In situ analysis of nitrifying bacteria in sewage treatment plants

1996 ◽  
Vol 34 (1-2) ◽  
pp. 237-244 ◽  
Author(s):  
Michael Wagner ◽  
Gabriele Rath ◽  
Hans-Peter Koops ◽  
Janine Flood ◽  
Rudolf Amann
Keyword(s):  
Activated Sludge ◽  
Sewage Treatment ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Microbiological Methods ◽  
Nitrite Oxidizing Bacteria ◽  
Identification Technique ◽  
Engineered Systems ◽  
Simultaneous Identification

Autotrophic microbial nitrification is the key process in the removal of ammonia from wastewater. To avoid the limitations of traditional microbiological methods an in situ identification technique for ammonia- and nitrite-oxidizing bacteria was developed. Based on comparative sequence analyses we designed a collection of 16S ribosomal RNA-targeted oligonucleotide probes for all validly described members of the genusNitrobacter . Whole cell hybridizations of target and reference cells with fluorescent probe derivatives were used to determine the optimal hybridization stringency for each of the probes. These probes were applied together with a recently developed probe for important members of the genus Nitrosomonas for simultaneous identification of ammonia- and nitrite-oxidizing bacteria in natural and engineered systems. Ammonia-oxidizing bacteria were identified in situ in river water, epiphytic biofilms from eutrophic wetlands, oligotrophic biofilms, a nitrifying trickling filter biofilm as well as in all analyzed nitrifying activated sludge samples. In none of these samples could Nitrobacter cells be detected in situ. However, all hitherto describedNitrobacter species and a strain of Nitrobacter sp. isolated from one of the analyzed nitrifying activated sludge samples showed bright hybridization signals with all Nitrobacter specific probes. Possible reasons for the absence of in situ detectable Nitrobacter cells are discussed.

Structure and function of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes

2000 ◽  
Vol 42 (12) ◽  
pp. 21-32 ◽  
Author(s):  
S. Okabe ◽  
Y. Watanabe
Keyword(s):  
In Situ Hybridization ◽  
Spatial Organization ◽  
Settling Tank ◽  
Outer Part ◽  
Domestic Wastewater ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Bacterial Populations ◽  
Nitrite Oxidizing Bacteria

Time dependent development of the spatial organization of NH4+- and NO2−-oxidizing bacterial populations in a domestic wastewater biofilm and in an autotrophic nitrifying biofilm were investigated by fluorescent in situ hybridization (FISH) with a set of 16S rRNA-targeted oligonucleotide probes. Population dynamics of nitrifying bacteria in the biofilms were correlated with the biofilm performance. In situ hybridization indicated that Nitrosomonas spp. (excluding probe NEU stained NH4+-oxidizing bacteria: i.e., N. marina-lineage, N. europaea-lineage, N. eutropha, and N. halophila) and Nitrospira-like bacteria were the numerically dominant nitrifying species in the domestic wastewater biofilm. However, probe NEU stained NH4+-inoxidizing bacteria became dominant populations in the autotrophic nitrifying biofilm (which were initially cultured with the primary settling tank effluent) after switching to the synthetic media. This population shift might be attributed to the effect of NO2−-–N accumulation and higher growth rates of N. europaea-lineage and N. eutropha, outcompeting other Nitrosomonas spp. in the synthetic medium. This evidence indirectly supports that N. europhaea has been most commonly isolated and studied in most of the previous researches. For the spatial organization of NH4+- and NO2−-oxidizing bacterial populations, bacteria of the genus Nitrobacter could not be detected, instead Nitrospira-like bacteria were found as the main nitrite-oxidizing bacteria in both biofilms. Whereas most of the ammonia-oxidizing bacteria were found throughout the biofilms, the location of nitrite-oxidizing bacteria was restricted to the active nitrite-oxidizing zone, which was detected in the inner part of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of NO2−-oxidizing bacteria, as determined with FISH. These observations have considerable significance to our understanding of microbial nitrification occurring in wastewater treatment processes and in the natural environment.

scholarly journals Microscale Distribution of Populations and Activities ofNitrosospira and Nitrospira spp. along a Macroscale Gradient in a Nitrifying Bioreactor: Quantification by In Situ Hybridization and the Use of Microsensors

