Enrichment and isolation of acidophilic sulfate-reducing bacteria from Tinto River sediments

Although some acidophilic and alkaliphilic species have been described recently, most of the known sulfate-reducing bacteria (SRB) grow optimally at neutral pH. In this study, sulfate reduction was studied with sediment samples from the extremely acidic Tinto River basin. Stable enrichments of SRB were obtained at pH 4 with glycerol, methanol and hydrogen; at pH 4.5 with lactate and at pH 5.5 with succinate as substrates. Inhibition of sulfate reduction by organic acids below their pKa was observed. Cloning and sequencing of 16S rRNA gene showed that fermentative bacteria (Paludibacter spp., Oscillibacter spp.) and SRB (Thermodesulfobium spp., Desulfosporosinus spp., Desulfitobacterium spp., Desulfotomaculum spp.) were co-enriched. By repeated serial dilutions and streaking on agar plates, four strains of SRB belonging to the Firmicutes phylum were obtained. Two of them show 96% 16S rRNA gene sequence similarity with Desulfosporosinus acidophilus, and a third one with Desulfosporosinus orientis. Another isolate has just 93% rRNA gene sequence similarity with the Desulfosporosinus/Desulfitobacterium cluster and might represent a novel species within a novel genus. One of the Desulfosporosinus strains was further investigated showing maximum growth at pH 5.5, and a pH-dependent inhibitory effect of organic acids and sulfide.