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

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).

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

• 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 into8 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.

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

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

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

Experimental setup for hydrogen productionindoor and outdoor setups

D D Androga, E Ozgur, I Eroglu, U Gunduz and M Yucel

(2012). Photofermentative Hydrogen Production in

Outdoor Conditions, Hydrogen Energy - Challenges and

Perspectives, Dragica Minic (Ed.), InTech, DOI:

10.5772/50390Abo-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

• 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!

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!

Overcoming the barrier:

Physiological Method - Microaerobic Fermentation by PNSB

DOE and RSM – H2 yield optimization

Variables: Inoculum size, Substrateconcentration, O2 concentration

O2 fed batch strategy – introducingO2 gradually (1.1 mol H2/mol lactate)

Immobilized biomass strategy – ↑ cells

1.4 mol H2/mol lactate

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: metaboliccomplementary microorganismscultivated 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

Co-culture: C. butyricum + R. palustris

Starch/glucose base medium

DOE -variables:

MO ratio (dark/photofermentativebacterium); 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

glucose53% SubstrateConvertion Efficiency

COD removal 25-58%

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

Efforts to increase the overall process efficiency

SEQUENTIAL SYSTEMS:metabolic complementarymicroorganisms growingseparately

Possibility to use varietyof substrates,

Possibility to set specificenvironmental andnutritional requirementsfor 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

•Low H2 yields

•Large amounts of side products (acetate, butyrate, lactate, ethanol, etc)

•No direct energy input needed

•Simple reactor technology

•Variety of wastestreams/energy crops can be used

Strategies for improving the yields