Elucidating Key Microbial Drivers for Methane Production during Cold Adaptation and Psychrophilic Anaerobic Digestion of Cattle Manure and Food Waste

At psychrophilic temperatures (<20 °C), anaerobic digestion produces less methane (CH₄). For psychrophilic anaerobic digestion (PAD) to be successful, investigation of cold-adapted microbial consortia involved in methane production is critical. This study aimed to investigate the microbial community driving enhanced methane production from the cold-adaptation process and bioaugmentation of PAD with cold-adapted inoculum (BI). Microbial consortia in cattle manure (CM) and food waste (FW) were adapted and applied during batch PAD of CM and FW to bioaugment methane production at 15 °C. Cold adaptation and PAD with BI resulted in cumulative specific methane yields of 0.874 ± 0.231 and 0.552 ± 0.089 L CH₄ g⁻¹ volatile solids, respectively, after 14 weeks, while the absence of BI (control) led to acidification and no methane production during PAD. Following 16S rRNA V4–V5 amplicon sequencing and metagenomic analyses, Methanosarcina was revealed as a key driver of methanogenesis during cold adaptation and PAD bioaugmentation. Furthermore, based on the predictive functional and metabolic analysis of the communities, possible synergies were proposed in terms of substrate production and utilization by the dominant microbial groups. For instance, during methane production, Bacteroides and Methanobrevibacter were possibly involved in a syntrophic relationship, which promoted methanogenesis by Methanosarcina. These findings provide insight into the prospective microbial synergies that can be harnessed and/or regulated in cold-adapted inoculum for the improvement of methane production during PAD.