Activated Sludge Microbial Community Research Articles

Quorum sensing bacteria improve microbial networks stability and complexity in wastewater treatment plants

Quorum-sensing bacteria (QSB) are crucial factors for microbial communication, yet their ecological role in wastewater treatment plants (WWTPs) remains unclear. Here, we developed a method to identify QSB by comparing 16S rRNA gene sequences. QSB in 388 activated sludge samples collected from 130 WWTPs across China primarily were identified as rare taxa and conditionally rare taxa. A co-occurrence network shared by all sludge communities revealed that QSB exhibited higher average clustering coefficient (0.46) than non-QSB (0.15). Individual sludge networks demonstrated that quorum sensing microbiomes were positively correlated with network robustness and network complexity, including average clustering coefficient and link density. We confirmed that QSB keystones and QSB nodes have a positive impact on network complexity by influencing network modularity through a structural equation model. Meanwhile, QSB communities directly contributed to maintaining network robustness (r = 0.29, P < 0.05). Hence, QSB play an important role in promoting network complexity and stability. Furthermore, QSB communities were positively associated with the functional composition of activated sludge communities (r = 0.33, P < 0.01), especially the denitrification capacity (r = 0.45, P < 0.001). Overall, we elucidated the ecological significance of QSB and provided support for QS-based regulation of activated sludge microbial communities.

Development of halotolerant multi-pesticides degraders for wastewater treatment

The biotreatment performance of high saline wastewater containing multiple pesticides is significantly reduced because the growth and metabolism of activated sludge microbial communities are severely impaired by high osmotic pressure and joint toxicity of multiple pesticides. In this study, Halomonas cupida J9 was metabolically engineered by chromosomal integration of three different pesticide hydrolase genes, to create a halotolerant multi-pesticides degrader. In batch degradation experiments, the finally constructed strain J9U-PVG was able to simultaneously degrade chlorpyrifos (organophosphate), carbaryl (carbamate) and α-cypermethrin (pyrethroid) in mineral salt medium supplemented with 4 g/L glucose and 60 g/L NaCl. Total cell lysate of J9U-PVG showed obvious degradation activity against chlorpyrifos, carbaryl and α-cypermethrin. Moreover, introduced Vitreoscilla hemoglobin (VHb) can enhance oxygen-sequestering capability of J9U-PVG and introduced green fluorescent protein (GFP) can be used as a biomarker to track the activity and movement of J9U-PVG in natural environments. More importantly, J9U-PVG could simultaneously and completely degrade chlorpyrifos, carbaryl and α-cypermethrin in high saline river water. Here, we demonstrate for the first time the successful construction of a halotolerant multi-pesticides degrader. This study underscores the value of H. cupida J9 as a promising chassis for the development of a halotolerant pesticide bioremediation platform. In the future, synthetic biology may serve as a powerful tool for expanding the catalytic repertoire of extremophiles for bioremediation of extreme environments.

Regulatory of salinity on assembly of activated sludge microbial communities and nitrogen transformation potential in industrial plants of the lower Yangtze River basin

This study aims to thoroughly investigate the impact mode of salinity carried by industrial wastewater on the anaerobic-anoxic-oxic (A2O) sludge in wastewater treatment plants (WWTPs). Through comprehensive investigation of the A2O stage activated sludge (AS) from 19 industrial WWTPs in the downstream area of the Yangtze River, China, A total of 38 samples of anaerobic sludge and oxic sludge were collected and analyzed. We found that salinity stress significantly inhibits the growth of the AS community, particularly evident in the anaerobic sludge community. Furthermore, the high-saline environment induces changes in the structure and functional patterns of the AS community, leading to intensive interactions and resource exchanges among microorganisms. Halophilic microorganisms may play a crucial role in this process, significantly impacting the overall community structure, especially in the oxic sludge community. Additionally, salinity stress not only suppresses the nitrogen transformation potential of the AS but also leads to the accumulation of nitrite, thereby increasing the emission potential of both NO and N2O, exacerbating the greenhouse effect of the A2O process in industrial WWTPs. The findings of this study provide necessary theoretical support for maintaining the long-term stable operation of the A2O sludge system in industrial WWTPs, reducing carbon footprint, and improving nitrogen removal efficiency.

Constructions of quorum sensing signaling network for activated sludge microbial community.

