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Urban black blooms that are primarily caused by organic carbon are deleterious environmental problems. However, detailed studies on the microbial characteristics that form urban black blooms are lacking. In this study, we observed the composition, diversity, and function of bacterial community in the overlying water and sediments during the occurrence and remediation of urban black blooms using high-throughput 16S rRNA gene amplicon sequencing analysis. First, we found that pivotal consortia in the overlying water increased significantly during the formation of black blooms, including the genera Acidovorax, Brevundimonas, Pusillimonas, and Burkholderiales involved in the degradation of refractory organics, as well as the genera Desulfovibrio, Dechloromonas, and Rhizobium related to the production of black and odorous substances. An RDA analysis revealed that chemical oxygen demand, dissolved oxygen, and oxidation reduction potential were related to the changes in microbial community composition. Furthermore, aeration was found to accelerate the removal of ammonia nitrogen and enhance the function of microbial community by stimulating the growth of order Planktomycetes during the remediation of black blooms, but aeration substantially damaged the microbial diversity and richness. Therefore, the health of the aquatic ecosystem should be comprehensively considered when aeration is applied to restore polluted waterbodies. Notably, we observed a large number of pathogenic bacteria in urban black blooms, which emphasizes the importance of treating domestic sewage so that it is harmless. Together, these findings provide new insights and a basis to prevent and manage urban black blooms.

External organic carbon sources are needed to provide electron donors for the denitrification of wastewater with a low COD/NO3−-N (C/N) ratio, increasing the treatment cost. The economic strategy is to enhance the bioactivity and/or biodiversity of denitrifiers to efficiently utilize organic substances in wastewater. In this study, novel zero-valent iron (ZVI) composite carriers were prepared and implemented in a suspended carrier biofilm reactor to enhance the bioactivity and/or biodiversity of denitrifiers. At the influent C/N ratio of 4 (COD was 179.5 ± 5.0 mg/L and TN was 44.2 ± 0.8 mg/L), COD and TN removal efficiencies were 85.1% and 66.4%, respectively, in the reactors filled with 3 wt% ZVI composite carriers. In contrast, COD and TN removal efficiencies were 70.4% and 55.3%, respectively, in the reactor filled with conventional high-density polyethylene (HDPE) biofilm carriers. The biofilm formation on the 3 wt% ZVI composite carriers was optimized due to its higher roughness (surface square roughness increased from 76.0 nm to 93.8 nm) and favorable hydrophilicity (water contact angle dropped to 72.5° ± 1.4° from 94.3° ± 3.2°) compared with the HDPE biofilm carriers. In addition, heterotrophic denitrifiers, Thauera and Dechloromonas, were enriched, whereas autotrophic denitrifiers, Raoultella and Thiobacillus, exhibited high relative abundance in the biofilm of ZVI composite carriers. The coexistence of heterotrophic denitrifiers and autotrophic denitrifiers on the surface of ZVI composite carriers provided mixotrophic metabolism of denitrification (including heterotrophic and iron-based autotrophic), thereby ensuring effective denitrification for wastewater with a low C/N ratio without external organic carbon source addition.

To explore the reasons for the excellent denitrification performance of the Micro-pressure double-cycle reactor (MPDR), the nitrogen removal mechanism of the reactor in the treatment of municipal wastewater was studied. Through analysis of flow simulation and dissolved oxygen (DO) distribution, it was determined that the reactor had a macroscopic biochemical reaction environment for simultaneous nitrification and denitrification (SND) because of the special structure of reactor. The result of sewage treatment showed that the average removal rates of COD, NH4+-N, TN, TP were 92.29%, 96.64%, 73.6% and 91.66% respectively, and the SND rate was 60.9%. Dechloromonas, Thermomonas, Micropruina, Tetrasphaera, etc. for nitrogen and phosphorus removal existed in the reactor at the same time to explain the excellent performance of the system. PICRUSt2 showed that the metabolic pathways related to nutrient degradation in the reactor were highly active and the abundance of denitrification functional genes was higher in the central zone and lower in the peripheral zone. The research results not only perfected the basic theory of the reactor, but more importantly, provided theoretical and technical support for the further application of the reactor.

Inoculating agricultural soils with nitrous oxide respiring bacteria (NRB) can reduce N2O-emission, but would be impractical as a standalone operation. Here we demonstrate that digestates obtained after biogas production are suitable substrates and vectors for NRB. We show that indigenous NRB in digestates grew to high abundance during anaerobic enrichment under N2O. Gas-kinetics and meta-omic analyses showed that these NRB’s, recovered as metagenome-assembled genomes (MAGs), grew by harvesting fermentation intermediates of the methanogenic consortium. Three NRB’s were isolated, one of which matched the recovered MAG of a Dechloromonas, deemed by proteomics to be the dominant producer of N2O-reductase in the enrichment. While the isolates harbored genes required for a full denitrification pathway and could thus both produce and sequester N2O, their regulatory traits predicted that they act as N2O sinks in soil, which was confirmed experimentally. The isolates were grown by aerobic respiration in digestates, and fertilization with these NRB-enriched digestates reduced N2O emissions from soil. Our use of digestates for low-cost and large-scale inoculation with NRB in soil can be taken as a blueprint for future applications of this powerful instrument to engineer the soil microbiome, be it for enhancing plant growth, bioremediation, or any other desirable function.

Nitrogen (N) shortage poses a great challenge to the implementation of in situ bioremediation practices in mining-contaminated sites. Diazotrophs can fix atmospheric N2 into a bioavailable form to plants and microorganisms inhabiting adverse habitats. Increasing numbers of studies mainly focused on the diazotrophic communities in the agroecosystems, while those communities in mining areas are still not well understood. This study compared the variations of diazotrophic communities in composition and interactions in the mining areas with different extents of arsenic (As) and antimony (Sb) contamination. As and Sb co-contamination increased alpha diversities and the abundance of nifH encoding the dinitrogenase reductase, while inhibited the diazotrophic interactions and substantially changed the composition of communities. Based on the multiple lines of evidence (e.g., the enrichment analysis of diazotrophs, microbe-microbe network, and random forest regression), six diazotrophs (e.g., Sinorhizobium, Dechloromonas, Trichormus, Herbaspirillum, Desmonostoc, and Klebsiella) were identified as keystone taxa. Environment-microbe network and random forest prediction demonstrated that these keystone taxa were highly correlated with the As and Sb contamination fractions. All these results imply that the above-mentioned diazotrophs may be resistant to metal(loid)s.

