Complete denitrification in coculture of obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacteria from a hypersaline soda lake

Eight anaerobic enrichment cultures with thiosulfate as electron donor and nitrate as electron acceptor were inoculated with sediment samples from hypersaline alkaline lakes of Wadi Natrun (Egypt) at pH 10; however, only one of the cultures showed stable growth with complete nitrate reduction to dinitrogen gas. The thiosulfate-oxidizing culture subsequently selected after serial dilution developed in two phases. Initially, nitrate was mostly reduced to nitrite, with a coccoid morphotype prevailing in the culture. During the second stage, nitrite was reduced to dinitrogen gas, accompanied by mass development of thin motile rods. Both morphotypes were isolated in pure culture and identified as representatives of the genus Thioalkalivibrio, which includes obligately autotrophic sulfur-oxidizing haloalkaliphilic species. Nitrate-reducing strain ALEN 2 consisted of large nonmotile coccoid cells that accumulated intracellular sulfur. Its anaerobic growth with thiosulfate, sulfide, or polysulfide as electron donor and nitrate as electron acceptor resulted in the formation of nitrite as the major product. The second isolate, strain ALED, was able to grow anaerobically with thiosulfate as electron donor and nitrite or nitrous oxide (but not nitrate) as electron acceptor. Overall, the action of two different sulfur-oxidizing autotrophs resulted in the complete, thiosulfate-dependent denitrification of nitrate under haloalkaliphilic conditions. This process has not yet been demonstrated for any single species of chemolithoautotrophic sulfur-oxidizing haloalkaliphiles.