Nutrient characteristics driven by multiple factors in large estuaries during summer: A case study of the Yangtze River Estuary.

Nutrients directly control the level of primary productivity and are crucial for the stability of marine ecosystems. Focusing on the survey results in August 2020 of the Yangtze River Estuary, this study elucidated the distribution characteristics and controlling factors of three nutrients: NO3-N, PO4-P, SiO3-Si. The results showed that the concentrations of NO3-N, PO4-P, SiO3-Si in the study area were generally higher near the shore than far shore, with average concentrations of 11.40, 0.70, and 23.73 µmol/L, respectively. The ocean currents drove the distribution of nutrients, and the transport of CDW and YSCC increased the nutrient levels. The resuspension of sediment caused by factors such as terrain and weather may lead to an abnormal increase in nutrients in the bottom waters. The main controlling factors of the three nutrients were different. NO3-N was significantly affected by human activities, PO4-P and SiO3-Si were mainly affected by natural factors.

Environmental pollution of fluoroquinolones and its relationship with nitrogen cycling mediated by microorganisms.

Fluoroquinolone antibiotics (FQs) are one of the most widely used antibiotics, which are new pollutants with 'pseudo persistence' in the environment, causing great ecological risks. FQs could change the structure and function of microbial communities and affect nitrogen cycling mediated by microorganisms. Consequently, FQs would change the composition of various types of nitrogen in the environment and exert a significant impact on the global nitrogen cycling. We encapsulated the distribution of FQs in the environment and its impacts on nitrogen cycling mediated by microorganisms, explained the role of FQs in each key process of nitrogen cycling, aiming to provide an important reference for revealing the ecological effects of FQs. Generally, FQs could be detected in various environmental media, with significant differences in the concentration and types of FQs in different environments. Ofloxacin, norfloxacin, ciprofloxacin, and enrofloxacin are the four types of FQs with the highest detection frequency and concentration. The effect of FQs on nitrogen cycling deeply depends on typical characteristics of concentration and species. FQs mainly inhibit nitrification by reducing the abundance of amoA gene related to ammoxidation process and the abundance and composition of ammoxidation bacteria. FQs inhibits nitrification by reducing the abundance and composition of microbial communities. The denitrification process is mainly inhibited due to the reduction of the activity of related enzymes and the abundance of genes such as narG, nirS, norB, and nosZ genes, as well as the abundance and composition of denitrifying functional microorganisms. The process of anammox is restricted due to the reduction of the abundance, composition and hzo gene abundance of anaerobic anammox bacteria. FQs lead to the reduction of active nitrogen removal and the increase of N2O release in the environment, with further environmental problems such as water eutrophication and greenhouse effect. In the future, we should pay attention to the effects of low concentration FQs and complex antibiotics on the nitrogen cycling, and focus on the effects of FQs on the changes of nitrogen cycle-related microbial monomers and communities.

Distribution patterns of six metals and their influencing factors in M4 seamount seawater of the Western Pacific.

Metals are crucial to the stability of marine ecosystems, and it is important to analyze their spatial heterogeneity. This study examined the distribution and influencing factors of six metals such as manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu) and cadmium (Cd) in M4 seamount of the Western Pacific. The results showed that the factors affecting the distribution of metals are complex. The concentration ranges of Mn, Fe, Co, Ni, Cu, and Cd in the M4 seamount were 0-0.05, 0-0.44, 0-0.0014, 0-0.082, 0.12-0.16, and 0-0.013 µg/L, respectively, roughly equivalent to those of other open seas, however, there were also some differences. Specifically, the distribution of ferromanganese nodules and Co-rich crusts, resulted in a significant increase in the concentration of metals such as Mn, Fe, and Co in the bottom. This study will significantly contribute to our understanding of the spatial heterogeneity of metals in seamount areas.

Behavioral and ecotoxicology of sulfonamide metabolites in the aquatic environment.

Sulfonamides (SAs) are the first broad-spectrum synthetic antimicrobial agents used in human health and veterinary medicine. The majority of SAs entering human body is discharged into aquatic environment in the form of parent material or metabolites. The residues of SAs and their metabolites in the aquatic environment and the development of drug resistance can be serious threats to ecosystems and human health. We summarized recent advances in the research of SAs. The main metabolite types of SAs and the distribution characteristics of metabolites in different aquatic environments were introduced. The ecotoxicology of SAs metabolites, especially the distribution and hazards of sulfonamide resistance genes (sul-ARGs), were discussed with emphasis. Finally, the future research works were proposed. This paper could provide basic information for further research on SAs.