To improve the nitrogen and phosphorus removal efficiency of surface flow constructed wetlands (SFCWs), biochar was added to an SFCW matrix. The effects of adding different amounts of biochar on water purification, the growth of Vallisneria natans (V. natans), and microbial mechanisms were explored through SFCW simulation experiments. The results showed that through the joint action of biochar and V. natans, the concentrations of total nitrogen, total phosphorus, and ammonia nitrogen in the effluent significantly decreased. The total biomass, relative growth rate, and chlorophyll content of V. natans were significantly reduced by adding biochar (≥20%, v/v), as the root activity and the root to leaf biomass ratio slightly increased at first and then decreased. The carbon and nitrogen contents of V. natans slightly increased with the addition of biochar (≥10%, v/v), but the phosphorus content slightly decreased. Moreover, the nitrogen content of the matrices decreased significantly over time (P

Made available in DSpace on 2022-04-28T19:41:21Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Microbial fuel cells are an eco-friendly technology that decontaminate wastewater and generate electricity by the action of exoelectrogenic microbes. However, mechanisms such as bioanode formation and stabilization and the electron transfer mechanism still have to be elucidated and enhanced before this technology can be scaled up for practical applications. Electrochemical characterization showed that a more electroactive biofilm (power density of 77 ± 8 mW m−2) emerged under a charged surface provided by a continuously applied electric current. Among the different molecules evaluated as electron transfer mediator, methylene green was the only molecule that improved cell performance. Methylene green addition increased the cell voltage by 20% and maintained the cell stable for six more days as compared to the control. Bioanode microbial community analysis revealed a high abundance of Arcobacter and Dechloromonas, which may play roles in electricity generation and methylene green discoloration. Microbial fuel cell voltage and stability were increased upon addition of methylene green, which was degraded in the bioanode over time. Identification of microbial members helps to understand MFC performance and to elucidate how methylene green improves MFC stability. Departamento de Química Faculdade de Filosofia Ciências e Letras de Ribeirão Preto FFCLRP Universidade de São Paulo, Av. Bandeirantes 3900 Departamento de Ciência do Solo Escola Superior de Agricultura Luiz de Queiroz Universidade de São Paulo Institute of Chemistry UNESP National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM), PO Box 355 Institute of Chemistry UNESP National Institute for Alternative Technologies of Detection Toxicological Evaluation and Removal of Micropollutants and Radioactives (INCT-DATREM), PO Box 355

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.

A continuous-flow Anaerobic/Anoxic/Oxic (A2/O) system was operated at different organic concentrations to systematically investigate the effect on the nutrient removal, secretion characteristics of extracellular polymer, phosphorus forms transformation and changes in functional flora in this system. The results showed that high organic loading was more conducive to promote the secretion of extracellular polymeric substance (EPS), the increase of polysaccharide content was more obvious compared with protein, the impact of organic loading on the components of loosely bound EPS (LB-EPS) was higher than that of tight-bound EPS (TB-EPS). Phosphorus in sludge floc mainly existed in the form of inorganic phosphorus (IP), and IP mainly existed in the form of apatite inorganic phosphorus (AP). High organic load showed higher phosphorus storage in EPS, and the phosphorus content in EPS was positively correlated with the content of EPS. Non-apatite phosphorus (NAIP) content played an important role in the extracellular dephosphorization. The abundance of Nitrosomonas and Nitrospira responsible for nitrification decreased with the increase in organic loading. The group of denitrifiers was large, and Azospira was the most abundant genus among them. Dechloromonas, Acinetobacter, Povalibacter, Chryseolinea and Pirellula were the functional genera closely associated with phosphorus removal. HIGHLIGHTS High organic loading promoted the secretion of EPS, the impact of organic loading on the components of LB-EPS was higher than that of TB-EPS.; NAIP played an important role in the extracellular phosphorus removal.; Influent organic loading shaped the bacterial community structure.; The core functional genera in N and P removal were identified by correlation analysis.

To comprehensively evaluate the impact of 2450 MHz electromagnetic wave (EW) loading on returned sludge (RS) for sludge reduction and pollutants removal in anaerobic/anoxic/oxic (A2O) process, the performance of the coupled process (EWRS-A2O) was investigated in current study. 250 W was the appropriate loading power when considering both biological effect and dissolution effect of electromagnetic wave on the returned sludge. After being loaded, floc structure of the returned sludge became looser, and the microbial composition changed. The continuous operation indicated that an 8.3 % improvement in total nitrogen removal and a 33.1 % of sludge reduction were achieved in EWRS-A2O, accompanied by an observed yield coefficient of 0.35 kg SS/kg COD. Although an increase in the total phosphorus concentration of the effluent was found, the impacts on the chemical oxygen demand and ammonia nitrogen of the effluent were minimal. When contrasted to conventional A2O, EWRS-A2O process exhibited higher microbial metabolic activity. Miseq sequencing results showed that the microbial richness increased in EWRS-A2O system with changing of the microbial community structure. Additionally, functional microorganism analysis revealed the enrichment of slow-growing bacteria (Dechloromonas), fermenting bacteria (Clostridium sensu stricto and Tolumonas), and heterotrophic denitrifying bacteria (Thermomonas).

Previous studies demonstrated that methane can be used as an electron donor to microbially remove various oxidized contaminants in groundwater. Natural gas, which is more widely available and less expensive than purified methane, is potentially an alternative source of methane. However, natural gas commonly contains a considerable amount of ethane (C2H6) and propane (C3H8), in addition to methane. It is important that these gaseous alkanes are also utilized along with methane to avoid emissions. Here, we demonstrate that perchlorate (ClO4-), a frequently reported contaminant in groundwater, can be microbially reduced to chloride (Cl-) driven by C2H6 or C3H8 under oxygen-limiting conditions. Two independent membrane biofilm reactors (MBfRs) supplied with C2H6 and C3H8, respectively, were operated in parallel to biologically reduce ClO4-. The continuous ClO4- removal during long-term MBfR operation combined with the concurrent C2H6/C3H8 consumption and ClO4- reduction in batch tests confirms that ClO4- reduction was associated with C2H6 or C3H8 oxidation. Polyhydroxyalkanoates (PHAs) were synthesized in the presence of C2H6 or C3H8 and were subsequently utilized for supporting ClO4- bio-reduction in the absence of gaseous alkanes. Analysis by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that transcript abundance of bmoX (encoding alpha hydroxylase subunit of C2H6/C3H8 monooxygenase) was positively correlated to the consumption rates of C2H6/C3H8, while pcrA (encoding a catalytic subunit of perchlorate reductase) was positively correlated to the consumption of ClO4-. High-throughput sequencing targeting 16S rRNA, bmoX, and pcrA indicated that Mycobacterium was the dominant microorganism oxidizing C2H6/C3H8, while Dechloromonas may be the major perchlorate-reducing bacterium in the biofilms. These findings shed light on microbial ClO4- reduction driven by C2H6 and C3H8, facilitating the development of cost-effective strategies for ex situ groundwater remediation.

