The document discusses various biological methods for hydrogen production, including reduction of CO2 with hydrogen, biomass processing through fermentation or reforming, and microbial routes like biophotolysis and dark fermentation. It summarizes different approaches to biophotolysis using cyanobacteria and photosynthetic bacteria. Efforts to improve process efficiency through co-cultures and sequential systems combining metabolic pathways are also outlined. The strategies aim to optimize variables like substrate concentration and organism ratios to increase hydrogen yield and removal of chemical oxygen demand while reducing byproducts.

1. 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
Overview of microbial hydrogen
production

2.  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).

3. 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).

4. Biophotolysis
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
culture of the microalga Chlamydomonas reinhardtii. J Biotechnol, 157(4), 620-
627.
Overview of the 50-L horizontal tubular photobioreactor
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
• Expensive hydrogen impermeable
photobioreactors required

5. • 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
8 mol of H2 (under the light)
Indirect biophotolysis by Nonheterocystous
Cyanobacteria
Source: Hallenbeck, P. C., & Benemann, J. R. (2002). Biological
hydrogen production; fundamentals and limiting processes.
International Journal of Hydrogen Energy, 27(11-12), 1185-1193.

6. Indirect biophotolysis
by Heterocystous
Cyanobacteria
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
• 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

7. 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
Photo-fermentation – basic information

8. Pros and Cons of Photo-fermentation
• Complete conversion of
organic acid wastes
• Potential waste treatment
credits – N-poor residues,
colorless
• Low light conversion efficiencies
• High energy demand by N2ase
• Expensive hydrogen impermeable
photobioreactors required

9. Experimental setup for hydrogen production
indoor and outdoor setups
D D Androga, E Özgür, I Eroglu, U Gündüz and M Yücel
(2012). Photofermentative Hydrogen Production in
Outdoor Conditions, Hydrogen Energy - Challenges and
Perspectives, Dragica Minic (Ed.), InTech, DOI:
10.5772/50390
Abo-Hashesh, M., Ghosh, D., Tourigny, A., Taous, A., &
Hallenbeck, P. C. (2011). Single stage photofermentative
hydrogen production from glucose: An attractive alternative to
two stage photofermentation or co-culture approaches. Int J
Hydrogen Energy, 36(21), 13889-13895.
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,
31(15), 2345-2355.
Combined light
source-Optical fiber
Tungsten bulbs
Sun light

10. • Metabolic engineering
- redirect metabolic flux to
N2ase by blocking pathways
What can be done for improving the yield?
• Physiological manipulation –
remove the need for light!

11. Overcoming the barrier:
Physiological Method - Microaerobic Fermentation by PNSB
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.
Diverse carbon
sources and
concentrations
Strategy to improve the
Yield!

12. Overcoming the barrier:
Physiological Method - Microaerobic Fermentation by PNSB
 DOE and RSM – H2 yield optimization
 Variables: Inoculum size, Substrate
concentration, O2 concentration
 O2 fed batch strategy – introducing
O2 gradually (1.1 mol H2/mol lactate)
 Immobilized biomass strategy – ↑
cells
1.4 mol H2/mol lactate

13. Substrate degradation and
byproducts consumption
simultaneously;
↑ H2 yields;
↑ COD removal;
↓ lag phase;
Resiliency to environmental
fluctuation ↑ stability of H2
production;
Efforts to increase the overall process efficiency
CO-CULTURES: metabolic
complementary microorganisms
cultivated in the same bioreactor
C6H12O6 + 2H2O → 4H2 + 2CO2 + 2CH3COOH
2CH3COOH + 4H2O + “light energy” → 8H2 + 4CO2
C6H12O6 → 2H2 + 2CO2 + C3H7COOH
C3H7COOH + 6H2O + “light energy” → 10H2 + 4CO2

14.  Co-culture: C. butyricum + R. palustris
 Starch/glucose base medium
 DOE -variables:
 MO ratio (dark/photofermentative
bacterium); Buffer concentration;
Substrate concentration;
 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.
6.4 mol H2/mol
glucose
53% Substrate
Convertion Efficiency
COD removal 25-58%
Efforts to increase the overall process efficiency

15.  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

16. 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
nutritional requirements
for microorganisms
Chen, C. Y., Yang, M. H., Yeh, K. L., Liu, C. H., &
Chang, J. S. (2008). Biohydrogen production using
sequential two-stage dark and photo fermentation
processes. Int J Hydrogen Energ, 33.

17. 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

18. Dark Fermentation
•Low H2 yields
•Large amounts of side
products (acetate,
butyrate, lactate,
ethanol, etc)
•No direct energy input
needed
•Simple reactor technology
•Variety of waste
streams/energy crops can
be used

19. Strategies for improving the yields