Viruses in deep-sea cold seep sediments harbor diverse survival mechanisms and remain genetically conserved within species.

Deep sea cold seep sediments have been discovered to harbor novel, abundant, and diverse bacterial and archaeal viruses. However, little is known about viral genetic features and evolutionary patterns in these environments. Here, we examined the evolutionary ecology of viruses across active and extinct seep stages in the area of Haima cold seeps in the South China Sea. A total of 338 viral operational taxonomic units are identified and linked to 36 bacterial and archaeal phyla. The dynamics of host-virus interactions are informed by diverse antiviral defense systems across 43 families found in 487 microbial genomes. Cold seep viruses are predicted to harbor diverse adaptive strategies to persist in this environment, including counter-defense systems, auxiliary metabolic genes, reverse transcriptases, and alternative genetic code assignments. Extremely low nucleotide diversity is observed in cold seep viral populations, being influenced by factors including microbial host, sediment depth, and cold seep stage. Most cold seep viral genes are under strong purifying selection with trajectories that differ depending on whether cold seeps are active or extinct. This work sheds light on the understanding of environmental adaptation mechanisms and evolutionary patterns of viruses in the sub-seafloor biosphere.

Unexpected genetic and microbial diversity for arsenic cycling in deep sea cold seep sediments.

Cold seeps, where cold hydrocarbon-rich fluid escapes from the seafloor, show strong enrichment of toxic metalloid arsenic (As). The toxicity and mobility of As can be greatly altered by microbial processes that play an important role in global As biogeochemical cycling. However, a global overview of genes and microbes involved in As transformation at seeps remains to be fully unveiled. Using 87 sediment metagenomes and 33 metatranscriptomes derived from 13 globally distributed cold seeps, we show that As detoxification genes (arsM, arsP, arsC1/arsC2, acr3) were prevalent at seeps and more phylogenetically diverse than previously expected. Asgardarchaeota and a variety of unidentified bacterial phyla (e.g. 4484-113, AABM5-125-24 and RBG-13-66-14) may also function as the key players in As transformation. The abundances of As cycling genes and the compositions of As-associated microbiome shifted across different sediment depths or types of cold seep. The energy-conserving arsenate reduction or arsenite oxidation could impact biogeochemical cycling of carbon and nitrogen, via supporting carbon fixation, hydrocarbon degradation and nitrogen fixation. Overall, this study provides a comprehensive overview of As cycling genes and microbes at As-enriched cold seeps, laying a solid foundation for further studies of As cycling in deep sea microbiome at the enzymatic and processual levels.

pH-Dependent Hydrogenotrophic Denitratation Based on Self-Alkalization.

Producing stable nitrite is a necessity for anaerobic ammonium oxidation (anammox) but remains a huge challenge. Here, we describe the design and operation of a hydrogenotrophic denitratation system that stably reduced >90% nitrate to nitrite under self-alkaline conditions of pH up to 10.80. Manually lowering the pH to a range of 9.00-10.00 dramatically decreased the nitrate-to-nitrite transformation ratio to <20%, showing a significant role of high pH in denitratation. Metagenomics combined with metatranscriptomics indicated that six microorganisms, including a Thauera member, dominated the community and encoded the various genes responsible for hydrogen oxidation and the complete denitrification process. During denitratation at high pH, transcription of periplasmic genes napA, nirS, and nirK, whose products perform nitrate and nitrite reduction, decreased sharply compared to that under neutral conditions, while narG, encoding a membrane-associated nitrate reductase, remained transcriptionally active, as were genes involved in intracellular proton homeostasis. Together with no reduction in only nitrite-amended samples, these results disproved the electron competition between reductions of nitrate and nitrite but highlighted a lack of protons outside cells constraining biological nitrite reduction. Overall, our study presents a stably efficient strategy for nitrite production and provides a major advance in the understanding of denitratation.

Phylogenetically and catabolically diverse diazotrophs reside in deep-sea cold seep sediments.