The paper assesses the impact of the duration of oxic and anoxic conditions on the selection of microbial populations in laboratory-scale integrated fixed-film sequencing batch reactors (IF-SBRs). IF-SBRs were fed anaerobically with glucose and sodium acetate based synthetic wastewater. Three IF-SBRs were operated with different lengths of oxic and anoxic periods, and a control IF-SBR was operated with continuous aeration during the react phase of the SBR cycle. Compact flocs were formed in the control bioreactor, whereas granular sludge developed rapidly within ten days in the IF-SBR operated with an extended anoxic phase (3.25 hours within the six-hour cycle). Filamentous bulking was observed in the IF-SBRs that were operated with relatively moderate anoxic periods. Confocal laser scanning microscopy of the granular sludge revealed that β-polysaccharide and amyloid adhesins were predominant in the periphery and core of the granules, respectively. 16S rRNA gene sequencing (Illumina MiSeq) revealed rapid selection and increases in the relative abundance of Flavobacterium, Caulobacter, Dechloromonas, Zoogloea, and bacterial genera related to the family Comamonadaceae, that were predominant in the granular sludge. The functional capabilities of the bacteria that were predominant in the granular sludge include quorum sensing, formation of storage polymers, and denitrification. Optimizing the length of oxic and anoxic conditions in sequencing batch reactors, and in the configuration in this study which was a hybrid system consisting of flocs, biofilm and granular sludge, is an important consideration in the selection of bacterial populations important to the development of microbial structures, and granular sludge in particular.

The discrepancy of c‐di‐GMP and EDR proved that TB‐EPS served as important component in the structure and growth of denitrification biofilm, denitrifiers could effectively receive electrons from both acetate and EPS, which was beneficial to maintain relatively high denitrification capability at low temperature. Pseudomonas played an important role to maintain stable structure by EPS production under shock organic loading rate and low temperature conditions, and the specialized communities suggested that good nitrate removal property could be enhanced not only by the interaction of unique OTUs but also through the high bioactivity of dominant species., Summary Microbial community may respond to different adverse conditions and result in the variation of extracellular polymeric substances (EPS) in denitrification biofilm; this study discovered the role of EPS in accordance with the analysis of cyclic diguanylate (c‐di‐GMP) and electron equilibrium (EE) under low organic loading rate, shock organic loading rate and low temperature conditions. Good nitrate removal performance could be achieved under shock organic loading rate and low temperature conditions; however, owing to the low organic loading rate, the carbon source was preferentially utilized for biomass growth. Tightly bound EPS (TB‐EPS) contents progressively increased and facilitated cell adhesion and biofilm formation. The stable TB protein (TB‐PN) content in TB‐EPS built a cross‐linked network to maintain internal biofilm structure and led to the rapid biosynthesis of polysaccharides, which could further enhance microbial adhesion and improve nitrate removal. C‐di‐GMP played an important role in biomass retention and biofilm formation, based on the correlation analysis of c‐di‐GMP and EPS. TB polysaccharide (TB‐PS) contents presented a significant positive correlation with c‐di‐GMP content, microbial adhesion and biofilm stabilization was further enhanced through c‐di‐GMP regulation. In addition, a remarkable negative correlation between electron deletion rate (EDR) and TB‐PN and TB‐PS was discovered, and TB‐PS was required to serve as energy source to enhance denitrification according to EE analysis. Surprisingly, dynamic microbial community was observed due to the drastic community succession under low temperature conditions, and the discrepancy between the dominant species for denitrification was found under shock organic loading rate and low temperature conditions. The notable increase in bacterial strains Simlicispira, Pseudomonas and Chryseobacterium was conducive to biofilm formation and denitrification under shock organic loading rate, while Dechloromonas and Zoogloea dramatically enriched for nitrate removal under low temperature conditions. The high abundance of Dechloromonas improved the secretion of EPS through the downstream signal transduction, and the c‐di‐GMP conserved in Pseudomonas concurrently facilitated to enhance exopolysaccharide production to shock organic loading rate and low temperature conditions.

Summary Microbial sulfate reduction and sulfur oxidation are vital processes to enhance organic matter degradation in sediments. However, the diversity and composition of sulfate‐reducing bacteria (SRB) and sulfur‐oxidizing bacteria (SOB) and their environmental driving factors are still poorly understood in aquaculture ponds, which received mounting of organic matter. In this study, bacterial communities, SRB and SOB from sediments of aquaculture ponds with different sizes of grass carp (Ctenopharyngodon idellus) were analysed using high‐throughput sequencing and quantitative real‐time PCR (qPCR). The results indicated that microbial communities in aquaculture pond sediments of large juvenile fish showed the highest richness and abundance of SRB and SOB, potentially further enhancing microbial sulfur cycling. Specifically, SRB were dominated by Desulfobulbus and Desulfovibrio, whereas SOB were dominated by Dechloromonas and Leptothrix. Although large juvenile fish ponds had relatively lower concentrations of sulfur compounds (i.e. total sulfur, acid‐volatile sulfide and elemental sulfur) than those of larval fish ponds, more abundant SRB and SOB were found in the large juvenile fish ponds. Further redundancy analysis (RDA) and linear regression indicated that sulfur compounds and sediment suspension are the major environmental factors shaping the abundance and community structure of SRB and SOB in aquaculture pond sediments. Findings of this study expand our current understanding of microbial driving sulfur cycling in aquaculture ecosystems and also provide novel insights for ecological and green aquaculture managements., In aquaculture pond sediments, sulfate‐reducing bacteria (SRB) produced AVS through sulfate reduction, and sulfur‐oxidizing bacteria was especially important to reduce AVS. High concentrations of sulfur compounds (TS, AVS, and ES) could inhibit SRB and sulfate reduction. Disturbance to sediments by fish activities could enhance the sulfate reduction and sulfur oxidation in sediments.

