Disinfectant-induced ammonia oxidation disruption in microbial N-cycling process in aquatic ecosystem after the COVID-19 outbreak.

Anthropogenic activities significantly impact the elemental cycles in aquatic ecosystems, with the N-cycling playing a critical role in potential nutrient turnover and substance cycling. We hypothesized that measures to prevent COVID-19 transmission profoundly altered the nitrogen cycle in riverine ecosystems. To investigate this, we re-analyzed metagenomic data and identified 60 N-cycling genes and 21 host metagenomes from four urban reaches (one upstream city, Wuhan, and two downstream cities) along the Yangtze River. Our analyses revealed a marked decrease in the abundance of bacterial ammonia monooxygenase genes, as well as in the host, ammonia-oxidizing autotrophic Nitrosomonas, followed by a substantial recovery post-pandemic. We posited that discharge of sodium hypochlorite (NaOCl) disinfectant may be a primary factor in the reduction of N-cycling process. To test this hypothesis, we exposed pure cultures of Nitrosomonas europaea to NaOCl to explore the microbial stress response. Results indicated that NaOCl exposure rapidly compromised the cell structure and inhibited ammonia oxidation of N. europaea, likely due to oxidative stress damage and reduced expression of nitrogen metabolism-related ammonia monooxygenase. Using the functional tagging technique, we determined that NaOCl directly destroyed the ammonia monooxygenase protein and DNA structure. This study highlights the negative impacts of chlorine disinfectants on the function of aquatic ecosystems and elucidates potential mechanisms of action.

Interspecific competition prevents the proliferation of social cheaters in an unstructured environment.

Bacterial communities are intricate ecosystems in which various members interact, compete for resources, and influence each other's growth. Antibiotics intensify this complexity, posing challenges in maintaining biodiversity. In this study, we delved into the behavior of kin bacterial communities when subjected to antibiotic perturbations, with a particular focus on how interspecific interactions shape these responses. We hypothesized that social cheating-where resistant strains shield both themselves and neighboring cheaters-obstructed coexistence, especially when kin bacteria exhibited varied growth rates and antibiotic sensitivities. To explore potential pathways to coexistence, we incorporated a third bacterial member, anticipating a shift in the dynamics of community coexistence. Simulations and experimental bacterial communities confirmed our predictions, emphasizing the pivotal role of interspecific competition in promoting coexistence under antibiotic interference. These insights are crucial for understanding bacterial ecosystem stability, interpreting drug-microbiome interactions, and predicting bacterial community adaptations to environmental changes.

Space aquatic chemistry: A roadmap for drinking water treatment in microgravity.

Rapid advancement in aerospace technology has successfully enabled long-term life and economic activities in space, particularly in Low Earth Orbit (LEO), extending up to 2000 km from the mean sea level. However, the sustainance of the LEO Economy and its Environmental Control and Life Support System (ECLSS) still relies on a regular cargo supply of essential commodities (e.g., water, food) from Earth, for which there still is a lack of adequate and sustainable technologies. One key challenge in this context is developing water treatment technologies and standards that can perform effectively under microgravity conditions. Solving this technical challenge will be a milestone in providing a scientific basis and the necessary support mechanisms for establishing permanent bases in outer space and beyond. To identify clues towards solving this challenge, we looked back at relevant scientific research exploring novel technologies and standards for deep space exploration, also considering feedback for enhancing these technologies on land. Synthesizing our findings, we share our outlook for the future of drinking water treatment in microgravity. We also bring up a new concept for space aquatic chemistry, considering the closed environment of engineered systems operating in microgravity.

Quorum-sensing gene regulates hormetic effects induced by sulfonamides in Comamonadaceae.

IMPORTANCE: Antibiotics can induce dose-dependent hormetic effects on bacterial cell proliferation, i.e., low-dose stimulation and high-dose inhibition. However, the underlying molecular basis has yet to be clarified. Here, we showed that sulfonamides play dual roles as a weapon and signal against Comamonas testosteroni that can modulate cell physiology and phenotype. Subsequently, through investigating the hormesis mechanism, we proposed a comprehensive regulatory pathway for the hormetic effects of Comamonas testosteroni low-level sulfonamides and determined the generality of the observed regulatory model in the Comamonadaceae family. Considering the prevalence of Comamonadaceae in human guts and environmental ecosystems, we provide critical insights into the health and ecological effects of antibiotics.