Microbially mediated nitrogen cycling in carbon-dominated cold seep environments remains poorly understood. So far anaerobic methanotrophic archaea (ANME-2) and their sulfate-reducing bacterial partners (SEEP-SRB1 clade) have been identified as diazotrophs in deep sea cold seep sediments. However, it is unclear whether other microbial groups can perform nitrogen fixation in such ecosystems. To fill this gap, we analyzed 61 metagenomes, 1428 metagenome-assembled genomes, and six metatranscriptomes derived from 11 globally distributed cold seeps. These sediments contain phylogenetically diverse nitrogenase genes corresponding to an expanded diversity of diazotrophic lineages. Diverse catabolic pathways were predicted to provide ATP for nitrogen fixation, suggesting diazotrophy in cold seeps is not necessarily associated with sulfate-dependent anaerobic oxidation of methane. Nitrogen fixation genes among various diazotrophic groups in cold seeps were inferred to be genetically mobile and subject to purifying selection. Our findings extend the capacity for diazotrophy to five candidate phyla (Altarchaeia, Omnitrophota, FCPU426, Caldatribacteriota and UBA6262), and suggest that cold seep diazotrophs might contribute substantially to the global nitrogen balance.

Coupled Aerobic Methane Oxidation and Arsenate Reduction Contributes to Soil-Arsenic Mobilization in Agricultural Fields.

Microbial oxidation of organic compounds can promote arsenic release by reducing soil-associated arsenate to the more mobile form arsenite. While anaerobic oxidation of methane has been demonstrated to reduce arsenate, it remains elusive whether and to what extent aerobic methane oxidation (aeMO) can contribute to reductive arsenic mobilization. To fill this knowledge gap, we performed incubations of both microbial laboratory cultures and soil samples from arsenic-contaminated agricultural fields in China. Incubations with laboratory cultures showed that aeMO could couple to arsenate reduction, wherein the former bioprocess was carried out by aerobic methanotrophs and the latter by a non-methanotrophic bacterium belonging to a novel and uncultivated representative of Burkholderiaceae. Metagenomic analyses combined with metabolite measurements suggested that formate served as the interspecies electron carrier linking aeMO to arsenate reduction. Such coupled bioprocesses also take place in the real world, supported by a similar stoichiometry and gene activity in the incubations with natural paddy soils, and contribute up to 76.2% of soil-arsenic mobilization into pore waters in the top layer of the soils where oxygen was present. Overall, this study reveals a previously overlooked yet significant contribution of aeMO to reductive arsenic mobilization.

A mixed blessing of viruses in wastewater treatment plants.

Activated sludge of wastewater treatment plants harbors a very high diversity of both microorganisms and viruses, wherein the latter control microbial dynamics and metabolisms by infection and lysis of cells. However, it remains poorly understood how viruses impact the biochemical processes of activated sludge, for example in terms of treatment efficiency and pollutant removal. Using metagenomic and metatranscriptomic deep sequencing, the present study recovered thousands of viral sequences from activated sludge samples of three conventional wastewater treatment plants. Gene-sharing network indicated that most of viruses could not be assigned to known viral genera, implying activated sludge as an underexplored reservoir for new viruses and viral diversity. In silico predictions of virus-host linkages demonstrated that infected microbial hosts, mostly belonging to bacteria, were transcriptionally active and able to hydrolyze polymers including starches, celluloses, and proteins. Some viruses encode auxiliary metabolic genes (AMGs) involved in carbon, nitrogen, and sulfur cycling, and antibiotic resistance genes (ARGs) for resistance to multiple drugs. The virus-encoded AMGs may enhance the biodegradation of contaminants like starches and celluloses, suggesting a positive role for viruses in strengthening the performance of activated sludge. However, ARGs would be disseminated to different microorganisms using viruses as gene shuttles, demonstrating the possibility for viruses to facilitate the spread of antibiotic resistance in the environment. Collectively, this study highlights the mixed blessing of viruses in wastewater treatment plants, and deciphers how they manipulate the biochemical processes in the activated sludge, with implications for both environmental protection and ecosystem security.

Biotic shortcut deselenization coupled to abiotic sulfide oxidation enabled pollutants co-removal and products recovery.