1999 ◽  
Vol 65 (8) ◽  
pp. 3690-3696 ◽  
Author(s):  
Andreas Schramm ◽  
Dirk de Beer ◽  
Johan C. van den Heuvel ◽  
Simon Ottengraf ◽  
Rudolf Amann
Keyword(s):  
In Situ Hybridization ◽  
Specific Activity ◽  
Reaction Rates ◽  
Bulk Water ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrite Oxidizing Bacteria ◽  
Water Gradient ◽  
Bacterial Aggregates

ABSTRACT The change of activity and abundance of Nitrosospiraand 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 μm 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 short-term 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 nitrite-oxidizing 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.

scholarly journals Operational modifications for the development of nitrifying bacteria in a large-scale biological aerated filter and its impact on wastewater treatment

2018 ◽  
Vol 78 (8) ◽  
pp. 1704-1714 ◽  
Author(s):  
François-René Bourgeois ◽  
Frédéric Monette ◽  
Daniel G. Cyr
Keyword(s):  
Large Scale ◽  
Oxygen Demand ◽  
Bacterial Biofilm ◽  
Nitrifying Bacteria ◽  
Nitrification Rate ◽  
Total Biomass ◽  
Ammonia Oxidizing Bacteria ◽  
Operational Conditions ◽  
Nitrite Oxidizing Bacteria ◽  
Aerated Filter

Abstract To develop a better understanding for fixed biomass processes, the development of a nitrifying bacterial biofilm, as well as the performance of treatment during modifications to operational conditions of a full-scale submerged biological filter were examined. The development of the nitrifying biofilm was investigated at four depth levels (1, 2, 4 and 5 feet). The result of bacterial subpopulations analyzed by qPCR relative to the physico-chemical parameters of the wastewater during the various tests (sustained aeration, modified backwash parameters and inflow restriction) revealed an increase of the relative presence of nitrifying microorganisms throughout the biofilm (especially for nitrite oxidizing bacteria (NOB)), but this was not necessarily accompanied by a better nitrification rate. The highest observed nitrification rate was 49% of removal in the test cell during backwashing conditions, whereas the relative ammonia oxidizing bacteria (AOB) population was 0.032% and NOB was 0.008% of the total biomass collected. The highest percentage of nitrifying bacteria observed (0.034% AOB and 0.18% NOB) resulted in a nitrification rate of 21%. The treatment of organic matter determined by measuring the chemical and biochemical oxygen demand (COD, CBOD5) was improved.

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

1998 ◽  
Vol 37 (4-5) ◽  
pp. 441-449 ◽  
Author(s):  
Michael Wagner ◽  
Daniel R. Noguera ◽  
Stefan Juretschko ◽  
Gabriele Rath ◽  
Hans-Peter Koops ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
In Situ Hybridization ◽  
Activated Sludge ◽  
Heterotrophic Bacteria ◽  
Sewage Treatment Plant ◽  
Laser Scanning Microscopy ◽  
Industrial Plant ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonia Oxidizers

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O2 within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters are not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 16S rDNA sequence analysis confirmed the affiliation suggested by FISH.

scholarly journals The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

2003 ◽  
Vol 69 (11) ◽  
pp. 6875-6887 ◽  
Author(s):  
Justyna Adamczyk ◽  
Martin Hesselsoe ◽  
Niels Iversen ◽  
Matthias Horn ◽  
Angelika Lehner ◽  
...  
Keyword(s):  
Community Structure ◽  
Activated Sludge ◽  
Fluorescence Labeling ◽  
Specific Substrate ◽  
Ammonia Oxidizing Bacteria ◽  
Microarray Hybridization ◽  
Independent Manner ◽  
And Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

Biological Treatment of High Ammonium Content Urban Landfill Leachate Using an Anaerobic-Aerobic Process

2013 ◽  
Vol 781-784 ◽  
pp. 2095-2099
Author(s):  
Hong Wei Sun ◽  
Yong Jun You ◽  
Ying Guo
Keyword(s):  
Landfill Leachate ◽  
Inhibitory Effect ◽  
Nitrifying Bacteria ◽  
Anaerobic Sludge ◽  
Nitrite Accumulation ◽  
Fish Analysis ◽  
Free Nitrous Acid ◽  
Nitrite Oxidizing Bacteria ◽  
Anaerobic Sludge Blanket