In wastewater treatment systems, the interactions among various microbes based on chemical signals, namely quorum sensing (QS), play critical roles in influencing microbial structure and function. However, it is challenging to understand the QS-controlled behaviors and the underlying mechanisms in complex microbial communities. In this study, we constructed a QS signaling network, providing insights into the intra- and interspecies interactions of activated sludge microbial communities based on diverse QS signal molecules. Our research underscores the role of diffusible signal factors in both intra- and interspecies communication among activated sludge microorganisms, and signal molecules commonly considered to mediate intraspecies communication may also participate in interspecies communication. QS signaling molecules play an important role as communal resources among the entire microbial group. The communication network within the microbial community is highly redundant, significantly contributing to the stability of natural microbial systems. This work contributes to the establishment of QS signaling network for activated sludge microbial communities, which may complement metabolic exchanges in explaining activated sludge microbial community structure and may help with a variety of future applications, such as making the dynamics and resilience of highly complex ecosystems more predictable.

Visualization of Polyhydroxyalkanoate Accumulated in Waste Activated Sludge.

Polyhydroxyalkanoates (PHAs) can be produced with municipal waste activated sludge from biological wastewater treatment processes. Methods of selective fluorescent staining with confocal laser scanning microscopy (CLSM) were developed and optimized to evaluate the distribution of PHA storage activity in this mixed culture activated sludge microbial communities. Selective staining methods were applied to a municipal activated sludge during pilot scale PHA accumulation in replicate experiments. Visualization of stained flocs revealed that a significant but limited fraction of the biomass was engaged with PHA accumulation. Accumulated PHA granules were furthermore heterogeneously distributed within and between flocs. These observations suggested that the PHA content for the bacteria storing PHAs was significantly higher than the average PHA content measured for the biomass as a whole. Optimized staining methods provided high acuity for imaging of PHA distribution when compared to other methods reported in the literature. Selective staining methods were sufficient to resolve and distinguish between distinctly different morphotypes in the biomass, and these observations of distinctions have interpreted implications for PHA recovery methods. Visualization tools facilitate meaningful insights for advancements of activated sludge processes where systematic observations, as applied in the present work, can reveal underlying details of structure-function relationships.

Absence of nitrogen and phosphorus in activated sludge: Impacts on flocculation characteristics and the microbial community

A sequencing batch reactor operated in anoxic/aerobic mode was used to investigate the effects of simultaneous nitrogen (N) and phosphorus (P) absence in activated sludge. Through controlling the N and P content in the influent water, we investigated changes in sludge concentration, settling performance, morphological structure, surface properties, extracellular polymer content, and microbial community structure and function. When N and P were simultaneously absent, the relative hydrophobicity of the activated sludge and the sludge concentration decreased. The specific oxygen uptake rate also decreased, the activated sludge did not bulk, and the flocs were larger and more concentrated. The content of extracellular polysaccharides did not significantly change. An increase in the concentration of extracellular proteins, particularly loose-binding layer proteins, improved activated sludge aggregation. The structure and function of the activated sludge microbial community changed significantly in the absence of both N and P, with a significant decrease in the relative abundance of species related to N and P metabolism. Tax4Fun analysis showed that the absence of both N and P decreased metabolism and genetic information processing of the microbial community. These results elucidate the effects of simultaneous N and P absence on activated sludge from the perspective of microbial community structure and function. These results will be useful for treatment of N and P deficient wastewater by the activated sludge method.

Cellular RNA levels define heterotrophic substrate-uptake rate sub-guilds in activated sludge microbial communities.

A heterotrophic-specialist model was proposed previously to divide wastewater treatment plant (WWTP) heterotrophs into sub-guilds of consumers of readily or slowly degradable substrates (RDS or SDS, respectively). The substrate degradation rate model coupled to metabolic considerations predicted that RNA and polyhydroxyalkanoate (PHA) levels would be positively correlated in the activated sludge communities with high RNA and PHA occurring in RDS-consumers, and low RNA with no PHA accumulation occurring in SDS-consumers because their external substrates are always present. This prediction was verified in previous studies and in the current one. Thus, RNA and PHA levels were used as biomarkers of the RDS- and SDS-consumer sub-guilds for cell sorting using flow cytometry of samples from three WWTPs. Subsequently, 16S rRNA gene amplicon sequencing revealed that the sorted groups were highly similar over time and among WWTPs, and demonstrated a clear segregation by RNA levels. Predicted ecophysiological traits based on 16S rRNA phylogeny suggested that the high-RNA population showed RDS-consumer traits such as higher rrn copy numbers per genome. Using a mass-flow immigration model, it appeared that the high-RNA populations exhibited high immigration rates more frequently than low-RNA populations, but the differences in frequencies were less with increasing solids residence times.

