Rapid quantification of total viral DNA in the supernatants of activated sludge samples with the fluorescent dye PicoGreen^®

Abstract

Aims: To develop a rapid and simple method for quantifying viral DNA concentrations and determining viral quantities in activated sludge. Methods and Results: Activated sludge samples were obtained from three full‐scale and one laboratory‐scale process. They were centrifuged and the supernatant was filtered through a 0·2‐μm membrane filter. Free DNA was removed by DNase‐I treatment; any DNA within the viral capsid was liberated by heat treatment and proteinase K, and viral DNA concentrations were determined using the dye PicoGreen®. To validate the method, we assessed the recovery of T4 phage added to filtered samples, which was 99% of those added. Viral DNA concentrations in samples from full‐scale plants ranged from 69 to 157 ng ml−1. Monitoring of laboratory‐scale reactor samples revealed that viral DNA concentrations varied with time. Our method involves a simple sample treatment protocol and allow rapid analysis of many samples. Conclusions: A simple, rapid and sensitive method was developed and successfully used to determine the viral DNA concentrations in activated sludge. Significance and Impact of the Study: This method provides a way to investigate impact of bacteriophages on the performance of wastewater treatment processes.

Dynamics of dsDNA Viruses Hosted by Indigenous Microorganisms in Activated Sludge in the Treatment of Synthetic Wastewater

“…In the original method to determine viral dsDNA concentrations developed by Otawa et al (2008), viral dsDNA concentrations were determined after treatment with DNase I. In the present study, the authors determined viral dsDNA concentrations without treatment with DNase I.…”

“…The concentrations of viral dsDNA were determined according to the method developed by Otawa et al (2008) with a modification. The original procedure by Otawa et al (2008) is briefly as follows activated sludge mixed liquor is centrifuged, the supernatant is filtered through a 0.2 μm membrane filter (Advantec, Tokyo, Japan), the filtrate is treated with DNase I (20 μg/mL, Funakoshi, Tokyo, Japan) for 30 min at 37°C, viral DNA is extracted from viral capsid by heating at 65°C for 60 min in the presence of 5 μg/mL proteinase K (MP Biomedicals LLC, USA) and 20 mM EDTA (Wako, Osaka, Japan), and finally, the concentrations of dsDNA are determined using a Quant-iT™ PicoGreen ® dsDNA reagent and kit (Invitrogen, USA) according to the manufacturer's instructions.…”

The Population Dynamics of Nitrifiers in Ammonium‐Rich Systems

“…High-ammonium-concentration wastewater typically also contains toxic substances; therefore, separate treatment is recommended instead of mixing with municipal wastewater. It is also suspected that nitrification suppression can be caused by meio-and microfaunal grazing (Ghyoot and Verstraere, 2000;Stief et al, 2003); allelopathic compounds excreted by heterotrophic bacteria (Stief et al, 2003); or viral infections (Otawa et al, 2009;Stief et al, 2003). Because nitrification (nitritation or nitratation) is a process of conversion of nitrogen form (not removal) and its products are used in further processes leading to nitrogen removal (denitrification, anammox), stability and undisturbed efficiency of nitrification are crucial for the entire nitrogen removal process.…”