Biogeochemical behavior and ecological environmental effects of fluoroquinolones.

Synthetic fluoroquinolones (FQs) are the third most commonly used antibiotics in the world and play an extremely important role in antibacterial drugs. The excessive use and discharge will alter ecological environment, with consequence on human health and global sustainable development. It is therefore of great significance for scientific use and management of FQs to systematically understand their biogeochemical behavior and eco-environmental effects. After drug administration in humans and animals, only a small part of FQs are transformed in vivo. The main transformation processes include formylation, acetylation, oxidation and cleavage of piperazine ring, defluorination and decarboxylation of aromatic core ring, etc. About 70% of the original drug and a small amount of transformed products would be migrated to the environment through excretion. After entering the environment, FQs and their transformation products mainly exist in environmental media such as water, soil and sediment, and undergo migration and transformation processes such as adsorption, photolysis and biodegradation. Adsorption facilitates transfer of FQs from medium to another. The photolysis mainly affects the C7-amine substituents of FQs, whereas the core structure of FQs remains intact. Biodegradation mainly refers to the degradation of FQs by microorganisms and microalgae, including piperazine modification of the piperazine ring such as acetylation and formylation, partial or complete ring cleavage, core structure decarboxylation, defluorination and conjugation formation. The migration and transformation processes of FQs cannot completely eliminate them from the environment. Instead, they would become "pseudo-persistent" pollutants, which seriously affect the behavior, growth and reproduction of algae, crustaceans and fish, change biogeochemical cycle, destroy aquatic environment, and stimulate microbial resistance and the generation of resistance genes. In the future, more in-depth studies should be conducted on the environmental behavior of FQs and their impacts on ecological environment, the risk assessment of microbial resistance and resistance genes of FQs, and the mechanism and effect of micro-biodegradation of FQs.

In situ reductive dehalogenation of groundwater driven by innovative organic carbon source materials: Insights into the organohalide-respiratory electron transport chain.

In situ bioremediation using organohalide-respiring bacteria (OHRB) is a prospective method for the removal of persistent halogenated organic pollutants from groundwater, as OHRB can utilize H2 or organic compounds produced by carbon source materials as electron donors for cell growth through organohalide respiration. However, few previous studies have determined the suitability of different carbon source materials to the metabolic mechanism of reductive dehalogenation from the perspective of electron transfer. The focus of this critical review was to reveal the interactions and relationships between carbon source materials and functional microbes, in terms of the electron transfer mechanism. Furthermore, this review illustrates some innovative strategies that have used the physiological characteristics of OHRB to guide the optimization of carbon source materials, improving the abundance of indigenous dehalogenated bacteria and enhancing electron transfer efficiency. Finally, it is proposed that future research should combine multi-omics analysis with machine learning (ML) to guide the design of effective carbon source materials and optimize current dehalogenation bioremediation strategies to reduce the cost and footprint of practical groundwater bioremediation applications.

The effects of seawater thermodynamic parameters on the oxygen minimum zone (OMZ) in the tropical western Pacific Ocean.

The continuous expansion of the oxygen minimum zone (OMZ) is a microcosm of marine hypoxia problem. Based on a survey in M4 seamount area of Tropical Western Pacific Ocean, the effects of thermodynamic parameters on OMZ were discussed. The study showed thermodynamic parameters mainly affect the upper oxycline of OMZ. The increase in temperature aggravates seawater stratification, which not only shallows oxycline but also increases the strength of DO stratification, promoting the expansion of OMZ. Based on relationships between thermodynamic parameters, water mass and DO, OMZ in this area is defined as follows: the water layer with low DO between the lower boundary of high-salt area and 1000 m. Moreover, the study showed that though there is no "seamount effect" on a scale of 3000 m, low-value areas of DO form at the bottom of seamount. This study will provide an evidence for expansion of OMZ exacerbated by global warming.

Improved performance of Zn-doped SnO2 modified g-C3N4 for visible light-driven photocatalysis.

