Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.     

Niche separation of ammonia-oxidizing bacteria across a tidal freshwater marsh

Like many functional groups or guilds of microorganisms, the group of ammonia-oxidizing bacteria (AOB) consists of a number of physiologically different species or lineages. These physiological differences suggest niche differentiation among these bacteria depending on the environmental conditions. Species of AOB might be adapted to different zones in the flooding gradient of a tidal marsh. This issue has been studied by sampling sediments from different sites and depths within a tidal freshwater marsh along the river Scheldt near the village of Appels in Belgium. Samples were taken in February, April, July and October 1998. Communities of AOB in the sediment were analysed on the basis of the 16S rRNA gene by application of polymerase chain reaction in combination with denaturing gradient gel electrophoresis (DGGE). In addition, moisture content and concentrations of ammonium and nitrate were determined as well as the potential ammonia-oxidizing activities. Six different DGGE bands belonging to the beta-subclass of the Proteobacteria were observed across the marsh. The community composition of AOB was determined by the elevation in the flooding gradient as well as by the sampling depth. The presence of plants was less important for the community composition of AOB. DGGE bands affiliated with the Nitrosospira lineage were mostly found in the upper part of the marsh and in the deeper layers of the sediment. Two of the three DGGE bands related to the Nitrosomonas oligotropha lineage were more broadly distributed over the marsh, but were predominantly found in the upper layers of the sediment. Members of the environmental Nitrosomonas lineage 5 were predominantly detected in the deeper layers in the lower parts of the marsh. Potential driving factors for niche differentiation are discussed.     

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Elevated salinity selects for a less diverse ammonia-oxidizing population in aquarium biofilters

The activity and changes in the structure of the community of the ammonia-oxidizing bacteria belonging to the Betaproteobacteria were monitored in freshwater and artificial seawater biofilters for two months after inoculation with a commercial nitrifying consortium. Both in freshwater and artificial seawater, ammonium oxidation proceeded immediately after addition of the inoculum, although initial activity in artificial seawater was lower than in freshwater. Denaturing gradient gel electrophoresis of the ammonia-oxidizing bacterial community of the inoculum and the freshwater and the artificial seawater aquaria as a function of time showed that initially only one dominant ammonia-oxidizer, closely related to Nitrosomonas marina, was detectable in all the systems. The fingerprint of the ammonia-oxidizing bacterial community in the artificial seawater biofilters continued to be dominated by this single band. In the freshwater aquaria, in contrast, the composition of the ammonia-oxidizer community became more diverse after one month, with 4-7 new bands appearing in the denaturing gradient gel fingerprint. Since the inoculum is cultivated at an average salinity of 11 gl(-1), it is argued that the elevated salinity selects for a less diverse ammonia-oxidizer community in the inoculum and the artificial seawater aquaria.     

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.     

Communities of ammonia-oxidizing bacteria in activated sludge of various sewage treatment plants in Tokyo

We investigated ammonia-oxidizing bacteria in activated sludge collected from 12 sewage treatment systems, whose ammonia removal and treatment processes differed, during three different seasons. We used real-time PCR quantification to reveal total bacterial numbers and total ammonia oxidizer numbers, and used specific PCR followed by denaturing gel gradient electrophoresis, cloning, and sequencing of 16S rRNA genes to analyze ammonia-oxidizing bacterial communities. Total bacterial numbers and total ammonia oxidizer numbers were in the range of 1.6 x 10(12) - 2.4 x 10(13) and 1.0 x 10(9) - 9.2 x 10(10)cellsl(-1), respectively. Seasonal variation was observed in the total ammonia oxidizer numbers, but not in the ammonia-oxidizing bacterial communities. Members of the Nitrosomonas oligotropha cluster were found in all samples, and most sequences within this cluster grouped within two of the four sequence types identified. Members of the clusters of Nitrosomonas europaea-Nitrosococcus mobilis, Nitrosomonas cryotolerans, and unknown Nitrosomonas, occurred solely in one anaerobic/anoxic/aerobic (A2O) system. Members of the Nitrosomonas communis cluster occurred almost exclusively in association with A2O and anaerobic/aerobic systems. Solid residence time mainly influenced the total numbers of ammonia-oxidizing bacteria, whereas dissolved oxygen concentration primarily affected the ammonia-oxidizing activity per ammonia oxidizer cell.     

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.     

Adaptation of ammonia-oxidizing microorganisms to environment shift of paddy field soil

Adaptation of microorganisms to the environment is a central theme in microbial ecology. The objective of this study was to investigate the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to a soil medium shift. We employed two rice field soils collected from Beijing and Hangzhou, China. These soils contained distinct AOB communities dominated by Nitrosomonas in Beijing rice soil and Nitrosospira in Hangzhou rice soil. Three mixtures were generated by mixing equal quantities of Beijing soil and Hangzhou soil (BH), Beijing soil with sterilized Hangzhou soil (BSH), and Hangzhou soil with sterilized Beijing soil (HSB). Pure and mixed soils were permanently flooded, and the surface-layer soil where ammonia oxidation occurred was collected to determine the response of AOB and AOA to the soil medium shift. AOB populations increased during the incubation, and the rates were initially faster in Beijing soil than in Hangzhou soil. Nitrosospira (cluster 3a) and Nitrosomonas (communis cluster) increased with time in correspondence with ammonia oxidation in the Hangzhou and Beijing soils, respectively. The 'BH' mixture exhibited a shift from Nitrosomonas at day 0 to Nitrosospira at days 21 and 60 when ammonia oxidation became most active. In 'HSB' and 'BSH' mixtures, Nitrosospira showed greater stimulation than Nitrosomonas, both with and without N amendment. These results suggest that Nitrosospira spp. were better adapted to soil environment shifts than Nitrosomonas. Analysis of the AOA community revealed that the composition of AOA community was not responsive to the soil environment shifts or to nitrogen amendment.     

