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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. [ABSTRACT FROM AUTHOR]

Aims: To isolate and characterize lytic-bacteriophages specific to Microlunatus phosphovorus, and prepare fluorescently labelled phages (FLPs) for the rapid detection of the host bacterium in activated sludge. Methods and Results: Isolation of bacteriophages lytic to M. phosphovorus was attempted by applying supernatants of activated sludge processes on the lawn of M. phosphovorus JCM9379 for plaque formation. Thirteen bacteriophage isolates were obtained. The restriction fragment length polymorphism analysis distinguished them into two different bacteriophages designated as φMP1 and φMP2. They were found to possess double-stranded DNA and host specificity. Morphological observations were done by electron microscopy. The bacteriophage particles stained by SYBR Green I was shown to be applicable to detect their host bacterial cells mixed with activated sludge. Conclusions: Two M. phosphovorus-specific bacteriophages were isolated and classified as Siphoviridae. FLPs of them were prepared, and successfully applied to detect the host bacterium added into the activated sludge. Significance and Impact of the Study: At least some of bacteria in activated sludge are susceptible to their related bacteriophages. Bacteriophages lytic to activated sludge bacteria could be affecting the bacterial population in activated sludge. The FLPs could be used for the easy-rapid detection of their host bacterium in activated sludge. [ABSTRACT FROM AUTHOR]

In the last decade, molecular biology has made significant progress, and innovative molecular methods have become available to analyze microbial community structures. Among them, we applied the FISH (Fluorescent in situ Hybridization) method to analyze activated sludge in wastewater treatment plants (WWTPs). As a result, domain- or division-level community structures in activated sludge were determined successfully without cultivation. We also applied the PCR (polymerase chain reaction) -DGGE (Denaturing Gradient Gel Electrophoresis) method for laboratory nitrifying sludge in order to investigate more detailed microbial community structure. By this method, genus- or species-level community structures were characterized well. This method was also found to be powerful for monitoring the change of microbial community structures. For example, the behavior of Nitrosomonas group was successfully detected in the reactor with nitrification by the PCR-DGGE method. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]