Enhanced biological phosphorus removal (EBPR) is used extensively in full-scale wastewater treatment plants for the removal of phosphorus. Despite previous evidence showing that glycine is a carbon source for a certain lineage of polyphosphate accumulating organisms (PAOs) such as Tetrasphaera, it is still unknown whether glycine can support EBPR. We observed an overall adverse effect of glycine on EBPR using activated sludge from both full-scale wastewater treatment plants and lab-scale reactors harboring distant and diverse PAOs and glycogen accumulating organisms (GAOs), including Candidatus Accumulibacter, Thiothrix, Tetrasphaera, Dechloromonas, Ca. Competibacter, and Defluviicoccus, among others. Glycine induced phosphorus (P) release under anaerobic conditions without being effectively taken up by cells. The induced P release rate correlated with glycine concentration in the range of 10 to 50 mg C/L. PAOs continued to release P in the presence of glycine under aerobic conditions without any evident P uptake. Under mixed carbon conditions, the occurrence of glycine did not seem to affect acetate uptake; however, it significantly reduced the rate of P uptake in the aerobic phase. Overall, glycine did not appear to be an effective carbon source for a majority of PAOs and GAOs in full-scale and lab-scale systems, and neither did other community members utilize glycine under anaerobic or aerobic conditions. Metatranscriptomic analysis showed the transcription of glycine cleavage T, P and H protein genes, but not of the L protein or the downstream genes in the glycine cleavage pathway, suggesting barriers to metabolizing glycine. The high transcription of a gene encoding a drug/metabolite transporter suggests a potential efflux mechanism, where glycine transported into the cells is in turn exported at the expense of ATP, resulting in P release without affecting the glycine concentration in solution. The ability of glycine to induce P release without cellular uptake suggests a way to effectively recover P from P-enriched waste sludge. Ministry of Education (MOE) National Research Foundation (NRF) Public Utilities Board (PUB) This research was supported by the National Natural Science Foundation of China (No. 51808297), the Natural Science Foundation of Guangdong Province, China (No. 2021A1515010494), the Science and Technology Program of Guangzhou, China (No. 202002030340), the Pearl River Talent Recruitment Program (No. 2019QN01L125), the Program for Science and Technology of Guangdong Province, China (No. 2018A050506009), and the Singapore National Research Foundation and the Ministry of Education under the Research Centre of Excellence Programme, and by a research grant from the National Research Foundation under its Environment and Water Industry Programme (project number 1102–IRIS–10–02), administered by Public Utilities Board, Singapore’s national water agency.

Large amounts of phosphorus (P) are present in activated sludge from municipal wastewater treatment plants, where it exists in the form of metal salt precipitates or biologically bound into the biomass as nucleic acids, cell membrane components, and the extracellular polymeric substances or, in special polyphosphate-accumulating organisms (PAOs), as intracellular polyphosphate. Only recently, methods that reliably allow an absolute quantification of the different P-fractions, such as sequential extraction, Raman microspectroscopy, solid-state 31P magic angle spinning (MAS) NMR, and solution state 31P NMR have been developed. This study combines these techniques to obtain a comprehensive P mass-balance of activated sludge from four wastewater treatment plants with enhanced biological phosphate removal (EBPR). The total content of P and various cations was measured by chemical analysis (ICP-OES), and different P fractions were extracted for chemical characterization. Chemically bound P constituted 38-69% of total P, most likely in the form of Fe, Mg, or Al minerals, while organically bound P constituted 7-9%. By using Raman microspectroscopy and solution state 31P NMR and 31P MAS NMR spectroscopy before and after anaerobic P-release experiments, poly-P was quantified and constituted 22-54% of total P in the activated sludges and was found in approx. 25% of all bacterial cells. Moreover, Raman microspectroscopy in combination with fluorescence in situ hybridization (FISH) was used to quantify the species-specific intracellular poly-P of known PAO genera (Tetrasphaera, Ca. Accumulibacter, Dechloromonas) and other microorganisms known to possess high level of poly-P, such as the filamentous Ca. Microthrix. They were all abundant, as measured by quantitative-FISH and amplicon sequencing, and accumulated large amount of poly-P, depending on their cell-size, contributing substantially to the P-removal. Interestingly, in all four EBPR plants investigated, only 1-13% of total poly-P was stored by unidentified PAO, highlighting that most PAOs in the full-scale EBPR plants investigated are now known.HighlightsExhaustive P mass-balance of main organic and inorganic P-species in four EBPR plantsQuantification of poly-P of FISH-defined PAO and other species with high P contentTotal P content was 36-50 mgP/gSS of which 31-62% was in biomass and as poly-PA high fraction of all cells (25-30%) contained a high content of poly-PKnown PAOs contained almost all poly-P in the EBPR plants investigated

Arsenic (As) mobilization in alluvial aquifers is facilitated by microbially catalyzed redox transformations that depend on the availability of electron acceptors (EAs). In this study, the response of an As-contaminated groundwater microbial community from West Bengal, India towards varied EAs was elucidated through microcosm based 16S rRNA gene amplicon sequencing. Acinetobacter, Deinococcus, Nocardioides, etc., and several unclassified bacteria (Ignavibacteria) and archaea (Bathyarchaeia, Micrarchaeia) previously not reported from As-contaminated groundwater of West Bengal, characterized the groundwater community. Distinct shifts in community composition were observed in response to various EAs. Enrichment of operational taxonomic units (OTUs) affiliated to Denitratisoma (NO3-), Spirochaetaceae (Mn4+), Deinococcus (As5+), Ruminiclostridium (Fe3+), Macellibacteroides (SO42-), Holophagae-Subgroup 7 (HCO3-), Dechloromonas and Geobacter (EA mixture) was noted. Alternatively, As3+ amendment as electron donor allowed predominance of Rhizobium. Taxonomy based functional profiling highlighted the role of chemoorganoheterotrophs capable of concurrent reduction of NO3-, Fe3+, SO42-, and As biotransformation in As-contaminated groundwater of West Bengal. Our analysis revealed two major aspects of the community, (a) taxa selective toward responding to the EAs, and (b) multifaceted nature of taxa appearing in abundance in response to multiple substrates. Thus, the results emphasized the potential of microbial community members to influence the biogeochemical cycling of As and other dominant anions/cations.

