Diversity study of nitrifying bacteria in full-scale municipal wastewater treatment plants.

We hypothesize that activated-sludge processes having stable and complete nitrification have significant and similar diversity and functional redundancy among its ammonia- and nitrite-oxidizing bacteria, despite differences in temperature, solids retention time (SRT), and other operating conditions. To evaluate this hypothesis, we examined the diversity of nitrifying bacterial communities in all seven water-reclamation plants (WRPs) operated by Metropolitan Water Reclamation District of Greater Chicago (MWRDGC). These plants vary in types of influent waste stream, plant size, water temperature, and SRT. We used terminal restriction fragment length polymorphism (T-RFLP) targeting the 16S rRNA gene and group-specific ammonia-monooxygenase functional gene (amoA) to investigate these hard-to-culture nitrifying bacteria in the full-scale WRPs. We demonstrate that nitrifying bacteria carrying out the same metabolism coexist in all WRPs studied. We found ammonia-oxidizing bacteria (AOB) belonging to the Nitrosomonas europaea/eutropha, Nitrosomonas oligotropha, Nitrosomonas communis, and Nitrosospira lineages in all plants. We also observed coexisting Nitrobacter and Nitrospira genera for nitrite-oxidizing bacteria (NOB). Among the factors that varied among the WRPs, only the seasonal temperature variation seemed to change the nitrifying community, especially the balance between Nitrosospira and Nitrosomonas, although both coexisted in winter and summer samples. The coexistence of various nitrifiers in all WRPs is evidence of functional redundancy, a feature that may help maintain the stability of the system for nitrification.

Discrimination of shifts in a soil microbial community associated with TNT-contamination using a functional ANOVA of 16S rRNA hybridized to oligonucleotide microarrays.

A functional ANOVA analysis of the thermal dissociation of RNA hybridized to DNA microarrays was used to improve discrimination between two soil microbial communities. Following hybridization of in vitro transcribed 16S rRNA derived from uncontaminated and 2,4,6-trinitrotoluene contaminated soils to an oligonucleotide microarray containing group- and species-specific perfect match (PM) probes and mismatch (MM) variants, thermal dissociation was used to analyze the nucleic acid bound to each PM-MM probe set. Functional ANOVA of the dissociation curves generally discriminated PM-MM probe sets when Td values (temperature at 50% probe-target dissociation) could not. Maximum discrimination for many PM and MM probes often occurred at temperatures greaterthan the Td. Comparison of signal intensities measured prior to dissociation analysis from hybridizations of the two soil samples revealed significant differences in domain-, group-, and species-specific probes. Functional ANOVA showed significantly different dissociation curves for 11 PM probes when hybridizations from the two soil samples were compared, even though initial signal intensities for 3 of the 11 did not vary.

Impact of prehybridization PCR amplification on microarray detection of nitrifying bacteria in wastewater treatment plant samples.

A gel-based microarray that included a set of 26 oligonucleotide probes targeting all nitrifying bacteria at varying levels of specificity suggested the presence of targeted microorganisms when hybridized to RNA isolated from a wastewater treatment plant, but could not discriminate between perfectly matched and mismatched sequences due in part to low signal intensity. To enhance sensitivity and improve discrimination, polymerase chain reaction was used to selectively amplify the 16S rRNA genes of specific nitrifier groups. RNA transcribed from these DNA templates was hybridized to the microarray and thermal dissociation analysis was used to characterize the specificity of hybridization. Amplification with Nitrospira-specific primers resulted in the selective amplification of this target group, confirmed by both a significant increase in signal intensity and a melting profile identical to the reference RNA. In contrast, Nitrobacter was not detected in the environmental samples with probe Nbac1000 despite pre-amplification with Nitrobacter-specific primers, indicating the absence of strains containing this Nitrobacter-specific sequence. Pre-amplification using primers specific for beta-Proteobacterial ammonia-oxidizing bacteria resulted in a significant increase in signal intensity for probe Nso190, but melting profiles for probe Nso190 showed a slight deviation between amplified RNA and the reference microorganism, suggesting that the amplification products contained some sequences that varied by a single nucleotide difference in the probe target region.

DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples.

A small scale DNA microarray containing a set of oligonucleotide probes targeting the 16S rRNAs of several groups of nitrifying bacteria was developed for the monitoring of wastewater treatment plant samples. The microarray was tested using reference rRNAs from pure cultures of nitrifying bacteria. Characterization of samples collected from an industrial wastewater treatment facility demonstrated that nitrifying bacteria could be detected directly by microarray hybridization without the need for PCR amplification. Specifically, the microarray detected Nitrosomonas spp. but did not detect Nitrobacter. The specificity and sensitivity of direct detection was evaluated using on-chip dissociation analysis, and by two independent analyses--an established membrane hybridization format and terminal restriction fragment length polymorphism fingerprinting (T-RFLP). The latter two analyses also revealed Nitrospira and Nitrobacter to be contributing populations in the treatment plant samples. The application of DNA microarrays to wastewater treatment systems, which has been demonstrated in the current work, should offer improved monitoring capabilities and process control for treatment systems, which are susceptible to periodic failures.