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• Methane and electron acceptors intersected in the groundwater table fluctuation (GTF) zone. • Anaerobic oxidation of methane (AOM) activity was notably high in the GTF zone. • Ca. Methanoperedens was highly abundant (85.4 % of Archaea) and active in the GTF zone. • Ca. Methanoperedens GTF50 oxidizes methane via the "reverse methanogenesis" pathway. Rice paddies are globally important sources of methane emissions and also active regions for methane consumption. However, the impact of fluctuating groundwater levels on methane cycling has received limited attention. In this study, we delved into the activity and microbial mechanisms underlying anaerobic oxidation of methane (AOM) in paddy fields. A comprehensive approach was employed, including 13C stable isotope assays, inhibition experiments, real-time quantitative reverse transcription PCR, metagenomic sequencing, and binning technology. Geochemical profiles revealed the abundant coexistence of both methane and electron acceptors in the groundwater table fluctuation (GTF) zone, at a depth of 40–60 cm. Notably, the GTF zone exhibited the highest rate of AOM, potentially linked to the reduction of iron oxides and nitrate. Within this zone, Candidatus Methanoperedens (belonging to the ANME-2d group) dominated the Archaea population, accounting for a remarkable 85.4 %. Furthermore, our results from inhibition experiments, RT-qPCR, and metagenome-assembled genome (MAG) analysis highlighted the active role of Ca. Methanoperedens GTF50 in the GTF zone. This microorganism could independently mediate AOM process through the intriguing "reverse methanogenesis" pathway. Considering the similarity in geochemical conditions across different paddy fields, it is likely that Ca. Methanoperedens-mediated AOM is prevalent in the GTF zones. [Display omitted] [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Degradation of organic contaminant by AOM microbes is first investigated. • High MO concentration inhibits its degradation in the AOM system. • Candidatus Methanoperedens can degrade MO but Candidatus Methylomirabilis cannot. • Candidatus Methanoperedens reduce MO alone or together with Pseudoxanthomonas. Abstract Anaerobic oxidation of methane (AOM) microorganisms widespread in nature and they are able to utilize methane as electron donor to reduce sulfate, nitrate, nitrite, and high valence metals. However, whether persistent organic contaminants can also be degraded remains unknown. In this study, the organic pollutant methyl orange (MO) was used to address this open question. The initial concentration of MO affected its degradation efficiency and higher concentration (>100 mg/L) caused considerable inhibition. A 13CH 4 isotope experiment indicated that methane oxidation was involved in MO degradation, which produced N , N -dimethyl- p -phenylenediamine, and 4-aminobenzenesulfonic acid corresponded stoichiometrically. During the long-term experiment, the maximum degradation rate was 47.91 mg/(L·d). The percentage of Candidatus Methanoperedens and Pseudoxanthomonas significantly increased after 30-d of MO degradation under CH 4 conditions; moreover, Candidatus Methanoperedens dominated (46.83%) the microbial community. Candidatus Methanoperedens , either alone or in combination with Pseudoxanthomonas , utilized methane as the sole carbon source to degrade MO via direct interspecies electron transfer or the syntrophy pathway. This study will add to our understanding of the functions and applications of AOM microorganisms. [ABSTRACT FROM AUTHOR]

Methane is a very potent greenhouse gas and can be oxidized aerobically or anaerobically through microbe-mediated processes, thus decreasing methane emissions in the atmosphere. Using a complementary array of methods, including phylogenetic analysis, physiological experiments, and light and electron microscopy techniques (including electron tomography), we investigated the community composition and ultrastructure of a continuous bioreactor enrichment culture, in which anaerobic oxidation of methane (AOM) was coupled to nitrate reduction. A membrane bioreactor was seeded with AOM biomass and continuously fed with excess methane. After 150 days, the bioreactor reached a daily consumption of 10 mmol nitrate · liter-1 · day-1. The biomass consisted of aggregates that were dominated by nitratedependent anaerobic methane-oxidizing "Candidatus Methanoperedens"-like archaea (40%) and nitrite-dependent anaerobic methane-oxidizing "Candidatus Methylomirabilis"-like bacteria (50%). The "Ca. Methanoperedens" spp. were identified by fluorescence in situ hybridization and immunogold localization of the methyl-coenzyme M reductase (Mcr) enzyme, which was located in the cytoplasm. The "Ca. Methanoperedens" sp. aggregates consisted of slightly irregular coccoid cells (-1.5-μm diameter) which produced extruding tubular structures and putative cell-to-cell contacts among each other. "Ca. Methylomirabilis" sp. bacteria exhibited the polygonal cell shape typical of this genus. In AOM archaea and bacteria, cytochrome c proteins were localized in the cytoplasm and periplasm, respectively, by cytochrome staining. Our results indicate that AOM bacteria and archaea might work closely together in the process of anaerobic methane oxidation, as the bacteria depend on the archaea for nitrite. Future studies will be aimed at elucidating the function of the cell-to-cell interactions in nitrate-dependent AOM. [ABSTRACT FROM AUTHOR]