Nitrogen removal and nitrous oxide (N2O) emission in a lab-scale constant-flow multiple anoxic (A) and aerobic (O) process, combined with the addition of suspended carriers, were investigated. Under steady state, 99.9% of ammonia nitrogen (NH4-N), 76.8% of orthophosphate, and 80.2% of total inorganic nitrogen removal efficiency was achieved. The N2O emission factor during nitrification was 0.8–1.9% of the oxidized NH4-N. The emission factor increased to 7.4–45.9% with the coexistence of heterotrophic activities. Extending the anoxic time from 30 to 90 min reduced the N2O emission factor from 1.6 to 1.0%. N2O emission was stimulated with nitrite (NO2-N) as the electron acceptor, with the N2O emission factor of 5.2–5.6%. Denitrification with internal organic carbon contributed 6.8% of reduced NO2-N to N2O. NO2-N might exert a crucial role in N2O emission independent of carbon source. For the acclimated microbial communities, Nitrospira and Nitrosospira were the dominant nitrifiers and responsible for the N2O emission during nitrification. Azospira, Dechloromonas, Flavobacterium, Pseudomonas, and unclassified genus of family Comamonadaceae might be responsible for N2O emission during denitrification. These findings may guide the design of multiple AO process for controlling N2O emission.

The peri-urban rivers are one of the critical interfaces between urban-rural symbiotic ecosystems and appear to be a reservoir of antibiotic resistance genes (ARGs) in the environment. To prevent the transmission risks of ARGs between peri-urban river and human, it is essential to explore the prevalence and source of ARGs in the environment for designing potential mitigation strategies. In this study, we focused on the characterization and source-tracking of ARGs in the sediments of a typical peri-urban river in Beijing, Chaobai River. Twenty-seven ARGs frequently reported in the environment, and two integrons (intI1 and intI2) were detected using high-throughput quantitative PCR. The profile of bacterial community was determined by performing 16S rRNA gene sequencing. Meanwhile, crAssphage, a novel recently-discovered DNA bacteriophage, was employed for tracking the contribution of human fecal pollution to the prevalence of ARGs. Results showed that the targeted ARGs were detected widely in the sediments of Chaobai River. Relatively, the abundances of ARGs in downstream were higher than those in the upstream, likely suggesting a gradient impact of anthropogenic activities along the river. Remarkably, the int1 gene was correlated significantly with most of the ARGs and might be the key factor influencing the shaping of ARGs in the river sediments. However, no significant correlations were observed between the ARGs and selective pressure factors, including antibiotics and metals. Of the identified 1039 genera, Escherichia-Shigella, Bacteroides, Arcobacter, Dechloromonas and Pseudomonas were the top most abundant organisms. Microbial source tracking based on the crAssphage annotation suggested that human sewage might be one of the potential sources of resistance bacteria in the river sediments. The study can advance our knowledge about ARGs in the peri-urban river and provides a management reference for ARG pollution control.

This study demonstrated a novel process configuration for sustaining mainstream anammox by integrating the anammox and endogenous partial denitrification-and-phosphorus removal (EPDPR) in two-stage sequencing batch reactors (SBRs). In the EPDPR-SBR, high nitrate-to-nitrite transformation (68.2%) and P removal (99.3%) were achieved by adjusting the anaerobic/anoxic/aerobic durations and influent nitrate concentration, providing a suitable NO2−-N/NH4+-N (∼1.37) for subsequent anammox reaction. In the Anammox-SBR, ∼95% of TN was removed without external carbon and oxygen demands. Satisfactory effluent quality (∼6 mgTN/L and 0.2 mgP/L) achieved in the integrated EPDPR/anammox opens a new window towards the energy-efficient wastewater treatment. Microbial analysis further revealed that Dechloromonas (1.6–9.6%) and Candidatus Competibacter (6.4–5.8%) respectively conducted P removal and NO2−-N production (79.2%) from NO3−-N denitrification in the EPDPR-SBR, whereas Candidatus Kuenenia (7.0–29.7%) dominated NO2−-N and NH4+-N removal (91.3% and 99.5%) in the Anammox-SBR, with 10 genera identified as denitrifying bacteria (0.6–8.1%) further reduced 18.9% of the produced NO3−-N.

A co-substrate strategy has the potential to contribute toward minimizing the poisoning of refractories on the anode respiring bacteria (ARB), which is a necessity for microbial fuel cell (MFC) to recover energy from toxic wastewater. However, little is known about the underlying mechanisms. This study employed 4-chlorophenol (4-CP) as target refractory pollutant and acetate as co-substrate. The co-substrate could stimulate 4-CP sensitive extracellular electron transfer (EET) enzymes’ activity under 4-CP stress, such as succinate dehydrogenase (complex II) and cytochromes c OmcA and OmcB. For the microbial community, ARB were still abundant during 4-CP toxification; Azospirillum and Dechloromonas were enriched to conduct aromatic ring breakage and dechlorination. Thus, a 4.3-fold increased power generation was achieved, and the 4-CP and chemical oxygen demand (COD) removals increased by ∼42% and ∼53%, respectively. An easy-to-degrade substrate could improve power re-coverage from refractory wastewater by enhancing EET enzyme activity and optimizing the microbial community.

The performances of anaerobic-anoxic-oxic processes with (A2O-B) and without (A2O-C) beneficial microorganisms were compared to provide valuable insights on how they are affected by changes in the microbial biomass and community composition. Although the A2O-B process showed lower concentrations of mixed liquor suspended solids and mixed liquor volatile suspended solids than the A2O-C process under identical operating conditions, the A2O-B process was more effective for the removal of organic materials and nutrients compared with the A2O-C process. Furthermore, the compressibility and settleability of the activated sludge were significantly better in the A2O-B process than in the A2O-C process due to the enhanced decomposition of extracellular polymeric substances. These results indicated that the inoculation of beneficial microorganisms may increase the proportions of microorganisms in relation to the removal of organic materials, nutrients (i.e., Zoogloea, Dechloromonas, Nitrospira, and Nitrosomonas) and the reduction of the excess sludge (i.e., Proteobacteria and Bacteroidetes).

In this study, a poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) supported denitrification system was developed for nitrate removal. Results showed that nitrate removal efficiency exceeded 95% and the maximal volumetric denitrification rate reached 0.77 kg m−3 day-1 with a hydraulic retention time (HRT) of 0.5 h. The effluent total organic carbon (TOC) decreased while the effluent pH increased with a shortage of HRT. Meanwhile, the weight-averaged molecular weight distribution analysis indicated that PHBV polymer’s intermediate degradation products was divided into three ranges, and the low-molecular-weight substances were mainly identified as acetate by a gas chromatography. The fourier transformation infrared spectra (FTIR) analysis indicated that these degradation products contained lots of C O and O H. Moreover, the microbial community structure of the PHBV biofilm was different with that of the inoculation sludge, and the abundant denitrifying bacteria mainly included Dechloromonas, Flavobacteriumand Stenotrophomonas. Functional protein, biological process and KEGG pathways were explored by Meta-proteomic approach, which further revealed the degradation and utilization mechanism of PHBV polymer.

