Metabolic coupling between soil aerobic methanotrophs and denitrifiers in rice paddy fields.

Paddy fields are hotspots of microbial denitrification, which is typically linked to the oxidation of electron donors such as methane (CH4) under anoxic and hypoxic conditions. While several anaerobic methanotrophs can facilitate denitrification intracellularly, whether and how aerobic CH4 oxidation couples with denitrification in hypoxic paddy fields remains virtually unknown. Here we combine a ~3300 km field study across main rice-producing areas of China and 13CH4-DNA-stable isotope probing (SIP) experiments to investigate the role of soil aerobic CH4 oxidation in supporting denitrification. Our results reveal positive relationships between CH4 oxidation and denitrification activities and genes across various climatic regions. Microcosm experiments confirm that CH4 and methanotroph addition promote gene expression involved in denitrification and increase nitrous oxide emissions. Moreover, 13CH4-DNA-SIP analyses identify over 70 phylotypes harboring genes associated with denitrification and assimilating 13C, which are mostly belonged to Rubrivivax, Magnetospirillum, and Bradyrhizobium. Combined analyses of 13C-metagenome-assembled genomes and 13C-metabolomics highlight the importance of intermediates such as acetate, propionate and lactate, released during aerobic CH4 oxidation, for the coupling of CH4 oxidation with denitrification. Our work identifies key microbial taxa and pathways driving coupled aerobic CH4 oxidation and denitrification, with important implications for nitrogen management and greenhouse gas regulation in agroecosystems.

Algal-bacterial shortcut nitrogen removal model with seasonal light variations.

The algal-bacterial shortcut nitrogen removal (ABSNR) process can be used to treat high ammonia strength wastewaters without external aeration. However, prior algal-bacterial SNR studies have been conducted under fixed light/dark periods that were not representative of natural light conditions. In this study, laboratory-scale photo-sequencing batch reactors (PSBRs) were used to treat anaerobic digester sidestream under varying light intensities that mimicked summer and winter conditions in Tampa, FL, USA. A dynamic mathematical model was developed for the ABSNR process, which was calibrated and validated using data sets from the laboratory PSBRs. The model elucidated the dynamics of algal and bacterial biomass growth under natural illumination conditions as well as transformation processes for nitrogen species, oxygen, organic and inorganic carbon. A full-scale PSBR with a 1.2 m depth, a 6-day hydraulic retention time (HRT) and a 10-day solids retention time (SRT) was simulated for treatment of anaerobic digester sidestream. The full-scale PSBR could achieve >90% ammonia removal, significantly reducing the nitrogen load to the mainstream wastewater treatment plant (WWTP). The dynamic simulation showed that ABSNR process can help wastewater treatment facilities meet stringent nitrogen removal standards with low energy inputs.

Nitrate increases the capacity of an aerobic moving-bed biofilm reactor (MBBR) for winery wastewater treatment.

We used bench-scale tests and mathematical modeling to explore chemical oxygen demand (COD) removal rates in a moving-bed biofilm reactor (MBBR) for winery wastewater treatment, using either urea or nitrate as a nitrogen source. With urea addition, the COD removal fluxes ranged from 34 to 45 gCOD/m2-d. However, when nitrate was added, fluxes increased up to 65 gCOD/m2-d, twice the amount reported for aerobic biofilms for winery wastewater treatment. A one-dimensional biofilm model, calibrated with data from respirometric tests, accurately captured the experimental results. Both experimental and modelling results suggest that nitrate significantly increased MBBR capacity by stimulating COD oxidation in the deeper, oxygen-limited regions of the biofilm. Our research suggests that the addition of nitrate, or other energetic and broadly used electron acceptors, may provide a cost-effective means of covering peak COD loads in biofilm processes for winery or another industrial wastewater treatment.

Effect of floating treatment wetland coverage ratio and operating parameters on nitrogen removal: toward design optimization.

