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- Materials and methods
A method based on the treatment of sludge with beef extract recovered, with similar efficiency, the three groups of bacteriophages studied from different kinds of sludges. The three groups of bacteriophages were found in high numbers in the different sludge types, the highest value being that of somatic coliphages in primary sludge of a biological treatment plant (1·1 × 105 pfu g−1) and the lowest being that of Bacteroides fragilis phages (110 pfu g−1) in de-watered, anaerobically, mesophilically-digested sludge. All phages studied accumulated in the sludges. In primary and activated sludges, all three types accumulated similarly but in lime-treated sludge and de-watered, anaerobically, mesophilically-digested sludge, the relative proportion of F-specific bacteriophages decreased significantly with respect to somatic coliphages and bacteriophages infecting B. fragilis. All phages survived successfully in stored sludge, depending on the temperature, and again, F-specific bacteriophages survived less successfully than the others.
As a consequence of this concern, methods to evaluate the presence of human viruses in sludge have been developed. First, it is necessary to elute the viruses from the sludges. The methods available are based on two categories of eluants (Hurst et al. 1989). The first category of eluants consists of proteinaceous materials, for example beef extract, that compete with viruses for binding sites. The second category includes solutions that contain various active substances, among which are chaotropic agents, like glycine, that alter the favourability of adsorption. Both kinds of eluants have been used to elute viruses from sludge (Berman et al. 1981; Goyal et al. 1984; Hurst & Goyke 1986; Albert & Schwartzbrod 1991). Once eluted, viruses are detected either by cell culture or by molecular techniques such as PCR, which have the disadvantage that they do not provide information on the infectivity of the viruses. Detecting viruses in eluates of sludge presents problems in addition to those presented by detection of viruses in water. These include the presence in the eluate of substances that are toxic for the cells (Hurst & Goyke 1983) and that inhibit PCR (Graff et al. 1993; Straub et al. 1994).
An investigation is described here which aimed to: (i) select an elution method valid for the three groups of phages and different types of sludges; (ii) determine the occurrence and levels of the three types of phages in different types of sludges; and (iii) obtain information on the persistence of the three groups of bacteriophages at different temperatures of storage in de-watered, mesophilically, anaerobically-digested sludges.
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- Materials and methods
All the methods tested for the extraction of bacteriophages from sludge showed similar efficiency of recovery for somatic coliphages and bacteriophages infecting B. fragilis. For these phages, the recommended method is that using glycine buffer as the eluant as it provided final suspensions of bacteriophages with less bacterial contamination than the other methods. Bacterial contamination impairs the performance of plaque assays used for the enumeration of bacteriophages. However, the method which uses glycine buffer as the eluant was clearly less efficient for the recovery of F-specific bacteriophages than the other two methods, probably because of the sensitivity of F-specific RNA bacteriophages to high pH values. All the methods gave similar results for the different types of sludges tested. Consequently, for this study, the method based on eluting phages with beef extract without acidification of the sludge samples, method 2, was used to evaluate the occurrence and levels of the different groups of phages.
No problems of toxicity to the host bacteria used for the detection of the bacteriophages were observed. This gives methods based on bacteriophages a clear advantage with respect to means of detecting animal viruses which are very sensitive to toxins accumulated in the sludges (Hurst & Goyke 1983; Graff et al. 1993; Straub et al. 1994).
In terms of the levels of bacterial indicators and bacteriophages, the raw sewage in the plants studied did not differ substantially from that of other western countries (Grabow et al. 1984; Havelaar et al. 1984; Nieuwstad et al. 1988). Consequently, the numbers of micro-organisms in the sludges should be similar to those found in similar studies elsewhere.
The viruses present in domestic sewage are mostly bound to sludge particles rather than being present as free suspensions (Lund & Ronne 1973). The removal of viruses from sewage after primary settling may only mean, in practical terms, a transfer of viruses from water to sludge, rather than actual inactivation. The data presented here indicate that the different groups of bacteriophages and bacterial indicators studied are transferred to sludge at a similar rate, as the numbers of the different micro-organisms in the sludge were 5–10-fold greater than in raw sewage. The data on indicator bacteria in the primary sludges studied here are similar to other primary sludge data described elsewhere (Berg & Berman 1980). Available data on bacteriophages are very scarce, but Williams et al. (1988) described studies in which the numbers of coliphages were very similar those described here. Therefore, it can be concluded that bacteriophages accumulate in primary sludge in the same way as bacteria and viruses.
The numbers of micro-organisms found in the sludge obtained after lime-aided settling were lower than those found in primary sludge, and the ratios between their concentrations were different to those for sewage and primary sludge. Bacterial indicators are inactivated more successfully than bacteriophages and as indicated by Metcalf (1978), faecal streptococci survive more successfully than faecal coliform bacteria. With regard to bacteriophages, F-specific bacteriophages appear to be more efficiently inactivated than either somatic coliphages or B. fragilis bacteriophages. Again, this may be explained by the different sensitivity of the phages to high pH. Different sensitivities of viruses to lime treatment have been described (Kock & Strauch 1981).
In activated sludge, the ratios between the concentrations of the bacteriophages studied were similar to those for sewage and primary sludge. Therefore, it may be supposed that the three groups of bacteriophages behave similarly. However, contrary to events which occurred during primary settling, the concentrations of bacteriophages did not increase with respect to their concentrations in sewage. In fact, this may indicate a significant inactivation of phages, as has been reported to occur in human viruses in this kind of waste-water treatment (Malina et al. 1975; Sanders et al. 1979). The relative numbers of bacterial indicators with respect to bacteriophages were lower that in treated sewage, which may indicate that bacterial indicators are inactivated more than bacteriophages in this kind of sewage treatment. However, these results should be interpreted with caution as no special treatment to disrupt the polysaccharidic matrix of the flocs was applied and therefore, bacterial levels described here may have been underestimated.
With regard to the effect of anaerobic, mesophilic digestion and de-watering of sludge, the data presented here indicate that faecal streptococci, somatic coliphages and phages infecting B. fragilis suffer a similar inactivation of less than one log. In contrast, F-specific bacteriophages suffer a decay of approximately 2 logs. Taking into consideration that in the process described here there was a reduction in the final volume of the sludge of 50%, the reduction in faecal streptococci is very similar to that described by Berg & Berman (1980) by anaerobic mesophilic digestion. In addition, the decay in the numbers of somatic coliphages and phages infecting B. fragilis are very similar to those described by these authors for enteroviruses.
The storage of de-watered sludge showed that bacteriophages persist for a long time depending on the temperature, with long periods of persistence at low temperatures, especially for somatic coliphages and phages infecting B. fragilis. These data cannot be compared with the survival of viruses under the same conditions. However, under cool conditions, viruses may persist for long periods in sludge lagoons (Sattar & Westwood 1979).
Although more experimentation is needed, it seems that bacteriophages may be potential model organisms for determining the fate of human viruses in different kinds of sludges, as in the different types of sludges studied, they seem to behave like viruses. Bacteriophages inactivate differently according to the various treatment conditions, as has been described for human viruses (Kock & Strauch 1981; Spillman et al. 1987; Straub et al. 1994). Therefore, the use of more resistant phages is likely to be recommended. Moreover, it has been shown in this paper that phage detection in sludges is easy and that the numbers of the different groups of bacteriophages found in the different sludge types are high enough to be used as model micro-organisms.