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Diversity of Activated Sludge Microbial Community Structure in Different Wastewater Treatment Plants

Insights into the microbial community structure in the biodegradation process of high-strength ammonia digestate liquid fraction in conventional activated sludge system

Biodegradation of digestate liquid fraction was performed in the activated sludge system with acetic acid, flume water, and molasses as external carbon sources. High-throughput sequencing was used to gain in-depth insight into the activated sludge microbial community. The type and amount of carbon source in influent (COD/TN ratio) significantly influenced microbial community structure, especially at the genus level, and thus the biodegradation performance of digestate liquid fraction. The highest total nitrogen and chemical oxygen demand removal efficiencies averaging 85.3% and 88.3%, respectively, were achieved in series with acetic acid and flume water and COD/TN ratio of 10.7 and 11.2, respectively. The microbial diversity in these series averaged at 3.08 and 3.65. The dominant bacteria at the phylum level in series with acetic acid were Proteobacteria and Bacteroidota, and at the genus level Azospira, while in series with flume water they were Bacteroidota and Firmicutes, and Macellibacteroides, respectively.

Deciphering the microbial community structures and functions of wastewater treatment at high-altitude area.

Introduction: The proper operation of wastewater treatment plants is a key factor in maintaining a stable river and lake environment. Low purification efficiency in winter is a common problem in high-altitude wastewater treatment plants (WWTPs), and analysis of the microbial community involved in the sewage treatment process at high-altitude can provide valuable references for improving this problem. Methods: In this study, the bacterial communities of high- and low-altitude WWTPs were investigated using Illumina high-throughput sequencing (HTS). The interaction between microbial community and environmental variables were explored by co-occurrence correlation network. Results: At genus level, Thauera (5.2%), unclassified_Rhodocyclaceae (3.0%), Dokdonella (2.5%), and Ferribacterium (2.5%) were the dominant genera in high-altitude group. The abundance of nitrogen and phosphorus removal bacteria were higher in high-altitude group (10.2% and 1.3%, respectively) than in low-altitude group (5.4% and 0.6%, respectively). Redundancy analysis (RDA) and co-occurrence network analysis showed that altitude, ultraviolet index (UVI), pH, dissolved oxygen (DO) and total nitrogen (TN) were the dominated environmental factors (p < 0.05) affecting microbial community assembly, and these five variables explained 21.4%, 20.3%, 16.9%, 11.5%, and 8.2% of the bacterial assembly of AS communities. Discussion: The community diversity of high-altitude group was lower than that of low-altitude group, and WWTPs of high-altitude aeras had a unique microbial community structure. Low temperature and strong UVI are pivotal factors contributing to the reduced diversity of activated sludge microbial communities at high-altitudes.

Responses of microbial interactions to elevated salinity in activated sludge microbial community

Responses of microbial interactions to elevated salinity in activated sludge microbial community

Hormetic effect of 17α-ethynylestradiol on activated sludge microbial community response.

Synthetic estrogen analogues are among the most potent estrogenic contaminants in effluents from wastewater treatment plants. Although its effects have been well elucidated in the feminization of male fish and interference with the endocrine systems in humans, it has not been fully explored in the activated sludge (AS) microbiome, particularly EE2 (17α-ethynylestradiol). Therefore, in this study, the bacterial community shift in a 6-day laboratory-scale reactor in environmental (0, 5, 10, and 100 ng/L) and predictive elevated concentrations (5, 10, and 100 mg/L) of EE2 was investigated using culture-based and metagenomics approaches. Results showed significant changes (t-test, all p < 0.05) between initial and final physicochemical parameters (pH, DO, and EC). Although environmental concentrations showed a slight decrease in microbial counts (5.6 × 106 to 4.6 × 106 CFU/ml) after a 24-h incubation for the culturable approach, the predictive elevated concentrations (5 to 100 mg/L) revealed a drastic microbial counts reduction (5.6 × 106 to 8 × 102 CFU/ml). The metagenomic data analysis uncovered that bacterial communities in the control sample were dominated by Proteobacteria, followed by Bacteroidetes and Firmicutes. The taxonomic classification after exposure of microbial communities in various concentrations revealed significant differences in community composition between environmental concentration (Shannon indices between 2.58 to 3.68) and predictive elevated concentrations (Shannon indices between 2.24 and 2.84; t-test, all p < 0.05). The EE2 enriched seven OTUs were Novosphingobium, Cloacibacterium, Stenotrophomonas, Enterobacteriaceae_unclassified, Stenotrophomonas, Enterobacteriaceae_unclassified and Rhodobacteraceae_unclassified. These results were supported by a dehydrogenase activity (DHA) test, which demonstrated less (about 40%) DHA in predictive elevated concentrations than in environmental concentrations. Notwithstanding, these findings suggest that EE2 may possess potent hormetic effect as evidenced by promotion of microbiome richness and dehydrogenase activity of AS in lower EE2 doses.