Floating treatment wetlands (FTWs) have the potential to improve the quality of wastewater discharges, yet design basics are unavailable to size these systems. This study investigates the effect of FTWs' coverage ratio and hydraulic retention time on agri-food wastewater treatment. This was studied in a pilot-scale experiment comprising four lagoons (6.5 m3 each) fed with real effluent from an existing tertiary treatment lagoon. An evaluation of FTW of different sizes (L24, L48, and L72 representing 24, 48, and 72% of pilot lagoons surface areas) and a control, L0 (without FTW), was performed over 16 months. Overall, L72 and L48 moderately improved total nitrogen (TN) mass removal compared to L0 (p < 0.05), while L24 exhibited similar TN mass removal (p = 0.196). The highest improvement was observed for L72, exhibiting up to 55% (mean of 13%) greater N mass removal than the control. The net increase in TN removal by FTWs was mainly related to denitrification, promoted by decreasing dissolved oxygen for increasing FTW coverage ratio. Residence time, temperature, and dissolved oxygen were the main parameters driving TN removal by FTWs. Retrofitting existing lagoons with FTW can facilitate N retrieval through plant harvesting, thereby reducing N remobilization from sediment (common in conventional lagoons).

Optimizing start-up strategies for the two-inflow nitritation/anammox process: Influence on biofilm microbial community composition.

Low-energy nitrogen removal from ammonium-rich wastewater is crucial in preserving the water environment. A one-stage nitritation/anammox process with two inflows treating ammonium-containing wastewater, supplied from inside and outside the wound filter, is expected to stably remove nitrogen. Laboratory-scale reactors were operated using different start-up strategies; the first involved adding nitritation inoculum after anammox biomass formation in the filter, which presented a relatively low nitrogen removal rate (0.171 kg N/m3 · d), at a nitrogen loading rate of 1.0 kg N/m3 · d. Conversely, the second involved the gradual cultivation of anammox and nitritation microorganisms, which increased the nitrogen removal rate (0.276 kg N/m3 · d). Furthermore, anammox (Candidatus Brocadia) and nitritation bacteria (Nitrosomonadaceae) coexisted in the biofilm formed on the filter surface. The abundance of nitritation bacteria (10.5%) in the reactor biofilm using the second start-up strategy was higher than that using the first (3.7%). Thus, the two-inflow nitritation/anammox process effectively induced habitat segregation using a suitable start-up strategy.

Comparing methanol and glycerol as carbon sources for mainstream partial denitrification/anammox in an IFAS process.

This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

Rapid start-up and operational characteristics of partial denitrification coupled with anammox driven by innovative strategies.

The Partial Denitrification-Anammox (PD/A) process established a low-consumption, efficient and sustainable pathway for complete nitrogen removal, which is of great interest to the industry. Rapid initiation and stable operation of the PD/A systems were the main issues limiting its engineering application in wastewater nitrogen removal. A PD/A system was initiated in a continuous stirred-tank reactors (CSTRs) in the presence of low concentration of organic matter, and the effects of organic matter types and COD/NO3--N ratios on the performance of the PD/A system, and microbial community characteristics were explored. The results showed that low concentrations of organic matter could promote the rapid initiation of the Anammox process and then the strategy of gradually replacing NO2--N with NO3--N could successfully initiate the PD/A system at 70 days. The type of organic matter had a significant effect on the initiation of the Anammox and the establishment of the PD/A system. Compared to glucose, sodium acetate was more favorable for rapid start-up and the synergy among microorganisms, and organic matter was lower, with an optimal COD/NO3--N ratio of 3.0. Microorganisms differed in their sensitivity to environmental factors. The relative abundance of Planctomycetota and Proteobacteria in R2 was 51 %, with the presence of three typical anammox bacteria, Candidatus_Brocadia, Candidatus_Kuenenia, and Candidatus_Jettenia in the system. This study provides a new strategy for the rapid initiation and stable operation of the PD/A process.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Nitrate denitrification rate response to temperature gradient change during river bank infiltration.