The low-cost composite of g-C3N4 modified by Zn-doped SnO2 nanoparticles was prepared for the first time in this work. The characterization results of XRD and SEM demonstrated that Zn was successfully doped into SnO2. The formed Sn-O-Zn bonds and interaction between the Zn-doped SnO2 sample and g-C3N4 in the composite were explored by FT-IR and XPS technologies. Photocatalytic degradation experiments showed that the as-prepared optimal composite photocatalyst displayed enhanced photocatalytic reactivity towards both dyes and antibiotics, which could degrade 85.6% of RhB and 86.8% of tetracycline within 30 and 90 min, respectively. The oxygen vacancies formed in SnO2 after Zn doping could capture the photogenerated electrons of g-C3N4, thereby promoting the separation of photogenerated electron-hole pairs, then more ·O2- and holes can be generated during the visible light-driven photocatalytic reaction, so that the composite of Zn-doped SnO2/g-C3N4 acquired higher photocatalytic activity and accelerated the degradation of target organics. Active species capturing experiments and ESR detection results also confirmed that ·O2- and holes were the main active species in the reaction process. This work developed a novel g-C3N4-based photocatalyst with no noble metal, low price, and high photocatalytic activity, which could provide a cost-effective and high-efficiency strategy for wastewater treatment.

Efficient photocatalytic degradation of doxycycline by coupling α-Bi2O3/g-C3N4 composite and H2O2 under visible light.

Antibiotic pollutants have posed a huge threat to the ecological environment and human health. In this work, α-Bi2O3/g-C3N4 composite was prepared and coupled with H2O2 for the rapid and efficient degradation of doxycycline (DOX) in water under visible light irradiation. The composite exhibited enhanced photocatalytic activity and 80.5% of DOX could be degraded in 120 min. The addition of H2O2 significantly improved the degradation efficiency of DOX under visible light, resulting in 79.0% of it degraded within 30 min, and the degradation rate constant of DOX was 3.6 times than that without H2O2. On the one hand, the Z-scheme heterojunction of α-Bi2O3/g-C3N4 promoted the separation rate of photogenerated electron-hole pairs, thereby enhancing the photocatalytic activity of the composite. On the other hand, the improvement of photocatalytic efficiency also benefited from the extra hydroxyl radicals generated by the reaction of photogenerated electrons with H2O2 in the photocatalytic system. Free radicals trapping experiments and electron spin resonance tests proved that played prominent role in the degradation process. After adding H2O2, OH also became important active species. Cyclic degradation experiments demonstrated the recyclability of the composite photocatalyst in DOX elimination applications. This work provides an efficient, clean, and recyclable purification strategy for removing antibiotic contaminants from water.

Influence of non-dechlorinating microbes on trichloroethene reduction based on vitamin B12 synthesis in anaerobic cultures.

In this study, the YH consortium, an ethene-producing culture, was used to evaluate the effect of vitamin B12 (VB12) on trichloroethene (TCE) dechlorination by transferring the original TCE-reducing culture with or without adding exogenous VB12. Ultra-high performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) was applied to detect the concentrations of VB12 and its lower ligand 5,6-dimethylbenzimidazole (DMB) in the cultures. After three successive VB12 starvation cycles, the dechlorination of TCE stopped mostly at cis-dichloroethene (cDCE), and no ethene was found; methane production increased significantly, and no VB12 was detected. Results suggest that the co-cultured microbes may not be able to provide enough VB12 as a cofactor for the growth of Dehalococcoides in the YH culture, possibly due to the competition for corrinoids between Dehalococcoides and methanogens. The relative abundances of 16 S rRNA gene of Dehalococcoides and reductive dehalogenase genes tceA or vcrA were lower in the cultures without VB12 compared with the cultures with VB12. VB12 limitation changed the microbial community structures of the consortia. In the absence of VB12, the microbial community shifted from dominance of Chloroflexi to Proteobacteria after three consecutive VB12 starvation cycles, and the dechlorinating genus Dehalococcoides declined from 42.9% to 13.5%. In addition, Geobacter, Clostridium, and Desulfovibrio were also present in the cultures without VB12. Furthermore, the abundance of archaea increased under VB12 limited conditions. Methanobacterium and Methanosarcina were the predominant archaea in the culture without VB12.

Addition of Shewanella oneidensis MR-1 to the Dehalococcoides-containing culture enhances the trichloroethene dechlorination.

