Laboratory-scale acidophilic nitrifying sequencing-batch reactors (ANSBRs) were constructed by seeding with sewage-activated sludge and cultivating with ammonium-containing acidic mineral medium (pH 4.0) with or without a trace amount of yeast extract. In every batch cycle, the pH varied between 2.7 and 4.0, and ammonium was completely converted to nitrate. Attempts to detect nitrifying functional genes in the fully acclimated ANSBRs by PCR with previously designed primers mostly gave negative results. 16S rRNA gene-targeted PCR and a subsequent denaturating gradient gel electrophoresis analysis revealed that a marked change occurred in the bacterial community during the overall period of operation, in which members of the candidate phylum TM7 and the class Gammaproteobacteria became predominant at the fully acclimated stage. This result was fully supported by a 16S rRNA gene clone library analysis, as the major phylogenetic groups of clones detected (>5% of the total) were TM7 (33%), Gammaproteobacteria (37%), Actinobacteria (10%), and Alphaproteobacteria (8%). Fluorescence in situ hybridization with specific probes also demonstrated the prevalence of TM7 bacteria and Gammaproteobacteria. These results suggest that previously unknown nitrifying microorganisms may play a major role in ANSBRs; however, the ecophysiological significance of the TM7 bacteria predominating in this process remains unclear.

The reductive dechlorinating abilities for chloroethenes of seven enrichment cultures from polychlorinated-dioxin-dechlorinating microcosm were investigated using culture-independent and -dependent methods. These cultures were constructed and maintained with 1,2,3-trichlorobenzene (1,2,3-TCB) or fthalide as an electron acceptor and hydrogen as an electron donor. Denaturing gradient gel electrophoresis (DGGE) analysis of the amplified fragments targeting the 16S rRNA gene showed one or two major bands, whose nucleotide sequences were then analyzed and were found to suggest that Dehalococcoides was one of the dominant bacteria in all enrichment cultures. The nucleotide sequence data revealed that the identity of the major band was 100% identical to the 16S rRNA gene sequence of the Pinellas subgroup of the Dehalococcoides clusters, that is, strains CBDB1 and FL2. Genetic diagnosis targeting the pceA, tceA, bvcA, vcrA and reductive dehalogenase homologous (rdh) gene was performed to investigate the potential for reductive chloroethene dechlorination of cultures. The required length of PCR-amplified fragments was not observed, suggesting that these cultures are not capable of reductively dechlorinating chloroethenes. However, a culture-dependent test indicated that two cultures, TUT1903 and TUT1952, reductively dechlorinated tetrachloroethene (PCE) to trichloroethene (TCE), although not completely. While, TUT2260 and TUT2264 completely converted PCE to TCE and dichloroethenes, but not further. These results suggest that these TUT cultures might include a novel type of bacteria belonging to the Dehalococcoides group and that currently available information on both the 16S rRNA gene and rdh gene sequences is insufficient to definitively evaluate the potential abilities for reductive dechlorination.

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