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)
• Expensive hydrogen impermeable production in a 50-L tubular photobioreactor by means of a sulfur-deprived
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 H2 (under the light) hydrogen production; fundamentals and limiting processes.
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,
31(15), 2345-2355.
Combined light
source-Optical fiber
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:
Abo-Hashesh, M., Ghosh, D., Tourigny, A., Taous, A., &
Hallenbeck, P. C. (2011). Single stage photofermentative 10.5772/50390
hydrogen production from glucose: An attractive alternative to
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;
C6H12O6 + 2H2O → 4H2 + 2CO2 + 2CH3COOH ↓ 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
Convertion Efficiency
 DOE -variables:
 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,
o H2 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
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.
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 H2 yields

needed
•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