Selenate and sulfide are both contaminants which severely pollute water bodies. Respective bioremediation of selenate- and sulfide-contaminated wastewaters requires abundant electron donors and acceptors. Here, we present a novel concept coupling biological selenate to selenite (shortcut deselenization) and chemical sulfide-driven selenite reduction, to remove multiple pollutants simultaneously. Vial tests showed that shortcut deselenization could save at least two thirds of operation time and one third of carbon source, compared to the complete deselenization to elemental selenium. Subsequent co-removal of sulfide and selenite was optimized at reaction pH of ∼10 and reactant molar ratio of ∼4. Using a newly-designed continuous flow system, >95% removal of both selenate and sulfide was achieved by coupling shortcut deselenization to sulfide oxidation. A series of characterization tools revealed that the final collected precipitates were comprised of high-purity hexagonal selenium (97.4%, wt) and inconsiderable sulfur (2.6%, wt). Superior over selenate-reducing solutions generally producing selenium mixed with reagents or microorganisms, the selenium products generated here were highly purified thus very favorable for further recovery and reuse. Overall, this proof-of-concept study provided a promising technology not only for co-removal of multiple pollutants, but also for substantial costs saving, as well as for valuable products recovery.

Methane-dependent selenate reduction by a bacterial consortium.

Methanotrophic microorganisms play a critical role in controlling the flux of methane from natural sediments into the atmosphere. Methanotrophs have been shown to couple the oxidation of methane to the reduction of diverse electron acceptors (e.g., oxygen, sulfate, nitrate, and metal oxides), either independently or in consortia with other microbial partners. Although several studies have reported the phenomenon of methane oxidation linked to selenate reduction, neither the microorganisms involved nor the underlying trophic interaction has been clearly identified. Here, we provide the first detailed evidence for interspecies electron transfer between bacterial populations in a bioreactor community where the reduction of selenate is linked to methane oxidation. Metagenomic and metaproteomic analyses of the community revealed a novel species of Methylocystis as the most abundant methanotroph, which actively expressed proteins for oxygen-dependent methane oxidation and fermentation pathways, but lacked the genetic potential for selenate reduction. Pseudoxanthomonas, Piscinibacter, and Rhodocyclaceae populations appeared to be responsible for the observed selenate reduction using proteins initially annotated as periplasmic nitrate reductases, with fermentation by-products released by the methanotrophs as electron donors. The ability for the annotated nitrate reductases to reduce selenate was confirmed by gene knockout studies in an isolate of Pseudoxanthomonas. Overall, this study provides novel insights into the metabolic flexibility of the aerobic methanotrophs that likely allows them to thrive across natural oxygen gradients, and highlights the potential role for similar microbial consortia in linking methane and other biogeochemical cycles in environments where oxygen is limited.

Making good use of methane to remove oxidized contaminants from wastewater.

Being an energetic fuel, methane is able to support microbial growth and drive the reduction of various electron acceptors. These acceptors include a broad range of oxidized contaminants (e.g., nitrate, nitrite, perchlorate, bromate, selenate, chromate, antimonate and vanadate) that are ubiquitously detected in water environments and pose threats to human and ecological health. Using methane as electron donor to biologically reduce these contaminants into nontoxic forms is a promising solution to remediate polluted water, considering that methane is a widely available and inexpensive electron donor. The understanding of methane-based biological reduction processes and the responsible microorganisms has grown in the past decade. This review summarizes the fundamentals of metabolic pathways and microorganisms mediating microbial methane oxidation. Experimental demonstrations of methane as an electron donor to remove oxidized contaminants are summarized, compared, and evaluated. Finally, the review identifies opportunities and unsolved questions that deserve future explorations for broadening understanding of methane oxidation and promoting its practical applications.

Will a Non-antibiotic Metalloid Enhance the Spread of Antibiotic Resistance Genes: The Selenate Story.

The rapid emergence of antibiotic resistance genes (ARGs) has become an increasingly serious threat to public health. Previous studies illustrate the antibiotic-like effect of many substances. However, whether and how commonly used or existing non-antibiotic metalloids (e.g., selenate) would enhance ARG spread remains poorly known. Here, we tracked the long-term operation of a bioreactor continuously fed with selenate for more than 1000 days. Metagenomic sequencing identified 191 different ARGs, of which the total abundance increased significantly after the amendment of selenate. Network analyses showed that ARGs resisting multiple drugs had very similar co-occurrence patterns, implying a potentially larger health risk. Host classification not only indicated multidrug-resistant species but also distinguished the mechanism of ARG enrichment for vertical transfer and horizontal gene transfer. Genome reconstruction of an ARG host suggested that selenate and its bioreduction product selenite could stimulate the overproduction of intracellular reactive oxygen species, which was confirmed by the direct measurement. Bacterial membrane permeability, type IV pilus formation, and DNA repair and recombination were also enhanced, together facilitating the horizontal acquirement of ARGs. Overall, this study for the first time highlights the ARG emergence and dissemination induced by a non-antibiotic metalloid and identifies ARG as a factor to consider in selenate bioremediation.

