Intermittent polarization: A promising strategy for microbial electricity driven reduction of DOM toxicity in actual industrial wastewater.

Dissolved organic matter (DOM) in actual industrial wastewater comprises various compounds that trigger toxicity in aquatic organisms; thus, advanced treatment for reducing DOM toxicity is urgently needed to ensure safe effluent discharge. Herein, we successfully reduced the toxicity of DOM in actual industrial wastewater without external chemical addition by applying intermittent polarization to electrochemical bioreactors. The bioreactor operated under intermittent polarization effectively reduced the toxicity of DOM by 76.7 %, resulting in the toxicity of effluent DOM (determined by malformation rate of zebrafish larvae) reaching less than 3.5 %. Notably, DOM compounds with high double-bond equivalence (DBE ≥ 8) were identified as the key components responsible for the toxicity of DOM through ultrahigh-resolution mass spectrometry analysis. Insight into microbe-DOM interactions revealed that intermittent polarization promoted the microbial consumption of high-DBE components of DOM by both affecting microbial composition (ß = -0.5421, p < 0.01) and function (ß = -0.4831, p < 0.01), thus regulating effluent DOM toxicity. The study findings demonstrate that intermittent polarization is a promising strategy for microbial electricity-driven reduction of DOM toxicity in actual industrial wastewater to meet the increasing safety requirements of receiving waters.

Weak magnetic field promotes denitrification by stimulating ferromagnetic ion-containing metalloprotein expression.

Weak magnetic field (WMF) has been recognized to promote biological denitrification processes; however, the underlying mechanisms remain largely unexplored, hindering the optimization of its effectiveness. Here, we systematically investigated the effects of WMF on denitrification performance, enzyme activity, microbial community, and metaproteome in packed bed bioreactors treating high nitrate wastewater under different WMF intensities and C:N ratios. Results showed that WMFs significantly promoted denitrification by consistently stimulating the activities of denitrifying reductases and NAD+/NADH biosynthesis across decreasing C:N ratios. Reductases and electron transfer enzymes involved in denitrification were overproduced due to the significantly enriched overexpression of ferromagnetic ion-containing (FIC) metalloproteins. We also observed WMFs' intensity-dependent selective pressure on microbial community structures despite the effects being limited compared to those caused by changing C:N ratios. By coupling genome-centric metaproteomics and structure prediction, we found the dominant denitrifier, Halomonas, was outcompeted by Pseudomonas and Azoarcus under WMFs, likely due to its structural deficiencies in iron uptake, suggesting that advantageous ferromagnetic ion acquisition capacity was necessary to satisfy the substrate demand for FIC metalloprotein overproduction. This study advances our understanding of the biomagnetic effects in the context of complex communities and highlights WMF's potential for manipulating FIC protein-associated metabolism and fine-tuning community structure.

Simultaneous efficient removal of tetracycline and mitigation of antibiotic resistance genes enrichment by a modified activated sludge process with static magnetic field.

To address the increasing issue of antibiotic wastewater, this study applied a static magnetic field (SMF) to the activated sludge process to increase the efficiency of tetracycline (TC) removal from swine wastewater and to reveal its enhanced mechanisms. The results demonstrated that the SMF-modified activated sludge process could achieve almost complete TC removal at sludge loading rates of 0.3 mg TC/g MLSS/d. Analysis of zeta potential and extracellular polymeric substances composition of the activated sludge revealed that SMF increased electrostatic interactions between TC and activated sludge and made activated sludge has much more binding sites, finally resulting in the increased TC biosorption. Metagenomic analysis showed that SMF promoted the enrichment of ammonia-oxidizing bacteria, TC-degrading bacteria, and aromatic compounds-degrading bacteria; it also enhanced ammonia monooxygenase- and cytochrome P450-mediated TC metabolism while upregulating functional genes associated with oxidase, reductase, and dehydrogenase - all contributing to increased TC biodegradation. Additionally, SMF mitigated the enrichment and spread of antibiotic resistance genes (ARGs) by decreasing the abundance of potential hosts of ARGs and inhibiting the upregulation of genes encoding ABC transporters and putative transposase. Based on these findings, this study demonstrates that magnetic field is an enhancement strategy with great potential to relieve the harmful impacts of the growing antibiotic wastewater problem on human health and the ecosystem.

