Microbial Production of Hydrogen Dr Bhavesh Patel Principal

Microbial Production of Hydrogen Dr. Bhavesh Patel Principal V. P. and R. P. T. P. Science College, Vallabh Vidyanagar Email- bhavesh 1968@rediffmail. com

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

Importance of Hydrogen as an Alternative Fuel • Increased levels of CO 2 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

Effects of Global Warming • Changes in climate • Sea level rise due to melting of polar icecaps – US seas rising 2. 5 -3. 0 mm/yr – By year 2050 it is most likely sea levels will rise by 15 cm, 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)

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 1970 s • Interest in hydrogen production again in 1990 s due to the awareness of Global Warming, etc.

Fermentative Production of Hydrogen • Clostridia species - Clostridia beijerincki – Used in fuel cell that produced 15 m. A 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

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

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

Hydrogenase • Many bacteria use it for H 2 dissimulation to use the electrons for electron transport • Two types of hydrogenases – Nickel-Iron centered - dissimulate hydrogen – Iron-only centered - evolve H 2

Mechanism of Hydrogenase • Hydrogen Dissimulation – H 2 Hydrogenase 2 H+ + 2 e- • Hydrogen Evolution – Fd-reduced + 2 H+ Hydrogenase H 2

Comparison of Hydrogenase Yields Chlamydomonas has the highest hydrogen yield

Advantages of Using C. reinhardtii • Cheap and easy to grow – Requires fluorescent light and 5% CO 2 – 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

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

Chlamydomonas reinhardtii

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 O 2, which is produced by C. reinhardtii during photosynthesis

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

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 O 2 binding and therefore higher H 2 production.

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.