A mixed consortium of methanotrophic archaea and bacteria boosts methane-dependent selenate reduction.

Though methane-based selenate reduction has been reported, neither the selenate load nor the removal rate could satisfy practical applications, thus limiting this technique to bio-remediate selenate pollution. In the present study, using a membrane biofilm batch reactor (MBBR), we successfully enriched a consortium performing methane-dependent selenate reduction, with enhanced reduction rates from 16.1 to 28.9 µM-day-1 under a comparable Se concentration to industrial wastewaters (i.e., ~500 µM). During active reduction, 16S rRNA gene copies of Archaea and Bacteria were both increased more than one order of magnitude. Clone library construction and high-throughput sequencing indicated that Methanosarcina and Methylocystis were the only methane-oxidizing microorganisms. The presence of 20 mM bromoethanesulphonate or 0.15 mM acetylene both significantly, but not completely, inhibited methane-dependent selenate reduction, indicating the concurrent contributions of methanotrophic archaea and bacteria. Fluorescence in situ hybridization (FISH) revealed that archaea directly adhered to the surface of the membrane while bacteria were in the outer layer, together forming the mature biofilm. This study highlights the crucial role of both methanotrophic archaea and bacteria in methane-dependent selenate reduction, and lays foundations in applying methane to bio-remediate practical selenate pollution.

Why does sulfate inhibit selenate reduction: Molybdenum deprivation from Mo-dependent selenate reductase.

Selenium pollution has become an increasingly serious global concern. Methane-fed selenate reduction has proven to be of great interest for the bioremediation of selenate-contaminated waters even with the coexistence of nitrate and dissolved oxygen. However, it is unclear if the common concurrent sulfate anion affects selenate removal. To address this question, we first introduced selenate (SeO42-) as the sole influent electron acceptor in a CH4-fed membrane biofilm reactor (CH4-MBfR); then we added different concentrations of sulfate (SO42-). The initial selenate removal efficiency (∼90%) was decreased by 50% in the presence of 15.6 µM of sulfate and completely inhibited after loading with 171.9 µM of sulfate. 16S rRNA gene sequencing showed that the selenate-reducing bacteria decreased after the addition of sulfate. Metagenomic sequencing showed that the abundance of genes encoding molybdenum (Mo)-dependent selenate reductase reduced by >50% when exposed to high concentrations of sulfate. Furthermore, the decrease in the total genes encoding all Mo-oxidoreductases was much greater than that of the genes encoding molybdate transporters, suggesting that the inhibition of selenate reduction by sulfate was most likely via the direct competition with molybdate for the transport system, leading to a lack of available Mo for Mo-dependent selenate reductases and thus reducing their activities. This result was confirmed by a batch test wherein the supplementation of molybdate mitigated the sulfate effect. Overall, this study shed light on the underlying mechanism of sulfate inhibition on selenate reduction and laid the foundation for applying the technology to practical wastewaters.

Characteristics and performances of microalgal-bacterial consortia in a mixture of raw piggery digestate and anoxic aerated effluent.

The piggery digestate of high ammonia was mixed with the anoxic aerated effluent of high nitrate and phosphorus, to cultivate a microalgal-bacterial consortium for simultaneous pollution removal and resource recovery. The highest removal of total inorganic nitrogen was achieved at 324.77 mg/L in 40% piggery digestate mixed with 60% anoxic aerated effluent, along with the most microalgae biomass production. The crude protein and fatty acids of C14-C20 in microalgae cells were 21.80% and 69.78%, indicating that this mixing strategy could produce abundant microalgal biomass suitable for biofuel generation and animal feed. High-throughput sequencing showed that microbial diversity increased and Paenibacillus, Thiopseudomonas and Pseudomonas were the dominant species promoting microalgal growth. Overall, these results provided a new insight of mixing two types of wastewaters for cultivating microalgal-bacterial consortia, to remove contamination and recover nutrients simultaneously.

Organic degrading bacteria and nitrifying bacteria stimulate the nutrient removal and biomass accumulation in microalgae-based system from piggery digestate.

