Stable-isotope probing (SIP) was used to identify acetate- or methanol-assimilating bacteria under nitrate-reducing conditions in activated sludge. A sludge sample obtained from wastewater treatment systems was incubated in a denitrifying batch reactor fed with synthetic wastewater containing [(13)C]acetate or [(13)C]methanol as the main carbon source and nitrate as the electron acceptor. We analyzed how growth of bacterial populations was stimulated by acetate or methanol as the external carbon source in nitrogen-removal systems. Most of the acetate- or methanol-assimilating bacteria identified by SIP have been known as denitrifiers in wastewater treatment systems. When acetate was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-labeled DNA were closely related to the 16S rRNA genes of Comamonadaceae (e.g., Comamonas and Acidovorax) and Rhodocyclaceae (e.g., Thauera and Dechloromonas) of the Betaproteobacteria, and Rhodobacteraceae (e.g., Paracoccus and Rhodobacter) of the Alphaproteobacteria. When methanol was used as the carbon source, 16S rRNA gene sequences retrieved from (13)C-DNA were affiliated with Methylophilaceae (e.g., Methylophilus, Methylobacillus, and Aminomonas) and Hyphomicrobiaceae. Rarefaction curves for clones retrieved from (13)C-DNA showed that the diversity levels for methanol-assimilating bacteria were considerably lower than those for acetate-assimilating bacteria. Furthermore, we characterized nitrite reductase genes (nirS and nirK) as functional marker genes for denitrifier communities in acetate- or methanol-assimilating populations and detected the nirS or nirK sequence related to that of some known pure cultures, such as Alcaligenes, Hyphomicrobium, and Thauera. However, most of the nirS or nirK sequences retrieved from (13)C-DNA were clustered in some unidentified groups. On the basis of 16S rRNA gene clone libraries retrieved from (13)C-DNA, these unidentified nir sequences might be identified by examining the nir gene in candidates for true denitrifiers (e.g., the families Comamonadaceae, Hyphomicrobiaceae, Methylophilaceae, and Rhodobacteraceae).

The doubling time of anaerobic ammonium-oxidizing (anammox) bacteria in an anaerobic biological filtrated (ABF) reactor was determined. Fluorescence in situ hybridization analysis was used to detect and count anammox bacteria cells in anammox sludge. As a result, the populations of anammox bacteria at 14th and 21st days were 1.1 x 10(6) and 1.7 x 10(7) cells/ml reactor, respectively. From these results, the doubling time of anammox bacteria was calculated as 1.8 days, and the specific growth rate (mu) was 0.39 day(-1). This result indicated that the anammox bacteria have higher growth rate than the reported value (doubling time, 11 days). Furthermore, it was clearly demonstrated that nitrogen conversion rate was proportional to the population of anammox bacteria. Maintaining the ideal environment for the growth of anammox bacteria in the ABF reactor might lead to faster growth. This is the first report of the growth rate of anammox bacteria based on the direct counting of anammox bacteria.

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Investigation of the diversity of nirK and nirS in denitrifying bacteria revealed that salinity decreased the diversity in a nitrate-containing saline wastewater treatment system. The predominant nirS clone was related to nirS derived from marine bacteria, and the predominant nirK clone was related to nirK of the genus Alcaligenes.Nitrogen removal from wastewater is accomplished by bacterial denitrification. Some types of industrial wastewater, such as metallurgic wastewater, contain large amounts of nitrate and saline (11). Although we have developed a nitrogen removal system for saline wastewater, nitrite often accumulates, particularly under relatively high-saline conditions (32). Therefore, it is important to understand the ecology of nitrite-reducing bacterial communities to determine stable operational conditions for denitrification processes.A few researchers and we have studied microbial communities in anaerobic reactors for the denitrification of saline wastewater (13,31,32) and used an approach based on taxonomic analysis of the 16S rRNA gene to identify all of the bacterial species within a community. These studies suggested that members of the gamma subdivision of the class Proteobacteria are important in such wastewater treatment systems. However, 16S rRNA gene-based approaches are unable to detect denitrifying bacteria particularly in such systems because bacterial groups possessing denitrifying abilities are phylogenetically diverse (33). Furthermore, although aerobic halophilic species have been reported (7,29), the microbial ecology of moderately halophilic denitrifying bacteria has hardly been reported.Recently, primer sets specific for functional genes involved in denitrification, namely, nirK, nirS (3, 9), and nosZ (22), have been developed. Thus, PCR-based approaches revealed denitrifying microbial communities in activated sludge (9, 27), marine sediment (4, 5, 15), and soil (17, 19) on the basis of nirK and nirS and in marine sediment on the basis of nosZ (23, 24). These approaches have contributed to the improvement of the complete or partial sequences of nitrite reductase genes and nitrous oxide reductase genes in the database. We expect that the information on these genes detected from various environments will contribute to further studies on the ecophysiology of denitrifying communities.In this study, we focused on the microbial ecology of nitritereducing bacteria in two series of metallurgic wastewater treatment systems (MWTSs) with different fluidity conditions; one of the MWTSs was composed of an anaerobic packed bed, and the other was composed of an anaerobic fluidized bed (31). The nirK and nirS heterogeneity in the anaerobic reactors of MWTSs was investigated by cloning, sequencing, and phylogenetic analysis to determine the actual denitrifying bacterial community. Furthermore, the anaerobic packed bed and the fluidized bed were compared for nirK and nirS diversity to investigate the influence of fluidity conditions on the denitrifying microbial community.Sludge samples and isola...

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