(86d) Microbial Community Structure of Sulfur-Oxidizing Cultures Enriched from Wastewater

Sulfur-oxidizing bacteria are important in transforming reduced sulfur species in wastewater conveyance systems and in off-gas treatment processes such as biofilters. The main goal of this study was to evaluate the microbial community structure of sulfur-oxidizing cultures enriched in aerated batch reactors with thiosulfate. The reactors were inoculated with either return activated sludge from a local wastewater treatment plant or compost media from a biofilter treating hydrogen sulfide emissions at a wastewater lift station. Both sets of reactors successfully converted 10 g/L thiosulfate to sulfate in 3-7 days. Two pH conditions were investigated: (1) in one set of activated sludge-inoculated reactors and one set of compost-inoculated reactors, the pH was allowed to naturally decrease from 7 to 3, and (2) in another set of compost-inoculated reactors, the pH was maintained at 7. In the reactors where the pH was allowed to drop from 7 to 3, sulfur oxidation activity ceased below pH 3.

Microbial community diversity in the reactors was analyzed using terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. In the activated sludge-inoculated reactors, T-RFLP profiles indicated that the microbial diversity decreased sharply within 5 days during which time the pH dropped from 7 to 4. The three operational taxonomic units (OTUs) that were dominant during active thiosulfate oxidation were not detected after the pH dropped below 3. T-RFLP coupled with 16S rRNA gene clone library results suggested that the dominant bacteria in the reactor were Thiobacillus spp. In the compost-inoculated reactors where the pH was allowed to drop naturally, T-RFLP and a 16S rRNA gene clone library suggested that the dominant bacteria in the reactor at pH 3 were Thiomonas spp. In the compost-inoculated reactors maintained at pH 7, T-RFLP profiles indicated the presence of two dominant OTUs during active thiosulfate oxidation: Thiomonas spp. and Stenotrophomonas spp. A Thiomonas sp. and a Stenotrophomonas sp. from the reactor community were subsequently isolated on agar plates. However, when these isolates were co-inoculated to an aerated batch reactor at pH 7, no thiosulfate oxidation was observed in 7 days, suggesting that the sulfur-oxidizing activity present in the original batch reactors could not be recreated from the cultured isolates. The results of this study highlight the strong selective pressure that is exerted on the microbial community under sulfur-oxidizing conditions and the limited microbial diversity that results. This research also demonstrates the limits of attempting to describe a microbial community via culture-based techniques.

In addition to describing microbial community structure in the thiosulfate-enriched reactors, the presence and expression of two sulfur-oxidizing genes, soxB and sqr, were investigated for the thiosulfate-enriched reactors described above and for a biofilter installed at the wastewater lift station. soxB was detected in DNA and RNA isolated from the compost-inoculated reactors enriched with thiosulfate; sqr was detected in the DNA but not in the RNA isolated from the same reactors. Similarly, sqr was detected in the DNA but not in the RNA isolated from the thiosulfate-enrichment reactors inoculated with activated sludge. In contrast, sqr was detected in both the DNA and RNA isolated from the biofilter treating hydrogen sulfide emissions at a wastewater lift station. These results suggest that sqr is expressed during hydrogen sulfide oxidation but not during thiosulfate oxidation. Studies are underway to further investigate this hypothesis.