Dehalococcoides is able to completely dehalogenate tetrachloroethene (PCE) and trichloroethene (TCE) to ethene (ETH). However, the dechlorination efficiency of Dehalococcoides is low and result in the accumulation of toxic intermediates. In this study, Shewanella oneidensis MR-1 (S. oneidensis MR-1) was added to the Dehalococcoides-containing culture and the complete TCE to ETH dechlorination was shortened from 24 days to 16 days. Dehalococcoides-targeted 16S rRNA gene and two model reductive dehalogenase (RDase) genes (tceA and vcrA), responsible for dechlorinating TCE to vinyl chloride (VC) and VC to ETH respectively, were characterized. Results showed that S. oneidensis MR-1 has no effect on the cell growth while the RDase genes expression was up-regulated and the RDase activity of Dehalococcoides was elevated. The mRNA abundance of vcrA increased approximately tenfold along with the increased concentration of vitamin B12 (cyanocobalamin). Interestingly, the addition of S. oneidensis MR-1 increased the concentration of vitamin B12 by affecting the microbial community structure. Therefore, the addition of S. oneidensis MR-1 might have a positive effect on regulating the activity of RDase of functional microorganisms and uptake of vitamin B12, and further provided a practical vision of chloroethene dechlorination by the Dehalococcoides-containing culture.

Histones and chymotrypsin-like elastases play significant roles in the antimicrobial activity of tongue sole neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are a form of extracellular antimicrobial structure of neutrophils observed in higher and lower vertebrates, the latter including the teleost fish tongue sole Cynoglossus semilaevis. However, the antimicrobial mechanism of fish NETs is unknown. In the present study, we examined the potential contribution of histones and elastases to the antibacterial effect of tongue sole NETs. For this purpose, two histones (CsH2B and CsH4) and two elastases (CsEla1 and CsEla2) of tongue sole were investigated. The histones and elastases possess the conserved domain structures characteristic of that of histones H2B/H4 and trypsin-like serine protease, respectively. Recombinant CsH2B, CsH4, CsEla1, and CsEla2 bound a wide range of Gram-negative and Gram-positive bacteria, and some of the bound bacteria were inhibited in growth by the bound histones/elastases. CsH2B, CsH4, CsEla1, and CsEla2 were all localized in NETs induced by various stimuli including bacterial pathogen. Treatment of NETs with antibodies targeting CsH2B, CsH4, CsEla1, and CsEla2 significantly reduced the antimicrobial effect of NETs. These results indicate that histones and chymotrypsin-like elastases are fundamental components of teleost NETs that play important roles in the antimicrobial activity of NETs.

Cytochemical identification of turbot myeloperoxidase-positive granulocytes by potassium iodide and oxidized pyronine Y staining.

Cytomorphological and cytochemical staining are important methods for the identification of cell types, in particular in fish which often lack biological tools such as specific antibodies. Myeloperoxidase (MPO) is usually used as an intracellular marker of neutrophil accumulation in tissues and a marker of neutrophil activity in plasma. In this study, we reported a potassium iodide and oxidized pyronine Y (KI-PyY) staining method for rapid and highly sensitive detection of MPO-positive cells in turbot blood, peritoneum, and tissues. MPO-positive cells, which mostly represented neutrophils, were stained brown and clearly distinguished from other cells, such as lymphocytes, monocytes, and macrophages, which were stained pink. Following bacterial stimulation, the proportions of neutrophils were 27.49% and 38.05% in peripheral blood leukocytes and peritoneum, respectively, judging by the stained MPO. Kidney granulocytes contained abundant MPO-positive cells which were probably immature neutrophils with low expression of MPO. It is noteworthy that MPO-positive cells were detected in the tissue sections of kidney, spleen, and gut, with distribution profiles specific to each tissue. However, the cell morphology was not distinct in the stained tissue sections. These results indicate that the KI-PyY staining method is highly sensitive, applicable to different types of samples, and will be useful for the study of neutrophils in different compartments of fish.

Effects of 1,1,1-Trichloroethane and Triclocarban on Reductive Dechlorination of Trichloroethene in a TCE-Reducing Culture.

Chlorinated compounds were generally present in the environment due to widespread use in the industry. A short-term study was performed to evaluate the effects of 1,1,1- trichloroethane (TCA) and triclocarban (TCC) on trichloroethene (TCE) removal in a reactor fed with lactate as the sole electron donor. Both TCA and TCC inhibited TCE reduction, but the TCC had a more pronounced effect compared to TCA. The TCE-reducing culture, which had never been exposed to TCA before, reductively dechlorinated TCA to 1,1-dichloroethane (DCA). Below 15 µM, TCA had little effect on the transformation of TCE to cis-dichloroethene (DCE); however, the reduction of cis-DCE and vinyl chloride (VC) were more sensitive to TCA, and ethene production was completely inhibited when the concentration of TCA was above 15 µM. In cultures amended with TCC, the reduction of TCE was severely affected, even at concentrations as low as 0.3 µM; all the cultures stalled at VC, and no ethene was detected. The cultures that fully transformed TCE to ethene contained 5.2-8.1% Dehalococcoides. Geobacter and Desulfovibrio, the bacteria capable of partially reducing TCE to DCE, were detected in all cultures, but both represented a larger proportion of the community in TCC-amended cultures. All cultures were dominated by Clostridium_sensu_stricto_7, a genus that belongs to Firmicutes with proportions ranging from 40.9% (in a high TCC (15 µM) culture) to 88.2%. Methanobacteria was detected at levels of 1.1-12.7%, except in cultures added with 15 and 30 µM TCA, in which they only accounted for ∼0.4%. This study implies further environmental factors needed to be considered in the successful bioremediation of TCE in contaminated sites.