The sludge in situ reduction process by inserting an anaerobic side-stream reactor (ASSR) in a sludge return line provides a cost-effective approach to reduce sludge production in activated sludge systems. In this study, four pilot-scale membrane bioreactors (MBRs), including an AO-MBR for control, ASSR coupled MBR (ASSR-MBR), a MBR with ASSR packed with carriers (AP-MBR) and an AP-MBR with part of sludge ultrasonicated before fed into ASSR (AUP-MBR) were operated in parallel to investigate enhancing effects of ultrasonication and packing carriers on sludge reduction and pollutants removal performance under both normal and low temperature. Low temperature showed negligible impact on COD removal, deteriorated NH4+ N and TN removal from 98.3% to 69.7% at 21.6 °C to 92.5% and 48.8% at 2.6 °C, and decreased sludge reduction efficiency (SRE) in ASSR-MBR. Packing carriers and ultrasonication both enhanced sludge reduction, especially under low temperature with SRE values increased from 8.2% of ASSR-MBR to 17.1% of AP-MBR and 32.6% of AUP-MBR at 4.5 ± 2.5 °C. Packing carriers and ultrasonication increased cell rupture by 11.1% and 14.5% in aerobic MBR, enhanced protease activity in ASSR by 60.0% and 116.3%, and reduced ATP content for heterotrophic metabolism by 31.4% and 7.3%, respectively. MiSeq sequencing results showed that packing carriers enriched hydrolytic bacteria (Terrimonas, Dechloromonas and Woodsholea), slow growers (Sulfuritalea, Thauera and Azospira) and predatory bacteria (Bdellovibrio and norank_Saprospiraceae), while ultrasonication further enriched hydrolytic bacteria (norank_Saccharibacteria and Ferruginibacter). Packing carriers is more cost-effective than ultrasonication to enhance sludge reduction by partial damage to bacterial cells and promoting better interaction between bacteria, enzymes and substrates to favor particles hydrolysis.

The influence of different pH conditions on the characteristics, nutrient removal efficiency and bacterial community structure of mature granules. Three identical sequencing batch reactors with stable granules were operated under controlled pH (≤7.0 and ≤7.5) and uncontrolled pH (initial pH = 7.5) conditions for two months. Results show that pH ≤ 7.0 significantly affected properties of the granules. Extracellular polymeric substance content decreased from 134 to 101 mg/gVSS, granules’ size decreased from 660 to 449 μm, and fraction of floccular biomass increased from 7.8 to 26.3%. However, maintaining pH ≤ 7.5 did not show significant impact on these parameters. Moreover, pH ≤ 7.0 deteriorated nitrogen removal (from 44.4 to 17.7%) by depleting the alkalinity and inhibiting the activity of nitrifying bacteria. Phosphorus removal was also significantly influenced by the pH control such that pH ≥ 7.5 improved the phosphorus removal from 90.8 to 99.6%, while pH ≤ 7.0 obtained 63.1% phosphorus removal. Results of DNA sequencing showed that pH control influenced bacterial community structure of the granules. The results confirmed the increase in the abundance of Dechloromonas, which were the main polyphosphate accumulating organisms at pH ≤ 7.5. Our results suggest that pH adjustment could alter bacterial communities of mature granules and therefore could be used to improve treatment functionality of the granules.

Advanced nutrient removal of municipal wastewater has insufficient carbon source, and resource recovery is neglected. In this study, a single-stage biofilter based on denitrifying phosphorus removal (DPR) was proposed for advanced nutrient removal and phosphorus recovery, which was operated under alternating anoxic/anaerobic mode with no extracellular carbon source in anoxic period. The results showed that the biofilter achieved efficient and stable performance with low carbon consumption (C/N ≈ 3.7). The average removal efficiency of NO3−-N, TN and PO43−-P were 74.81%, 71.08% and 91.15%, respectively. DPR primarily occurred in the middle of the filtration bed and nutrient removal was driven by intracellular polymers, which was the main carbon source. High-throughput sequencing indicated that Dechloromonas was enriched and contributed to DPR while Zoogloea was responsible for endogenous denitrification. Denitrifying polyphosphate accumulating organisms and endogenous denitrifiers synergistically enhanced the nutrient removal capacity. The study further provides research perspectives for improving nutrient removal.

This study investigated the direct microbial reduction of perchlorate using domesticated activated sludge and showed the interaction effect of water quality factors on perchlorate bio-reduction. The dominant perchlorate-reducing bacteria belong to Dechloromonas sp., which was successfully acclimated from regular activated sludge after 20 days domestication. Batch reduction experiments indicated that high bio-reduction efficiency of perchlorate could be achieved at 303–313 K and pH = 6.5–7.5. Perchlorate-reducing bacteria can more efficiently utilize acetate and butanedioic to reduce perchlorate compared with the other electron donors that significantly affect the microbial community structure and dominant bacteria of activated sludge. The three-level and three-variable Box-Behnken Design response surface method predicted a maximum perchlorate reduction efficiency of 100% under the following optimal conditions: pH = 7.5, 308 K, and 0.51 mg/L of acetate. Furthermore, the influence of two factors on perchlorate reduction was in this order: interaction between pH and acetate > interaction between pH and temperature > interaction between temperature and acetate.

