Quantification of enteric viruses, pathogen indicators, and Salmonella bacteria in class B anaerobically digested biosolids by culture and molecular methods

Abstract

The most common class B biosolids in the United States are generated by mesophilic anaerobic digestion (MAD), and MAD biosolids have been used for land application. However, the pathogen levels in MAD biosolids are still unclear, especially with respect to enteric viruses. In this study, we determined the occurrence and the quantitative levels of enteric viruses and indicators in 12 MAD biosolid samples and of Salmonella enterica in 6 MAD biosolid samples. Three dewatered biosolid samples were also included in this study for purposes of comparison. Human adenoviruses (HAdV) had the highest gene levels and were detected more frequently than other enteric viruses. The gene levels of noroviruses (NV) reported were comparable to those of enteroviruses (EV) and human polyomaviruses (HPyV). The occurrence percentages of HAdV, HAdV species F, EV, NV GI, NV GII, and HPyV in MAD samples were 83, 83, 42, 50, 75, and 58%, respectively. No hepatitis A virus was detected. Infectious HAdV was detected more frequently than infectious EV, and all infectious HAdV were detected when samples were propagated in A549 cells. Based on most-probable-number (MPN) analysis, A549 cells were more susceptible to biosolid-associated viruses than BGM cells. All indicator levels in MAD biosolids were approximately 10(4) MPN or PFU per gram (dry), and the dewatered biosolids had significantly higher indicator levels than the MAD biosolids. Only two MAD samples tested positive for Salmonella enterica, where the concentration was below 1.0 MPN/4 g. This study provides a broad comparison of the prevalence of different enteric viruses in MAD biosolids and reports the first detection of noroviruses in class B biosolids. The observed high quantitative and infectivity levels of adenoviruses in MAD biosolids indicate that adenovirus is a good indicator for the evaluation of sludge treatment efficiency.

FIG. 1.

The methodology of enteric virus determination by cell culture, ICC-PCR, and qPCR.

FIG. 2.

Indicator levels in biosolid samples (n = 3 dewatered biosolid samples; n = 12 MAD biosolid samples). Error bars represent the standard deviations of the measurement values of samples collected from different sampling events.

FIG. 3.

Enteric virus levels in biosolid samples (n = 3 dewatered biosolid samples; n = 12 MAD biosolid samples). Error bars represent the standard deviations of the measurement values of samples collected from different sampling events.

FIG. 4.

MPN values for enteric viruses propagated in BGM and A549 cells (n = 2 dewatered biosolid samples; n = 8 MAD biosolid samples). Error bars represent the standard deviations of the measurement values of samples collected from different sampling events.