Biological system consisting of an up-flow anaerobic sludge blanket (UASB) and anoxic-oxic (A/O) reactor was applied to treat high ammonium content urban landfill leachate. Inhibitory effect of free ammonia (FA) and free nitrous acid (FNA) on nitrifying bacteria activity was used to achieve nitrogen removal via nitrite pathway in the A/O. Results demonstrated that removed efficiencies of COD, total nitrogen (TN) and NH4+-N were 95.3%, 84.6 %and 99.2%, respectively. Stable nitrite pathway with above 90% nitrite accumulation was successfully achieved in the A/O reactor by synergetic inhibition of FA and FNA on the activity of nitrite oxidizing bacteria (NOB). Moreover, Fluorescence in situ hybridization (FISH) analysis showed that AOB was dominant microorganism.

scholarly journals Functional degeneracy in translationally active Nitrospira populations confers resilience to out-selection in partial nitritation activated sludge

2021 ◽  
Author(s):  
Maxwell B.W. Madill ◽  
Yaqian Luo ◽  
Pranav Sampara ◽  
Ryan M. Ziels
Keyword(s):  
Activated Sludge ◽  
Ecological Niches ◽  
Nitrite Accumulation ◽  
Partial Nitritation ◽  
Physiological Diversity ◽  
Translational Activity ◽  
Subsequent Decrease ◽  
Nitrite Oxidizing Bacteria ◽  
Canonical Amino Acid

AbstractEnergy-efficient nitrogen removal in activated sludge wastewater treatment systems could be achieved by oxidizing a part of the influent ammonium to nitrite (partial nitritation; PN) to provide growth substrates for anammox. However, the metabolic and physiological diversity of nitrite oxidizing bacteria (NOB) makes their out-selection a challenge in mainstream activated sludge, warranting measurements of their in situ physiology and activity under selective growth pressures. Here, we examined the long-term (~100 day) stability of PN in mainstream activated sludge obtained by treating a portion of return activated sludge with a sidestream flow containing free ammonia (FA) at 200 mg NH3-N/L. The nitrite accumulation ratio peaked at 42% by day 40 in the reactor with sidestream FA-treatment, while it did not increase in the control reactor without FA added to the sidestream. A subsequent decrease in nitrite accumulation within the FA-treated reactor coincided with shifts in dominant Nitrospira 16S rRNA amplicon sequence variants (ASVs). We applied bioorthogonal non-canonical amino acid tagging (BONCAT) coupled with fluorescence activated cell sorting (FACS) to investigate changes in the translational activity of NOB populations throughout sidestream exposure to FA. BONCAT-FACS confirmed that the single Nitrospira ASV out-selected in the FA-treated reactor had reduced translational activity following exposure to FA, whereas the two Nitrospira ASVs that emerged after process acclimation were not impacted by FA. Thus, functionally degenerate Nitrospira populations likely provided resilience to the nitrite-oxidizing community during FA-treatment by shifting activity to physiologically resistant members. These results highlight how in situ physiology approaches like BONCAT can resolve ecological niches in the activated sludge microbiome, and how functional degeneracy observed within Nitrospira populations likely necessitates a combination of out-selection strategies to achieve stable mainstream PN.

scholarly journals Nitrification mainly driven by ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in an anammox-inoculated wastewater treatment system

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jing Lu ◽  
Yiguo Hong ◽  
Ying Wei ◽  
Ji-Dong Gu ◽  
Jiapeng Wu ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Activated Sludge ◽  
Nitrogen Removal ◽  
High Throughput Sequencing ◽  
High Efficiency ◽  
Ammonia Oxidation ◽  
Ammonia Oxidizing Bacteria ◽  
Ammonium Oxidation ◽  
Strong Activity ◽  
Nitrite Oxidizing Bacteria