Nitrate attenuation during river bank infiltration is the key process for reducing nitrogen pollution. Temperature is considered to be an important factor affecting nitrate attenuation. However, the magnitude and mechanism of its impact have not been clear for a long time. In this study, the effects of temperature and temperature gradient on the nitrate denitrification rate were investigated via static batch and dynamic soil column simulation experiments. The results showed that temperature had a significant effect on the denitrification rate. Temperature effects were first observed in denitrifying bacteria. At low temperatures, the microorganism diversity was low, resulting in a lower denitrification rate constant. The static experimental results showed that the denitrification rate at 19 °C was approximately 2.4 times that at 10 °C. The dynamic soil column experiment established an exponential positive correlation between the nitrate denitrification decay kinetic constant and temperature. The affinity of denitrifying enzymes for nitrate in the reaction substrate was ordered as follows: decreasing temperature gradient (30 °C → 10 °C) > zero temperature gradient (10 °C) > increasing temperature gradient condition (0 °C → 10 °C). This study provides a theoretical basis for the biogeochemical processes underlying river bank infiltration, which will help aid in the development and utilization of groundwater resources.

Enhancement of electron transfer via magnetite in nitrite-dependent anaerobic methane oxidation system.

Nitrite-dependent anaerobic methane oxidation (n-DAMO) is a novel denitrification process that simultaneously further removes and utilizes methane from anaerobic effluent from wastewater treatment plants. However, the metabolic activity of n-DAMO bacteria is relative low for practical application. In this study, conductive magnetite was added into lab-scale sequencing batch reactor inoculated with n-DAMO bacteria to study the influence on n-DAMO process. With magnetite amendment, the nitrogen removal rate could reach 34.9 mg N·L-1d-1, nearly 2.5 times more than that of control group. Magnetite significantly facilitated the interspecies electron transfer and built electrically connected community with high capacitance. Enzymatic activities of electron transport chain were significantly elevated. Functional gene expression and enzyme activities associated with nitrogen and methane metabolism had been highly up-regulated. These results not only propose a useful strategy in n-DAMO application but also provide insights into the stimulating mechanism of magnetite in n-DAMO process.

CH4 control and nitrogen removal from constructed wetlands by plant combination.

Global warming trend is accelerating. This study proposes a green and economical methane (CH4) control strategy by plant combination in constructed wetlands (CWs). In this study, a single planting of Acorus calamus L. hybrid constructed wetland (HCW-A) and a mixed planting of Acorus calamus L. and Eichhornia crassipes (Mart.) Solms hybrid constructed wetland (HCW-EA) were constructed. The differences in nitrogen removal performance and CH4 emissions between HCW-A and HCW-EA were compared and analyzed. The findings indicated that HCW-EA demonstrated significant improvements over HCW-A, with NH4+-N and TN removal rates increasing by 21.61% and 16.38% respectively, and CH4 emissions decreased by 43.36%. The microbiological analysis results showed that plant combination promoted the enrichment of Proteobacteria, Alphaproteobacteria and Bacillus. More nitrifying bacteria carrying nxrA genes and denitrifying bacteria carrying nirK genes accelerated the nitrogen transformation process. In addition, the absolute abundance ratio of pmoA/mcrA increased, reducing the release of CH4.

Unveiling Microbial Nitrogen Metabolism in Rivers using a Machine Learning Approach.

Microbial nitrogen metabolism is a complicated and key process in mediating environmental pollution and greenhouse gas emissions in rivers. However, the interactive drivers of microbial nitrogen metabolism in rivers have not been identified. Here, we analyze the microbial nitrogen metabolism patterns in 105 rivers in China driven by 26 environmental and socioeconomic factors using an interpretable causal machine learning (ICML) framework. ICML better recognizes the complex relationships between factors and microbial nitrogen metabolism than traditional linear regression models. Furthermore, tipping points and concentration windows were proposed to precisely regulate microbial nitrogen metabolism. For example, concentrations of dissolved organic carbon (DOC) below tipping points of 6.2 and 4.2 mg/L easily reduce bacterial denitrification and nitrification, respectively. The concentration windows for NO3--N (15.9-18.0 mg/L) and DOC (9.1-10.8 mg/L) enabled the highest abundance of denitrifying bacteria on a national scale. The integration of ICML models and field data clarifies the important drivers of microbial nitrogen metabolism, supporting the precise regulation of nitrogen pollution and river ecological management.

