Pathogen Characterization of Fresh and Stored Mesophilic Anaerobically Digested Biosolids

Abstract

Culturable bacterial pathogens (Campylobacter, Salmonella, Listeria, Yersinia) and indicators (E. coli, enterococci, Clostridium perfringens) were quantified at six water resource recovery facilities that land apply anaerobically digested biosolids in Ontario, Canada. Cryptosporidium parvum and Giardia lamblia were also quantified by polymerase chain reaction (PCR). Salmonella and Listeria were frequently detected in sludge and liquid biosolids (70-100% of samples) but less often in fresh dewatered cake biosolids (50-60%); with low levels in fresh cake (<100 cells/g dw). Yersinia were in 20 to 30% of samples, typically at very low levels (<10 cell/g dw). Giardia and Cryptosporidium were detected in 80 and 20% of cake biosolids at geometric means of 270 cysts/g dw and 70 oocysts/g dw, respectively. E. coli reduction was typically >2-log10 while pathogen reduction was variable. "Sudden increase" of pathogens was not observed, however, Salmonella and E. coli showed regrowth (at 1 to 3 orders of magnitude) after 2- to 3-day storage at 30 °C.

Pathogen Survey of Pulp and Paper Mill Biosolids Compared with Soils, Composts, and Sewage Biosolids

“…Mesophilic anaerobically digested municipal sewage biosolids (MAD; digested at 36–37°C for 14–15 d) were also investigated from four generators (TS 2% [liquid] to 24% [dewatered] dry wt. ; n = 12) that are land applied in Ontario (Facilities 3, 4, and 5 in Flemming et al, 2017).…”

“…Verotoxigenic E. coli was screened in 10% of E. coli colonies per sample using a Vero cell assay (Konowalchuck et al, 1977). Enterococci were enumerated by plate counts as described in American Public Health Association (1998) on mEnterococcus agar, and C. perfringens were enumerated by spread plating using a modification of USEPA (1996) method on mClostridium Perfringens agar as described by Flemming et al (2017). Salmonella spp.…”

“…Salmonella spp. were enumerated from four or more decimal dilutions in a three‐tube most probable number (MPN) assay (USDA, 1998a), as described by Flemming et al (2017). Campylobacter were enumerated by a modified three‐tube MPN method (USDA, 1998b) that included Cefoperazone (Sigma) at 30 mg L −1 before incubation and cultured anaerobically on modified campylobacter charcoal differential agar (Flemming et al, 2017).…”

“…DNA was extracted using the QIAamp Stool Kit protocol with modifications (QIAGEN Inc.) as described in Flemming et al (2017). The same DNA extraction was used to enumerate all bacterial pathogens tested in a sample.…”

“…The modified semiquantitative PCR MPN method (as described above) was performed using primers HB‐1 and HB‐2 (Chung et al, 1999) for C. parvum and primers GGLa, GGRa, GGLa‐1, GGLa‐2, and GGRa‐1 (Goulden, 2003) for Giardia intestinalis as described by Flemming et al (2017).…”