Effects of salinity on simultaneous reduction of perchlorate and nitrate in a methane-based membrane biofilm reactor.

This study builds upon prior work showing that methane (CH4) could be utilized as the sole electron donor and carbon source in a membrane biofilm reactor (MBfR) for complete perchlorate (ClO4-) and nitrate (NO3-) removal. Here, we further investigated the effects of salinity on the simultaneous removal of the two contaminants in the reactor. By testing ClO4- and NO3- at different salinities, we found that the reactor performance was very sensitive to salinity. While 0.2 % salinity did not significantly affect the hydrogen-based MBfR for ClO4- and NO3- removals, 1 % salinity completely inhibited ClO4- reduction and significantly lowered NO3- reduction in the CH4-based MBfR. In salinity-free conditions, NO3- and ClO4- removal fluxes were 0.171 g N/m2-day and 0.091 g/m2-day, respectively, but NO3- removal fluxes dropped to 0.0085 g N/m2-day and ClO4- reduction was completely inhibited when the medium changed to 1 % salinity. Scanning electron microscopy (SEM) showed that the salinity dramatically changed the microbial morphology, which led to the development of wire-like cell structures. Quantitative real-time PCR (qPCR) indicated that the total number of microorganisms and abundances of functional genes significantly declined in the presence of NaCl. The relative abundances of Methylomonas (methanogens) decreased from 31.3 to 5.9 % and Denitratisoma (denitrifiers) decreased from 10.6 to 4.4 % when 1 % salinity was introduced.

Selenate and Nitrate Bioreductions Using Methane as the Electron Donor in a Membrane Biofilm Reactor.

Selenate (SeO4(2-)) bioreduction is possible with oxidation of a range of organic or inorganic electron donors, but it never has been reported with methane gas (CH4) as the electron donor. In this study, we achieved complete SeO4(2-) bioreduction in a membrane biofilm reactor (MBfR) using CH4 as the sole added electron donor. The introduction of nitrate (NO3(-)) slightly inhibited SeO4(2-) reduction, but the two oxyanions were simultaneously reduced, even when the supply rate of CH4 was limited. The main SeO4(2-)-reduction product was nanospherical Se(0), which was identified by scanning electron microscopy coupled to energy dispersive X-ray analysis (SEM-EDS). Community analysis provided evidence for two mechanisms for SeO4(2-) bioreduction in the CH4-based MBfR: a single methanotrophic genus, such as Methylomonas, performed CH4 oxidation directly coupled to SeO4(2-) reduction, and a methanotroph oxidized CH4 to form organic metabolites that were electron donors for a synergistic SeO4(2-)-reducing bacterium.

Interaction of perchlorate and trichloroethene bioreductions in mixed anaerobic culture.

This work evaluated the interaction of perchlorate and trichloroethene (TCE), two common co-contaminants in groundwater, during bioreduction in serum bottles containing synthetic mineral salts media and microbial consortia. TCE at concentrations up to 0.3mM did not significantly affect perchlorate reduction; however, perchlorate concentrations higher than 0.1mM made the reduction of TCE significantly slower. Perchlorate primarily inhibited the reduction of vinyl chloride (VC, a daughter product of TCE) to ethene. Mechanistic analysis showed that the inhibition was mainly because perchlorate reduction is thermodynamically more favorable than reduction of TCE and its daughter products and not because of toxicity due to accumulation of dissolved oxygen produced during perchlorate reduction. As the initial perchlorate concentration increased from 0 to 600mg/L in a set of serum bottles, the relative abundance of Rhodocyclaceae (a putatively perchlorate-reducing genus) increased from 6.3 to 80.6%, while the relative abundance of Dehalococcoides, the only known genus that is able to reduce TCE all the way to ethene, significantly decreased. Similarly, the relative abundance of Proteobacteria (a phylum to which most known perchlorate-reducing bacteria belong) increased from 22% to almost 80%.