Diversity, community structure, and abundance of nirS-type denitrifying bacteria on suspended particulate matter in coastal high-altitude aquaculture pond water.

Denitrifying bacteria harboring the nitrate reductase S (nirS) gene convert active nitrogen into molecular nitrogen, and alleviate eutrophication in aquaculture water. Suspended particulate matter (SPM) is an important component of aquaculture water and a carrier for denitrification. SPM with different particle sizes were collected from a coastal high-altitude aquaculture pond in Maoming City, China. Diversity, community structure, abundance of nirS-type denitrifying bacteria on SPM and environmental influencing factors were studied using high-throughput sequencing, fluorescence quantitative PCR, and statistical analysis. Pseudomonas, Halomonas, and Wenzhouxiangella were the dominant genera of nirS-type denitrifying bacteria on SPM from the ponds. Network analysis revealed Pseudomonas and Halomonas as the key genera involved in the interaction of nirS-type denitrifying bacteria on SPM in the ponds. qPCR indicated a trend toward greater nirS gene abundance in progressively larger SPM. Dissolved oxygen, pH, temperature, and SPM particle size were the main environmental factors influencing changes in the nirS-type denitrifying bacterial community on SPM in coastal high-altitude aquaculture pond water. These findings increase our understanding of the microbiology of nitrogen cycle processes in aquaculture ecosystem, and will help optimize aquatic tailwater treatment strategies.

Thauera sp. for efficient nitrate removal in continuous denitrifying moving bed biofilm reactor.

Thauera is the most widely found dominant denitrifying genus in wastewater. In earlier study, MBBR augmented with a specially developed denitrifying five-membered bacterial consortium (DC5) where Thauera was found to be the most abundant and persistent genus. Therefore, to check the functional potential of Thauera in the removal of nitrate-containing wastewater in the present study Thauera sp.V14 one of the member of the consortium DC5 was used as the model organism. Thauera sp.V14 exhibited strong hydrophobicity, auto-aggregation ability, biofilm formation and denitrification ability, which indicated its robust adaptability short colonization and nitrate removal efficiency. Continuous reactor studies with Thauera sp.V14 in 10 L dMBBR showed 91% of denitrification efficiency with an initial nitrate concentration of 620 mg L-1 within 3 h of HRT. Thus, it revealed that Thauera can be employed as an effective microorganism for nitrate removal from wastewater based on its performance in the present studies.

The effect of reflux ratio on sulfur disproportionation tendency in anaerobic baffled reactor with the heterotrophic combining sulfur autotrophic processes under high concentration perchlorate stress.

