The present study showed that there was a linear increase in the sulfide concentration of spring water in a bottle with the uncultivated
Thermodesulfobacteria-dominated SMSS (consortium I) which occurred in spring water containing low levels of sulfide in May and September 2001 (Fig.
7A). The
35SO
42− tracer experiment provided evidence that there was reduction of sulfate to sulfide through anaerobic sulfate respiration by microbes dominant in the SMSS at Nakabusa and Yumata. The SRR was much higher than that of the molybdate-amended control, since the molybdate greatly inhibited the SRR. In addition, RT-PCR-DGGE analysis combined with the FISH assay with probe S-*-Tdes-0830-a-A-20 indicated that as-yet-uncultivated microbes in the
Thermodesulfobacteria were dominant in the SMSS (Fig.
2D and E). Moreover, phylogenetic analysis revealed the dominance of DSR sequences NSdsr3-2-2, NSdsr3-2-4, YSdsr1, and YSdsr2, which form a branch separate from the known
Thermodesulfobacterium lineage (Fig.
6). Therefore, these results demonstrated that as-yet-uncultivated microbes belonging to the
Thermodesulfobacteria, which were dominant in the SMSS, constituted a numerically important population and played a critical role in the biological sulfide production in the SMSS in the hot springs at Nakabusa and Yumata. Thermophilic SRB, whose 16S rDNA genes are closely related to 16S rDNA genes of strains of
T. commune and
T. hveragerdense, formed an individual branch (branch B), as shown in Fig.
5, and were enriched from the SMSS from the Nakabusa hot spring. The DSR gene fragments of the thermophilic SRB, which are closely related to those of a strain of
T. commune, were detected in the enrichment cultures (data not shown). However, the contribution of this organism to in situ sulfide production in the SMSS was probably less than that of the as-yet-uncultivated
Thermodesulfobacteria that were dominant there, since no 16S rRNA or DSR gene closely related to the 16S rRNA or DSR gene of the
T. commune strain was found in the in situ samples.
Although the function of microbes corresponding to the archaeal DGGE bands in geothermal hot springs is unknown, the archaeal community seems to play a minor role in sulfide production in the SMSS based on the lack of detection of archaeal RT-PCR amplification with a primer set for the domain
Archaea. Similarly, it has been reported that based on a FISH assay performed with the 16S rRNA-targeted specific oligodeoxynucleotide probe, archaeal members are in the minority in black filamentous communities dominated by phylotype pBB in the
Aquificae associated with hot springs at Calcite Springs in Yellowstone National Park (
38).