Microbial community compositions in different functional zones of Carrousel oxidation ditch system for domestic wastewater treatment
© The Author(s) 2017
Received: 28 January 2017
Accepted: 31 January 2017
Published: 15 February 2017
The microbial community diversity in anaerobic-, anoxic- and oxic-biological zones of a conventional Carrousel oxidation ditch system for domestic wastewater treatment was systematically investigated. The monitored results of the activated sludge sampled from six full-scale WWTPs indicated that Proteobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Verrucomicrobia, Acidobacteria and Nitrospirae were dominant phyla, and Nitrospira was the most abundant and ubiquitous genus across the three biological zones. The anaerobic-, anoxic- and oxic-zones shared approximately similar percentages across the 50 most abundant genera, and three genera (i.e. uncultured bacterium PeM15, Methanosaeta and Bellilinea) presented statistically significantly differential abundance in the anoxic-zone. Illumina high-throughput sequences related to ammonium oxidizer organisms and denitrifiers with top50 abundance in all samples were Nitrospira, uncultured Nitrosomonadaceae, Dechloromonas, Thauera, Denitratisoma, Rhodocyclaceae (norank) and Comamonadaceae (norank). Moreover, environmental variables such as water temperature, water volume, influent ammonium nitrogen, influent chemical oxygen demand (COD) and effluent COD exhibited significant correlation to the microbial community according to the Monte Carlo permutation test analysis (p < 0.05). The abundance of Nitrospira, uncultured Nitrosomonadaceae and Denitratisoma presented strong positive correlations with the influent/effluent concentration of COD and ammonium nitrogen, while Dechloromonas, Thauera, Rhodocyclaceae (norank) and Comamonadaceae (norank) showed positive correlations with water volume and temperature. The established relationship between microbial community and environmental variables in different biologically functional zones of the six representative WWTPs at different geographical locations made the present work of potential use for evaluation of practical wastewater treatment processes.
KeywordsCarrousel oxidation ditch Biologically functional zone Wastewater treatment Microbial community structure Illumina high-throughput sequencing
Oxidation ditches are widely applied in wastewater treatment, especially in the small-scale and medium-scale wastewater treatment plants (WWTPs) in China (Zhang et al. 2016). As a modified activated sludge process, oxidation ditches have distinct advantages in simple construction, flexible operating mode, low sludge production, and special capability of nitrification and denitrification within the same tank. An oxidation ditch is often used to achieve simultaneous nitrification and denitrification because of the alternation of aerobic and anoxic zones within a channel, which can be formed by regulating the oxygen supply with surface aeration equipment or surface aeration combined with microporous aeration (Ammary and Radaideh 2005; Liu et al. 2010; Jin et al. 2015; Zhou et al. 2015).
The Carrousel oxidation ditch system, usually with an external pre-anaerobic zone and an internal pre-anoxic zone, is one of the most commonly used type of oxidation ditches (Peng et al. 2008; Jin et al. 2014). Carrousel oxidation ditch is designed to achieve an excellent condition for denitrifying and nitrifying bacteria in alternating anoxic–oxic zones in the looped channel system. In recent decades, operational conditions (e.g. temperature, hydraulic retention time, dissolved oxygen and operational mode), ditch geometry, aerator design and mode of aeration have been extensively investigated to optimize the treatment process and enhance nitrogen removal (Liu et al. 2010; Saida et al. 2010; Jin et al. 2015). Meanwhile, mathematical models have also been developed for simulating hydrodynamics, oxygen mass transfer, carbon oxidation, nitrification and denitrification processes, and optimizing the complicated alternating aerobic and anoxic conditions in oxidation ditches (Saida et al. 2010; Xie et al. 2011; Yang et al. 2013; Lei and Ni 2014).
As an important microbially mediated process for wastewater treatment and nitrogen removal, the efficiency and stability of Carrousel oxidation ditch system is entirely dependent upon the concerted and syntrophic activity of microorganisms belonging to different function performing nitrification, anammox and denitrification (Vanwonterghem et al. 2014; Rodríguez et al. 2015). Recently, several molecular technologies based on microbial 16S rRNA have been used to investigate the microbial community structures in oxidation ditch system (Zheng et al. 2015; Xia et al. 2016). For instance, Guo et al. (2013) studied the community structure of the nitrifying bacteria using fluorescence in situ hybridization (FISH). Zhou et al. (2015) identified the simultaneous nitrification and denitrification in an oxidation ditch by observing coexistence of nitrifying and denitrifying bacteria as well as some microaerophilic microorganisms using FISH and polymerase chain reaction–denaturing gradient gel electrophoresis methods. Jin et al. (2015) investigated the effect of different aeration modes on microbial communities of a Carrousel oxidation ditch by high-throughput 454 Pyrosequencing, of which activated sludge samples were sampled from anoxic and oxic zones and mixed with same proportion.