AbstractAnaerobic ammonium oxidation (anammox) process has been acknowledged as an environmentally friendly and time-saving technique capable of achieving efficient nitrogen removal. However, the community of nitrification process in anammox-inoculated wastewater treatment plants (WWTPs) has not been elucidated. In this study, ammonia oxidation (AO) and nitrite oxidation (NO) rates were analyzed with the incubation of activated sludge from Xinfeng WWTPs (Taiwan, China), and the community composition of nitrification communities were investigated by high-throughput sequencing. Results showed that both AO and NO had strong activity in the activated sludge. The average rates of AO and NO in sample A were 6.51 µmol L−1 h−1 and 6.52 µmol L−1 h−1, respectively, while the rates in sample B were 14.48 µmol L−1 h−1 and 14.59 µmol L−1 h−1, respectively. The abundance of the nitrite-oxidizing bacteria (NOB) Nitrospira was 0.89–4.95 × 1011 copies/g in both samples A and B, the abundance of ammonia-oxidizing bacteria (AOB) was 1.01–9.74 × 109 copies/g. In contrast, the abundance of ammonia-oxidizing archaea (AOA) was much lower than AOB, only with 1.28–1.53 × 105 copies/g in samples A and B. The AOA community was dominated by Nitrosotenuis, Nitrosocosmicus, and Nitrososphaera, while the AOB community mainly consisted of Nitrosomonas and Nitrosococcus. The dominant species of Nitrospira were Candidatus Nitrospira defluvii, Candidatus Nitrospira Ecomare2 and Nitrospira inopinata. In summary, the strong nitrification activity was mainly catalyzed by AOB and Nitrospira, maintaining high efficiency in nitrogen removal in the anammox-inoculated WWTPs by providing the substrates required for denitrification and anammox processes.

scholarly journals Study on the diversity of denitrification bacteria treating with wastewater by using PPGC filler on SBMBBR at low temperature

2020 ◽  
Vol 158 ◽  
pp. 04002
Author(s):  
Jinxiang Fu ◽  
Zhe Zhang ◽  
Jinghai Zhu
Keyword(s):  
Waste Water ◽  
Low Temperature ◽  
Activated Sludge ◽  
Removal Efficiency ◽  
Relative Abundance ◽  
High Throughput Sequencing ◽  
Denitrifying Bacteria ◽  
Nitrifying Bacteria ◽  
Ammonia Oxidizing Bacteria ◽  
Biological Nitrogen

Aiming at the problem of the low removal efficiency of biological nitrogen-removing of low temperature waste-water, using Polyurethane Porous Gel Carrier (PPGC)-SBMBBR treated low temperature sewage, in compared with conventional SBR,and viaing Miseq high-throughput sequencing technology in analysis of the differences of microbial diversity and abundance of structure on the two reactors of activated sludge, revealed dominant nitrogen-removing bacterium improving the treatment efficiency of low temperature sewage. The results shows that the removal efficiency of the effluent nitrogen and the sludge sedimentation rate of (PPGC)-SBMBBR reactor are significantly improved under the water temperature (6.5±1℃). Adding the filler can contribute to improvement of bacterial diversity and relative abundance of nitrification and denitrification bacterium in the activated sludge system. The main relative abundance of ammonia oxidizing bacteria (AOB),nitrite oxidizing bacteria (NOB),anaerobic denitrifying bacteria, and aerobic denitrifying bacteria in (PPGC)-SBMBBR(R2) are significantly better than SBR (R1),and the R2 reactor can independently enrich the nitrifying bacteria and the aerobic denitrifying bacteria, such as Nitrospira, Hydrogens, Pseudomonas, and Zoogloea. The total relative abundance of dominant and nitrifying denitrifying bacterium increases from 28.65% of R1 to 60.23% of R2, providing a microbiological reference for improving the efficiency of biological nitrogen removal in low temperature waste-water.

Transition of bacterial spatial organization in a biofilm monitored by FISH and subsequent image analysis

2004 ◽  
Vol 49 (11-12) ◽  
pp. 365-370 ◽  
Author(s):  
Y. Aoi ◽  
S. Tsuneda ◽  
A. Hirata
Keyword(s):  
Image Analysis ◽  
Outer Layer ◽  
Spatial Organization ◽  
Heterotrophic Bacteria ◽  
Great Influence ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrite Oxidizing Bacteria ◽  
Growth Activity ◽  
Do Concentration

The dynamic transition of bacterial community structure in a biofilm was monitored by the fluorescence in situ hybridization (FISH) technique and subsequent image analysis. Heterotrophic bacteria that had occupied the outer layer were gradually decreased whereas ammonia-oxidizing bacteria (AOB) gradually increased their growth activity and extended their existence area to the outer layer of the biofilm through the gradual reduction of the C/N ratio. The spatial organization of AOB in the biofilm dynamically changed responding to the environmental conditions such as pH fluctuation and lack of dissolved oxygen (DO) and had great influence on the nitrification activity. The accumulation of nitrite was observed at lower DO concentration, which might be due to the property that nitrite-oxidizing bacteria (NOB) possess of higher Km values for oxygen than AOB.

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