Activated sludge microbial communities and hydrolytic potential in a full-scale SBR system treating landfill leachate

Landfill leachate, due to its recalcitrant nature and toxicity, poses a serious environmental threat, which requires the implementation of effective treatment processes. In this work, a full-scale treatment system consisting of two Sequencing Batch Reactors (SBRs) was used for the processing of landfill leachate of intermediate to mature age (BOD/COD ratio of 0.16). Biosystem operation resulted in BOD5, COD and TKN removal efficiencies of 81%, 39% and 76%, respectively, whereas the low residual NO3 --N concentration in the effluent (4.01 ± 0.10 mg/L) was indicative of the efficient denitrification process. Assessment of hydrolytic potential of activated sludge revealed high endocellular and extracellular lipase activities, which reached values up to 206 and 141 U/g protein respectively, possibly as the consequence of plastics degradation during maturation process. Implementation of Illumina sequencing indicated the predominance of Alphaproteobacteria, accompanied by members of Bacteroidetes, Betaproteobacteria and Chloroflexi. Paracoccus was the predominant genus identified, followed by representatives of the genera Bellilinea, Flavobacterium, Thauera and Truepera. Nitrosomonas was the major ammonia-oxidizing bacterium (AOB), while nitrite oxidation was mainly achieved by the uncultured nitrite-oxidizing bacterium (NOB) Candidatus Nitrotoga.

Recovery of High Quality Metagenome-Assembled Genomes From Full-Scale Activated Sludge Microbial Communities in a Tropical Climate Using Longitudinal Metagenome Sampling.

The analysis of metagenome data based on the recovery of draft genomes (so called metagenome-assembled genomes, or MAG) has assumed an increasingly central role in microbiome research in recent years. Microbial communities underpinning the operation of wastewater treatment plants are particularly challenging targets for MAG analysis due to their high ecological complexity, and remain important, albeit understudied, microbial communities that play ssa key role in mediating interactions between human and natural ecosystems. Here we consider strategies for recovery of MAG sequence from time series metagenome surveys of full-scale activated sludge microbial communities. We generate MAG catalogs from this set of data using several different strategies, including the use of multiple individual sample assemblies, two variations on multi-sample co-assembly and a recently published MAG recovery workflow using deep learning. We obtain a total of just under 9,100 draft genomes, which collapse to around 3,100 non-redundant genomic clusters. We examine the strengths and weaknesses of these approaches in relation to MAG yield and quality, showing that co-assembly may offer advantages over single-sample assembly in the case of metagenome data obtained from closely sampled longitudinal study designs. Around 1,000 MAGs were candidates for being considered high quality, based on single-copy marker gene occurrence statistics, however only 58 MAG formally meet the MIMAG criteria for being high quality draft genomes. These findings carry broader broader implications for performing genome-resolved metagenomics on highly complex communities, the design and implementation of genome recoverability strategies, MAG decontamination and the search for better binning methodology.

Effects of perfluorooctanoic acid (PFOA) on activated sludge microbial community under aerobic and anaerobic conditions.

