Quantification of viable but nonculturable Salmonella spp. and Shigella spp. during sludge anaerobic digestion and their reactivation during cake storage

The presence of viable but nonculturable (VBNC) bacterial pathogens which often fail to be detected by cultivation and can regain the cultivability if the living conditions improve were reported. The objective of this study was to determine the occurrence of VBNC Salmonella spp. and Shigella spp. in the biosolids during anaerobic digestion and its reactivation during the cake storage.The occurrence of VBNC Salmonella spp. and Shigella spp. during mesophilic, temperature-phased, thermophilic anaerobic digestion of sewage sludge and the subsequent storage were studied by RT-qPCR and most probable number (MPN) method. The VBNC incidence of Salmonella spp. and Shigella spp. during thermophilic digestion was four orders of magnitude higher than those of mesophilic digestion. Accordingly, higher resuscitation ratio of VBNC pathogens was also achieved in thermophilic digested sludge. As a result, the culturable Salmonella typhimurium contents in thermophilic digested sludge after cake storage were two orders of magnitude higher than mesophilic digestion. Both quantitative PCR and reverse transcription quantitative PCR assay results showed the two bacterial counting numbers remained stable throughout the cake storage.The results indicate that the increase in the culturable Salmonella spp. and Shigella spp. after centrifugal dewatering was attributed to the resuscitation from the VBNC state to the culturable state.Thermophilic anaerobic digestion mainly induced Salmonella spp. and Shigella spp. into VBNC state rather than killed them, suggesting that the biological safety of sewage sludge by temperature-phased anaerobic digestion should be carefully assessed.