Enumeration of Bacteriophages and Host Bacteria in Sewage and the Activated-Sludge Treatment Process

Abstract

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 x 107, 9.5 x 107, and 8.4 x 107/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 x 107 and 1.3 x 109/ml, respectively, which represented only 3 and 6.8%

A century of bacteriophage research and applications: impacts on biotechnology, health, ecology and the economy!

“…133 They are abundantly present on Earth, at total numbers estimated at >10 31 for aquatic environments, >10 7 per gram of soil and >10 8 -10 10 phages per millilitre of sewage. [134][135][136][137][138] The pioneering work of d'Herelle at the Institut Pasteur in Paris that continued at the now EIBMV in Tbilisi, Georgia, led to the successful application of phage therapy. About 120 scientists and 700 technicians worked at the Eliava Institute.…”

Metagenomic analysis of viruses in reclaimed water

“…In addition, high VLP concentrations in reclaimed water samples may reflect the abundance of phages in treated effluent. A study investigating bacteriophage populations in an activated sludge system found that there was a net production of phages in the system, with total phage concentrations in the supernatant of an activated-sludge reactor and the unchlorinated effluent measuring as high or higher than those in sewage entering the reactor (Ewert and Paynter, 1980). The abundance of phages in reclaimed water was also supported by TEM of viral concentrates, where an abundance of phage-like particles was observed compared with other VLPs (examples of VLPs shown in Fig.…”

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

“…Viruses are known to be important components of marine and freshwater microbial communities (Paul et al 1991;Suttle and Chan 1994;Suttle 1994;Fuhrman and Noble 1995;Fuhrman 1999;Wommack and Colwell 2000). Furthermore, surprisingly large numbers of viruses (such as bacteriophage) have been detected in activated sludge samples [10 7 -10 8 particles ml )1 (Ewert and Paynter 1980) and 10 8 -10 9 particles ml )1 (Otawa et al 2007)], at levels comparable to or greater than their numbers in any other aquatic environments. Thus, it is probable that viruses exert considerable effects on population dynamics of the bacteria in activated sludge (Otawa et al 2005(Otawa et al , 2007Lee et al 2006a,b).…”

“…Metagenomic analyses suggest a high diversity of viral species in aquatic environments and the existence of previously unknown viral genes (Breitbart et al 2002;Zhang et al 2006). Viral community dynamics in natural environments have been determined mainly with the techniques like plate assays (Adams 1959;Khan et al 2002a,b;Lee et al 2004Lee et al , 2006a, pulse-field gel electrophoresis (PFGE) (Wommack et al 1999a,b;Otawa et al 2007), direct counting by transmission electron microscopy (Ewert and Paynter 1980), fluorescence microscopy (Noble and Fuhrman 1998;Otawa et al 2007), or flow cytometry (Marie et al 1999). However, no simple tools for monitoring viral dynamics have been reported in the literature.…”