Microbial communities assembly in wastewater treatment plants in China.

Community assembly processes determine community structure. Deterministic processes are essential for optimizing activated sludge (AS) bioreactor performance. However, the debate regarding the relative importance of determinism versus stochasticity remains contentious, and the influencing factors are indistinct. This study used large-scale 16S rRNA gene data derived from 252 AS samples collected from 28 cities across China to explore the mechanism of AS community assembly. Results showed that the northern communities possessed lower spatial turnover and more significant dispersal limitation than those in the south, whereas the latter had more substantial deterministic processes than the former (14.46 % v.s. 9.12 %). Meanwhile, the communities in the south exhibited lower network complexity and stability. We utilized a structural equation model to explore the drivers of deterministic processes. Results revealed that low network complexity (r = -0.56, P < 0.05) and high quorum sensing bacteria abundance (r = 0.25, P < 0.001) promoted deterministic assembly, which clarifies why determinism was stronger in southern communities than northern ones. Furthermore, total phosphorus and hydraulic retention time were found to be the primary abiotic drivers. These findings provide evidence to understand the community deterministic assembly, which is crucial for resolving community structure and improving bioreactor performance.

Changes in characteristics and risk of freshwater microplastics under global warming.

Microplastics present a significant threat to freshwater ecosystems. However, the impact of global warming on their characteristics and associated risks remains uncertain. This study collected 2793 sample sites from literature and datasets to create a new risk assessment and rank methodology, known as the Multi-characteristics Potential Ecological Risk Index (MPERI), which incorporates various microplastic characteristics, such as concentration, size distribution, color, shape, and polymer diversity. Using regression random forest models (RRF), this study predicted that a 10 °C increase would raise microplastic concentration from 12,465.34 ± 68,603.87 to 13,387.17 ± 60,692.96 particles/m3. The percentage of small-size microplastics initially decreased (from 69.10 % to 68.72 %) and then increased (from 68.72 % to 68.78 %), while the diversity of color, shape, and polymer decreased by 0.29 %, 3.24 %, and 0.17 %, respectively. Furthermore, global warming could increase the rank of microplastic risks from high (405.25 ± 528.9) to dangerous (535.37 ± 582.03) based on the MPERI method. Most countries would experience an increase in risk values, with Indonesia and Vietnam transitioning from low to medium risk, and China and Malaysia transitioning from high to dangerous risk. The feature importance assessment of the RRF model indicated that concentration was the most influential variable in determining the change in risk values. While other microplastic characteristics had a lesser impact compared to concentration, they still influenced the risk ranking. This study highlights the role of global warming in shaping microplastic risks.

Degradation of conjugated estrogen in visible light-driven intimately coupled photocatalysis and biodegradation system.

Visible light-driven intimately coupled photocatalysis and biodegradation (VDICPB) is an efficient technology for removing recalcitrant contaminants, but the degradation pathway on 17ß-estradiol 3-Sulfate (E2-3S) is still not clear. In this study, VDICPB based on N-doped TiO2 as a photocatalyst was established to investigate the removal and transformation of E2-3S in synthetic wastewater. VDICPB showed a satisfactory removal efficiency of 97.8 ± 0.4 %, which was much higher than that of independent photocatalysis (84.0 ± 2.2 %) or biodegradation system (71.4 ± 1.8 %). Steroid C/D-rings of E2-3S was broken in VDICPB since the transformation process reached terminal central pathway. Primary metabolites did not accumulate in VDICPB, resulting in a low expression of functional genes. E2-3S was mainly removed by cooperative interaction of photocatalysis and co-metabolism of biofilm. Photocatalysis led to deconjugation and microbes acted to mineralization. This study provides technical reference and theoretical support for the removal of new pollutants.

