Identifying diazotrophs by incorporation of nitrogen from 15N2 into RNA

The diversity and abundance of active diazotrophs was investigated in a New Zealand pulp and paper wastewater by enrichment with N-15(2). Purified N-15-RNA was analysed by reverse transcription, molecular cloning and sequence analysis of 16S rRNA to reveal a diverse community of bacteria as indicated by a Shannon Weaver Index value of > 2.8. The major class represented in the enriched culture were the gamma-Proteobacteria at 85% with a secondary group of the phylum Firmicutes present at 8.2%, the remaining sequences were affiliated with the alpha- and beta-Proteobacterial classes (1.4% and 4.3%, respectively). Three dominant genera, Aeromonas, Pseudomonas and Bacillus, were identified by comparison with published sequences and phylogenetic analysis. To confirm that representatives of the taxonomic groups identified from the active enriched nitrogen-fixing community were capable of fixing nitrogen Aeromonas and Pseudomonas species were cultivated and shown to possess nifH genes. In wastewater, fluorescence in situ hybridisation probing revealed that the dominant nitrogen-fixing population identified in this study were present in the population, but at lower levels. The population is, therefore, reliant on a small sub-population of diazotrophs to supply the community's nitrogen needs above that already present in the wastewater.