In a laboratory scale, an anaerobic baffled reactor (ABR) consisting of eight compartments, the heterotrophic combining sulfur autotrophic processes under different reflux ratios were constructed to achieve effective perchlorate removal and alleviate sulfur disproportionation reaction. Perchlorate was efficiently removed with effluent perchlorate concentration below 0.5 µg/L when the influent perchlorate concentration was 1030 mg/L during stages I ~ V, indicating that heterotrophic combining sulfur autotrophic perchlorate reduction processes can effectively achieve high concentration perchlorate removal. Furthermore, the 100% reflux ratio could reduce the contact time between sulfur particles and water; thus, the sulfur disproportionation reaction was inhibited. However, the inhibition effect of reflux on sulfur disproportionation was attenuated due to dilute perchlorate concentration when a reflux ratio of 150% and 200% was implemented. Meanwhile, the content of extracellular polymeric substances (EPS) in the heterotrophic unit (36.79 ~ 45.71 mg/g VSS) was higher than that in the sulfur autotrophic unit (22.19 ~ 25.77 mg/g VSS), indicating that high concentration perchlorate stress in the heterotrophic unit promoted EPS secretion. Thereinto, the PN content of sulfur autotrophic unit decreased in stage III and stage V due to decreasing perchlorate concentration in the autotrophic unit. Meanwhile, the PS content increased with increasing reflux in the autotrophic unit, which was conducive to the formation of biofilm. Furthermore, the high-throughput sequencing result showed that Proteobacteria, Chloroflexi, Firmicutes, and Bacteroidetes were the dominant phyla and Longilinea, Diaphorobacter, Acinetobacter, and Nitrobacter were the dominant genus in ABR, which were associated with heterotrophic or autotrophic perchlorate reduction and beneficial for effective perchlorate removal. The study indicated that reflux was a reasonable strategy for alleviating sulfur disproportionation in heterotrophic combining sulfur autotrophic perchlorate removal processes.

Effect of salinity on biological nitrogen removal from wastewater and its mechanism.

The nitrogen discharge from saline wastewater will cause significant pollution to the environment. As a high-efficiency and low-cost treatment method, biological treatment has a promising application prospect in the removal of nitrogen from high-salt wastewater. However, the inhibitory effect of high salt on microorganisms increases the difficulty of its treatment. This review discusses the influence of salinity on the nitrogen removal process, considering both traditional and novel biological techniques. Common methods to enhance the effectiveness of biological nitrogen removal processes and their mechanisms of action in engineering practice and research, including sludge acclimation and inoculation of halophilic bacteria, are also introduced. An outlook on the future development of biological nitrogen removal processes for high-salt wastewater is provided to achieve environmentally friendly discharge of high-salt wastewater.

Effect of ciprofloxacin on the one-stage partial nitrification and anammox biofilm system: A multivariate analysis focusing on size-fractionated organic components.

The impact of ciprofloxacin (CIP) in the partial nitrification and anammox biofilm system was investigated by multivariate analysis, focusing on size-fractionated organic components. The CIP dose of 10 µg/L did not inhibit the total nitrogen (TN) removal efficiency, even though the abundance of antibiotic resistant genes (ARGs) (i.e., qnrD, qnrB, qnrA, qnrS, and arcA) was elevated. However, a gradual higher CIP dosing up to 100 µg/L inhibited the TN removal efficiency, while the abundance of ARGs was still increased. Moreover, both the TN removal efficiency and the abundant ARGs were dwindled at 470 µg/L of CIP. As the CIP dose increased from 0 to 100 µg/L, the abundance of high molecular weight (MW) fractions (14,000 to 87,000 Da; 1000 to 14,000 Da) and humic/fulvic acid-like components in the soluble extracellular polymeric substances (HSS) decreased, with more increases of low MW (84-1000 Da; less than 84 Da) fractions and soluble microbial by-products in soluble extracellular polymeric substances (SMPS). Continuously increasing the CIP dose till 470 µg/L, an inverse trend of the changes of these organic components was noted, along with clear reductions of the microbial diversity and richness, and the abundance of key functional genes responsible for nitrogen removal. The predominance of functional gene amoA (related with ammonia oxidizing bacteria) was more significantly with more distribution of SMPS with relatively low MW and less distribution of HSS with relatively high MW, as well as polymer decomposing microorganisms such as Bryobacteraceae and the unclassified Saprospirales.

Effect of sludge redistribution strategy on stability of partial nitrification-anammox process: Further exploration of the potential value of sludge.

