DOI: 10.5061/dryad.m905qfv67
title: Data for: Coupled anaerobic methane oxidation and metal reduction in soil under elevated CO2

description: 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.
description: 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
 (NO<sub>3</sub><sup>-</sup>), manganese
 (Mn<sub>(IV)</sub>), iron (Fe<sub>(III)</sub>) or
 sulfate (SO<sub>4</sub><sup>2-</sup>) in
 soils (exp. 1). Soil slurries were anaerobically incubated following a
 completely randomized design (CRD), in which two main factors were
 <sup>13</sup>CH<sub>4</sub> (no
 <sup>13</sup>CH<sub>4</sub> addition vs
 +<sup>13</sup>CH<sub>4</sub>) and e-acceptor (no
 e-acceptor addition, +NO<sub>3</sub><sup>-</sup>,
 +Mn<sub>(IV)</sub>, +Fe<sub>(III)</sub>, and
 +SO<sub>4</sub><sup>2-</sup>). Each treatment
 replicated 3 times. We measured
 <sup>13</sup>CH<sub>4</sub> consumption,
 <sup>13</sup>CO<sub>2</sub> production and
 Mn<sup>2+</sup>/Fe<sup>2+</sup> production after
 incubation. File 2: We
 incubated the soils from the rice FACE system to investigate the influence
 of eCO<sub>2</sub> 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 CO<sub>2</sub> treatment
 (aCO<sub>2</sub> vs eCO<sub>2</sub>) was
 considered the whole-plot factor, while the two nested treatments
 <sup>13</sup>CH<sub>4</sub> addition (no
 <sup>13</sup>CH<sub>4</sub> addition and
 +<sup>13</sup>CH<sub>4</sub>) and e-acceptor
 identity (no e-acceptor addition, +Mn<sub>(IV)</sub> and
 +Fe<sub>(III)</sub>) constituted a 2×3 factorial structure.
 Each treatment replicated 3 times (exp. 2). We measured
 <sup>13</sup>CH<sub>4</sub> consumption and
 Mn<sup>2+</sup>/Fe<sup>2+</sup> production after
 incubation. Also, We carried out a field study to examine the influence of
 eCO<sub>2</sub> on the anaerobic transformation of
 <sup>13</sup>CH<sub>4</sub> in the paddy system.
 The experiment was a split-plot design with whole-plots arranged in a RCBD
 (3 blocks). The CO<sub>2</sub> treatment
 (aCO<sub>2</sub> vs eCO<sub>2</sub>) was the
 whole-plot treatment, whereas the e-acceptor identity (no e-acceptor
 addition, +Mn<sub>(IV)</sub> and
 +Fe<sub>(III)</sub>) was considered the split-plot factor.
 There were 3 replicates per treatment (exp. 3).  We measured
 <sup>13</sup>CH<sub>4</sub> production and
 <sup>13</sup>CO<sub>2</sub> 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 CH<sub>4</sub> cycling
 in the field to explore the possible biological mechanisms underlying the
 eCO<sub>2</sub> effect on the transformation of
 CH<sub>4</sub>. Genomic DNA, for soil metagenomic sequencing,
 was extracted from 5 g aliquots of field soils sampled at the heading
 stage of rice plants from aCO<sub>2</sub> and
 eCO<sub>2</sub> 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 <sup>30</sup>N<sub>2</sub> and
 <sup>29</sup>N<sub>2</sub> production following
 the addition
 of <sup>15</sup>NO<sub>3</sub><sup>-</sup> 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<sub>(IV)</sub> and +Fe<sub>(III)</sub>). 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 CO<sub>2</sub> on the coupling of anaerobic oxidation of methane to iron reduction. Soil slurries were incubated following the additions of CH<sub>4</sub>, CH<sub>4</sub>+Fe<sub>(III)</sub>, CH<sub>4</sub>+CO<sub>2</sub> or +CH<sub>4</sub>+Fe<sub>(III)</sub>+CO<sub>2</sub>. There were 3 replicates per treatment.  We measured CH<sub>4</sub> consumption after incubation.