Denaturing gradient gel electrophoretic analysis of ammonia-oxidizing bacterial community structure in the lower Seine River: impact of Paris wastewater effluents

The Seine River is strongly affected by the effluents from the Acheres wastewater treatment plant (WWTP) downstream of the city of Paris. We have shown that the effluents introduce large amounts of ammonia and inoculate the receiving medium with nitrifying bacteria. The aim of the present study was to investigate the diversity of the ammonia-oxidizing bacterial population by identifying autochthonous bacteria from upstream and/or allochthonous ammonia-oxidizing bacteria from the WWTP effluents. Measurements of potential nitrifying activity, competitive PCR, and denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments specific to ammonia-oxidizing bacteria (AOB) were used to explore the succession and shifts of the ammonia-oxidizing community in the lower Seine River and to analyze the temporal and spatial functioning of the system at several different sampling dates. A major revelation was the stability of the patterns. The CTO primers used in this study (G. A. Kowalchuk, J. R. Stephen, W. D. Boer, J. I. Prosser, T. M. Embley, and J. W. Woldendorp, Appl. Environ. Microbiol. 63:1489-1497, 1997) were shown not to be completely specific to AOB of the beta subclass of Proteobacteria. We further demonstrated that when DGGE patterns are interpreted, all the different bands must be sequenced, as one major DGGE band proved to be affiliated with a group of non-AOB in the beta subclass of Proteobacteria. The majority of AOB (75 to 90%) present in the lower Seine river downstream of the effluent output belong to lineage 6a, represented by Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria. This dominant lineage was represented by three bands on the DGGE gel. The major lineage-6a AOB species, introduced by the WWTP effluents, survived and might have grown in the receiving medium far downstream, in the estuary; it represented about 40% of the whole AOB population. The other two species belonging to lineage 6a seem to be autochthonous bacteria. One of them developed a few kilometers downstream of the WWTP effluent input in an ammonia-enriched environment, and the other appeared in the freshwater part of the estuary and was apparently more adapted to estuarine conditions, i.e., an increase in the amount of suspended matter, a low ammonia concentration, and high turnover of organic matter. The rest of the AOB population was represented in equal proportions by Nitrosospira- and Nitrosococcus mobilis-like species.     

Changes in the community structure and activity of betaproteobacterial ammonia-oxidizing sediment bacteria along a freshwater-marine gradient

To determine whether the distribution of estuarine ammonia-oxidizing bacteria (AOB) was influenced by salinity, the community structure of betaproteobacterial ammonia oxidizers (AOB) was characterized along a salinity gradient in sediments of the Ythan estuary, on the east coast of Scotland, UK, by denaturant gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rRNA gene fragments. Ammonia-oxidizing bacteria communities at sampling sites with strongest marine influence were dominated by Nitrosospira cluster 1-like sequences and those with strongest freshwater influence were dominated by Nitrosomonas oligotropha-like sequences. Nitrosomonas sp. Nm143 was the prevailing sequence type in communities at intermediate brackish sites. Diversity indices of AOB communities were similar at marine- and freshwater-influenced sites and did not indicate lower species diversity at intermediate brackish sites. The presence of sequences highly similar to the halophilic Nitrosomonas marina and the freshwater strain Nitrosomonas oligotropha at identical sampling sites indicates that AOB communities in the estuary are adapted to a range of salinities, while individual strains may be active at different salinities. Ammonia-oxidizing bacteria communities that were dominated by Nitrosospira cluster 1 sequence types, for which no cultured representative exists, were subjected to stable isotope probing (SIP) with 13C-HCO3-, to label the nucleic acids of active autotrophic nitrifiers. Analysis of 13C-associated 16S rRNA gene fragments, following CsCl density centrifugation, by cloning and DGGE indicated sequences highly similar to the AOB Nitrosomonas sp. Nm143 and Nitrosomonas cryotolerans and to the nitrite oxidizer Nitrospira marina. No sequence with similarity to the Nitrosospira cluster 1 clade was recovered during SIP analysis. The potential role of Nitrosospira cluster 1 in autotrophic ammonia oxidation therefore remains uncertain.     

Seasonal variation in communities of ammonia-oxidizing bacteria based on polymerase chain reaction - denaturing gradient gel electrophoresis in a biofilm reactor for drinking water pretreatment

The diversity and variation of total and active ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment were characterized by clone libraries and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA and its gene during a whole year. Sequences obtained from clone libraries affiliated with the Nitrosomonas oligotropha lineage and the Nitrosomonas communis lineage. An uncultured subgroup of Nitrosomonas communis lineage was also detected. Seasonal variations in both total and active ammonia-oxidizing bacteria communities were observed in the DGGE profiles, but an RNA-based analysis reflected more obvious dynamic changes in ammonia-oxidizer community than a DNA-based approach. Statistical study based on canonical correspondence analysis showed that a community shift of active ammonia oxidizers was significantly influenced by temperature and pH, but no significant correlation was found between environmental variables and total ammonia-oxidizer community shift.     