In this study, Illumina high-throughput sequencing was employed to reveal the microbial community diversity and structure of the activated sludge sampled from six full-scale WWTPs with Carrousel oxidation ditch systems. The core microbial populations and distribution of ammonium oxidizer organisms and denitrifiers in different functional zones in Carrousel oxidation ditch systems were studied. Differentially abundant features of the core microorganisms between the three biological functional zones and between the six different geographically located WWTPs were evaluated. More importantly, relationship between microbial community and environmental variables were established, which is of significance to diagnosis of practical wastewater treatment processes.
Materials and methods
Sample collection and determination
The investigated six full-scale Carrousel oxidation ditch systems (all equipped with an external pre-anaerobic zone and an internal pre-anoxic zone) respectively belong to six WWTPs at geographically different location in China, represented by XJYQ, ZZLQ, HBLY, BJYF, GDHZ and MYYX. The descriptions of these WWTPs were shown in Additional file 1: Table S1. The anaerobic-(A1), anoxic-(A2) and oxic-(O) activated sludge samples were collected respectively from the corresponding biologically functional zones in Carrousel oxidation ditch systems. Each activated sludge sample was fixed on site by mixing with 50% ethanol (v/v) on site, kept in an ice box for transport and stored at −20 °C in laboratory before DNA extraction. Concentrations of chemical oxygen demand (COD) and ammonium nitrogen in the influent/effluent of the WWTPs were measured according to standard analytical procedures (Clesceri et al. 1998). The level of pH and DO was determined on site by a pH sensor (pHS-25) and a DO sensor (WTW Oxi 340i), respectively.
Genomic DNA was extracted from each activated sludge sample using the PowerWater DNA Isolation Kit (MO BIO, CA, USA) according to manufacturer’s protocols. The extracted DNA samples were stored at −20 °C for subsequent assays. The intact DNA was confirmed on 1.5% agarose gel electrophoresis. The concentration and quality of the extracted DNA were assessed with a Nanodrop2000 microspectrophotometry (Thermo Scientific, DE, USA).
Polymerase chain reaction (PCR) amplification and high-throughput sequencing
The hypervariable V3–V4 region of 16S rRNA genes were amplified from all DNA extracts with barcoded primers 340F (CCTACGGGNBGCASCAG) and 805R (GACTACNVGGGTATCTAATCC) under following conditions: initial denaturation at 95 °C for 3 min, followed by 30 cycles at 95 °C for 30 s, 50 °C for 30 s and 72 °C for 60 s and final extension at 72 °C for 7 min by the LabCycler PCR (Sensoquest, Germany). The 50 µL PCR mixture contained 5 µL of 10× buffer, 38.8 µL of 5 ddH2O, 1 µL of dNTP (10 mM), 2 µL of each primer, 0.2 µL of KAPA Taq polymerase (KAPA Biosystems, USA) and 1 µL of genomic DNA. After confirmed by 1.5% agarose gel electrophoresis, the PCR products were mixed to get equal concentration of DNA fragment for each sample and purified using MinElute PCR Purification Kit (QIAGEN, Germany), and then were sequenced using the Illumina Hiseq2500 PE250 platform.
High-throughput sequencing data analysis
All the obtained paired-end reads of 16S rRNA gene PCR amplicons were quality filtered and denoised to remove low quality or ambiguous reads. Then the treated forward and reverse reads were merged with PANDAseq (Masella et al. 2012). The putative chimeric sequences were identified and excluded with USEARCH61 pipeline (Edgar et al. 2011) in QIIME (Caporaso et al. 2010). The remaining set of high quality sequences were clustered into operational taxonomic units (OTUs) at a 97% similarity threshold using UCLUST methods (Edgar 2010) embedded in QIIME. The taxonomic identities of the representative sequences from each OTU were classified via the Ribosomal Database Project (RDP) classifier with the SILVA databases (Wang et al. 2007). The Illumina sequencing raw data obtained from this study were deposited in the NCBI Sequence Read Archive with accession No. SRP093686 (PRJNA354474).