The microalgae-based system has been applied in anaerobic digestate treatment for nutrient removal and biomass production. To optimize its performance in treating piggery digestate, here, commercial bacterial agents, including organic degrading bacteria (Cb) and nitrifying bacteria (Nb), were inoculated into the microalgae-based system dominated by Desmodesmus sp. CHX1 (D). Reactor DN (inoculated with D and Nb) and DCN (inoculated with D, and Cb to Nb at a ratio of 1:2) have better performance on NH4+-N removal, with a final efficiency at 40.26% and 39.87%, respectively, and no NO3--N or NO2--N accumulations. The final total chlorophyll concentration, an indicator of microalgal growth, reached 4.74 and 5.47 mg/L in DN and DCN, respectively, three times more than that in D. These results suggested that high NH4+-N removal was achieved by the assimilation into high microalgal biomass after the inoculation with functional bacteria. High-throughput sequencing showed that the richness of microbial community decreased but the evenness increased by inoculating functional microorganisms. Microalgae aggregating bacteria were Cellvibrio, Sphingobacterium, Flavobacterium, Comamonas, Microbacterium, Dyadobacter, and Paenibacillus. This study revealed that the inoculation with functional bacteria reconstructed the microbial community which benefited for the microalgal growth and nutrient removal, providing a promising strategy for treating highly-concentrated digestate.

How nitrate affects perchlorate reduction in a methane-based biofilm batch reactor.

Nitrate (NO3-) affected perchlorate (ClO4-) reduction in a membrane batch biofilm reactor (MBBR), even though the electron donor, CH4, was available well in excess of its demand. For example, the perchlorate-reduction rate was 1.7 mmol/m2-d when perchlorate was the sole electron acceptor, but it dropped to 0.64 mmol/m2-d when nitrate also was present. The perchlorate-reduction rate returned to 1.60 mmol/m2-d after all nitrate was consumed. Denitratisoma and Azospirillum were main genera involved in perchlorate and nitrate reduction, and both could utilize NO3- and ClO4- as electron acceptors. Results of the reverse transcription-polymerase chain reaction (RT-PCR) showed that transcript abundances of nitrate reductase (narG), nitrite reductase (nirS), and perchlorate reductase (pcrA) increased when the perchlorate and nitrate concentrations were higher. Specifically, pcrA transcripts correlated to the sum of perchlorate and nitrate, rather than perchlorate individually. Analysis based on Density Functional Theory (DFT) suggests that bacteria able to utilize both acceptors, preferred NO3- over ClO4- due to nitrate reduction having lower energy barriers for proton and electron transfers.

Multi-omics reveal various potential antimonate reductases from phylogenetically diverse microorganisms.

While previous work has demonstrated that antimonate (Sb(V)) can be bio-reduced with methane as the sole electron donor, the microorganisms responsible for Sb(V) reduction remain largely uncharacterized. Inspired by the recently reported Sb(V) reductase belonging to the dimethyl sulfoxide reductase (DMSOR) family, this study was undertaken to use metagenomics and metatranscriptomics to unravel whether any DMSOR family genes in the bioreactor had the potential for Sb(V) reduction. A search through metagenomic-assembled genomes recovered from the microbial community found that some DMSOR family genes, designated sbrA (Sb(V) reductase gene), were highly transcribed in four phylogenetically disparate assemblies. The putative catalytic subunits were found to be representatives of two distinct phylogenetic clades of reductases that were most closely related to periplasmic nitrate reductases and respiratory arsenate reductases, respectively. Putative operons containing sbrA possessed many other components, including genes encoding c-type cytochromes, response regulators, and ferredoxins, which together implement Sb(V) reduction. This predicted ability was confirmed by incubating the enrichment culture with 13C-labeled CH4 and Sb(V) in serum bottles, where Sb(V) was reduced coincident with the production of 13C-labeled CO2. Overall, these results increase our understanding of how Sb(V) can be bio-reduced in environments.

Methane oxidation coupled to vanadate reduction in a membrane biofilm batch reactor under hypoxic condition.

This study shows vanadate (V(V)) reduction in a methane (CH4) based membrane biofilm batch reactor when the concentration of dissolved oxygen (O2) was extremely low. V(IV) was the dominant products formed from V(V) bio-reduction, and majority of produced V(IV) transformed into precipitates with green color. Quantitative polymerase chain reaction and Illumina sequencing analysis showed that archaea methanosarcina were significantly enriched. Metagenomic predictive analysis further showed the enrichment of genes associated with reverse methanogenesis pathway, the key CH4-activating mechanism for anaerobic methane oxidation (AnMO), as well as the enrichment of genes related to acetate synthesis, in archaea. The enrichment of aerobic methanotrophs Methylococcus and Methylomonas implied their role in CH4 activation using trace level of O2, or their participation in V(V) reduction.

