Molecular identification of nitrifying bacteria in activated sludge (original) (raw)
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Water science and technology : a journal of the International Association on Water Pollution Research, 2003
Nitrification was assessed in two full-scale wastewater treatment plants (WWTPs) over time using molecular methods. Both WWTPs employed a complete-mix suspended growth, aerobic activated sludge process (with biomass recycle) for combined carbon and nitrogen treatment. However, one facility treated primarily municipal wastewater while the other only industrial wastewater. Real time PCR assays were developed to determine copy numbers for total 16S rDNA (a measure of biomass content), the amoA gene (a measure of ammonia-oxidizers), and the Nitrospira 16S rDNA gene (a measure of nitrite-oxidizers) in mixed liquor samples. In both the municipal and industrial WWTP samples, total 16S rDNA values were approximately 2-9 x 10(13) copies/L and Nitrospira 16S rDNA values were 2-4 x 10(10) copies/L. amoA gene concentrations averaged 1.73 x 10(9) copies/L (municipal) and 1.06 x 10(10) copies/L (industrial), however, assays for two distinct ammonia oxidizing bacteria were required.
Real-Time PCR Quantification of Nitrifying Bacteria in a Municipal Wastewater Treatment Plant
Environmental Science & Technology, 2003
Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were loglinear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotrophalike amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 ( 2.0 × 10 11 for bacteria, 3.7 ( 3.2 × 10 10 for Nitrospira, 1.2 ( 0.9 × 10 10 for all AOB, and 7.5 ( 6.0 × 10 9 for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitriteoxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 ( 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 ( 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.
In situ characterization of nitrifiers in an activated sludge plant: detection of Nitrobacter Spp
Journal of Applied Microbiology, 2002
The purpose of this work was to investigate microbial ecology of nitrifiers at the genus level in a typical full-scale activated sludge plant. Methods and Results: Grab samples of mixed liquor were collected from a plug-flow reactor receiving domestic wastewater. Fluorescent in situ hybridization technique (FISH) was used to characterize both ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in combination with Confocal Scanning Laser Microscope (CSLM). Fluorescently labelled, 16S rRNA-targeted oligonucleotide probes were used in this study. Both Nitrosomonas and Nitrosospira genera as AOB and Nitrobacter and Nitrospira genera as NOB were sought with genus specific probes Nsm156, Nsv443 and NIT3 and NSR1156, respectively. Conclusions: It was shown that Nitrosospira genus was dominant in the activated sludge system studied, although Nitrosomonas is usually assumed to be the dominant genus. At the same time, Nitrobacter genus was detected in activated sludge samples. Significance and Impact of the Study: Previous studies based on laboratory scale pilot plants employing synthetic wastewater suggested that only Nitrospira are found in wastewater treatment plants. We have shown that Nitrobacter genus might also be present. We think that these kinds of studies may not give a valid indication of the microbial diversity of the real fullscale plants fed with domestic wastewater.
Applied and Environmental Microbiology, 2001
Uncultivated Nitrospira-like bacteria in different biofilm and activated-sludge samples were investigated by cultivation-independent molecular approaches. Initially, the phylogenetic affiliation of Nitrospira-like bacteria in a nitrifying biofilm was determined by 16S rRNA gene sequence analysis. Subsequently, a phylogenetic consensus tree of the Nitrospira phylum including all publicly available sequences was constructed. This analysis revealed that the genus Nitrospira consists of at least four distinct sublineages. Based on these data, two 16S rRNA-directed oligonucleotide probes specific for the phylum and genus Nitrospira, respectively, were developed and evaluated for suitability for fluorescence in situ hybridization (FISH). The probes were used to investigate the in situ architecture of cell aggregates of Nitrospira-like nitrite oxidizers in wastewater treatment plants by FISH, confocal laser scanning microscopy, and computer-aided three-dimensional visualization. Cavities and a network of cell-free channels inside the Nitrospira microcolonies were detected that were water permeable, as demonstrated by fluorescein staining. The uptake of different carbon sources by Nitrospira-like bacteria within their natural habitat under different incubation conditions was studied by combined FISH and microautoradiography. Under aerobic conditions, the Nitrospira-like bacteria in bioreactor samples took up inorganic carbon (as HCO 3 ؊ or as CO 2 ) and pyruvate but not acetate, butyrate, and propionate, suggesting that these bacteria can grow mixotrophically in the presence of pyruvate. In contrast, no uptake by the Nitrospira-like bacteria of any of the carbon sources tested was observed under anoxic or anaerobic conditions.
Applied and Environmental Microbiology, 2002
Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotrophalike ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ؎ 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ؎ 0.28% of the biosludge population in the municipal WWTP and 0.37% ؎ 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.