Heatmap of the top 50 genera in each sample was conducted using R packages. The open source software Cytoscape v3.3.0 (Shannon et al. 2003) was employed for clustering network analysis to visualize the most abundant OTUs and to compare their abundance among the different samples. The linear discriminant analysis (LDA) effect size (LEfSe) pipeline (http://huttenhower.sph.harvard.edu/galaxy/) (Segata et al. 2011) was used to identify differentially abundant features among the different biological functional zones of Carrousel system and different sampling WWTPs. The differential features were identified on the OTU level (relative abundance >1%). The nonparametric factorial Kruskal–Wallis (KW) rank sum test was used to detect taxa with significant differential abundances. LDA was used to evaluate the effect size of each differentially abundant trait. The LEfSe analysis was performed under the alpha value for the Kruskal–Wallis test is <0.05, and the threshold on the logarithmic LDA score for discriminative features is >2.0 (Zhang et al. 2013). Principal component analysis (PCA) was conducted using Canoco 4.5 (Microcomputer Power, USA). Redundancy analysis (RDA), a form of constrained ordination, was employed to analyze the relationships between the abundance of core genera and environmental variables using R software with the vegan and ggplot2 packages.
Biodiversity and microbial community profiles in Carrousel system
Using Illumina Sequencing of 16S rRNA Gene Amplicons, 8017-10681 OTUs obtained from the individual activated sludge sample. The OTUs, Good’s coverage, Chao1, ACE, Shannon and Simpson of each sample could be seen in Additional file 1: Table S2. Forty-eight bacterial and 7 archaeal phyla were retrieved from 164,591 Illumina effective sequences obtained from the 18 activated sludge samples, while the reads for archaeal phyla only accounted 1.22% of the total phyla.
The most abundant genus is Nitrospira with a relative abundance varying from 0.8 to 6.1% in individual samples, while the second is Gemmatimonadaceae (belonging to Gemmatimonadetes phylum) with a relative abundance varying from 1.1 to 3.6% in individual samples. The other top 10 abundant genera detected in all samples were Comamonadaceae (no rank), Thauera, uncultured Nitrosomonadaceae, Dechloromonas and Rhodocyclaceae (no rank) belonging to β-proteobacteria, Candidatus Microthrix belonging to Actinobacteria pyhlum, OPB35 soil group (no rank) belonging to Verrucomicrobia pyhlum, and Chloroflexi (no rank and uncultured) belonging to Chloroflexi phylum.
Distribution of ammonium oxidizer organisms and denitrifiers
Five denitrifying groups, Dechloromonas, Thauera, Denitratisoma, Rhodocyclaceae (norank) and Comamonadaceae (norank) with relative abundance ranged from 0.2 to 4.8% were detected in all the samples. Dechloromonas, Thauera, Denitratisoma and Rhodocyclaceae (norank) belongs to Rhodocyclaceae family.
PAC revealed that samples of anaerobic-(A1), anoxic-(A2) and oxic-(O) zone from each WWTP were grouping together, but the relative abundance of the ammonium oxidizers and denitrifers differed apparently with different WWTPs (Fig. 3b). Among the six WWTPs, the most relative abundant genera involved in nitrogen transformation in BJYF were Dechloromonas (3.1–3.8%), Nitrosomonadaceae (uncultured) (2.7–3.8%) and Denitratisoma (1.8%), and in XJYQ were Thauera (2.9–4.8%) and Comamonadaceae (norank) (3.7–4.3%), while that in ZZLQ and GDHZ exhibited Nitrospira (4.8–6.1%) and Rhodocyclaceae (norank) (2.5–3.4%), respectively. XJYQ presented the highest relative abundance of the sum of the five denitrifers among the six WWTPs, while MYYX was the lowest. The differences in wastewater quality and environmental factors may result in the bacterial abundant discrepancies among the WWTPs.
