Continued current emissions of carbon dioxide (CO₂) and methane (CH₄) by human activities will increase global atmospheric CO₂ and CH₄ concentrations and surface temperature significantly. Fields of paddy rice, the most important form of anthropogenic wetlands, account for about 9% of anthropogenic sources of CH₄. Elevated atmospheric CO₂ may enhance CH₄ production in rice paddies, potentially reinforcing the increase in atmospheric CH₄. However, what is not known is whether and how elevated CO₂ influences CH₄ consumption under anoxic soil conditions in rice paddies, as the net emission of CH₄ is a balance of methanogenesis and methanotrophy. In this study, we used a long-term free-air CO₂ enrichment experiment to examine the impact of elevated CO₂ on the transformation of CH₄ in a paddy rice agroecosystem. We demonstrate that elevated CO₂ substantially increased anaerobic oxidation of methane (AOM) coupled to manganese and/or iron oxides reduction in the calcareous paddy soil. We further show that elevated CO₂ may stimulate the growth and metabolism of Candidatus Methanoperedens nitroreducens, which is actively involved in catalyzing AOM when coupled to metal reduction, mainly through enhancing the availability of soil CH₄. These findings suggest that a thorough evaluation of climate-carbon cycle feedbacks may need to consider the coupling of methane and metal cycles in natural and agricultural wetlands under future climate change scenarios.

File 1 and File 5: We incubated the soils from the two paddy systems to examine whether anaerobic oxidation of methane could be coupled to the reduction of nitrate (NO₃^-), manganese (Mn_(IV)), iron (Fe_(III)) or sulfate (SO₄^2-) in soils (exp. 1). Soil slurries were anaerobically incubated following a completely randomized design (CRD), in which two main factors were ¹³CH₄ (no ¹³CH₄ addition vs +¹³CH₄) and e-acceptor (no e-acceptor addition, +NO₃^-, +Mn_(IV), +Fe_(III), and +SO₄^2-). Each treatment replicated 3 times. We measured ¹³CH₄ consumption, ¹³CO₂ production and Mn²⁺/Fe²⁺ production after incubation.

File 2: We incubated the soils from the rice FACE system to investigate the influence of eCO₂ on the coupling of anaerobic oxidation of methane to metal reduction. Soil slurries were incubated following a split-plot experiment with factorial structure, and whole-plots were organized as a RCBD (3 blocks). The CO₂ treatment (aCO₂ vs eCO₂) was considered the whole-plot factor, while the two nested treatments ¹³CH₄ addition (no ¹³CH₄ addition and +¹³CH₄) and e-acceptor identity (no e-acceptor addition, +Mn_(IV) and +Fe_(III)) constituted a 2×3 factorial structure. Each treatment replicated 3 times (exp. 2). We measured ¹³CH₄ consumption and Mn²⁺/Fe²⁺ production after incubation. Also, We carried out a field study to examine the influence of eCO₂ on the anaerobic transformation of ¹³CH₄ in the paddy system. The experiment was a split-plot design with whole-plots arranged in a RCBD (3 blocks). The CO₂ treatment (aCO₂ vs eCO₂) was the whole-plot treatment, whereas the e-acceptor identity (no e-acceptor addition, +Mn_(IV) and +Fe_(III)) was considered the split-plot factor. There were 3 replicates per treatment (exp. 3).  We measured ¹³CH₄ production and ¹³CO₂ production after incubation.

File 3: We performed qPCR tests using a specific primer set targeting the mcrA gene of Ca. M. nitroreducens to quantify the role of Ca. M. nitroreducens in mediating anaerobic oxidation of methane coupled to metal reduction in soils. We measured the absolute abundance of Ca. M. nitroreducens by qPCR.

File 4: We used metagenomic sequencing to analyze microbial functional genes involved in CH₄ cycling in the field to explore the possible biological mechanisms underlying the eCO₂ effect on the transformation of CH₄. Genomic DNA, for soil metagenomic sequencing, was extracted from 5 g aliquots of field soils sampled at the heading stage of rice plants from aCO₂ and eCO₂ plots in 2015 using PowerSoil® kits, and was then sequenced on the Illumina HiSeq 2500 platform. We analyzed the functional genes using KEGG database.

File 6: We carried out a field study to examine the potential of denitrification in the paddy system. We measured in-situ ³⁰N₂ and ²⁹N₂ production following the addition of ¹⁵NO₃^- after one day incubation in the field. Values are means (n = 3) ± SEM.

File 7: We conducted a sterilization test to determine the involvement of soil microbes in the coupling of anaerobic oxidation of methane to metal reduction. Soil slurries were incubated following a CRD, in which two factors were the sterilization treatment (non-sterilized and sterilized) and the e-acceptor identity (no e-acceptor addition, +Mn_(IV) and +Fe_(III)). There were 3 replicates per treatment.  We measured 13CO2 production after incubation. We also sequenced the 16S rRNA genes of genomic DNA in the calcareous soil sampled from exp. 1.

File 8: We conducted an incubation using the calcareous soil to investigate the influence of CO₂ on the coupling of anaerobic oxidation of methane to iron reduction. Soil slurries were incubated following the additions of CH₄, CH₄+Fe_(III), CH₄+CO₂ or +CH₄+Fe_(III)+CO₂. There were 3 replicates per treatment.  We measured CH₄ consumption after incubation.