Paddy soil fertilization, organic and inorganic alternative electron acceptors shape microbial community network and determine competitive pathways of Anaerobic Oxidation of Methane

Anaerobic oxidation of methane (AOM) is a globally important CH₄ sink that is offsetting potential CH₄ emission into the atmosphere. The AOM depends on the availability of the alternative to oxygen electron acceptors (AEAs) which can be of inorganic (e.g. NO₃^-, Fe³⁺, SO₄^2-), and organic (e.g. humic acids) origin. Flooded paddy soils are among the ecosystems with pronounced AOM. Due to a variety of fertilization practices, including combinations of mineral (NPK) and organic (pig manure, biochar) fertilizers, there is a range of AEAs available in paddy soil under anaerobic conditions. However, it remains unclear whether (i) AOM has a preferential pathway in paddy soil, and (ii) how do AEAs and fertilization type affect anaerobic microbial interactions. Therefore, we tested the effects of key AEAs – NO₃^-, Fe³⁺, SO₄^2-, and humic acids – on bacterial community structure (by 16s rRNA gene sequencing) in paddy soil with ongoing AOM experiment under mineral and organic fertilization. We hypothesized that incorporation of labeled ¹³C-CH₄ during AOM into CO₂ and phospholipid fatty acid biomarkers (PLFA) along with co-occurrence bacterial network analysis will reveal the preferential AOM pathway as related to a type of fertilization.

Bacterial alpha-diversity was significantly increased after 84-day anaerobic incubation. Pig manure significantly increased the microbial biomass as compared with NPK and Biochar, but the AEAs amendment did not affect the biomass. Anaerobic incubation, fertilization treatments specific biochar and NPK, and AEAs amendments specific SO₄^2- and humic acids were factors contributing to microbiome variation. Network analysis indicated that microbial communities involved in CH₄ cycling (i.e. NC10, sulfate-reducing bacteria, Geobacter, syntrophic bacteria with methanogens and ANME-2) had non-random co-occurrence patterns and was modularized. There were 16 ¹³C-enriched PLFA biomarkers confirming the incorporation of C-CH₄ into bacteria. AOM and ¹³C-PLFA were significantly higher under Pig manure relative to other fertilizations. AOM was more intensive under NO₃^- than Fe³⁺ and humic acids, but was close to zero under SO₄^2- amendment. However, the relative abundance of NC10 phylum which includes organisms performing AOM, and sulfate-reducing bacteria were higher under SO₄^2-. The relative abundance of Geobacter was highest under biochar and NPK fertilization with SO₄^2- and humic acids amendments. Taken together, NO₃^--driven AOM is the most potent AOM pathway in paddy soil, which however co-exists with the AOM pathways via reduction of NO₂^- by NC10 bacteria and reduction of Fe³⁺ and humic acids by consortia of ANME with Geobacter. Consequently, the co-occurrence network and evidence from ¹³C incorporation into CO₂ and PLFAs indicate the multiple competitive pathways of AOM in paddy soil.