Reduction of Escherichia coli load and pathogenic risk in high-solid pig manure anaerobic digestion: a comparative study of plug-flow Reactor and CSTR systems.

Anaerobic digestion has been widely used for animal manure treatment, yet important questions remain regarding its effectiveness in pathogen inactivation and reduction of pathogenic risk. In particular, the influence of reactor configuration on pathogen inactivation remains insufficiently understood, as continuous stirred tank reactors (CSTRs) have shown variable effectiveness, while the potential of plug flow reactors (PFRs) has received far less attention. Therefore, this study compared long-term anaerobic digestion systems operated as CSTRs and PFRs to investigate their performance in reducing the abundance, pathogenicity, and overall pathogenic risk of Escherichia coli. Pathogenicity was evaluated by combining Caenorhabditis elegans infection assays with virulence factor gene profiling, enabling assessment of both phenotypic virulence and its underlying genetic determinants. The results showed that the PFR reduced E. coli levels by 3.3 log10, compared to 2.6 log10 in the CSTR. Crucially, the C. elegans assay indicated that E. coli isolates from the PFR exhibited lower virulence potential than those from the CSTR. Among the 88 virulence factor genes identified, 12 were significantly associated with pathogenicity in the infection assay. Consequently, the overall pathogenic risk, estimated by integrating bacterial abundance with isolate-specific pathogenicity, decreased in both reactors, with the PFR demonstrating superior efficiency. Furthermore, these findings emphasize the importance of integrating both genotypic and phenotypic pathogenicity assessments to achieve more reliable risk evaluation and control in manure treatment systems.