The potential correlation between the succession of microflora and volatile flavor compounds during the production of Zhenba bacon.

Microbial composition plays an important role in the quality and flavor of bacon. The aims of this study were to detect bacterial community succession using high-throughput sequencing (HTS) and volatile flavor compound changes using gas chromatography-ion mobility spectrometry (GC-IMS) during the production of Zhenba bacon. The results showed that a total of 70 volatile compounds were detected. Among them, ketones, hydrocarbons, aldehydes, esters and alcohols were the main substances in the curing and smoking stages. In addition, the fungal abundance was greater than the bacterial abundance, and there was obvious succession of the microbial community with changes in fermentation time and processing technology. The main functional bacterial genera in the curing and smoking stages were Staphylococcus, Psychrobacter and Latilactobacillus, and the main fungal genera were Fusarium and Debaryomyces. Through correlation analysis, we found that pyrrole, 2-pentanol, methyl isobutyl ketone (MIBK) and ethyl acetate (EA) were significantly correlated with Staphylococcus, Psychrobacter, Pseudomonas and Myroides (p < 0.01), and it is speculated that they contribute significantly to flavor formation. The results of this study are helpful for understanding the microbial dynamics and characteristic volatile flavor compounds in Zhenba bacon, and provide new insights into the relationship between microorganisms and flavor through potential correlations.

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.

Soil Geobacteraceae are the key predictors of neurotoxic methylmercury bioaccumulation in rice.

Contamination of rice by the potent neurotoxin methylmercury (MeHg) originates from microbe-mediated Hg methylation in soils. However, the high diversity of Hg methylating microorganisms in soils hinders the prediction of MeHg formation and challenges the mitigation of MeHg bioaccumulation via regulating soil microbiomes. Here we explored the roles of various cropland microbial communities in MeHg formation in the potentials leading to MeHg accumulation in rice and reveal that Geobacteraceae are the key predictors of MeHg bioaccumulation in paddy soil systems. We characterized Hg methylating microorganisms from 67 cropland ecosystems across 3,600 latitudinal kilometres. The simulations of a rice-paddy biogeochemical model show that MeHg accumulation in rice is 1.3-1.7-fold more sensitive to changes in the relative abundance of Geobacteraceae compared to Hg input, which is recognized as the primary parameter in controlling MeHg exposure. These findings open up a window to predict MeHg formation and accumulation in human food webs, enabling more efficient mitigation of risks to human health through regulations of key soil microbiomes.

A data-driven analysis to discover research hotspots and trends of technologies for PFAS removal.

The frequent detection of persistent per- and polyfluoroalkyl substances (PFAS) in organisms and environment coupled with surging evidence for potential detrimental impacts, have attracted widespread attention throughout the world. In order to reveal research hotspots and trends of technologies for PFAS removal, herein, we performed a data-driven analysis of 3975 papers and 436 patents from Web of Science Core Collection and Derwent Innovation Index databases up to 2023. The results showed that China and the USA led the way in the research of PFAS removal with outstanding contributions to publications. The progression generally transitioned from accidental discovery of decomposition, to experimentation with removal effects and mechanisms of existing methods, and finally to enhanced defluorination and mechanism-driven design approaches. The keywords co-occurrence network and technology classification together revealed the main knowledge framework, which was constructed and correlated through contaminants, substrates, materials, processes and properties. Moreover, adsorption was demonstrated to be the dominant removal process among the current studies. Subsequently, we concluded the principles, advances and drawbacks of enrichment and separation, biological methods, advanced oxidation and reduction processes. Further exploration indicated the hotspots such as alternatives and precursors for PFAS ("genx": 1.258, "f-53b": 0.337), degradable mineralization technologies ("photocatalytic degrad": 0.529, "hydrated electron": 0.374), environment-friendly remediation technologies ("phytoremedi": 0.939, "constructed wetland": 0.462) and combination with novel materials ("metal-organic framework": 1.115, "layered double hydroxid": 0.559) as well as computer science ("molecular dynamics simul": 0.559, "machine learn"). Furthermore, the future direction of technological innovation might lie in high-performance processes that minimize secondary pollution, the development of recyclable and renewable treatment agents, and collaborative control strategies for multiple pollutants. Overall, this study offers comprehensive and objective review for researchers and industry professionals in this field, enabling rapid access to knowledge guidance and insights into research frontiers.

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.

Comparative evaluation of SPE methods for biotoxicity assessment of water and wastewater: Linkage between chemical extracting efficiency and biotoxicity outcome.

Biotoxicity assessment results of environmental waters largely depend on the sample extraction protocols that enrich pollutants to meet the effect-trigger thresholds of bioassays. However, more chemical mixture does not necessarily translate to higher combined biotoxicity. Thus, there is a need to establish the link between chemical extracting efficiency and biotoxicity outcome to standardize extraction methods for biotoxicity assessment of environmental waters. This study compares the performance of five different extraction phases in solid phase extraction (SPE), namely HLB, HLB+Coconut, C18 cartridge, C18 disk and Strata-X, and evaluated their chemical extracting efficiencies and biotoxicity outcomes. We quantitatively assessed cytotoxicity, acute toxicity, genotoxicity, estrogenic activity, and neurotoxicity of the extracts using in vitro bioassays and characterized the chemical extracting efficiencies of the SPE methods through chemical recoveries of 23 model compounds with different polarities and total organic carbon. Using Pareto ranking, we identified HLB+Coconut as the optimal SPE method, which exhibited the highest level of water sample biotoxicity and recovered the most chemicals in water samples. We found that the biotoxicity outcomes of the extracted water samples significantly and positively correlated with the chemical extracting efficiencies of the SPE methods. Moreover, we observed synchronous changing patterns in biotoxicity outcome and chemical extracting efficiencies in response to increasing sample volumes per cartridge (SVPC) during SPE. Our findings underscore that higher chemical extracting efficiency of SPE corresponds to higher biotoxicity outcome of environmental water samples, providing a scientific basis for standardization of SPE methods for adequate assessment of biotoxicities of environmental waters.

Inter-plasmid transfer of antibiotic resistance genes accelerates antibiotic resistance in bacterial pathogens.

Antimicrobial resistance is a major threat for public health. Plasmids play a critical role in the spread of antimicrobial resistance via horizontal gene transfer between bacterial species. However, it remains unclear how plasmids originally recruit and assemble various antibiotic resistance genes (ARGs). Here, we track ARG recruitment and assembly in clinically relevant plasmids by combining a systematic analysis of 2420 complete plasmid genomes and experimental validation. Results showed that ARG transfer across plasmids is prevalent, and 87% ARGs were observed to potentially transfer among various plasmids among 8229 plasmid-borne ARGs. Interestingly, recruitment and assembly of ARGs occur mostly among compatible plasmids within the same bacterial cell, with over 88% of ARG transfers occurring between compatible plasmids. Integron and insertion sequences drive the ongoing ARG acquisition by plasmids, especially in which IS26 facilitates 63.1% of ARG transfer events among plasmids. In vitro experiment validated the important role of IS26 involved in transferring gentamicin resistance gene aacC1 between compatible plasmids. Network analysis showed four beta-lactam genes (blaTEM-1, blaNDM-4, blaKPC-2, and blaSHV-1) shuffling among 1029 plasmids and 45 clinical pathogens, suggesting that clinically alarming ARGs transferred accelerate the propagation of antibiotic resistance in clinical pathogens. ARGs in plasmids are also able to transmit across clinical and environmental boundaries, in terms of the high-sequence similarities of plasmid-borne ARGs between clinical and environmental plasmids. This study demonstrated that inter-plasmid ARG transfer is a universal mechanism for plasmid to recruit various ARGs, thus advancing our understanding of the emergence of multidrug-resistant plasmids.

Scientometric analysis of electrocatalysis in wastewater treatment: today and tomorrow.