Enhancement of micropollutant biotransformation by adding manganese sand in constructed wetlands.

Recent investigations have shown that the addition of manganese (Mn) sand to constructed wetlands (i.e., Mn-amended CWs) can improve the performance of organic micropollutants (MPs) removal. In addition to the direct oxidation and adsorption of Mn oxides, the indirect role of Mn oxides in MP biotransformation is crucial to the removal of MPs but has seldom been referred to. Herein, we constructed lab-scale CWs with or without the addition of natural Mn sand (∼35% Mn oxides) to decipher the influence of Mn oxides on the biotransformation of the six selected MPs which commonly existed in the wastewater. The experimental results showed that the addition of Mn sand to CWs can improve the removal of MPs (8.48% atrazine, 13.16% atenolol, and 6.27% sulfamethoxazole [pairwise Wilcoxon test p < 0.05]). Combining the detection of transformation products and metagenomic sequencing, we found that the enhanced removal of atrazine in the Mn-amended CWs was mainly due to the bioaugmented hydroxylation process. The enrichment of biotransformation-related genes and associated microbes of atenolol and sulfamethoxazole in Mn-amended CWs indicated that the addition of Mn sand to CWs can strengthen the biotransformation of MPs. Furthermore, we found that these MP-biodegrading microbes were widely present in the full-scale CWs. Overall, our research provides fundamental information and insights for further application of Mn-amended CWs in MP removal.

High efficient removal of 4-aminophenylarsonic acid from aqueous solution via enhanced FeOOH using Mn(VII).

Efficient removal of 4-aminophenylarsonic acid from contaminated water sources is essential to mitigate arsenic pollution. We proposed a competent technique to achieve 4-aminophenylarsonic acid removal via adsorption on enhanced α-FeOOH using various concentrations of Mn(VII). The elimination rate of 4-aminophenylarsonic acid applying FeOOH with Mn(VII) was dependent on acidic conditions. More than 99.9% of 4-aminophenylarsonic acid was eliminated in a 6-min reaction time under acidic conditions. The reaction of 4-aminophenylarsonic acid was fast at 4.0 and 5.0 pH, with its complete oxidation into arsenate and the liberation of manganese Mn(II) in the initial stage of the reaction. Similarly, the reaction rate constant (kobs) decreased from 0.7048 ± 0.02 to 0.00155 ± 0.00007 as the pH increased from 4.0 to 9.0. Oxidation capacity was considerably enhanced via the removal of electrons from 4-aminophenylarsonic acid to Mn(VII) after the creation of its radical intermediate and further change in Mn(III) to Mn(II) in the solution. The results showed that Mn(VII) played a crucial role in 4-aminophenylarsonic acid degradation at a low pH (e.g., 4.0), and the oxidation process proceeded in different manners, namely, electron transfer, hydroxylation, and ring-opening. These results illustrated that Mn(VII) is an effective, economic purification process to mitigate 4-aminophenylarsonic acid generated from poultry waste.

A method for simulating spatial fates of chemicals in flowing lake systems: Application to phthalates in a lake.

In order to describe spatio-temporal distribution of chemicals in flowing lake systems, a dynamic multimedia fate model of chemicals with spatial differentiation was constructed by coupling the level IV fugacity model with lake hydrodynamics. It was successfully applied to four phthalates (PAEs) in a lake recharged by reclaimed water and its accuracy was verified. Results show that under the long-term influence of flow field, the distributions of PAEs in both lake water and sediment have significant spatial heterogeneity of 2∼5 orders of magnitude, but present different distribution rules, which was explained by analysis of PAE transfer fluxes. The spatial distribution of PAEs in the water column depends on hydrodynamic conditions and whether the primary source is reclaimed water or atmospheric input. Slow water exchange and flow speed promote the migration of PAEs from water to sediment, causing them to always accumulate in sediments far away from the recharging inlet. Uncertainty and sensitivity analysis show that the PAE concentrations in water phase are mainly affected by emission and physicochemical parameters, while those in sediment phase are also sensitive to environmental parameters. The model can provide important information and accurate data support for the scientific management of chemicals in flowing lake systems.

Dam construction alters planktonic microbial predator‒prey communities in the urban reaches of the Yangtze River.