Molecular microbial diversity in a nitrifying reactor system without sludge retention
Fems Microbiology Ecology, 1998
Recently, the single reactor system for high activity ammonia removal over nitrite (SHARON) process was developed for the removal of ammonia from wastewater with high ammonia concentrations. In contrast to normal systems, this nitrifying reactor system is operated at relatively high temperatures (35³C) without sludge retention. Classical methods to describe the microbial community present in the reactor failed and, therefore, the microorganisms responsible for ammonia removal in this single reactor system were investigated using several complementary molecular biological techniques. The results obtained via these molecular methods were in good agreement with each other and demonstrated successful monitoring of microbial diversity. Denaturing gradient gel electrophoresis of 16S rRNA PCR products proved to be an effective technique to estimate rapidly the presence of at least four different types of bacteria in the SHARON reactor. In addition, analysis of a 16S rRNA gene library revealed that there was one dominant (69%) clone which was highly similar (98.8%) to Nitrosomonas eutropha. Of the other clones, 14% could be assigned to new members of the Cytophaga/Flexibacter group. These data were qualitatively and quantitatively confirmed by two independent microscopic methods. The presence of about 70% ammonia oxidizing bacteria was demonstrated using a fluorescent oligonucleotide probe (NEU) targeted against the 16S rRNA of the Nitrosomonas cluster. Electron microscopic pictures showed the typical morphology of ammonia oxidizers in the majority of the cells from the SHARON reactor. z
Molecular detection of microbial community in a nitrifying–denitrifying activated sludge system
International Biodeterioration & Biodegradation, 2013
Presence of different forms of ammonia in the wastewater treatment plants are toxic to aquatic life and promote eutrophication in receiving water bodies. It becomes imperative to lower the ammonia concentration in the effluents for which characterization of microbial diversity is foremost objective. Study was planned to identify the diversity of nitrifiers and denitrifiers from activated sludge using PCR technique. Cultural techniques revealed high bacterial diversity was found in the aeration tank where majority of the isolates belongs to class g proteobacteria followed by b proteobacteria suggesting the adaptability of these groups. Presence of Nitrosomonas europaea revealed better ammonium oxidation condition at longer SRT of 6 and 7 d with DO concentration of 2.5 mg/L. As a part of NOB, Nitrospira sp. found to be more dominant than Nitrobacter winogradskyi as its detection was achieved in all samples of 6 and 7 d SRT, while later was detected only on 7 d SRT. Use of cultural techniques in combination with PCR analysis with 16S rRNA approach revealed Pseudomonas aeruginosa as important denitrifier. Highest removal efficiencies of COD, BOD, TN and NH þ 4 eN were found to be 81, 78, 70 and 69% respectively at optimized SRT of 7 d.
Journal of Bioremediation & Biodegradation, 2014
Nitrifying bacteria plays a major role in converting the waste water to valuable renewable resources for the society. Ammonia is the toxic excretory product of most aquatic organisms and nitrite formed by the oxidation of ammonia is also toxic. Ammonia oxidizing bacteria converts ammonia to nitrite and Nitrite oxidizing bacteria convert nitrite to nitrate. Among them, Nitrosomonas sp. and Nitrobacter sp. has been the most widely studied organisms. An advanced molecular technique made it possible to explore the nitrifying bacteria in the environment and to enhance our knowledge of its functioning. In view of this it would be of prime importance for rapid detection of these strains/ isolates using molecular techniques. The present study was undertaken to develop and validate 16S rDNA sequence analysis of Nitrosomonas sp. and Nitrobacter sp. collected from different bheries in East Kolkata Wetland, West Bengal.
2007
In order to improve wastewater treatment processes, a need exists for tools that rapidly give detailed insight into the community structure of activated sludge, supplementary to chemical and physical data. In this study, the advantages of microarrays and quantitative polymerase chin reaction (PCR) methods were combined into a real-time PCR assay that allows the simultaneous quantification of phylogenetic and functional genes involved in nitrification and denitrification processes. Simultaneous quantification was possible along a 5-log dynamic range and with high linear correlation (R 2 > 0.98). The specificity of the assay was confirmed by cloning and sequencing analyses of PCR amplicons obtained from activated sludge. The real-time assay was validated on mixed liquid samples of different treatment plants, which varied in nitrogen removal rate. The abundance of ammonia oxidizers was in the order of magnitude of 106 down to 104 ml−1, whereas nitrite oxidizers were less abundant (103–101 order of magnitude). The results were in correspondence with the nitrite oxidation rate in the sludge types. As for the nirS, nirK, and nosZ gene copy numbers, their abundance was generally in the order of magnitude of 108–105. When sludge samples were subjected to lab-scale perturbations, a decrease in nitrification rate was reflected within 18 h in the copy numbers of nitrifier genes (decrease with 1 to 5 log units), whereas denitrification genes remained rather unaffected. These results demonstrate that the method is a fast and accurate tool for the analysis of the (de)nitrifying community structure and size in both natural and engineered environmental samples.
Environmental Microbiology, 2006
Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira -like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospiralike bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 m m m m g ml ----1 . The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira -like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as ' Candidatus Nitrospira defluvii'.