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.     

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.     

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.     

PCR profiling of ammonia-oxidizer communities in acidic soils subjected to nitrogen and sulphur deposition

Communities of ammonia-oxidizing bacteria (AOB) were characterized in two acidic soil sites experimentally subjected to varying levels of nitrogen and sulphur deposition. The sites were an acidic spruce forest soil in Deepsyke, Southern Scotland, with low background deposition, and a nitrogen-saturated upland grass heath in Pwllpeiran, North Wales. Betaproteobacterial ammonia-oxidizer 16S rRNA and ammonia monooxygenase (amoA) genes were analysed by cloning, sequencing and denaturing gradient gel electrophoresis (DGGE). DGGE profiles of amoA and 16S rRNA gene fragments from Deepsyke soil in 2002 indicated no effect of nitrogen deposition on AOB communities, which contained both Nitrosomonas europaea and Nitrosospira. In 2003, only Nitrosospira could be detected, and no amoA sequences could be retrieved. These results indicate a decrease in the relative abundance of AOB from the year 2002 to 2003 in Deepsyke soil, which may be the result of the exceptionally low rainfall in spring 2003. Nitrosospira-related sequences from Deepsyke soil grouped in all clusters, including cluster 1, which typically contains only sequences from marine environments. In Pwllpeiran soil, 16S rRNA gene libraries were dominated by nonammonia oxidizers and no amoA sequences were detectable. This indicates that autotrophic AOB play only a minor role in these soils even at high nitrogen deposition.     

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.     

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.     

南美白对虾养殖底泥中氨氧化细菌与氨氧化古菌多态性分析

【目的】本研究皆在了解虾养殖底泥中氨氧化细菌与氨氧化古菌群落多态性。【方法】以功能基因为基础,构建氨氧化细菌(AOB)与氨氧化古菌(AOA)的氨单加氧酶α亚基基因(amoA)克隆文库。利用限制性片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术将克隆文库阳性克隆子进行归类分析分成若干个可操作分类单元(Operational Taxa Units,OTUs)。【结果】通过序列多态性分析,表明AOB amoA基因克隆文库中所有序列都属于变形杆菌门β亚纲(β-Proteobacteria)中的亚硝化单细胞菌属(Nitrosomonas)及Nitrosomonas-like,未发现亚硝化螺旋菌属(Nitrosospira)。AOA amoA基因克隆文库中只有一个OTU序列属于未分类的古菌(Unclassified-Archaea),其余序列都属于泉古菌门(Crenarchaeote)。AOA群落结构单一且存在一个绝对优势类群OTU3,其克隆子数目占克隆文库的57.45%。AOB和AOA amoA基因克隆文库分别包括13个OTUs和9个OTUs,其文库覆盖率分别为73.47%和90.43%。AOB amoA基因克隆文库的Shannon-Wiener指数、Evenness指数、Simpson指数、Richness指数均高于AOA。【结论】虾养殖塘底泥中存在氨氧化古菌的amoA基因,且多态性低于氨氧化细菌,表明氨氧化古菌在虾养殖塘底泥的氮循环中可能具有重要的作用。     

Comparison of the community structures of ammonia-oxidizing bacteria and archaea in rhizoplanes of floating aquatic macrophytes

Some common floating aquatic macrophytes could remove nutrients, such as nitrogen, from eutrophic water. However, the relationship between these macrophytes and the ammonia-oxidizing microorganisms on their rhizoplanes is still unknown. In this study, we examined communities of ammonia-oxidizing archaea (AOA) and bacteria (AOB) on the rhizoplanes of common floating aquatic macrophytes (Eichhornia crassipes, Pistia stratiotes and Ipomoea aquatic) in a eutrophic reservoir.The results show that AOB were the predominant ammonia-oxidizer on the three rhizoplanes. The principal AOB were Nitrosomonas europaea and Nitrosomonas ureae clades. The principal group of AOA was most similar to the clone from activated sludge. The ratio of AOB amoA gene copies to AOA varied from 1.36 (on E. crassipes) to 41.90 (on P. stratiotes). Diversity of AOA was much lower than that of AOB in most samples, with the exception of P. stratiotes.     

High levels of nitrifying bacteria in intermittently aerated reactors treating high ammonia wastewater

Changes in the fractions of ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in two laboratory-scale reactors were investigated using 16S rRNA probe hybridizations. The reactors were operated in intermittent aeration mode and different aeration cycles to treat anaerobically digested swine wastewater with ammonia concentrations up to 175 mg NH(3)-N/L. High ammonia removals (>98.8%) were achieved even with increased nitrogen loads and lower aeration: non-aeration time ratios of 1h:3h. Nitrosomonas/Nitrosococcus mobilis were the dominant ammonia-oxidizing bacteria in the reactors. Nitrospira-like organisms were the dominant nitrite-oxidizing bacteria during most of the investigation, but were occasionally outcompeted by Nitrobacter. High levels of nitrifiers were measured in the biomass of both reactors, and ammonia-oxidizing bacteria and nitrite-oxidizing bacterial levels adjusted to changing aeration: non-aeration time ratios. Theoretical ammonia-oxidizer fractions, determined by a mathematical model, were comparable to the measured values, although the measured biomass fractions were different at each stage while the theoretical values remained approximately constant. Stable ammonia removals and no nitrite accumulation were observed even when rRNA levels of ammonia oxidizers and nitrite-oxidizers reached a minimum of 7.2% and 8.6% of total rRNA, respectively. Stable nitrogen removal performance at an aeration: non-aeration ratio of 1h:3h suggests the possibility of significant savings in operational costs.     

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms

The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial wastewater were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify.