Microscale distribution of populations and acitivities of Nitrosospira and Nitrospira ssp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors (original) (raw)
Related papers
1999
The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 m on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a shortterm incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitriteoxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.
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.
Water Research, 2008
AQUASIM FISH Juxta-positioning Model Nitrite oxidation Real-time PCR a b s t r a c t The autotrophic two-species biofilm from the packed bed reactor of a life-support system, containing Nitrosomonas europaea ATCC 19718 and Nitrobacter winogradskyi ATCC 25391, was analysed after 4.8 years of continuous operation performing complete nitrification. Realtime quantitative polymerase chain reaction (Q-PCR) was used to quantify N. europaea and N. winogradskyi along the vertical axis of the reactor, revealing a spatial segregation of N. europaea and N. winogradskyi. The main parameters influencing the spatial segregation of both nitrifiers along the bed were assessed through a multi-species one-dimensional biofilm model generated with AQUASIM software. The factor that contributed the most to this distribution profile was a small deviation from the flow pattern of a perfectly mixed tank towards plug-flow. The results indicate that the model can estimate the impact of specific biofilm parameters and predict the nitrification efficiency and population dynamics of a multispecies biofilm.
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.
Competition betweenNitrospira spp. andNitrobacter spp. in nitrite-oxidizing bioreactors
Biotechnology and Bioengineering, 2006
In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying-activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area (RCA) of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization (FISH). The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira, caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira. An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population. ß
Water Environment Research, 2001
Fluorescent in situ hybridization (FISH) was used to quantify the ammonia-oxidizing populations within intact biofilm samples collected from a full-scale nitrifying trickling filter (NTF). Ammonia, nitrite, and nitrate concentrations were measured for aqueous samples taken in conjunction with biofilm samples at multiple filter depths. Correlation coefficients for individual sampling events, calculated by simple linear regression of FISH signal area and ammonia removal rates, ranged from 0.558 when using probe NEU23a to 0.982 when using probe Nso190. The improved correlations with Nso190 suggest that genera other than Nitrosomonas are present in this system. Percent biofilm coverage, as determined by 4Ј,6-diamidino-2phenylindole counterstaining and dry weight biomass measurements, did not change throughout the NTF. This indicates that biofilm growth is fairly uniform throughout the filter even if nitrifier growth is not. Water Environ. Res., 73, 329 (2001).
A quantitative measure of nitrifying bacterial growth
Water Research, 2006
Nitrifying bacteria convert ammonia (NH 3) to nitrate (NO 3 À) in a nitrification reaction. Methods to quantitatively separate the growth rate of these important bacterial populations from that of the dominant heterotrophic bacteria are important to our understanding of the nitrification process. The changing concentration of ammonia is often used as an indirect measure of nitrification but ammonification processes generate ammonia and confound this approach while heterotrophs remove nitrate via denitrification. Molecular probe methods can tell us what proportion of the microbial community is nitrifying bacteria but not their growth rate. The technique proposed here was able to quantify the growth rate of the nitrifying bacterial populations amidst complex ecological processes. The method incubates [methyl-3 H] thymidine with water samples in the presence and absence of an inhibitor of nitrification-thiourea. The radioactively labeled DNA in the growing bacteria was extracted. The rate of incorporation of the label into the dividing bacterial DNA was used to determine bacterial growth rate. Total bacterial community growth rates in full-scale and pilot-scale fixed-film nitrifying reactors and an activated sludge reactor were 2.1 Â 10 8 , 4.1 Â 10 8 and 0.4 Â 10 8 cell ml À1 d À1 , respectively; the growth rate of autotrophic-nitrifying bacteria was 0.7 Â 10 8 , 2.6Â 10 8 and 0.01 Â 10 8 cell ml À1 d À1 , respectively. Autotrophic-nitrifying bacteria contributed 30% and 60% of the total bacterial community growth rate in the nitrifying reactors whereas only 2% was observed in the activated sludge reactor that was not designed to nitrify. The rates of ammonia loss from the nitrifying reactors corresponded to the rate of growth of the nitrifying bacteria. This method has the potential to more often identify factors that enhance or limit nitrifying processes in both engineered and natural aquatic environments.
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.
Journal of Bioscience and Bioengineering, 2000
The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16s rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic wastewater, while heterotrophic bacteria and ammoniaoxidizing bacteria were localized at different positions in the biofilm in organic wastewater. The dynamics of the microbial ecology in the biofilm with regard to the spatial distribution of ammonia-oxidizing bacteria and heterotrophic bacteria caused by a gradual change in substrate composition was successfully monitored by FISH analysis.
Nitrite concentration influences the population structure of Nitrospira-like bacteria
Environmental Microbiology, 2006
Nitrospira are a monophyletic but diverse group of organisms, are widely distributed in many natural habitats, and play a key role in nitrogen elimination during biological wastewater treatment. Phylogenetic analyses of cloned 16S rRNA genes and fluorescence in situ hybridization with newly developed rRNA-targeted oligonucleotide probes revealed coexistence of uncultured members of sublineages I and II of the genus Nitrospira in biofilm and activated sludge samples taken from nitrifying wastewater treatment plants. Quantitative microscopic analyses of their spatial arrangement relative to ammonia oxidizers in the biofilm and activated sludge flocs showed that members of the Nitrospira sublineage I occurred significantly more often in immediate vicinity to ammonia oxidizers than would be expected from random community assembly while such a relationship was not observed for Nitrospira sublineage II. This spatial distribution suggested a niche differentiation of these coexisting Nitrospira populations with respect to their preferred concentrations of nitrite. This hypothesis was tested by mathematical modelling of nitrite consumption and resulting nitrite gradients in nitrifying biofilms and by quantifying the abundance of sublineage I and II Nitrospira in activated sludge during incubations with nitrite in different concentrations. Consistent with the observed localization patterns, a higher nitrite concentration selected for sublineage I but suppressed sublineage II Nitrospira .