1. Microbial Production of
Hydrogen
Dr. Bhavesh Patel
Principal
V.P. and R.P.T.P. Science College, Vallabh Vidyanagar
Email- bhavesh1968@rediffmail.com
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 2.5-3.0mm/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 $270-475
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
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
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.