Per- and polyfluoroalkyl substances (PFASs) have received increasing attention due to their widespread presence in diverse environments including wastewater treatment plants (WWTPs) and their potential adverse health effects. Perfluorooctanoic acid (PFOA) is one of the most detected forms of PFASs in WWTPs. However, there is still a paucity of knowledge about the effect of PFASs on microorganisms of the key component of WWTP, activated sludge. In this study, lab-scale microcosm experiments were established to evaluate the influences of PFOA on activated sludge microbes under aerobic and anaerobic conditions. The diversity, structure, and microbe-microbe interaction of microbial community were determined by 16S rRNA gene amplicon sequencing and co-occurrence network analysis. After 90days of exposure to PFOA, activated sludge microbial richness decreased under both aerobic and anaerobic conditions. Specifically, under aerobic condition, Rhodopseudomonas (mean relative abundance 3.6%), Flavobacterium (2.4%), and Ignavibacterium (6.6%) were enriched in PFOA-spiked activated sludge compared with that in the unspiked sludge (2.6%, 0.1%, and 1.9%, respectively). By contrast, after 90days of exposure to PFOA, Eubacterium (2.1%), Hyphomicrobium (1.8%), and Methyloversatilis (1.2%) were enriched under anaerobic condition, and more abundant than that in the control sludge (0.4%, 1.5%, and 0.6%, respectively). These genera were the potential PFOA-resistant members. In addition, Azospirillum and Sporomusa were the most connected taxa in PFOA-aerobic and PFOA-anaerobic networks, respectively. Prediction of the functional gene showed that PFOA inhibited some gene expression of sludge microbes, such as transcription, amino acid transport and metabolism, and energy production and conversion. In summary, continued exposure to PFOA induced substantial shifts of the sludge bacterial diversity and composition under both aerobic and anaerobic conditions.

Petrochemical and municipal wastewater treatment plants activated sludge each own distinct core bacteria driven by their specific incoming wastewater

Microorganisms in activated sludge from wastewater treatment plants (WWTPs) form complex networks to convert a wide variety of pollutants, thus ensuring water purification and environmental protection. In this study, activated sludge samples were collected from three full-scale WWTPs: a petrochemical WWTP (PWWTP), a municipal WWTP treating domestic wastewater (MWWTP_D), and a municipal WWTP treating a mixture of domestic wastewater and multiple industrial effluents (MWWTP_I+D). These samples were analyzed by high-throughput sequencing of 16S rRNA gene PCoA and CPCoA indicated that the samples from three WWTPs were separated, suggesting that each WWTP had unique microbiome characters (P < 0.05). This was also evidenced by the different predominant bacteria (PDB), biomarkers, and key nodes of co-occurrence network in the three WWTPs. Microorganisms with all three above mentioned characteristics were defined the core bacteria, specifically: Georgfuchsia, Thauera and GP4 in PWWTP, Phaeodactylibacter and Hyphomicrobiuml in MWWTP_D, and Otheakwangia, Terrimonas, Phenylobacterium, etc. in MWWTP_I + D. Furthermore, in accordance with the functional profile prediction, the functional groups in PWWTP metabolized aromatic compound, sulfur compounds and heavy metal typically present in petrochemical wastewater. In contrast, the microbiome in MWWTP_D was represented by the population breaking down macromolecular biodegradable organic matter and the nitrogen nutrients that constitute the vast majority of domestic wastewater pollutants. Both functional groups coexist in MWWTP_I + D. These results revealed that the specific composition of incoming wastewaters produced distinct ecological niches and modulated the ecological structure of activated sludge microbial communities in real-world WWTPs. However, the generalization of the results of this study will require further research.

Microbial communities across activated sludge plants show recurring species-level seasonal patterns

Microbial communities in activated sludge (AS) are the core of sanitation in wastewater treatment plants (WWTPs). Microbial communities in AS have shown seasonal changes, however, long-term experiments (>2 years) are rarely conducted, limiting our understanding of the true seasonal dynamics in WWTPs. In this study, we resolved the microbial seasonal dynamics at the species level in four municipal full-scale WWTPs, sampled every 7–10 days, during 3–5 consecutive years. By applying a new time-series analysis approach, we revealed that the seasonal pattern was species-specific, where species belonging to the same functional guild or genus may show different seasonal dynamics. Species could be grouped into cohorts according to their seasonal patterns, where seasonal cohorts showed repeatable annual dynamics across years and plants. Species were also grouped according to their net growth rate in the AS (i.e., growing species and disappearing species). Growing species were more prevailing in spring and autumn cohorts, while disappearing species, which were only present due to the continuous immigration from influent wastewater, were mostly associated with winter and spring cohorts. Most known process-critical species, such as nitrifiers, polyphosphate accumulating organisms and filamentous organisms, showed distinct species-specific patterns. Overall, our study showed that overarching seasonal patterns affected microbial species in full-scale AS plants, with similar seasonal patterns across plants for many dominant species. These recurrent seasonal variations should be taken into account in the operation, understanding and management of the WWTPs.