While dam construction supports social and economic development, changes in hydraulic conditions can also affect natural aquatic ecosystems, especially microbial ecosystems. The compositional and functional traits of multi-trophic microbiota can be altered by dam construction, which may result in changes in aquatic predator-prey interactions. To understand this process, we performed a large-scale sampling campaign in the urban reaches of the dam-impacted Yangtze River (1 995 km) and obtained 211 metagenomic datasets and water quality data. We first compared the compositional traits of planktonic microbial communities upstream, downstream, and in a dam reservoir. Results showed that Bacteroidetes (R-strategy) bacteria were more likely to survive upstream, whilst the reservoir and downstream regions were more conducive to the survival of K-strategy bacteria such as Actinobacteria. Eukaryotic predators tended to be enriched upstream, whilst phototrophs tended to be enriched in the reservoir and downstream regions. Based on bipartite networks, we inferred that the potential microbial predator-prey interactions gradually and significantly decreased from upstream to the downstream and dam regions, affecting 56% of keystone microbial species. Remarkably, functional analysis showed that the abundance of the photosynthetic gene psbO was higher in the reservoir and downstream regions, whilst the abundance of the KEGG carbohydrate metabolic pathway was higher upstream. These results indicate that dam construction in the Yangtze River induced planktonic microbial ecosystem transformation from detritus-based food webs to autotroph-based food webs.

Influenza A(H7N9) Pandemic Preparedness: Assessment of the Breadth of Heterologous Antibody Responses to Emerging Viruses from Multiple Pre-Pandemic Vaccines and Population Immunity.

Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 µg hemagglutinin (HA)/dose + AS03A; 2. inactivated vaccine with 5.75 µg HA/dose + AS03A; 3. inactivated vaccine with 11.5 µg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 µg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).

Role of ammonia-oxidizing microorganisms in the removal of organic micropollutants during simulated riverbank filtration.

Biodegradation plays an important role in the removal of organic micropollutants (OMPs) during riverbank filtration (RBF) for drinking water production. The ability of ammonia-oxidizing microorganisms (AOM) to remove OMPs has attracted increasing attention. However, the distribution of AOM in RBF and its role in the degradation of OMPs remains unknown. In this study, the behavior of 128 selected OMPs and the distribution of AOM and their roles in the degradation of OMPs in RBF were explored by column and batch experiments simulating the first meter of the riverbank. The results showed that the selected OMPs were effectively removed (82/128 OMPs, >70% removal) primarily by biodegradation and partly by adsorption. Inefficiently removed OMPs tended to have low molecular weights, low log P, and contain secondary amides, secondary sulfonamides, secondary ketimines, and benzyls. In terms of the microbial communities, the relative abundance of AOM increased from 0.1%-0.2% (inlet-sand) to 5.3%-5.9% (outlet-sand), which was dominated by ammonia-oxidizing archaea whose relative abundance increased from 23%-72% (inlet-sand) to 97% (outlet-sand). Comammox accounted for 23%-64% in the inlet-sand and 1% in the outlet-sand. The abundances of AOM amoA genes kept stable in the inlet-sand of control columns, while decreased by 78% in the treatment columns, suggesting the inhibition effect of allylthiourea (ATU) on AOM. It is observed that AOM played an important role in the degradation of OMPs, where its inhibition led to the corresponding inhibition of 32 OMPs (5/32 were completely suppressed). In particular, OMPs with low molecular weights and containing primary amides, secondary amides, benzyls, and secondary sulfonamides were more likely to be removed by AOM. This study reveals the vital role of AOM in the removal of OMPs, deepens our understanding of the degradation of OMPs in RBF, and offers valuable insights into the physiochemical properties of OMPs and their AOM co-metabolic potential.

Metal(loid) flux change in Dongting Lake due to the operation of Three Gorges Dam, China.