Electrocatalytic methods are valuable tools for addressing water pollution and scarcity, offering effective pollutant removal and resource recovery. To investigate the current status and future trends of electrocatalysis in wastewater treatment, a detailed analysis of 9417 papers and 4061 patents was conducted using scientometric methods. China emerged as the leading contributor to publications, and collaborations between China and the USA have emerged as the most frequent partnerships. Primary article co-citation clusters focused on oxygen evolution reaction and electrochemical oxidation, transitioning towards advanced oxidation processes ("persulfate activation"), and electrocatalytic reduction processes ("nitrate reduction"). Bifunctional catalysts, theoretical calculations, electrocatalytic combination technologies, and emerging contaminants were identified as current research hotspots. Patent analysis revealed seven types of electrochemical technologies, which were compared using SWOT analysis, highlighting electrochemical oxidation as prominent. The technological evolution presented the pathway of electro-Fenton to combined electrocatalytic technologies with biochemical processes, and finally to coupling with electrocoagulation. Standardized evaluation systems, waste resource utilization, and energy conservation were important directions of innovation in electrocatalytic technologies. Overall, this study provided a reference for researchers to understand the framework of electrocatalysis in wastewater treatment and also shed light on potential avenues for further innovation in the field.

Advanced electrolysis sulfur-based biofiltration for simultaneous total nitrogen removal and estrogen toxicity reduction from low carbon-to-nitrogen ratio wastewater.

A sulfur-based biofilter enhanced by phosphate modified activated carbon as particle electrodes was constructed to simultaneously remove total nitrogen (TN) and estrogen from low carbon-to-nitrogen ratio (C/N) wastewater containing 1 mg/L 17-alpha-ethinylestradiol (EE2). Results showed that the enhanced biofilter achieved outstanding performance in EE2 removal (93.2 %) and TN reduction (effluent < 5 mg/L), demonstrating robustness against C/N fluctuations. It was noteworthy that it successfully reduced both acute toxicity (59.5 %) and estrogenic activity (88.6 %). Comprehensive characterization investigations and microbial community structure analysis revealed that enhanced electron transfer and increased microbial abundance likely contributed to improved biofilter performance. Core microorganisms, such as Pseudomonas and Chryseobacterium were identified as key contributors to synergistic estrogen degradation and denitrification. This study presented a feasible and promising strategy of combined process with three-dimensional electrodes and sulfur-based biofilter, highlighting substantial potential for advanced purification and safe reuse of wastewater.

Electrocatalytic denitrification biofilter for advanced purification of chlorophenols via ceramsite-based Ti/SnO2-Sb particle electrode: Performance, microbial community structure and mechanism.

In response to the demand for advanced purification of industrial secondary effluent, a new method has been developed for treating chlorophenol wastewater using the novel ceramsite-based Ti/SnO2-Sb particle electrodes (Ti/SnO2-Sb/CB) enhanced electrocatalytic denitrification biofilter (EDNBF-P) to achieve removal of chlorophenols (CPs), denitrification, and reduction of effluent toxicity. The results showed that significantly improved CPs and TN removal efficiency at low COD/N compared to conventional denitrification biofilter, with CPs removal rates increasing by 0.33%-59.27% and TN removal rates increasing by 12.53%-38.92%. Under the conditions of HRT = 2h, 3V voltage, charging times = 12h, and 25 °C, the concentrations of the CPs in the effluent of EDNBF-P were all below 1 mg/L, the TN concentration was below 15 mg/L, while the effluent toxicity reached the low toxicity level. Additionally, the Ti/SnO2-Sb/CB particle electrodes effectively alleviated the accumulation of NO2--N caused by applied voltage. The Silanimonas, Pseudomonas and Rhodobacter was identified as the core microorganism for denitrification and toxicity reduction. This study validated that EDNBF-P could achieve synergistic treatment of CPs and TN through electrocatalysis and microbial degradation, providing a methodological support for achieving advanced purification of chlorophenol wastewater with low COD/N in industrial applications.