Anaerobic methane oxidation coupled to chromate reduction in a methane-based membrane biofilm batch reactor.

Chromate can be reduced by methanotrophs in a membrane biofilm reactor (MBfR). In this study, we cultivated a Cr(VI)-reducing biofilm in a methane (CH4)-based membrane biofilm batch reactor (MBBR) under anaerobic conditions. The Cr(VI) reduction rate increased to 0.28 mg/L day when the chromate concentration was ≤ 2.2 mg/L but declined sharply to 0.01 mg/L day when the Cr(VI) concentration increased to 6 mg/L. Isotope tracing experiments showed that part of the 13C-labeled CH4 was transformed to 13CO2, suggesting that the biofilm may reduce Cr(VI) by anaerobic methane oxidation (AnMO). Microbial community analysis showed that a methanogen, i.e., Methanobacterium, dominated in the biofilm, suggesting that this genus is probably capable of carrying out AnMO. The abundance of Methylomonas, an aerobic methanotroph, decreased significantly, while Meiothermus, a potential chromate-reducing bacterium, was enriched in the biofilm. Overall, the results showed that the anaerobic environment inhibited the activity of aerobic methanotrophs while promoting AnMO bacterial enrichment, and high Cr(VI) loading reduced Cr(VI) flux by inhibiting the methane oxidation process.

Dissolved oxygen has no inhibition on methane oxidation coupled to selenate reduction in a membrane biofilm reactor.

Methane oxidation coupled to selenate reduction has been suggested as a promising technology to bio-remediate selenium contaminated environments. However, the effect of dissolved oxygen (DO) on this process remained unclear. Here, we investigate the feasibility of selenate removal at two distinct DO concentrations. A membrane biofilm reactor (MBfR) was initially fed with ∼5 mg Se/L and then lowered to ∼1 mg Se/L of selenate, under anoxic condition containing ∼0.2 mg/L of influent DO. Selenate removal reached approximately 90% without selenite accumulation after one-month operation. Then 6-7 mg/L of DO was introduced and showed no apparent effect on selenate reduction in the subsequent operation. Electron microscopy suggested elevated oxygen exposure did not affect microbial shapes. 16S rDNA sequencing showed the aerobic methanotroph Methylocystis increased, while possible selenate reducers, Ignavibacterium and Bradyrhizobium, maintained stable after oxygen boost. Gene analysis indicated that nitrate/nitrite reductases positively correlated with selenate removal flux and were not remarkably affected by oxygen addition. Reversely, enzymes related with aerobic methane oxidation were obviously improved. This study provides a potential technology for selenate removal from oxygenated environments in a methane-based MBfR.

Methane oxidation coupled to perchlorate reduction in a membrane biofilm batch reactor.

A specially designed CH4-based membrane biofilm batch reactor (MBBR) was applied to investigate anaerobic methane oxidation coupled to perchlorate reduction (AnMO-PR). The 0.21 mM ClO4- added in the first stage of operation was completely reduced in 28 days, 0.40 mM ClO4- was reduced within 23 days in stage 2, and 0.56 mM of ClO4- was reduced within 30 days in stage 3. Although some chlorate (ClO3-) accumulated, the recovery of Cl- was over 92%. Illumina sequencing of the 16S rRNA gene documented that the bacterial community was mainly composed by perchlorate-reducing bacteria (PRB), methanotrophic bacteria, and archaea. Real-time quantitative PCR showed the archaeal 16S rRNA and mcrA genes increased as more ClO4- was reduced, and the predominant archaea belonged to Methanosarcina mazei, which is related to ANME-3, an archaeon able to perform reverse methanogenesis. Several pieces of evidence support that ClO4- reduction by the MBBR biofilm occurred via a synergism between Methanosarcina and PRB: Methanosarcina oxidized methane through reverse methanogesis and provided electron donor for PRB to reduce ClO4-. Because methanotrophs were present, we cannot rule out that they also were involved in AnMO-PR if they received O2 generated by disproportionation of ClO2- from the PRB.