Effect of some physiological factors on nitrogenase activity and nitrogenase mediated hydrogen evolution by mixed microbial culture.

Fermentative H2 evolution, nitrogenase activity (acetylene reduction) and nitrogenase mediated H2 evolution was studied in free cells of mixed microbial population of H2 producers. At 3% glucose level, the cells produced 8.35 l H2/mol glucose utilized. The role of nitrogenase system in H2 generation was evident by derepressed nitrogenase activity (0.46 nmoles C2H4 produced/mg protein/h) under defined in vitro conditions. For maximum expression of the activity, the cells required preactivation under anaerobic conditions by incubating at 40 degrees C for 20-24 h with 0.2% glucose in the culture medium. At an O2 level of more than 0.25%, the acetylene reduction activity decreased significantly and could not be detected at a level of 20%. Nitrogenase activity development was higher at acetylene: inoculum ratio between 4.2-6.25. H2 evolution was lower when the mixed cells were incubated under an atmosphere of 10% C2H2 and 5% CO gas. This decrease in H2 evolution was also evident at 2.5-6.5 mM NaNO3 and KNO3 concentrations in the liquid culture medium thus establishing more than 50% H2 evolution through nitrogenase.