Influence of operating conditions on the persistence of E. coli, enterococci, Clostridium perfringens and Clostridioides difficile in semi-continuous mesophilic anaerobic reactors

International audience; This study examined the combined effect of hydraulic retention time (HRT), organic loading rate (OLR) and heat pretreatment of manure (70 degrees C, 1 h) on the fate of E. coli, enterococci, C. perfringens, C. difficile, and on chemical parameters (volatile fatty acids and ammonia) that may inactivate pathogens. Semi-continuous mesophilic anaerobic reactors were fed with pig manure and horse feed. The operating conditions were 2, 3, 4 COD.L-1.d(-1) (OLR), 24, 35, 46 days (HRT) and use or not of a thermal pretreatment. The levels of the chemical parameters did not reach concentrations capable of inactivating the four bacteria. Anaerobic digestion led to a Log io removal > 3 (E. coli), 0.9-2.1 (enterococci), 0.1-0.6 (C. perfringens) and 0-1 (C. difficile). Increasing HRT only reduced the concentration of E. coli in the digestate. Increasing OLR reduced the Log(10) removal of enterococci and C. difficile. The heat pretreatment led to non-detection of E. coli in the digestate, reduced the concentration of C. perfringens by 0.8-1.3 Log(10) and increased the concentration of C. difficile by 0.04-0.7 Log(10). Enterococci, not detected in the heated manure, were present in the digestate. The distribution of genes encoding virulence factors of C. difficile (tcdA and tcdB) and C. perfringens (cpa, cpb2 and cpb) was not impacted by anaerobic digestion or by the heat pretreatment. Enterococci, C. perfringens, C. difficile were present in the digestate at relatively stable concentrations regardless of the operating conditions, indicating that even with heat pretreatment, the biosafety of digestate cannot be guaranteed in mesophilic conditions.