Determination of virus abundance, diversity and distribution in a municipal wastewater treatment plant

Abstract

Procedures to extract and count sludge viruses-like particles from municipal sewage treatment plant were optimized by epifluorescence microscopy using SYBR Green I as a stain. The highest indigenous virus yields from the bulk of the anaerobic digestion sludge and influent (solid) were obtained by utilizing 10 mM sodium pyrophosphate as eluant solution with vortex and 1 min of sonication. The use of 1x phosphate buffered saline as eluant with vortex and 1 min of sonication yields highest indigenous virus from activated sludge. The efficiency of extracting indigenous viruses by sodium pyrophosphate-ultrasound treatment was about 62% of the extractable virus particles from activated sludge and 87% for anaerobic digestion sludge, respectively. Samples treated with DNase had decreased, but not significant, virus counts, suggesting a minor effect of extracellular DNA on virus count. Following the optimized procedure, we investigated the abundance and diversity of virus particles in the wastewater stream of a municipal treatment plant. The concentrations of virus particles ranged from 0.28 x 10(9) ml(-1) to 27.04 x 10(9) ml(-1). Transmission electron microscopy (TEM) showed a high variety of virus morphotypes in sludge. Pulsed-field gel electrophoresis (PFGE) revealed a diverse and dynamic viral community in different stages of the system with genome sizes ranging from 33 kb to >350 kb with most of the viral DNA in the 30-80 kb and 200-350 kb size ranges. Collectively, our study suggested that indigenous viruses are abundant and dynamic in the municipal wastewater treatment system and may play an important role in functioning of the system.