Viability and removal assessment of Escherichia coli and Salmonella spp. by real-time PCR with propidium monoazide in the hygienization of sewage sludge using three anaerobic processes

Abstract

Sewage sludge should be stabilized for its beneficial use and pathogens, among other factors, should comply with environmental regulations. Three sludge stabilization process were compared to assess their suitability for producing Class A biosolids: MAD-AT (mesophilic (37 °C) anaerobic digestion (MAD) followed by an alkaline treatment (AT)); TAD (thermophilic (55 °C) anaerobic digester); and TP-TAD (mild thermal (80 °C, 1 h) pretreatment (TP) followed by a TAD). E. coli and Salmonella spp. were determined, differentiating three possible states: total cells (qPCR), viable cells using the propidium monoazide method (PMA-qPCR), and culturable cells (MPN).Culture techniques followed by the confirmative biochemical tests identified the presence of Salmonella spp. in PS and MAD samples, while the molecular methods (qPCR and PMA-qPCR) showed negative results in all samples. The TP + TAD arrangement reduced the concentration of total and viable E. coli cells in a greater extent than the TAD process. However, an increase of culturable E. coli was observed in the corresponding TAD step, indicating that the mild thermal pretreatment induced the viable but non-culturable state in E. coli. In addition, the PMA technique did not discriminate viable from non-viable bacteria in complex matrices. The three processes produced Class A biosolids (fecal coliforms < 1000 MPN/gTS and Salmonella spp, < 3 MPN/gTS) maintaining compliance after a 72 h storage period. It appears that the TP step favors the viable but not culturable state in E. coli cells, a finding that should be considered when adopting mild thermal treatment in sludge stabilization process arrangements.