Effect of salinity on temporal and spatial dynamics of ammonia-oxidising bacteria from intertidal freshwater sediment

Temporal and spatial dynamics within an ammonia-oxidising community from intertidal, freshwater sediments were studied in microcosms simulating flooding twice a day with fresh, brackish and marine waters. The microcosms had been filled with the upper 5 cm of intertidal freshwater sediment from the river Scheldt. Changes in community composition were examined by denaturing gradient gel electrophoresis of amplified DNA from the community. In the first week of incubation the initially present members of the Nitrosomonas oligotropha lineage were replaced by other members of the same lineage in the top layer of the sediment subjected to flooding with freshwater. Prolonged incubation extended niche differentiation to a depth of 5 cm. In the microcosms flooded with saline media, the initially present members of the N. oligotropha lineage were replaced by strains belonging to the Nitrosomonas marina lineage, but only in the top 1cm. Shift in community composition occurred earlier in the marine microcosms than in the brackish microcosms and was slower than the change in the freshwater microcosms. Irrespective of the nature of the flooding medium, shifts in community composition were always consistent among replicate microcosms. We conclude that salinity is an important steering factor in niche differentiation among ammonia-oxidising bacteria and also that changes within the community of this functional group of bacteria may occur at different rates.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.     

Epiphyton as a niche for ammonia-oxidizing bacteria: detailed comparison with benthic and pelagic compartments in shallow freshwater lakes

Next to the benthic and pelagic compartments, the epiphyton of submerged macrophytes may offer an additional niche for ammonia-oxidizing bacteria in shallow freshwater lakes. In this study, we explored the potential activities and community compositions of ammonia-oxidizing bacteria of the epiphytic, benthic, and pelagic compartments of seven shallow freshwater lakes which differed in their trophic status, distribution of submerged macrophytes, and restoration history. PCR-denaturing gradient gel electrophoresis analyses demonstrated that the epiphytic compartment was inhabited by species belonging to cluster 3 of the Nitrosospira lineage and to the Nitrosomonas oligotropha lineage. Both the ammonia-oxidizing bacterial community compositions and the potential activities differed significantly between compartments. Interestingly, both the ammonia-oxidizing bacterial community composition and potential activity were influenced by the restoration status of the different lakes investigated.     

太湖竺山湾沉积物中氨氧化原核生物的垂直分布与多样性

原核生物驱动的氨氧化过程对于富营养化湖泊的氮循环具有重要意义。为了解太湖藻型湖区沉积物中氨氧化原核生物的垂直分布和多样性特征,采用分子生态学方法,对竺山湾沉积物剖面中氨单加氧酶基因(amoA)或16S rRNA基因等特征分子标记的变化和序列特征进行了分析。结果表明,氨氧化细菌(ammonia-oxidizing bacteria,AOB)和氨氧化古菌(ammonia-oxidizing archaea,AOA)共存于沉积物各层。AOB的优势种在5cm深度以下发生明显改变,这可能与沉积物氧化还原电位及铵态氮的变化有关;所有细菌amoA序列均属亚硝化单胞菌(Nitrosomonas)。AOA群落结构自表层至7cm深度变化不大,所有古菌amoA序列分属泉古菌CG1.1b和CG1.1a两大类群,这可能与太湖形成历史上的海陆交替过程有关。此外,沉积物各层均未发现典型厌氧氨氧化(anaerobic ammonium oxidation,anammox)细菌16S rRNA基因序列。这些发现丰富了对太湖藻型湖区氨氧化原核生物分布、多样性及环境调控原理的认识,对理解富营养化湖泊氨氧化规律、认识湖泊生态系统氮循环功能具有借鉴意义。     

Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam

The abundance and composition of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) communities under different long-term (17 years) fertilization practices were investigated using real-time polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). A sandy loam with pH (H(2)O) ranging from 8.3 to 8.7 was sampled in years 2006 and 2007, including seven fertilization treatments of control without fertilizers (CK), those with combinations of fertilizer nitrogen (N), phosphorus (P) and potassium (K): NP, NK, PK and NPK, half chemical fertilizers NPK plus half organic manure (1/2OMN) and organic manure (OM). The highest bacterial amoA gene copy numbers were found in those treatments receiving N fertilizer. The archaeal amoA gene copy numbers ranging from 1.54 x 10(7) to 4.25 x 10(7) per gram of dry soil were significantly higher than those of bacterial amoA genes, ranging from 1.24 x 10(5) to 2.79 x 10(6) per gram of dry soil, which indicated a potential role of AOA in nitrification. Ammonia-oxidizing bacteria abundance had significant correlations with soil pH and potential nitrification rates. Denaturing gradient gel electrophoresis patterns revealed that the fertilization resulted in an obvious change of the AOB community, while no significant change of the AOA community was observed among different treatments. Phylogenetic analysis showed a dominance of Nitrosospira-like sequences, while three bands were affiliated with the Nitrosomonas genus. All AOA sequences fell within cluster S (soil origin) and cluster M (marine and sediment origin). These results suggest that long-term fertilization had a significant impact on AOB abundance and composition, while minimal on AOA in the alkaline soil.     

Community structure and abundance of ammonia-oxidizing archaea and bacteria after conversion from soybean to rice paddy in albic soils of Northeast China

Community composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the albic soil grown with soybean and rice for different years was investigated by construction of clone libraries, denaturing gradient gel electrophoresis (DGGE), and quantitative polymerase chain reaction (q-PCR) by PCR amplification of the ammonia monooxygenase subunit A (amoA) gene. Soil samples were collected at two layers (0–5 and 20–25 cm) from a soybean field and four rice paddy fields with 1, 5, 9, and 17 years of continuous rice cultivation. Both the community structures and abundances of AOA and AOB showed detectable changes after conversion from soybean to rice paddy judged by clone library, DGGE, and q-PCR analyses. In general, the archaeal amoA gene abundance increased after conversion to rice cultivation, while bacterial amoA gene abundance decreased. The abundances of both AOA and AOB were higher in the surface layer than the bottom one in the soybean field, but a reverse trend was observed for AOB in all paddy samples regardless of the duration of paddy cultivation. Phylogenetic analysis identified nine subclusters of AOA and seven subclusters of AOB. Community composition of both AOA and AOB was correlated with available ammonium and increased pH value caused by flooding in multiple variance analysis. Community shift of AOB was also observed in different paddy fields, but the two layers did not show any detectable changes in DGGE analysis. Conversion from soybean to rice cultivation changed the community structure and abundance of AOA and AOB in albic agricultural soil, which requires that necessary cultivation practice be followed to manage the N utilization more effectively.     

Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.     

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

Deep biosphere-related bacteria within the subsurface of tidal flat sediments

Biogeochemical and microbiological processes in the upper sediment layers of tidal flats were analysed in many investigations, while deeper zones remained largely unexplored. Therefore, denaturant gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments along the depth profile of up to 5.5 m-long sediment cores was performed in comparison with lithological and geochemical parameters. The investigation revealed that different compartments of the sediment columns were characterized by specific microbial communities. These compartments were analysed by sequencing of 113 DGGE bands. The upper layers down to 160-200 cm were dominated by gamma- and delta-Proteobacteria representing more than 60% of the total number of phylotypes. Underneath, a striking shift in community composition was observed, as the Proteobacteria were replaced by Chloroflexi with more than 60% of all sequences. As sulfate was still available as an electron acceptor in these layers, the abundance of Chloroflexi might be promoted by the electron donor or the quality of the carbon source. The dominance of this group, previously known as green non-sulfur bacteria, indicates the presence of a typical deep-biosphere microbial community in relatively young subsurface sediments. Thus, tidal flats might offer a convenient possibility to study and understand certain aspects of the deep biosphere in general.     

Changes in Community Structure of Sediment Bacteria Along the Florida Coastal Everglades Marsh–Mangrove–Seagrass Salinity Gradient

Community structure of sediment bacteria in the Everglades freshwater marsh, fringing mangrove forest, and Florida Bay seagrass meadows were described based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) patterns of 16S rRNA gene fragments and by sequencing analysis of DGGE bands. The DGGE patterns were correlated with the environmental variables by means of canonical correspondence analysis. There was no significant trend in the Shannon–Weiner index among the sediment samples along the salinity gradient. However, cluster analysis based on DGGE patterns revealed that the bacterial community structure differed according to sites. Not only were these salinity/vegetation regions distinct but the sediment bacteria communities were consistently different along the gradient from freshwater marsh, mangrove forest, eastern-central Florida Bay, and western Florida Bay. Actinobacteria- and Bacteroidetes/Chlorobi-like DNA sequences were amplified throughout all sampling sites. More Chloroflexi and members of candidate division WS3 were found in freshwater marsh and mangrove forest sites than in seagrass sites. The appearance of candidate division OP8-like DNA sequences in mangrove sites distinguished these communities from those of freshwater marsh. The seagrass sites were characterized by reduced presence of bands belonging to Chloroflexi with increased presence of those bands related to Cyanobacteria, γ-Proteobacteria, Spirochetes, and Planctomycetes. This included the sulfate-reducing bacteria, which are prevalent in marine environments. Clearly, bacterial communities in the sediment were different along the gradient, which can be explained mainly by the differences in salinity and total phosphorus.     

