Enhancing E. coli removal in mesophilic anaerobic digestion: New insights from inoculum comparison and metagenomic analysis

Anaerobic digestion (AD) process removes most pathogenic bacteria. However, the dominant mechanisms of E. coli removal remain unclear in AD systems. This study employed granular sludge (GS), crushed granular sludge (CGS), and anaerobic sludge (AS) as inocula to investigate E. coli removal mechanisms during mesophilic AD. The results indicated that E. coli removal rate was the highest at CGS groups (90–92 %). Although GS has strong stress resistance of environment, crushing treatment could break archaea distribution structure, thus decreasing negative correlations among archaea. The removal effect of E. coli of acetoclastic methanogen (Methanothrix) was significantly stronger than that of hydrogenotrophic methanogen (Methanoculleus). Methanogenic predicted functional genes upregulate (acetoclastic > hydrogenotrophic > methylotrophic pathways) at CGS and GS groups could effectively increase E. coli removal rate. This study demonstrates that enhancing acetoclastic methanogen activity through inoculum structural optimization is a critical strategy for maximizing E. coli removal in mesophilic AD systems.