Chloroform (CF) can undergo reductive dechlorination to dichloromethane, chloromethane, and methane. However, competition for hydrogen (H2 ), the electron-donor substrate, may cause poor dechlorination when multiple electron acceptors are present. Common acceptors in anaerobic environments are nitrate (NO3 - ), sulfate (SO4 2- ), and bicarbonate (HCO3 - ). We evaluated CF dechlorination in the presence of HCO3 - at 1.56 e- Eq/m2 -day, then NO3 - at 0.04-0.15 e- Eq/m2 -day, and finally NO3 - (0.04 e- Eq/m2 -day) along with SO4 2- at 0.33 e- Eq/m2 -day in an H2 -based membrane biofilm reactor (MBfR). When the biofilm was initiated with CF-dechlorination conditions (no NO3 - or SO4 2- ), it yielded a CF flux of 0.14 e- Eq/m2 -day and acetate production via homoacetogenesis up to 0.26 e- eq/m2 -day. Subsequent addition of NO3 - at 0.05 e- Eq/m2 -day maintained full CF dechlorination and homoacetogenesis, but NO3 - input at 0.15 e- Eq/m2 -day caused CF to remain in the reactor's effluent and led to negligible acetate production. The addition of SO4 2- did not affect CF reduction, but SO4 2- reduction significantly altered the microbial community by introducing sulfate-reducing Desulfovibrio and more sulfur-oxidizing Arcobacter. Dechloromonas appeared to carry out CF dechlorination and denitrification, whereas Acetobacterium (homoacetogen) may have been involved with hydrolytic dechlorination. Modifications to the electron acceptors fed to the MBfR caused the microbial community to undergo changes in structure that reflected changes in the removal fluxes.

Electrostimulated hydrolysis acidification (eHA) has been used as an efficient biological pre-treatment of refractory industrial wastewater. However, the effects of electrostimulation on the function and assembly of planktonic anaerobic sludge microbial communities are poorly understood. Using 16S rRNA gene and metagenomic sequencing, we investigated planktonic sludge microbial community structure, composition, function, assembly, and microbial interactions in response to electrostimulation. Compared with a conventional hydrolysis acidification (HA) reactor, the planktonic sludge microbial communities selected by electrostimulation promoted biotransformation of the azo dye Alizarin Yellow R. The taxonomic and functional structure and composition were significantly shifted upon electrostimulation with azo dyes degraders (e.g. Acinetobacter and Dechloromonas) and electroactive bacteria (e.g. Pseudomonas) being enriched. More microbial interactions between fermenters and decolorizing and electroactive bacteria, as well as fewer interactions between different fermenters evolved in the eHA microbial communities. Moreover, the decolorizing bacteria were linked to the higher abundance of genes encoding for azo- and nitro-reductases and redox mediator (e.g. ubiquinone) biosynthesis involved in the transformation of azo dye. Microbial community assembly was more driven by deterministic processes upon electrostimulation. This study offers new insights into the effects of electrostimulation on planktonic sludge microbial community function and assembly, and provides a promising strategy for the manipulation of anaerobic sludge microbiomes in HA engineering systems.

Xenobiotics are generally known as man-made refractory organic pollutants widely distributed in various environments. For exploring the bioremediation possibility of xenobiotics, two novel xenobiotics-degrading strains affiliated with Azonexaceae were isolated. We report here the phylogenetics, genome, and geo-distribution of a novel and ubiquitous Azonexaceae species that primarily joins in the cometabolic process of some xenobiotics in natural communities. Strains s22 and t15 could be proposed as a novel species within Dechloromonas based on genomic and multi-phylogenetic analysis. Pan-genome analysis showed that the 63 core genes in Dechloromonas include genes for dozens of metabolisms such as nitrogen fixation protein (nifU), nitrogen regulatory protein (glnK), dCTP deaminase, C4-dicarboxylate transporter, and fructose-bisphosphate aldolase. Strains s22 and t15 have the ability to metabolize nitrogen, including nitrogen fixation, NirS-dependent denitrification, and dissimilatory nitrate reduction. Moreover, the novel species possesses the EnvZ-OmpR two-component system for controlling osmotic stress and QseC-QseB system for quorum sensing to rapidly sense environmental changes. It is intriguing that this new species has a series of genes for the biodegradation of some xenobiotics such as azathioprine, 6-Mercaptopurine, trinitrotoluene, chloroalkane, and chloroalkene. Specifically, glutathione S-transferase (GST) and 4-oxalocrotonate tautomerase (praC) in this novel species play important roles in the detoxification metabolism of some xenobiotics like dioxin, trichloroethene, chloroacetyl chloride, benzo[a]pyrene, and aflatoxin B1. Using data from GenBank, DDBJ and EMBL databases, we also demonstrated that members of this novel species were found globally in plants (e.g. rice), guts (e.g. insect), pristine and contaminated regions. Given these data, Dechloromonas sp. strains s22 and t15 take part in the biodegradation of some xenobiotics through key enzymes.

Ecological purification in a reservoir is an important strategy to control the level of nutrients in water. The bacterial community of such a reservoir is the main agent for pollutant degradation, which has not been fully documented. Taking the Jinze Reservoir, a freshwater source for Shanghai, China as the case, its spatial distributions of water and sediment bacteria were determined using 16S rRNA gene-based Illumina MiSeq sequencing, and the environmental parameters were analyzed. The reservoir takes natural river water and functions as an ecological purification system, consisting of three functional zones, i.e., pretreatment zone, ecological purification zone, and ecological sustaining zone. Results show that the concentrations of both total nitrogen (TN) and total phosphorus (TP) decreased considerably after the ecological treatment, and the concentration of dissolved oxygen (DO) in the ecological purification zone was boosted from that before pretreatment. In addition, patterns of bacterial communities in both water and sediment were similar and consisted of mainly Proteobacteria, Actinobacteria, and Bacteroidetes. However, difference in water bacterial composition was distinct in each functional zone, whereas the bacterial communities in sediment changed only slightly among sediment samples. Network analysis revealed nonrandom co-occurrence patterns of bacterial community composition in water and sediment, and Pseudomonas, unclassified Comamonadaceae, Variovorax, and Dechloromonas were the key taxa in the co-occurrence network. The bacterial taxa from the same module of the network had strong ecological connections, participated in C-cycles, and shared common trophic properties. PICRUSt analysis showed that bacteria were involved potentially in various essential processes; and the abundance of predicted xenobiotic biodegradation genes showed a decreasing trend in water samples from the inlet to the outlet of the reservoir. These results improve our current knowledge of the spatial distribution of bacteria in water and sediment in ecological purification reservoirs.