Indole metabolism by phenol-stimulated activated sludges: Performance, microbial communities and network analysis

Indole and phenol often coexist in the coking wastewater, while the effects of phenol on microbial communities of indole metabolism were less explored. In this study, the microbial interactions within activated sludge microbial communities stimulated by indole (group A) or by indole and phenol (group B) were systematically investigated in sequencing batch reactors (SBRs). The results showed that the removal of indole was increased by adding phenol. By using high-throughput sequencing technology, it was found that α-diversity was reduced in both groups. According to the relative abundance analysis, the indole-degrading genus Comamonas was the core genus in both groups (33.94% and 61.40%). But another indole-degrading genus Pseudomonas was only enriched in group A with 12.22% relative abundance. Meanwhile, common aromatic degrading genus Dyella and Thermomonas were enriched only in group B. It was found that the relative abundance of cytochrome P450 and styrene degradation enzymes were increased in group B by PICRUSt analysis. Based on the phylogenetic molecular ecological networks (pMENs), module hub OTU_1149 (Burkholderia) was only detected in group B, and the positive interactions between the key functional genus Burkholderia and other bacteria were increased. This study provides new insights into our understanding of indole metabolism communities stimulated by phenol, which would provide useful information for practical coking wastewater treatment.

Effects of long-term perfluorooctane sulfonate (PFOS) exposure on activated sludge performance, composition, and its microbial community

The widespread presence and persistence of perfluorooctane sulfonate (PFOS) in wastewater treatment plants, as well as its toxicity and bioaccumulation potential, necessitates the investigation on their impact on bioreactor performance. A 48-day exposure test was adopted to study the effects of low (10 μg L-1) and high (1000 μg L-1) PFOS concentrations in a sequencing batch reactor on the performance, composition, and microbial community of activated sludge. The results suggested that adding PFOS at low and high concentrations lowered the removal efficiency of total nitrogen by 22.48% (p < 0.01) and 16.30% (p < 0.01) respectively, while enhanced that of total phosphorus by 1.87% (p > 0.05) and 7.07% (p < 0.05) respectively, compared with the control group. The addition of PFOS also led to the deterioration of activated sludge dewatering performance. Composition and spectroscopic measurements revealed that the PFOS dosage changed the composition of the activated sludge. Furthermore, the PFOS altered the structure and function of the activated sludge microbial community as well as key enzyme activities.

Effects of influent immigration and environmental factors on bacterial assembly of activated sludge microbial communities

The functional mechanism of microbial assembly of activated sludge (AS) in urban wastewater treatment plants (UWTPs) remains unclear. A comprehensive quantitative evaluation of the contribution of influent immigration and environmental factors to AS community composition requires investigation. In this study, the microbial characteristics of six full-scale UWTPs with different influent compositions and environmental factors (altitude, temperature, dissolved oxygen (DO), pH, chemical oxygen demand (COD), total nitrogen (TN), ammonia nitrogen (NH4+-N), and total phosphorus (TP)) were analyzed to determine the main forces affecting the bacterial assembly of AS microbial communities. Abundant and core taxa were screened out based on the abundance and frequency of operational taxonomic units (OTUs) occurrence in all samples. Abundant OTUs (18.7% occurrence) accounted for 87.7% of the total 16S rRNA sequences, while rare OTUs (71.7% occurrence) accounted for only 7.8% of the total 16S rRNA sequences. A total of 135 OTUs were identified as core taxa, accounting for 14.6–26.2% of the total reads, of which 83 OTUs belonged to abundant taxa. The richness and uniformity of the influent community were significantly lower than those of the AS system. The community composition in influent varied from that in AS. Moreover, about 89.7% (86.5% of 16S rRNA sequences) OTUs in AS samples showed positive growth rates, indicating that immigration of influent communities had a limited effect on the microbial composition of AS. Redundancy analysis (RDA) combined with co-occurrence network showed that the bacterial assembly of microbial communities was significantly correlated with altitude, pH, and TN (P < 0.05), and these three parameters could explain 23.3%, 21.1%, and 17.7% of the bacterial assembly of AS microbial communities in UWTPs, respectively.