A drastic decrease in the suspended sediment of Dongting Lake (DTL) has been observed due to Three Gorges Dam (TGD) impoundment operation since 2003. However, the relationship between sediment loads and metal fluxes has not been studied. This study comprehensively analyzed the content characteristics of seven metal(loid)s (As, Cd, Cr, Cu, Hg, Pb, and Zn) in the surface sediment of DTL from 2000 to 2019. The period of 2005-2009 corresponded to a metal(loid) enrichment stage in the sediment of DTL. The metal(loid) cumulative input of DTL from 2000 to 2019 reached 153 × 103 t, and the increasing rate was gradually diminished because of TGD operation, while the metal(loid) cumulative output reached 132 × 103 t. Undergoing an input-output state transition, the metal(loid) cumulative deposition of DTL in 2019 was only 42% of its peak in 2007. Especially, the metal(loid) fluxes of DTL all became negative for the first time in 2006. It is worth noting that Cd in DTL has shifted to a net export during the study period. Finally, the assessment results of pollution, risk, and toxicity indicated that metal(loid) effects on sediment quality were weakening in recent years. This study confirmed that DTL has shifted from metal(loid) deposition to export, providing new information for future DTL management options.

Siderophores provoke extracellular superoxide production by Arthrobacter strains during carbon sources-level fluctuation.

Superoxide and other reactive oxygen species (ROS) shape microbial communities and drive the transformation of metals and inorganic/organic matter. Taxonomically diverse bacteria and phytoplankton produce extracellular superoxide during laboratory cultivation. Understanding the physiological reasons for extracellular superoxide production by aerobes in the environment is a crucial question yet not fully solved. Here, we showed that iron-starving Arthrobacter sp. QXT-31 (A. QXT-31) secreted a type of siderophore [deferoxamine (DFO)], which provoked extracellular superoxide production by A. QXT-31 during carbon sources-level fluctuation. Several other siderophores also demonstrated similar effects to A. QXT-31. RNA-Seq data hinted that DFO stripped iron from iron-bearing proteins in electron transfer chain (ETC) of metabolically active A. QXT-31, resulting in electron leakage from the electron-rich (resulting from carbon sources metabolism by A. QXT-31) ETC and superoxide production. Considering that most aerobes secrete siderophore(s) and undergo carbon sources-level fluctuation, the superoxide-generation pathway is likely a common pathway by which aerobes produce extracellular superoxide in the environment, thus influencing the microbial community and cycling of elements. Our results pointed that the ubiquitous siderophore might be the potential driving force for the microbial generation of superoxide and other ROS and revealed the important role of iron physiology in microbial ROS generation.

Disruption and recovery of river planktonic community during and after the COVID-19 outbreak in Wuhan, China.

During the COVID-19 outbreak in Wuhan, large amounts of anti-coronavirus chemicals, such as antiviral drugs and disinfectants were discharged into the surrounding aquatic ecosystem, causing potential ecological damage. Here, we investigated plankton in the Wuhan reaches of the Yangtze River, before, during, and after COVID-19, with the river reaches of three adjacent cities sampled for comparison. During the COVID-19, planktonic microbial density declined significantly. Correspondingly, the eukaryotic and prokaryotic community compositions and functions shifted markedly, with increasing abundance of chlorine-resistant organisms. Abundance of antibiotic resistance genes, virulence factor genes, and bacteria containing both genes increased by 2.3-, 2.7-, and 7.9-fold, respectively, compared to other periods. After COVID-19, all measured plankton community compositional and functional traits recovered in the Yangtze River.

Profiling microbial removal of micropollutants in sand filters: Biotransformation pathways and associated bacteria.

Although there is growing evidence that micropollutants can be microbially converted in rapid sand filters of drinking water treatment plants (DWTPs), little is known about the biotransformation pathways and associated microbial strains in this process. Here, we constructed sand filter columns filled with manganese or quartz sand obtained from full-scale DWTPs to explore the biotransformation of eight micropollutants. Under seven different empty bed contact times (EBCTs), the column experiments showed that caffeine and atenolol were easily removed (up to 92.1% and 97.6%, respectively) with adsorption and microbial biotransformation of the filters. In contrast, the removal of other six micropollutants (i.e., naproxen, carbamazepine, atrazine, trimethoprim, sulfamethoxazole, and sulfadiazine) in the filters were less than 27.1% at shorter EBCTs, but significantly increased at EBCT = 4 h, indicating the dominant role of microbial biotransformation in these micropollutants removal. Integrated analysis of metagenomic reads and transformation products of micropollutants showed a shift in caffeine oxidation and demethylation pathways at different EBCTs, simultaneous occurrence of atrazine hydrolysis and oxidation pathways, and sulfadiazine and sulfamethoxazole oxidation in the filters. Furthermore, using genome-centric analysis, we observed previously unidentified degrading strains, e.g., Piscinibacter, Hydrogenophaga, and Rubrivivax for caffeine transformation, and Methylophilus and Methyloversatilis for atenolol transformation.