A double-chamber microbial fuel cell (MFC) with Ru/Fe-modified-biocathode was constructed for simultaneous mineralization of 2-anilinophenylacetate (APA) and denitrification. The factors on performance of simultaneous APA degradation and denitrification were explored. The contributions of ROS to APA degradation were evaluated by EPR and quenching experiments. The microbial community of Ru/Fe-modified-biocathode was determined by high-throughput sequencing. Results showed that low resistance accelerated APA degradation by Ru/Fe-modified-biocathode, while higher initial APA concentration inhibited microbial activity of the biocathode. The optimum ammonia concentration was 50 mg L−1, while too high or too low ammonia concentration did not favor APA degradation. The APA degradation efficiency of Ru/Fe-modified-biocathode-MFC was higher than that of other modified-cathode-MFCs. The APA degradation process confirmed to the pseudo-first-order kinetic model, and APA degradation kinetic constant, the maximum removal efficiency of TOC, ammonia and TN were 2.15d−1, 59.70%, 99.20% and 44.56% respectively, signifying a simultaneous APA mineralization and denitrification performance of Ru/Fe-modified-biocathode-MFC. The coulombic efficiency decreased with APA concentration increase. OH was the primary radical in APA degradation progress. Eight kinds of intermediates were measured, and two APA degradation pathways were proposed, among which APA hydroxylation was the main pathway. The microbial community of Ru/Fe-modified-biocathode was dominated with Nitrosomonas at genus level, and enriched with various APA-degraders, nitrifiers, and denitrifiers such as Pseudomonas, Nitrospira, Nitrobacter, Paracoccus, Thermomonas, Dechloromonas, and Clostridium_Sutra_stricto_1. COG analysis showed the redox reaction of Ru/Fe might affect signal transduction and environment adaptation, while FAPROTAX analysis suggested that Ru/Fe-modified-biocathode exhibited higher nitrification activity than that of carbon-felt-biocathode. The synergistic mechanism of simultaneous APA mineralization and denitrification was mainly redox reaction of Ru/Fe and supplemented by aerobic biodegradation.

Background Denitrifying phosphorus removal sludge (DPRS) is widely adopted for nitrogen and phosphorus removal in wastewater treatment but faces threats from heavy metals. However, a lack of understanding of the taxon-specific heavy metal-resistance mechanisms hinders the targeted optimization of DPRS’s robustness in nutrient removal. Results We obtained 403 high- or medium-quality metagenome-assembled genomes from DPRS treated by elevating cadmium, nickel, and chromium pressure. Then, the proteomic responses of individual taxa under heavy metal pressures were characterized, with an emphasis on functions involving heavy metal resistance and maintenance of nutrient metabolism. When oxygen availability was constrained by high-concentration heavy metals, comammox Nitrospira overproduced highly oxygen-affinitive hemoglobin and electron-transporting cytochrome c-like proteins, underpinning its ability to enhance oxygen acquisition and utilization. In contrast, Nitrosomonas overexpressed ammonia monooxygenase and nitrite reductase to facilitate the partial nitrification and denitrification process for maintaining nitrogen removal. Comparisons between phosphorus-accumulating organisms (PAOs) demonstrated different heavy metal-resistance mechanisms adopted by Dechloromonas and Candidatus Accumulibacter, despite their high genomic similarities. In particular, Dechloromonas outcompeted the canonical PAO Candidatus Accumulibacter in synthesizing polyphosphate, a potential public good for heavy metal detoxification. The superiority of Dechloromonas in energy utilization, radical elimination, and damaged cell component repair also contributed to its dominance under heavy metal pressures. Moreover, the enrichment analysis revealed that functions involved in extracellular polymeric substance formation, siderophore activity, and heavy metal efflux were significantly overexpressed due to the related activities of specific taxa. Conclusions Our study demonstrates that heavy metal-resistance mechanisms within a multipartite community are highly heterogeneous between different taxa. These findings provide a fundamental understanding of how the heterogeneity of individual microorganisms contributes to the metabolic versatility and robustness of microbiomes inhabiting dynamic environments, which is vital for manipulating the adaptation of microbial assemblages under adverse environmental stimuli.

Selenate (SeO4 2- ) reduction in hydrogen (H2 )-fed membrane biofilm reactors (H2 -MBfRs) was studied in combinations with other common electron acceptors. We employed H2 -MBfRs with two distinctly different conditions: R1, with ample electron-donor availability and acceptors SeO4 2- and sulfate (SO4 2- ), and R2, with electron-donor limitation and the presence of electron acceptors SeO4 2- , nitrate (NO3 - ), and SO4 2- . Even though H2 was available to reduce all input SeO4 2- and SO4 2- in R1, SeO4 2- reduction was preferred over SO4 2- reduction. In R2, co-reduction of NO3 - and SeO4 2- occurred, and SO4 2- reduction was mostly suppressed. Biofilms in all MBfRs had high microbial diversity that was influenced by the "rare biosphere" (RB), phylotypes with relative abundance less than 1%. While all MBfR biofilms had abundant members, such as Dechloromonas and Methyloversatilis, the bacterial communities were significantly different between R1 and R2. For R1, abundant genera were Methyloversatilis, Melioribacter, and Propionivibrio; for R2, abundant genera were Dechloromonas, Hydrogenophaga, Cystobacter, Methyloversatilis, and Thauera. Although changes in electron-acceptor or -donor loading altered the phylogenetic structure of the microbial communities, the biofilm communities were resilient in terms of SeO4 2- and NO3 - reductions, because interacting members of the RB had capacity of respiring these electron acceptors. This article is protected by copyright. All rights reserved.

Denitrification is an important part of the nitrogen cycle and the key step to removal of nitrogen in surface-flow wetlands. In this study, we explored space–time analysis with high-throughput sequencing to elucidate the relationships between denitrifying bacteria community structures and environmental factors during different seasons. Our results showed that along the flow direction of different processing units, there were dynamic changes in physical and chemical indicators. The bacterial abundance indexes (ACEs) in May, August, and October were 686.8, 686.8, and 996.2, respectively, whereas the Shannon-Weiner indexes were 3.718, 4.303, and 4.432, respectively. Along the flow direction, the denitrifying bacterial abundance initially increased and then decreased subsequently during the same months, although diversity tended to increase. The abundance showed similar changes during the different months. Surface flow wetlands mainly contained the following denitrifying bacteria genus: unclassified Bacteria (37.12%), unclassified Proteobacteria (18.16%), Dechloromonas (16.21%), unranked environmental samples (12.51%), unclassified Betaproteobacteria (9.73%), unclassified Rhodocyclaceae (2.14%), and Rhodanobacter (1.51%). During different seasons, the same unit showed alternating changes, and during the same season, bacterial community structures were influenced by the second genus proportion in different processing units. ACEs were strongly correlated with temperature, dissolved oxygen, and pH. Bacterial diversity was strongly correlated with temperature, electrical conductivity, pH, and oxidation reduction potential. Denitrifying bacteria are greatly affected by environmental factors such as temperature and pH.