Isolation and characterization of the N2-fixing symbiotic cyanobacterium Anabaena azollae

Abstract

Expression of nitrogenase activity (C2H2 reduction) has been studied in the symbiotic N2-fixing cyanobacterium Anabaena azollae within the intact Azolla plant and after its isolation from the host. The cyanobacterium (blue-green alga) has been isolated successfully from the host plant by standard microbiological techniques. Optimum growth occurred in semi-solid (0.1% agar) Allen and Anion’s medium devoid of combined nitrogen. The alga has a generation time of 8–12 h when grown on atmospheric nitrogen. Addition of NO3 - or NH4 + did not change the growth rate. Actively N2-fixing filaments have a 15–18% heterocyst frequency. Under aerobic conditions, laboratory-grown A. azollae showed nitrogenase activity of 80–120 n mol C2H4 µgChla -1 h-1. The activity was linear for 2h in the light. The presence of high nitrogenase activity correlates with the observed high heterocyst frequency. Fresh intact Azolla plants showed a C2H2-reduction rate of 1.8–2 µmol C2H4g (fresh wt)-1h-1 and the activity was almost equal in light and dark, but, after 2 h dark incubation, the activity began to decline. The repression of nitrogenase activity by combined nitrogen sources viz., NO3 - or NH4 + was more pronounced in isolated A. azollae than in the whole Azolla plants.