Unraveling the Dual Role in Enhancing Methane Production and Mitigating Antibiotic Resistance Gene Spread in Anaerobic Co-digestion of Microalgae and Waste Activated Sludge

Waste activated sludge (WAS) is a double-edged sword – a recognized repository for antibiotic resistance genes (ARGs) but also a renewable substrate for methane production. Developing effective WAS treatment strategies is therefore of both ecological and practical importance. In this study, we proposed an anaerobic co-digestion approach of WAS and microalgae Chlorella sp. at a 1:2 ratio (MAcoD-1:2). Results showed that MAcoD-1:2 notably increased cumulative methane production by 52.7%. Co-digestion also demonstrated a significant increase in the abundance of hydrolyzing acidifying bacteria Candidatus_Promineofilum (12.25%) and methanogenic archaea Methanothrix (61.2%). This microbial shift suggested that cosubstrates availability fostered a stable bacterial community structure and synergistic metabolic interactions, thus enhancing methane production. Metagenomic analysis revealed a significant reduction in both ARGs and mobile genetic elements in MAcoD-1:2. Notably, substrate level regulation was found to drive restructuring of microbial communities and metabolic patterns. Investigation showed that the Embden-Meyerhof-Parnas pathways were significantly inhibited while the pentose phosphate pathway was promoted, which constrained the cellular energy budget available for ARG horizontal transfer. Partial least squares path modelling (PLS-PM) further substantiated these findings, revealing methane metabolism negatively affected ARGs (-4.52), whereas confirming its positive correlation with methane production (0.22). Our findings provided distinctive perspectives on WAS resource utilization and novel technologies to inhibit the spread of ARGs.