potential waste treatment credits – N-poor residues, • Expensive hydrogen impermeable colorless photobioreactors required Experimental setup for hydrogen production indoor and outdoor setups Sun light Tungsten bulbs Chen, C. Y., Lee, C. M., & Chang, J. S. (2006). Feasibility study on bioreactor strategies for enhanced photohydrogen production from R. palustris WP3-5 using optical-fiber-assisted illumination systems. Int J Hydrogen Energy, D D Androga, E Özgür, I Eroglu, U Gündüz and M Yücel 31(15), 2345-2355. (2012). Photofermentative Hydrogen Production in Outdoor Conditions, Hydrogen Energy - Challenges and Abo-Hashesh, M., Ghosh, D., Tourigny, A., Taous, A., & Perspectives, Dragica Minic (Ed.), InTech, DOI: Combined light Hallenbeck, P. C. (2011). Single stage photofermentative 10.5772/50390 hydrogen production from glucose: An attractive alternative to source-Optical fiber two stage photofermentation or co-culture approaches. Int J Hydrogen Energy, 36(21), 13889-13895. What can be done for improving the yield? • Metabolic engineering - redirect metabolic flux to N2ase by blocking pathways • Physiological manipulation – remove the need for light! Overcoming the barrier: Physiological Method - Microaerobic Fermentation by PNSB Diverse carbon sources and concentrations Strategy to improve the Yield! Abo-Hashesh, M., Hallenbeck, P.C. 2012. Microaerobic dark fermentative hydrogen production by the photosynthetic bacterium, R. capsulatus JP91. International Journal of Low-Carbon Technologies. Overcoming the barrier: Physiological Method - Microaerobic Fermentation by PNSB DOE and RSM – H2 yield optimization Variables: Inoculum size, Substrate 1.4 mol H2/mol lactate concentration, O2 concentration O2 fed batch strategy – introducing O2 gradually (1.1 mol H2/mol lactate) Immobilized biomass strategy – ↑ cells Efforts to increase the overall process efficiency CO-CULTURES: metabolic Substrate degradation and complementary microorganisms byproducts consumption cultivated in the same bioreactor simultaneously; ↑ H2 yields; ↑ COD removal; C H O + 2H O → 4H + 2CO + 2CH COOH 6 12 6 2 2 2 3 ↓ lag phase; 2CH3COOH + 4H2O + “light energy” → 8H2 + 4CO2 Resiliency to environmental fluctuation ↑ stability of H2 C6H12O6 → 2H2 + 2CO2 + C3H7COOH production; C3H7COOH + 6H2O + “light energy” → 10H2 + 4CO2 Efforts to increase the overall process efficiency Co-culture: C. butyricum + R. palustris 6.4 mol H2/mol Starch/glucose base medium glucose 53% Substrate DOE -variables: Convertion Efficiency MO ratio (dark/photofermentative bacterium); Buffer concentration; Substrate concentration; COD removal 25-58% Responses: o H2 Yield, H2 Production, COD removal Hitit, Z. Y., Lazaro, C. Z., & Hallenbeck, P. C. (2017). Hydrogen production by co-cultures of C. butyricum and R. palustris: Optimization of yield using response surface methodology. Int J Hydrogen Energy, 42(10), 6578-6589. Efforts to increase the overall process efficiency Co-culture: Cellulomonas fimi + R. palustris DOE - variables: MO ratio (cellulolytic/photofermentative bacterium); carbon and nitrogen source concentration Responses: o Cellulose degradation, H2 Yield, oH2 Production, COD removal Hitit, Z. Y., Lazaro, C. Z., & Hallenbeck, P. C. (2017b). Single stage hydrogen production from cellulose through photo- fermentation by a co-culture of C. fimi and R. palustris. Int J Hydrogen Energy, 42(10), 6556-6566. Efforts to increase the overall process efficiency SEQUENTIAL SYSTEMS: metabolic complementary microorganisms growing separately Possibility to use variety of substrates, Possibility to set specific environmental and Chen, C. Y., Yang, M. H., Yeh, K. L., Liu, C. H., & nutritional requirements Chang, J. S. (2008). Biohydrogen production using sequential two-stage dark and photo fermentation for microorganisms processes. Int J Hydrogen Energ, 33. Dark Fermentation – another way to get hydrogen Anaerobic metabolism of substrates Two basic types of H2 fermentations: - Driven by need to produce ATP (thru acetate) - Driven by need to reoxidize NADH Mainly Clostridium and Enterobacter Dark Fermentation •No direct energy input •Low H yields needed 2 •Large amounts of side •Simple reactor technology products (acetate, butyrate, lactate, •Variety of waste ethanol, etc) streams/energy crops can be used Strategies for improving the yields .