Induction and persistence of VBNC pathogens during high solid pig manure anaerobic digestion, dewatering, and...

Sanitization is a critical foundation for the safe treatment of animal manure. High-temperature anaerobic digestion (AD) is often recommended for pathogen inactivation. However, heat stress may induce microorganisms into a viable-but-non-culturable (VBNC) state rather than truly inactivating them. This study therefore investigated the fate of Escherichia coli, Salmonella, and Enterococcus in pig manure during biothermal hydrolysis-enhanced AD (55 °C followed by 37 °C) and compared it with single-phase mesophilic AD (37 °C). In the two-phase process, culturable E. coli and Salmonella were reduced to below the detection limit (< 1 CFU/g fresh matter) at 55 °C, while Enterococcus achieved a > 5 log₁₀ reduction. Conversely, high levels of culturable E. coli and Enterococcus persisted in the single-phase mesophilic AD. However, propidium monoazide-quantitative PCR revealed substantial VBNC counts (5.6 log₁₀ - 7.8 log₁₀ cells/g fresh matter) in both systems' effluents. Imaging flow cytometry, excitation-emission matrix fluorescence spectroscopy, and ATP measurements further confirmed that these VBNC cells retained membrane integrity and energy metabolism. VBNC cells also showed reduced cell size and amino acid/protein-related signals, indicating a low-metabolic but persistent state. During 129-day storage at ambient temperatures rising from 24 to 30 °C, culturable Salmonella increased to 2 log₁₀-4 log₁₀ CFU/g fresh matter in both digestates while culturable E. coli increased only in the single-phase digestate, likely due to proliferation of residual culturable cells or resuscitation of VBNC cells. These findings reveal that conventional culture-based methods underestimate the actual pathogen load. Optimizing the AD process is therefore necessary to truly eliminate VBNC pathogens for safe land application.