Thiobacillus thiophilus became the best competitor in enriched communities
The community of control sediments was serially diluted and cultured anaerobically in the NS media where nitrate was the sole electron acceptor and sulfide was the sole electron donor. This is to enrich bacteria having a complete or partial respiratory chain from sulfide, the initial e-donor to nitrate, the terminal e-acceptor. The dominance of Thiobacillus reappeared in 20 out of 38 communities (Figure 5A) though it was initially sparse (<1.3% relative abundance). This enrichment suggested the competent metabolic fitness ofThiobacillus . Linear regression also identified positive correlations between its relative abundance and nitrate consumption, nitrite production and sulfate production (Figure 5B-5D).
Community enrichments of different dilutabilities were streaked on NS and NTS plates (anaerobic at room temperature). However, no colony formed on the NS plates. Using solid NTS medium, 14 strains were isolated from all community enrichments and all were members of phylum Proteobacteria (Table 1). Unlike community enrichments, these pure isolates must a complete respiratory chain from thiosulfate to nitrate since interspecies cross-feeding was excluded. We then determined their metabolic characteristics using liquid NS and NTS medium, and found that strain S5643 (Thiobacillus thiophilus ) was the sole sulfide oxidizer. PCR of selected functional genes involved in nitrogen and sulfur cycling found that (i) all strains except T83 (Sphingopyxis terrae ) and T546 (Rhodanobacter lindaniclasticus ) had nitrate reductase narG or napA ; (ii) all strains had nitrite reductase nirK or nirS ; and (iii) thiosulfate oxidasesox B appeared only in 6 strains (Table 1), including S5643. These results confirmed the metabolic fitness of Thiobacillus, which was the sole sulfide-oxidizing denitrifier among the 14 isolates.