Decipher syntrophies and adaptive response towards enhancing conversion of propionate to methane under psychrophilic condition

Propionate is a key intermediate in anaerobic digestion (AD) under low operational temperatures, which can destabilize the process. In this study, the supplementation of syntrophic cold-tolerant consortia and trace elements significantly improved the performance of psychrophilic (20 °C) reactor, increasing methane production to 91 % of mesophilic reactor levels and reducing propionate concentrations to less than 2 % of those in untreated psychrophilic reactors. Multi-omics analyses revealed that psychrophilic conditions downregulated the methylmalonyl-CoA and aceticlastic methanogenesis pathways. Electron paramagnetic resonance analyses detected 2.6E-05mol/L reactive oxygen species as stress metabolites in the inhibited psychrophilic reactors. Conversely, supplementation with syntrophic cold-tolerant consortia and trace elements enhanced the abundance of Smithellaceae, Syntrophobacteraceae, and Methanothrix by fivefold in the bioenhanced reactors. This supplementation broadened the propionate degradation pathways from relying solely on the methylmalonyl-CoA pathway to also incorporating the dismutation pathway, while upregulating both pathways. These changes enhanced methanogenesis from propionate through improved activity of the syntrophic cold-tolerant consortia. Genome-centric metatranscriptomic analysis identified the upregulation of key antioxidant genes (sod, kat, grx), temperature regulation genes (cspA), and cryoprotective genes (pslF, pslH, cysE) within the syntrophic cold-tolerant consortia. Additionally, extracellular polymeric substance (EPS) yield per cell increased in the bioenhanced reactors by up to 1.07-fold compared to RC-P. These metabolic traits emphasize the critical roles in mitigating oxidative stress, adapting to low temperatures, and supporting efficient methanogenesis under psychrophilic conditions. These findings offer insights into the transcriptional responses and adaptive mechanisms of propionate-degrading consortia in response to psychrophilic stress.