The stability of the two-stage partial nitrification-anammox (PN/A) system was compromised by the inappropriate conversion of insoluble organic matter. In response, a sludge redistribution strategy was implemented. Through the redistribution of PN sludge and anammox sludge in the two-stage PN/A system, a transition was made to the Anammox-single stage PN/A (A-PN/A) system. This specific functional reorganization, facilitated by the rapid reorganization of microbial communities, has the potential to significantly decrease the current risk of suppression. The results of the study showed that implementing the sludge redistribution strategy led to a substantial enhancement in the total nitrogen removal rate (TNRR) by 87.51%, accompanied by a significant improvement of 34.78% in the chemical oxygen demand removal rate (CRR). Additionally, this approach resulted in a remarkable two-thirds reduction in the aeration requirements. High-throughput sequencing revealed that the strategy enriched anammox and ammonia-oxidizing bacteria while limiting denitrifying bacteria, as confirmed by quantitative polymerase chain reaction analysis. Furthermore, the principal component analysis revealed that the location and duration of aeration had direct and indirect effects on functional gene expression and the evolution of microbial communities. This study emphasizes the potential benefits of restructuring microbial communities through a sludge redistribution strategy, especially in integrated systems that encounter challenges with suppression.

Anammox process in anaerobic baffled biofilm reactors with columnar packings: Characteristics of flow field and microbial community.

The enrichment of anammox bacteria is a key issue in the application of anammox processes. A new type of reactor － anaerobic baffle biofilm reactor (ABBR) developed from anaerobic baffle reactor (ABR) was filled with columnar packings and established for effective enrichment of anammox bacteria. The flow field analysis showed that, compared with ABR, ABBR narrowed the dead zone so as to improve the substrate transferring performances. Two ABBRs with different types of columnar packings (Packings 1 and Packings 2) were constructed to culture anammox biofilms. Packings 1 consisted of the single-form honeycomb carriers while Packings 2 was modular composite packings consisting of non-woven fabric and honeycomb carriers. The effects of different types of columnar packings on microbial community and nitrogen removal were studied. The ABBR filled with Packings 2 had a higher retention rate of biomass than the ABBR filled with Packings 1, making the anammox start-up period be shortened by 21.28%. The enrichment of anammox bacteria were achieved and the dominant anammox bacteria were Candidatus Brocadia in both R1 and R2. However, there were four genera of anammox bacteria in R2 and one genus of anammox bacteria in R1, and the cell density of anammox bacteria in R2 was 95% higher than that in R1. R2 has the advantage of maintaining excellent and stable nitrogen removal performance at high nitrogen loading rate. The results revealed that the packings composed of two types of carriers may have a better enrichment effect on anammox bacteria. This study is of great significance for the rapid enrichment of anammox bacteria and the technical promotion of anammox process.

Evaluation of ecological impacts with ferrous iron addition in constructed wetland under perfluorooctanoic acid stress.

In this study, ferrous ion (Fe(II)) had the potential to promote ecological functions in constructed wetlands (CWs) under perfluorooctanoic acid (PFOA) stress. Concretely, Fe(II) at 30 mg/L and 20-30 mg/L even led to 11.37% increase of urease and 93.15-243.61% increase of nitrite oxidoreductase respectively compared to the control. Fe(II) promotion was also observed on Nitrosomonas, Nitrospira, Azospira, and Zoogloea by 1.00-6.50 folds, which might result from higher expression of nitrogen fixation and nitrite redox genes. These findings could be explanation for increase of ammonium removal by 7.47-8.75% with Fe(II) addition, and reduction of nitrate accumulation with 30 mg/L Fe(II). Meanwhile, both Fe(II) stimulation on PAOs like Dechloromonas, Rhodococcus, Mesorhizobium, and Methylobacterium by 1.58-2.00 folds, and improvement on chemical phosphorus removal contributed to higher total phosphorus removal efficiency under high-level PFOA exposure. Moreover, Fe(II) raised chlorophyll content and reduced the oxidative damage brought by PFOA, especially at lower dosage. Nevertheless, combination of Fe(II) and high-level PFOA caused inhibition on microbial alpha diversity, which could result in decline of PFOA removal (by 4.29-12.83%). Besides, decrease of genes related to nitrate reduction demonstrated that enhancement on denitrification was due to nitrite reduction to N2 pathways rather than the first step of denitrifying process.