Abundance differences between the 3 biological functional zones
Abundance differences between the geographically distributed Carrousel oxidization ditches
The most differentially abundant taxa in ZZLQ belonged to two bacterial phyla of Nitrospirae and Actinobacteria, while that in MYYX belonged to two bacterial phyla of Chloroflexi and Gemmatimonadales, and that in MYYX belonged to a bacterial phylum of Planctomycetes (Fig. 7b). At family level, 7 differentially abundant taxonomic clades were detected in ZZLQ (including Hyphomicrobiaceae, Planctomycetaceae, Nitrospiraceae, uncultured soil bacterium JG30-KF-CM45, uncultured bacterium KD4-96, DS-100 and GR-WP33-58), while that of 2 in MYYX (Gemmatimonadaceae and I-10) and one in GDZH (Haliangiaceae). At genus level, also 7 differentially abundant taxonomic clades were detected in ZZLQ (including Hyphomicrobium, Planctomyces, Nitrospira, uncultured soil bacterium JG30-KF-CM45, uncultured bacterium KD4-96, DS-100 and uncultured XanthomonadalesIncertaeSedis), while 2 differentially abundant taxonomic clades were detected in MYYX (uncultured Gemmatimonadaceae and uncultured bacterium I-10) and one in GDZH (Haliangium). Except the genera within the above phyla, 4 differentially abundant genera of Hyphomicrobium (within Hyphomicrobiaceae family and Rhizobiales order and α-proteobacteria), uncultured bacterium I-10 (within I-10 family and Rhodospirillales order and α-proteobacteria), Haliangium (within Haliangiaceae family and δ-Proteobacteria calss) and uncultured XanthomonadalesIncertaeSedis (within Xanthomonadales order, γ-proteobacteria) were also detected.
The relationships between environmental variables and microbial community
Proteobacteria, Chloroflexi, Bacteroidetes, Actinobacteria, Verrucomicrobia, Acidobacteria and Nitrospirae were dominating phyla across the anaerobic-, anoxic- and oxic-zones in Carrousel oxidation ditch system, and accounted 75.2–85.8% of all sequences of the six investigated full-scale WWTPs. Proteobacteria (mainly including α-, β-, γ-, δ-proteobacteria) was the most dominant phylum and accounted 33.9–50.9% of all the monitored phyla. Previous reports on activated sludge from municipal WWTPs have showed that the above phyla are common and Proteobacteria, Actinobacteria, Bacteroidetes and Verrucomicrobia dominated in both DNA and cDNA sets (Yu and Zhang 2012; Cydzik-Kwiatkowska and Zielińska 2016); also Proteobacteria commonly dominated 21–65% of all phyla and the β-proteobacteria is the most abundant class which is mainly responsible for organic and nutrient removal (Nielsen et al. 2010; Hu et al. 2012; Wang et al. 2014b; Cydzik-Kwiatkowska and Zielińska 2016). Compared to common wastewater treatment activated sludge system, the phylum Nitrospirae, of which members are usually found as predominant NOB in WWTPs (Nielsen et al. 2010; Rodríguez et al. 2015), presented in the subdominant phyla in the Carrousel oxidation ditch system for nitrogen removal function.
The genus Nitrospira was the most abundant and ubiquitous bacterial genus across the anaerobic-, anoxic- and oxic-zones in the Carrousel oxidation ditch systems. The second most abundant genus is Gemmatimonadaceae, which family members had also been detected in activated sludge sewage treatment systems, and could grow by both aerobic and anaerobic respiration (Zhang et al. 2003; Shu et al. 2015). The other subdominant genera were Comamonadaceae (no rank), Thauera, uncultured Nitrosomonadaceae, Dechloromonas, Rhodocyclaceae (no rank) Candidatus Microthrix, OPB35 soil group (no rank) and Chloroflexi (no rank and uncultured). Candidatus Microthrix (ranging from 0.5 to 4.1% in individual samples) commonly occurred at the water–air interface of biological WWTPs, where they are considered notorious in causing foaming and bulking (Muller et al. 2012). Also the Candidatus Microthrix parvicella is been considered as a specialized lipid consumer with a physiological potential analogous to polyphosphate-accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) (Nielsen et al. 2002). The Verrucomicrobia OPB35 soil group, the most abundant prokaryotic taxa (Lanzén et al. 2015), were also detected in the 18 activated samples with the abundance ranged from 0.5 to 3.4%. The relative abundance of Chloroflexi (no rank and uncultured) ranged from 0.2 to 4.9% of the samples. Members of phylum Chloroflexi are commonly detected from sediment and involve carbon cycling, organohalide respiration, fermentation, CO2 fixation, and acetogenesis with ATP formation by substrate-level phosphorylation (Hug et al. 2013).
