Fate of pathogenic bacteria in five full-scale biogas plants monitored using cultivation, dPCR, and shotgun...

Current global standards for quantification of pathogenic or indicator bacteria in biogas plants primarily rely on culture-based methods using specific media. However, molecular techniques such as quantitative PCR, digital PCR (dPCR), and shotgun metagenomics are increasingly employed in research and may offer more effective pathogen monitoring for industrial applications. This study analyzed samples from five full-scale biogas plants using traditional culture-based methods, dPCR and shotgun metagenomics to monitor indicator bacteria (Escherichia coli, Enterococcus spp. and Clostridium perfringens) and pathogenic species (Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus and Clostridium botulinum). The DNA extraction protocol was optimized to achieve quantification limits of 1.1 copies of gene g^-1 wet weight, compatible with regulatory thresholds. Comparing the three methods revealed that shotgun metagenomics detected a greater diversity of pathogenic species in biowaste, including S. aureus and C. botulinum. Acidophilic conditions in hydrolysis tank effectively hygienized the biowaste. In contrast, the four agricultural biogas plants showed limited effect on the three indicator bacteria, as indicated by dPCR. This study demonstrates, for the first time, the added value of combining dPCR and shotgun metagenomics to assess pathogen dynamics in biogas plants. Together, these methods provide a more comprehensive and specific view of microbial contaminants, as illustrated by the detection of Enterococcus cecorum in digestates.