Population turnover and food-web reorganization sustain acetoclastic methanogenesis at 45 °C in tropical paddy soil

Abstract

Moderately thermophilic temperatures (45 °C) are widely expected to suppress acetoclastic methanogenesis in paddy soils because thermodynamic constraints favor syntrophic acetate oxidation coupled with hydrogenotrophic pathways. However, tropical paddy soils often deviate from this prediction. Here, we used an integrated multi-omics approach combining metabolite profiling, metagenomics, metatranscriptomics, and absolute quantification (qPCR/RT-qPCR) to investigate how methanogenesis is sustained in Philippine paddy soil at 45 °C over 120 days. Contrary to the thermodynamic predictions, methane production remained active, and acetoclastic methanogenesis persisted throughout the incubation, even though transcript abundance declined in the late phase. This resilience was not driven by increased per-copy transcriptional activity but by the population expansion of thermotolerant Methanosarcina lineages affiliated with M. flavescens and M. barkeri , which replaced thermolabile Methanosarcina spp. and Methanotrichaceae . Concurrently, the upstream community reorganized toward Firmicutes- dominated hydrolytic guilds, showing increased expression of carbohydrate-active enzymes that accelerated substrate turnover. Syntrophy-associated modules were activated but did not lead to dominant syntrophic acetate oxidation; instead, acetate turnover remained closely linked to acetoclastic methanogenesis, while propionate oxidation was likely mediated by a Heliobacteriaceae - Methanocellaceae axis. Within dominant Methanosarcina populations, methanogenic pathways shifted over time, with acetoclastic activity prevailing early and methylotrophic expression increasing later, indicating metabolic flexibility. These results demonstrate that moderate warming can trigger soil-specific reorganization of methanogenic food web without universally suppressing acetoclastic methane production, highlighting the need to incorporate community-level thermal adaptation into models of methane emissions from paddy soils.