Illumina sequences associated with ammonium oxidizer organisms and denitrifiers included Nitrospira (including Candidatus Nitrospira defluvii spp. and other two uncultured Nitrospira organisms), uncultured Nitrosomonadaceae, Dechloromonas, Thauera, Denitratisoma, Rhodocyclaceae (norank) and Comamonadaceae (norank), which presented top50 abundant genera in all samples. Nitrospira as the most diverse known group of NOB in classical two-step nitrification theory (oxidation of ammonium via nitrite to nitrate), in which of Candidatus Nitrospira inopinata is recently discovered to be complete ammonium oxidizer (Comammox) organisms i.e., novel one step nitrification theory which completely oxidize ammonium to nitrate (Daims et al. 2015; van Kessel et al. 2015). Candidatus Nitrospira defluvii spp., a predominant nitrite oxidizer in WWTP, possesses a periplasmically oriented enzyme nitrite oxidoreductase (NXR), which differs from other known nitrite oxidizers as Nitrobacter and Nitrococcus (Lucker et al. 2010; Nielsen et al. 2010; Rodríguez et al. 2015). The periplasmic forms of NXR are considered to be more efficient because of more proton-motive force produced by per oxidized nitrite and no nitrite/nitrate transporting across the cytoplasmic membrane (Lucker et al. 2010; Rodríguez et al. 2015). Nitrosomonadaceae (uncultured) presented a relative abundance varying from 0.27 to 3.8% in individual samples. Also, genus Nitrosomonas was detected in all the samples with relative low abundance of 0.03–0.35% (out of the top 50 genera). The genera within family Nitrosomonadaceae as Nitrosomonas and Nitrosospira are the most detected AOB in WWTPs (Li et al. 2016). The other dominant genera of Comamonadaceae (no rank), Thauera, Dechloromonas and Rhodocyclaceae (no rank) are capable of denitrification (Hwang et al. 2006; Lu et al. 2014). Rhodocyclaceae and Comamonadaceae (both within β-proteobacteria) were reported as the core families with responsibility for denitrifying and aromatic degrading processes in wastewater treatment activated sludge systems (Loy et al. 2005; Ma et al. 2015), of which Thauera is one of the most often detected genera and functionally important denitrifier in activated sludge systems (Lu et al. 2014; Ma et al. 2015). Furthermore, Dechloromonas-related bacteria have been demonstrated with the capability of nitrate/nitrite reduction, acetate uptake and polyphosphate and polyhydroxyacids storage (Cydzik-Kwiatkowska and Zielińska 2016). Among the anaerobic-(A1), anoxic-(A2) and oxic-(O) function zones, the abundances of AOB, NOB and denitrifiers clustered together within each Carrousel oxidation ditch system (Fig. 3b), because Carrousel oxidation ditch is a looped channel system, and the most of the activated sludge separated from the final clarifier is returned to the anaerobic-zone to maintain the proper biomass level in the system.
Furthermore, the phylogeny-based UniFrac analysis (Fig. 1), clustering network analysis (Fig. 4), and LEfSe analysis (Fig. 5) demonstrated that the activated sludge samples were certainly similar between those taken from the same type of plant (i.e. the different biological zones). The clustering network analysis showed that anaerobic-, anoxic- and oxic-zones shared approximately similar percentages across the 50 most abundant OTUs. The standard deviation (SD, calculated by weighted-degree of the three zones at a certain OTU) of the top 50 OTUs ranged from 0.2 to 14.3, and Coefficient of variation (CV, calculated as the ratio of the SD to the mean and multiplied by 100), evaluating dispersion of a probability distribution among the three zones, ranged from 3.0 to 19. Only 6 OTUs (referred to the genera of Terrimonas, Caldilineaceae (uncultured), β-proteobacteria (no rank), uncultured B1-7BS, Denitratisoma, Caldithrix and uncultured Hydrogenophilaceae) presented the CV value higher than 15, which indicated that the probability distributions of the 6 OTUs exhibited relative higher dispersion degree among the 3 biological functional zones. The LEfSe analysis result indicated only13 microbial clades within an archaeal phylum of Euryarchaeota and two bacterial classes of γ-proteobacteria (with the highest LDA score) and Actinobacteria, showed statistically significant differences in the anoxic-zone among the 3 biological zones (Fig. 5). At genus level, there were 3 differential abundant taxa (i.e. uncultured bacterium PeM15, Methanosaeta and Bellilinea) in the anoxic-zone among the 3 biological zones. The members of Actinobacteria phylum have been verified as important PAOs in enhanced biological phosphorus removal systems, and also some may contain nitrite reductase genes involved in denitrification (Rodríguez et al. 2015). Methanosaeta within Methanosarcinales is an obligate aceticlastic methanogen (Rodríguez et al. 2015). Methanosarcinales showed statistically significant differences in the anoxic-zone among the 3 biological zones might be caused by the high acetate concentrations (Anderson et al. 2009), which were added into the anoxic-zone as external carbon source to enhance the denitrification process.
The 6 studied biological WWTPs with Carrousel oxidization ditches process shared the major phyla (Fig. 1) and the most abundant genera (top50) (Fig. 2), but were dissimilar in the microbial abundance distribution. These results were complying with previous reported microbial community structure distribution in geographically distributed biological WWTPs (Xia et al. 2010; Shu et al. 2015). The distribution of detected AOB, NOB and denitrifiers also exhibited apparently differences among the 6 WWTPs (Fig. 3). The significant differential abundances occurred in three WWTPs i.e. GDZH, MYYX and ZZLQ by the LEfSe analysis (Fig. 7), which indicated that 36 microbial clades within 7 bacterial phyla of Nitrospira, Planctomycetes, Gemmatimonadales, Chloroflexi, Acidobacteria and Proteobacteria presented statistically significant differences. At genus level, there were 10 significantly differential abundant taxa, of which genera Nitrospira, uncultured Gemmatimonadaceae and Haliangium showed the highest LDA score in the activated sludge samples from the ZZLQ, MYYX and GDHZ, respectively. Such discrepancies in microbial community structures among the geographically distributed WWTPs harbored the same biological treatment process might be correlated with the different environmental conditions and operating parameters among the different geographically located WWTPs (Hu et al. 2012; Xia et al. 2016).
The RDA analytical results (Fig. 8) showed that the abundance of Nitrospira, uncultured Nitrosomonadaceae and Denitratisoma presented strong positive correlations with the northern latitude of the WWTP, and influent/effluent concentration of COD and ammonium nitrogen. Dechloromonas, Thauera, Rhodocyclaceae (norank) and Comamonadaceae (norank) presented positive correlations with the water volume and temperature. Among the tested environmental variables, the temperature and water quality play important parts in the microbial metabolisms, nitrifying process and denitrification (Ju et al. 2014; Lu et al. 2014; Wang et al. 2014a). The temperature and influent ammonium nitrogen can impact the abundance of AOB and the balance between AOB and NOB in activated sludge systems (Cydzik-Kwiatkowska and Zielińska 2016). Also, Nitrospira and Nitrosomonas have been verified more susceptible to seasonal variations and displayed higher abundances in relative higher temperature (Wan et al. 2011; Ju et al. 2014). In the present study, the abundance of Nitrospira, Nitrosomonadaceae (uncultured) and Denitratisoma were greatly influenced by not only the water quality, but also the northern latitude of the WWTP. Shanks et al. (2013) reported that city latitude could result in the variation of the sewage infrastructure community composition among cities with a wide range of geographic locations. Zhang et al. (2012) found that although some detected microbial genera existed in all WWTPs, the microbial composition of biomass differed with different geographically location. Thus, city latitude might impact the microbial community structures of untreated wastewaters, which further influence the microbial community of biological treated system of WWTP. Also, the effects of city latitude on microbial composition might due to the fact that the temperatures, technological system and water quality of wastewater treatment vary in different locations (Cydzik-Kwiatkowska and Zielińska 2016). The northern latitude of the WWTP presented significant correlation with the water quality (i.e. influent/effluent concentration of COD and ammonium nitrogen) in this study (Fig. 8). However, further study on spatial and temporal dynamics of ammonium oxidizer organisms and denitrifiers in activated sludge system is needed. In general, the established relationship between microbial community and environmental variables in different biologically functional zones of the six representative WWTPs at different geographical locations made the present work of potential use for evaluation of practical wastewater treatment processes.
wastewater treatment plants
fluorescence in situ hybridization
chemical oxygen demand
polymerase chain reaction
operational taxonomic units
linear discriminant analysis
linear discriminant analysis effect size pipeline
principal component analysis
ammonium oxidizing bacteria
DX performed the research, analyzed data and wrote this manuscript. QC helped to draft the manuscript. SL and JN designed the research and polished the manuscript. All authors read and approved the final manuscript.
We thanks for the financial support by National Natural Science Foundation of China (Grant No. 51539001).
The authors declare that they have no competing interests.
Availability of data and materials
The data are included within the manuscript, and all data are fully available without restriction.
Consent for publication
This manuscript does not contain any individual person’s data.
Ethics approval and consent to participate
This article does not contain any studies with human participants or animals performed by any of the authors.
Financial support was from National Natural Science Foundation of China (Grant No. 51539001).
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