Bioreduction of Chromate in a Methane-Based Membrane Biofilm Reactor.

For the first time, we demonstrate chromate (Cr(VI)) bioreduction using methane (CH4) as the sole electron donor in a membrane biofilm reactor (MBfR). The experiments were divided into five stages lasting a total of 90 days, and each stage achieved a steady state for at least 15 days. Due to continued acclimation of the microbial community, the Cr(VI)-reducing capacity of the biofilm kept increasing. Cr(VI) removal at the end of the 90-day test reached 95% at an influent Cr(VI) concentration of 3 mg Cr/L and a surface loading of 0.37g of Cr m(-2) day(-1). Meiothermus (Deinococci), a potential Cr(VI)-reducing bacterium, was negligible in the inoculum but dominated the MBfR biofilm after Cr(VI) was added to the reactor, while Methylosinus, a type II methanotrophs, represented 11%-21% of the total bacterial DNA in the biofilm. Synergy within a microbial consortia likely was responsible for Cr(VI) reduction based on CH4 oxidation. In the synergy, methanotrophs fermented CH4 to produce metabolic intermediates that were used by the Cr(VI)-reducing bacteria as electron donors. Solid Cr(III) was the main product, accounting for more than 88% of the reduced Cr in most cases. Transmission electron microscope (TEM) and energy dispersive X-ray (EDS) analysis showed that Cr(III) accumulated inside and outside of some bacterial cells, implying that different Cr(VI)-reducing mechanisms were involved.

Quantitative detection of selenate-reducing bacteria by real-time PCR targeting the selenate reductase gene.

We designed a primer set to target selenate reductase (SerA) for detecting selenate reducing bacteria (SeRB). Our serA gene-based PCR primer set has high specificity in that it and positively amplified some SeRB, but not denitrifying bacteria (DB). Phylogenetic analysis of serA clone sequences of environmental samples from selenate-reducing membrane biofilm reactor (MBfR) biofilms showed that these sequences were closely grouped and had high similarity to selenate reductase gene sequences from SeRB Thauera selenatis and DB Dechloromonas; however, they were distant to other genes from dimethylsulfoxide (DMSO) enzyme family. Constructing a standard curve targeting the serA gene, we found that the good linearity for the qPCR assay when applied it to quantify SeRB in MBfR biofilms, and the gene copies of SeRB correlated well to the selenate removal percentages. Our results demonstrated the feasibility of using the serA gene-based PCR primer set to detect and quantify SeRB in environmental samples.

Evolution of the microbial community of the biofilm in a methane-based membrane biofilm reactor reducing multiple electron acceptors.

Previous work documented complete perchlorate reduction in a membrane biofilm reactor (MBfR) using methane as the sole electron donor and carbon source. This work explores how the biofilm's microbial community evolved as the biofilm stage-wise reduced different combinations of perchlorate, nitrate, and nitrite. The initial inoculum, carrying out anaerobic methane oxidation coupled to denitrification (ANMO-D), was dominated by uncultured Anaerolineaceae and Ferruginibacter sp. The microbial community significantly changed after it was inoculated into the CH4-based MBfR and fed with a medium containing perchlorate and nitrite. Archaea were lost within the first 40 days, and the uncultured Anaerolineaceae and Ferruginibacter sp. also had significant losses. Replacing them were anoxic methanotrophs, especially Methylocystis, which accounted for more than 25 % of total bacteria. Once the methanotrophs became important, methanol-oxidizing denitrifying bacteria, namely, Methloversatilis and Methylophilus, became important in the biofilm, probably by utilizing organic matter generated by the metabolism of methanotrophs. When methane consumption was equal to the maximum-possible electron-donor supply, Methylomonas, also an anoxic methanotroph, accounted for >10 % of total bacteria and remained a major part of the community until the end of the experiments. We propose that aerobic methane oxidation coupled to denitrification and perchlorate reduction (AMO-D and AMO-PR) directly oxidized methane and reduced NO3 (-) to NO2 (-) or N2O under anoxic condition, producing organic matter for methanol-assimilating denitrification and perchlorate reduction (MA-D and MA-PR) to reduce NO3 (-). Simultaneously, bacteria capable of anaerobic methane oxidation coupled to denitrification and perchlorate reduction (ANMO-D and ANMO-PR) used methane as the electron donor to respire NO3 (-) or ClO4 (-) directly. Graphical Abstract ᅟ.