Isolation and properties of obligately chemolithoautotrophic and extremelyalkali-tolerant ammonia-oxidizing bacteria from Mongolian soda lakes

Five mixed samples prepared from the surface sediments of 20 north-east Mon golian soda lakes with total salt contents from 5 to 360 g/l and pH values from 9.7 to 10.5 were used to enrich for alkaliphilic ammonia-oxidizing bac teria. Successful enrichments at pH 10 were achieved on carbonate mineral m edium containing 0.6 M total Na+ and less than or equal to4 mM NH4Cl. Five isolates (ANs1-ANs5) of ammonia-oxidizing bacteria capable of growth at pH 10 were obtained from the colonies developed on bilayered gradient plates. The cells were motile and coccoid, with well-developed intracytoplasmic mem branes (ICPM) and carboxysomes. At pH 10.0, ammonia was toxic for growth at concentrations higher than 5 mM NH4Cl. The bacteria were able to grow with in the salinity range of 0.1-1.0 M of total Na+ (optimum 0.3 M). In media c ontaining 0.3-0.6 M total Na+, optimal growth in batch cultures occurred in the presence of a bicarbonate/carbonate buffer system within the pH range 8.5-9.5, with the highest pH limit at pH 10.5. At pH lower than 8.0, growth was slower, most probably due to decreasing free ammonia. The pH profile o f the respiratory activity was broader, with limits at 6.5-7.0 and 11.0 and an optimum at 9.5-10.0. In pH-controlled, NH3-limited continuous culture, isolate ANs5 grew up to pH 11.3, which is the highest pH limit known for am monia-oxidizing bacteria so far. This showed the existence of extremely alk ali-tolerant ammonia-oxidizing bacteria in the soda lakes. Comparative 16S rDNA sequence analysis of the five isolates demonstrated that they possess identical 16S rDNA genes and that they are closely related to Nitrosomonas halophila (sequence similarity 99.3%), a member of the beta -subclass of th e Proteobacteria. This affiliation was confirmed by comparative sequence an alysis of the amoA gene, encoding the active-site subunit of the ammonia-mo noxygenase, of one of the isolates. DNA-DNA hybridization data further supp orted that the soda lake isolates are very similar to each other and repres ent an alkali-tolerant subpopulation of N. halophila whose species descript ion is herewith amended.