Dark Fermentation (4 H2 /Glucose + 2 Acetates);

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 photofermentation 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