1. Microbial Production of Hydrogen
Dr. Bhavesh Patel
Principal
V.P. and R.P.T.P. Science College, Vallabh Vidyanagar -

2. Outline Importance of hydrogen production Types of hydrogen production
Fermentative
Photosynthetic
The hydrogenase enzyme
Hydrogen production by Chlamydomonas reinhardtii
Research proposal

3. Importance of Hydrogen as an Alternative Fuel
Increased levels of CO2 from fossil fuels cause an increase in the Greenhouse Effect
One of the detrimental effects of the Greenhouse Effect is Global Warming
Combustion of Hydrogen produces water, which is not detrimental to the environment

4. Effects of Global Warming
Changes in climate
Sea level rise due to melting of polar icecaps
US seas rising mm/yr
By year 2050 it is most likely sea levels will rise by 15cm, but 1% chance they will rise one meter (Titus et al 1991)
One meter rise in sea level will cause $ billion dollars in damage (Titus and Narayanam 1995)

5. Microbial Hydrogen Production
Types of microbial hydrogen production
Fermentative
Photosynthetic (aerobic/anaerobic)
Most interest in hydrogen production research in US during the Energy Crisis of the 1970s
Interest in hydrogen production again in 1990s due to the awareness of Global Warming, etc.

6. Fermentative Production of Hydrogen
Clostridia species - Clostridia beijerincki
Used in fuel cell that produced 15mA over 20 days using waste from alcohol distillery (Taguchi et al 1992)
Methanogens -Methanotrix soehngenii
Archeabacteria -Pyrococcus furiosus (hyperthermophile)
Eschericia coli - Formate Hydrogen Lyase pathway, which is inefficient

7. Photosynthetic Production of Hydrogen
Purple Sulfur bacteria (Thiocapsa and Chromantium)
Non-Sulfur bacteria (Rhodospirillum and Rhodopseudomonas)
Green Algae (Chlamydomonas reinhardtii)
Advantages:
Photosynthetic organsims produce more hydrogen than fermentative organisms (Nandi and Sengupta 1998)
Photosynthetic organisms only require light and water

8. Photosynthetic Hydrogen Production Mechanisms
The purple sulfur bacteria and the non-sulfur bacteria produce hydrogen through a reversible hydrogenase and as a by-product of denitrification.
Chlamydomonas reinhardtii a green algae, produces hydrogen through hydrogenase.
Hydrogenase is induced under anaerobic conditions

9. Hydrogenase
Many bacteria use it for H2 dissimulation to use the electrons for electron transport
Two types of hydrogenases
Nickel-Iron centered - dissimulate hydrogen
Iron-only centered - evolve H2

10. Mechanism of Hydrogenase
Hydrogen Dissimulation
H2 Hydrogenase H e-
Hydrogen Evolution
Fd-reduced + 2H+ Hydrogenase H2

11. Comparison of Hydrogenase Yields
Chlamydomonas has the highest hydrogen yield

12. Advantages of Using C. reinhardtii
Cheap and easy to grow
Requires fluorescent light and 5% CO2
Grows at room temp. in water
Mutants have been isolated that are more energetically efficient (lack Photosystem I)
Research into its life cycle and flagella have yielded useful molecular techniques for studying the organism

13. Chlamydomonas reinhardtii
Green-alga (eukaryotic)
Biflagellated, unicellular, photosynthetic
Reproduces asexually or sexually under adverse conditions

14. Chlamydomonas reinhardtii

15. C. reinhardtii Hydrogenase
Enzyme located in the chloroplast
Receives electrons from reduced ferrodoxin
Hydrogenase stimulated under anaerobic conditions (Happe et el 1994)
Hydrogenase inhibited by O2, which is produced by C. reinhardtii during photosynthesis

16. Research Needs Investigate hydrogenase oxygen inhibition
Investigate production of hydrogen by the Photosystem I mutant

17. Research Proposal
Problem: Hydrogenase in C. reinhardtii inhibited by oxygen, which is made by the organism.
Solution: Determine Oxygen binding site on hydrogenase and create a clone with lower O2 binding and therefore higher H2 production.

18. Research Approach
Isolate gene for hydrogenase (BAC library has recently become available)
Sequence the hydrogenase gene and determine the oxygen binding site.
Create hydrogenase clone that does not bind oxygen through site-directed mutagenesis.
Isolate hydrogenase deficient C. reinhardtii mutant through UV-mutagenesis.
Introduce mutant hydrogenase into hydrogenase deficient clone.