Environmental heterogeneity determines the response patterns of microbially mediated N-reduction processes to sulfamethoxazole in river sediments.

The widespread occurrence of antibiotics in aquatic ecosystems leads to potential ecological risks to organisms, in turn affecting microbially mediated processes. Here, we investigated the response of dominant N-reduction processes to the frequently detected antibiotic sulfamethoxazole (SMX) along the Chaobai River with regional environmental heterogeneity, including denitrification, anaerobic ammonium oxidation (anammox), dissimilatory nitrate reduction to ammonium (DNRA), and nitrous oxide (N2O) release. We found two divergent SMX response patterns for denitrification in contrasting scenarios of geochemical properties. In the context of low nitrate and carbon, SMX weakened denitrification with a slightly stimulation first. Whereas SMX directly inhibited denitrification when nitrate and carbon were sufficient. High SMX concentration suppressed anammox (26-72%) and DNRA activities (48-84%) via restraining the activities of anammox and DNRA bacteria. Notably, SMX increased the contribution of denitrification to N-reduction at the expense of DNRA to N-reduction, leading to a shift in nitrogen conversion towards denitrification. Additionally, SMX stimulated N2O emission (up to 91%) due to superior restraint on process of N2O reduction to N2 and an incline for N-reduction towards denitrification, thereby exacerbating greenhouse effect. Our results advance the understanding of how nitrogen cycling is affected by SMX in aquatic ecosystems with environmental heterogeneity.

Mixing regime shapes the community assembly process, microbial interaction and proliferation of cyanobacterial species Planktothrix in a stratified lake.

Lake mixing influences aquatic chemical properties and microbial community composition, and thus, we hypothesized that it would alter microbial community assembly and interaction. To clarify this issue, we explored the community assembly processes and cooccurrence networks in four seasons at two depths (epilimnion and hypolimnion) in a mesotrophic and stratified lake (Chenghai Lake), which formed stratification in the summer and turnover in the winter. During the stratification period, the epilimnion and hypolimnion went through contrary assembly processes but converged to similar assembly patterns in the mixing period. In a highly homogeneous selection environment, species with low niche breadth were filtered, resulting in decreased species richness. Water mixing in the winter homogenized the environment, resulting in a simpler microbial cooccurrence network. Interestingly, we observed a high abundance of the cyanobacterial genus Planktothrix in the winter, probably due to nutrient redistribution and Planktothrix adaptivity to the winter environment in which mixing played important roles. Our study provides deeper fundamental insights into how environmental factors influence microbial community structure through community assembly processes.

Manganese oxides in Phragmites rhizosphere accelerates ammonia oxidation in constructed wetlands.

Phragmites reeds are widely used in constructed wetlands (CWs) for treating wastewater. The enrichment of microorganisms and Fe/Mn plaque in Phragmites rhizospheres largely contributes to pollutant removal. However, their interactions and potential synergistic roles in water purification are poorly understood. To address the issue, we first compared the microbial community traits in the Phragmites rhizosphere and adjacent bulk soil in six long-term operated CWs. Results showed that enriched microbes and functional genes in the Phragmites rhizosphere were largely involved in Mn oxidation, resulting in a two to three times enrichment of Mn oxides in the rhizosphere. In turn, the enriched Mn oxides played significant roles in driving microbial community composition and function. To further understand the biological manganese oxidation in the rhizosphere, we identified Mn-oxidizing bacteria using genome-centric analysis and found that 92% of identified Mn-oxidizing bacteria potentially participated in nitrogen cycling. We then conducted relationships between Mn-oxidizing genes and different nitrogen cycling genes and found Mn-oxidizing gene abundance was significantly correlated with ammonia oxidation gene amoA (R = 0.65). Remarkably, complete ammonia oxidation (comammox) Nitrospira, accounting for 39.11% of ammonia oxidizers, also positively correlated with Mn-oxidizing microbes. Based on the above observations, we inferred that the use of Mn oxides as a substrate in CWs may enhance ammonia oxidation. To apply this to actual engineering, we explored treatment performance in a pilot-scale Mn-amending CW. As expected, ammonia removal capacity improved by 23.34%, on average, in the Mn-amending CW. In addition, the abundance of amoA genes increased significantly in the Mn-amending CW, indicating improved biological processes rather than chemical reactions.