Methylmercury photodegradation in paddy water: An overlooked process mitigating methylmercury risks.

Photodegradation is critical to reduce the potent neurotoxic methylmercury (MeHg) in water and its subsequent accumulation along food chains. However, this process has been largely ignored in rice paddies, which are hotspots of MeHg production and receive about a quarter of the world's developed freshwater resources. Here, we reported that significant MeHg photodegradation, primarily mediated by hydroxyl radicals, occurs in the overlying water during rice growth. By incorporating field-measured light interception into a rice paddy biogeochemistry model, as well as photodegradation rates obtained from 42 paddy soils stretching ∼3500 km across China, we estimated that photodegradation reduced MeHg concentrations in paddy water and rice by 82 % and 11 %, respectively. Without photodegradation, paddy water could be a significant MeHg source for downstream ecosystems, with an annual export of 178 - 856 kg MeHg to downstream waters in China, the largest rice producer. These findings suggest that photodegradation in paddy water is critical for preventing greater quantities of MeHg entering human food webs.

Chemical characteristics of dissolved organic matter in effluent from sludge alkaline fermentation liquid-fed sequencing batch reactors.

Sludge alkaline fermentation liquid (SAFL) is a promising alternative to acetate for improving biological nitrogen removal (BNR) from wastewater. SAFL inevitably contains some refractory compounds, while the characteristics of dissolved organic matter (DOM) in effluent from SAFL-fed BNR process remain unclear. In this study, the molecular weight distribution, fluorescent composition and molecular profiles of DOM in effluent from SAFL and acetate-fed sequencing batch reactors (S-SBRs and A-SBRs, respectively) at different hydraulic retention time (12 h and 24 h) was comparatively investigated. Two carbon sources resulted in similar effluent TN, but a larger amount of DOM, which was bio-refractory or microorganisms-derived, was found in effluent of S-SBRs. Compared to acetate, SAFL increased the proportion of large molecular weight organics and humic-like substances in effluent DOM by 74.87%-101.3% and 37.52%-48.35%, respectively, suggesting their bio-refractory nature. Molecular profiles analysis revealed that effluent DOM of S-SBRs exhibited a more diverse composition and a higher proportion of lignin-like molecules. Microorganisms-derived molecules were found to be the dominant fraction (71.51%-72.70%) in effluent DOM (<800 Da) of S-SBRs. Additionally, a prolonged hydraulic retention time enriched Bacteroidota, Haliangium and unclassified_f_Comamonadaceae, which benefited the degradation of DOM in S-SBRs. The results help to develop strategies on reducing effluent DOM in SAFL-fed BNR process.

Carbon Source in Tertiary Denitrification Regulates Dissolved Organic Nitrogen in Wastewater Effluent.

With global eutrophication and increasingly stringent nitrogen discharge restrictions, dissolved organic nitrogen (DON) holds considerable potential to upgrade advanced wastewater denitrification because of its large contribution to low-nitrogen effluents and stronger stimulation effect for algae. Here, we show that DON from the postdenitrification systems dominates effluent eutrophication potential under different carbon sources. Methanol resulted in significantly lower DON concentrations (0.84 ± 0.03 mg/L) compared with the total nitrogen removal-preferred acetate (1.11 ± 0.02 mg/L) (p < 0.05, ANOVA). With our well-developed mathematical model (R2 = 0.867-0.958), produced DON instead of shared (persist in both influent and effluent) and/or removed DON was identified as the key component for effluent DON variation (Pearson r = 0.992, p < 0.01). The partial least-squares path modeling analysis showed that it is the microbial community (r = 0.947, p < 0.01) rather than the predicted metabolic functions (r = 0.040, p > 0.1) that affected produced DON. Carbon sources rebuild the microorganism-DON interaction by affecting the structure of microbial communities with different abilities to generate and recapture produced DON to finally regulate effluent DON. This study revalues the importance of carbon source selection and overturns the current rationality of pursuing only the total nitrogen removal efficiency by emphasizing DON.