Factors regulating nitrogenase activity and hydrogen evolution in Azolla-anabaena symbiosis

Abstract

Nitrogenase activity and H₂ production capacity have been studied in intact Azolla plants. Under aerobic conditions the plants showed a C₂H₂ reduction rate of 6.65 nmoles C₂H₄ mg^-1 fresh weight in light at 48h. Considerable activity was also present in the dark. Though H₂ evolution was detected under aerobic conditions there was multifold stimulation under anaerobic conditions. There was no significant change in nitrogenase activity under anaerobic conditions. Increasing concentrations of O₂ inhibited nitrogenase activity but 5% O₂ proved stimulatory for H₂ evolution in light. In the dark, there was a gradual stimulation in H₂ evolution even up to 20% O₂. Addition of combined nitrogen sources, namely NH₄Cl or KNO₃ (10 mM) resulted in complete inhibition of C₂H₂-reduction activity within 48 h, but H₂ evolution was not inhibited. Indeed these combined nitrogen sources stimulated H₂ evolution. Though nitrogenase activity was affected, the heterocyst frequency remained unaltered. Phosphate addition resulted in significant stimulation of nitrogenase and H₂ evolution activity. These results suggest that on nitrogenase and H₂ evolution activity in Azolla are affected by a number of factors which show a differential effect on nitrogenase and H₂ evolution. Furthermore, our results indicate the presence of a soluble reversible hydrogenase in Azolla.