[Enhanced Removal of Pollutants in Constructed Wetlands with Manganese Sands].

Manganese (Mn) sands have been widely used in water purification due to their strong oxidation and adsorption abilities. However, there are few reports on the use of manganese sands as filler material in constructed wetlands. Based on previous studies, we speculated that the addition of manganese sands in constructed wetlands would enhance the removal of pollutants from the source water, and the resulting Mn(Ⅱ) could then be oxidized by the rhizosphere and soil microorganisms in the wetlands. To test this hypothesis, this study explored the enhanced removal of pollutants in wetlands constructed with manganese sands as substrates and Phragmites as plants, and also examined the role of Phragmites rhizosphere microorganisms in water purification. By comparing the treatment effects between the wetlands constructed with and without manganese sands (control), we found that the wetland containing manganese sands exhibited significantly improved removal of dissolved organic carbon and total nitrogen, as well as removal of ammonia nitrogen during periods of lower temperature. The 16S rRNA sequencing showed that the addition of manganese sands could increase the richness and diversity of Phragmites rhizosphere microorganisms, but had limited impacts on the microbial community structure, which might be an important factor for enhancing the water treatment performance of constructed wetlands. This study provides a new method for the technological optimization of constructed wetlands.

Machine learning approach identifies water sample source based on microbial abundance.

Water quality can change along a river system due to differences in adjacent land use patterns and discharge sources. These variations can induce rapid responses of the aquatic microbial community, which may be an indicator of water quality characteristics. In the current study, we used a random forest model to predict water sample sources from three different river ecosystems along a gradient of anthropogenic disturbance (i.e., less disturbed mountainous area, wastewater discharged urban area, and pesticide and fertilizer applied agricultural area) based on environmental physicochemical indices (PCIs), microbiological indices (MBIs), and their combination. Results showed that among the PCI-based models, using conventional water quality indices as inputs provided markedly better prediction of water sample source than using pharmaceutical and personal care products (PPCPs), and much better prediction than using polycyclic aromatic hydrocarbons (PAHs) and substituted PAHs (SPAHs). Among the MBI-based models, using the abundances of the top 30 bacteria combined with pathogenic antibiotic resistant bacteria (PARB) as inputs achieved the lowest median out-of-bag error rate (9.9%) and increased median kappa coefficient (0.8694), while adding fungal inputs reduced the kappa coefficient. The model based on the top 30 bacteria still showed an advantage compared with models based on PCIs or the combination of PCIs and MBIs. With improvement in sequencing technology and increase in data availability in the future, the proposed method provides an economical, rapid, and reliable way in which to identify water sample sources based on abundance data of microbial communities.

Recovery trajectories and community resilience of biofilms in receiving rivers after wastewater treatment plant upgrade.

Wastewater treatment plant (WWTP) upgrades can reduce both nutrient and micropollutant emissions into receiving rivers, thus modifying the composition and function of biological communities. However, how microbial communities vary and whether they can be restored to levels found in less-polluted rivers remains uncertain. Aquatic biofilms are sensitive to environmental change and respond rapidly to bottom-up pressure. Thus, we used 12 flumes configured in three experimental treatments to mimic the dynamic processes of biofilm microbial communities occurring in a wastewater-receiving river following WWTP upgrade, with rivers containing two levels of nutrients and micropollutants used as references. We compared the biofilm microbial biomass, carbon source utilization, and community composition among the three "blocks". Results showed that the metabolic patterns of the carbon sources and composition of the biofilm bacterial communities in the flumes mimicking a receiving river with WWTP upgrade recovered over time to those mimicking a less-disturbed river. The restoration of potential carboxylic acid-consuming denitrifying bacteria (i.e., Zoogloea, Comamonas, Dechloromonas, and Acinetobacter) likely played a significant role in this process. Combining quantitative analysis of the denitrification genes nirS and nosZ, we confirmed that the denitrification function of the river biofilms recovered after WWTP upgrade, consistent with our previous field investigation.