Diversity and activity of culturable nitrogen fixing heterotrophic bacteria from estuarine and coastal environments of Southeastern Arabian Sea (SEAS)

Abstract

Nitrogen fixation by diazotrophic bacteria serve as an important source of fixed nitrogen in an aquatic ecosystem and thereby directly influence the carbon flux and primary production. Currently there is little information about the cultivable heterotrophic diazotrophs and the eco-physiological roles of nitrogen fixing bacteria in aquatic environments. The focus of the present study was to understand the diversity of cultivable heterotrophic diazotrophs and to evaluate their nitrogen fixation capability in the estuarine and coastal environments of the Southeastern Arabian Sea. The heterotrophic diazotrophic bacteria were isolated on nitrogen-free media and the potential activity of nitrogen fixation was estimated by acetylene reduction assay. The molecular basis of the nitrogen fixation capability among the above isolates was analyzed testing for the presence of dinitrogenase reductase (nifH) gene. The 16S rRNA gene based identity revealed that the cultivable heterotrophic diazotrophs belonged to α, β, γ-Proteobacteria and Firmicutes. The results also revealed that α-Proteobacterium Nitratireducter kimnyeongensis was found to be a potential diazotroph (190.3 ± 4.55 nmol C2H4/mg protein/day), which was isolated from the coastal ecosystem. Various strains of γ-Proteobacteria such as Klebsiella pneumonia, Klebsiella quasipneumonia and Klebsiella variicola also exhibited relatively high nitrogen-fixing activity compared to that of Pseudomonas flavescens and Halomonas meridiana Other cultivable diazotrophs encountered in the study area were Oceanobacillus iheyencis, Bacillus aerius, Exiguobacterium profundum, Staphylococcus warneri, Bacillus amyloliquefaciens, Staphylococcus arlettae and Staphylococcus caprae. The results revealed that Gram-negative bacterial strains possessed relatively superior nitrogen fixation activity over Gram-positive isolates (p<0.01). In the light of our observations, we hypothesize that heterotrophic diazotrophs play key role in nitrogen fixation process and contribute to primary production in estuarine and coastal environments of Southeastern Arabian Sea.