Effect of monensin feeding and withdrawal on populations of individual bacterial species in the rumen of lactating dairy cows fed high-starch rations

Population dynamics of ammonia-oxidizing bacteria (AOB) in a full-scale aerated submerged biofilm reactor for micropolluted raw water pretreatment was investigated using molecular techniques for a period of 1 year. The ammonia monooxygenase (amoA) gene fragments were amplified from DNA and RNA extracts of biofilm samples. Denaturing gradient gel electrophoresis (DGGE) profile based on the amoA messenger RNA approach exhibited a more variable pattern of temporal dynamics of AOB communities than the DNA-derived approach during the study. Phylogenetic analysis of excised DGGE bands revealed three AOB groups affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage, and an unknown Nitrosomonas group. The population size of betaproteobacterial AOB, quantified with 16S ribosomal RNA gene real-time polymerase chain reaction assay, ranged from 6.63 × 10⁵ to 2.67 × 10⁹ cells per gram of dry biofilm and corresponded to 0.23–1.8% of the total bacterial fraction. Quantitative results of amoA gene of the three specific AOB groups revealed changes in competitive dominance between AOB of the N. oligotropha lineage and N. communis lineage. Water temperature is shown to have major influence on AOB population size in the reactor by the statistic analysis, and a positive correlation between AOB cell numbers and ammonia removal efficiency is suggested (r = 0.628, P < 0.05).     

Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 10(5) bacteria ml(-1). The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) active AOB ml(-1) were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml(-1) detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.     

Influence of effluent irrigation on community composition and function of ammonia-oxidizing bacteria in soil

The effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the alpha-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period, Nitrosospira-like populations were dominant in soils irrigated with FAW, while Nitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of the amoA gene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.     

Mediating N2O emissions from municipal solid waste landfills: Impacts of landfill operating conditions on community structure of ammonia-oxidizing bacteria in cover soils

Chemolithotrophic ammonia-oxidizing bacteria (AOB) can produce N₂O, a highly potent greenhouse gas. Denaturing gradient gel electrophoresis (DGGE) analyses of the ammonia monooxygenase structural gene (amoA) and 16S rDNA gene were used to investigate the AOB community structure in the cover soils of municipal solid waste (MSW) landfills under three operating conditions: (a) MSW with soil cover, (b) MSW with soil cover, irrigation piping and vegetation, and (c) MSW covered with high-density polyethylene (HDPE) liner, soil cover, irrigation piping and vegetation. AOB species in MSW cover soils were significantly distinguished by the operation of HDPE liner isolation. The community structures of the Nitrosomonas europaea-like AOB species dominated in soils without HDPE liner isolation, whether vegetation and irrigation with landfill leachate existed or not, whereas Nitrospira-like AOB species dominated in soils with HDPE liner isolation. Lower N₂O flux from the soils with HDPE liner isolation would be partially related to these special community structures.     

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.     

Use of quantitative real-time PCR to monitor population dynamics of ammonia-oxidizing bacteria in batch process

A quantitative real-time PCR (QPCR) assay with the TaqMan system was used to quantify 16S rRNA genes of β-proteobacterial ammonia-oxidizing bacteria (AOB) in a batch nitrification bioreactor. Five different sets of primers, together with a TaqMan probe, were used to quantify the 16S rRNA genes of β-proteobacterial AOB belonging to the Nitrosomonas europaea, Nitrosococcus mobilis, Nitrosomonas nitrosa, and Nitrosomonas cryotolerans clusters, and the genus Nitrosospira. We also used PCR followed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing of their 16S rRNA genes to identify the AOB species. Seed sludge from an industrial wastewater treatment process controlling high-strength nitrogen wastewater (500 mg/L NH₄ ⁺–N) was used as the inoculum for subsequent batch experiment. The Nitrosomonas nitrosa cluster was the predominant AOB (2.3 × 10⁵ copies/mL) in the start-up period of the batch experiment. However, from the exponential growth period, the Nitrosomonas europaea cluster was the most abundant AOB, and its 16S rRNA gene copy number increased to 8.9 × 10⁶ copies/mL. The competitive dominance between the two AOB clusters is consistent with observed differences in ammonia tolerance and substrate affinity. Analysis of the DGGE results indicated the presence of Nitrosomonas europaea ATCC19718 and Nitrosomonas nitrosa Nm90, consistent with the QPCR results.