Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5-10, and within a salt range from 0.2 to 4 M Na⁺, with an optimum at 0.5-1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles.