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Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences

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.

Urban sewage treatment is accompanied by large-scale sludge production and numerous micro-organisms are present in this sludge. The presence of micro-organisms in sludge is a potential health risk as a consequence of the current ultimate disposal and utilization practices of sludge (i.e. ocean disposal, lagoons, land application, landfill and incineration). Human enteric viruses appear among the pathogens found in sludges. Indeed, examinations of raw sludges by different authors in various developed countries of different geographical areas show that the numbers of enteric viruses may range from 1000 to more than 50 000 plaque forming units per litre (pfu l−1) (Berg & Berman 1980; Schwartzbrod & Mathieu 1986; Williams & Hurst 1988) with numbers of adenovirus (Williams et al. 1988) and rotavirus (Bosch et al. 1986) even higher. Concern has been expressed over the destruction of viruses after sludge processing by anaerobic digestion, heat treatment, irradiation, composting and lime treatment (Ward & Ashley 1976, 1978; Ward 1977; Berg & Berman 1980; Epp & Metz 1980; Kock & Strauch 1981; Bosch et al. 1986; Williams et al. 1988; Straub et al. 1994). Concern also exists over the fate of viruses in actual sludge application sites (Sattar & Westwood 1979; Bitton & Farrah 1980).

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).

Surrogate indicators provide an alternative approach. For water, phages of enteric bacteria have been proposed (IAWPRC Study Group 1991). The phages being studied for this purpose are somatic coliphages (Hilton & Stotzky 1973), F-specific RNA bacteriophages (Havelaar et al. 1984) and phages infecting Bacteroides fragilis (Tartera & Jofre 1987). Information on bacteriophages in sludges is scarce. However, the data available indicate that they may be a useful tool with which to model the fate of human enteric viruses in sludges (Ohgaki et al. 1986; Traub et al. 1986; Williams et al. 1988; Ketranakul & Ohgaki 1989; Ketranakul et al. 1991). In addition, information on the elution of bacteriophages from either sludges or sediments is rare, although both kinds of eluants mentioned above have been used (Williams et al. 1988; Jofre et al. 1989; Ketranakul & Ohgaki 1989).

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences

Phage detection

Escherichia coli CN13, grown on nutrient agar containing nalidixic acid (1 × 10−1g l–l), was used for the quantification of somatic coliphages (Payment & Franco 1993). Escherichia coli HS (pF ampR), grown on tryptone agar supplemented with streptomycin (15 × 10–3g l–l) and ampicillin (15 × 10–3 g l–l), was used for the quantification of F-specific bacteriophages (Debartolomeis & Cabelli 1991). Bacteroides fragilis HSP40, grown on Bacteroides phage recovery medium (BPRM) (Tartera et al. 1992) was used in the quantification of B. fragilis phages. All phages were quantified by the double agar layer method (Adams 1959).

Bacteriological determinations

Faecal bacteria were quantified in 10 g of sludge sample after suspension in 0·1 litre 1/4 Ringer's solution and 30 min magnetic stirring at room temperature. After convenient dilutions had been made, membrane filter assays for faecal coliforms and faecal streptococci were done according to standard methods (Anonymous 1992).

Sludge samples

Different kinds of sludges obtained from two water treatment plants were studied. The levels of bacterial indicators and bacteriophages from the incoming raw sewage from the two plants are shown in Table 1.

Table 1.  Geometric mean (standard deviation) of counts of the indicated micro-organisms 0·1 l−1 of incoming raw sewage in the two water treatment plants studied
 Plant A (n = 16)Plant B (n = 12)
Faecal coliforms2·6×107 (2·1×107)2·1×107 (2·1×107)
Faecal streptococci1·2×106 (8·6×105)1·2×106 (7·4×106)
Somatic coliphages5·3×105 (3·3×105)7·0×105 (4·2×106)
F-specific phages8·1×104 (9·6×104)2·1×105 (3·3×105)
Bacteroides fragilis phages2·7×103 (4·3×103)2·9×103 (4·8×103)

Plant A is a physicochemical plant (1 400 000 equivalent inhabitants) with two alternative treatments. During the winter, treatment at this plant consists of a primary settling whereas in the summer, it consists of lime-aided settling. Two kinds of sludge were studied from this plant: primary sludge and the sludge obtained after lime treatment.

Plant B is a biological plant (400 000 equivalent inhabitants) that includes a primary settling and an aerated activated sludge step followed by an anaerobic, mesophilic sludge digestion. Three kinds of sludges were studied from this plant: primary, obtained after a double sedimentation, activated, and de-watered, anaerobically, mesophilically digested. The latter was obtained as follows. A mixture of primary (two thirds) and activated sludge (one third) underwent anaerobic digestion at 35 °C for about 25 d. It was then treated with a solution of synthetic organic polyelectrolyte flocculant prior to mechanical dehydration by means of centrifugation to reach a moisture content of about 75%, with a reduction in volume of approximately 50%.

Sludge samples were transported to the laboratory within 2 h of collection and hand shaken until well mixed; appropriate volumes were then dispensed into beakers and stored, for a maximum of overnight, at 4 °C until processing.

Water samples

Incoming raw sewage from the two plants was collected in sterile containers, transported to the laboratory within 2 h of collection and stored for a maximum of overnight at 4 °C until testing for bacteria and bacteriophages.

Elution of bacteriophages from sludges

Three methods were assayed for the isolation of bacteriophages from primary and activated sludges.

Method 1.

Method 1 was that described by Williams & Hurst (1988) for the elution of somatic coliphages from sludges, with slight modifications. Briefly, each sludge sample was adjusted to pH 4·5 with 1 mol l−1 HCl; 0·05 mol l−1 AlCl3 was then added to a final concentration of 0·0005 mol l−1. Each sample was centrifuged at 1400 g for 15 min and the supernatant fluid discarded. The sediment was resuspended using a volume of a solution of 10% beef extract in water, pH 7. The volume of beef extract solution used for suspending the sediment was equal to 10 times the sediment volume. After 15 min of magnetic stirring, the suspension was centrifuged at 10 000 g at 4 °C for 30 min. The supernatant fluid was then assayed for phages without any further decontaminating treatment.

Method 2.

Method 2 was as method 1, but without acidification of the sludge to pH 4·5. Instead, the sludge was neutralized to pH 7·2.

Method 3.

Method 3 is that described by Jofre et al. (1989) for the elution of bacteriophages infecting Bacteroides fragilis and somatic coliphages from marine sediments. Briefly, each sludge was centrifuged at 1400 g for 15 min and the supernatant fluid discarded. The pellet was resuspended using a volume of 0·25 mol l−1 glycine buffer, pH 10·5. The volume of glycine buffer used for suspending the sediment was equal to 10 times the sediment volume. After adjusting the pH of the suspension to pH 10·5 with 1 mol l−1 NaOH, and 15 min of magnetic stirring, the suspension was centrifuged at 10 000 g at 4 °C for 30 min. The supernatant fluid was immediately neutralized to pH 7·2 with 1 mol l−1 HCl and assayed for phages without any further decontaminating treatment.

For the isolation of bacteriophages from de-watered, anaerobically-digested sludge, two variants of methods 2 and 3 were used. As the sludge was already de-watered, it was processed in the same way as the sediment (pellet) of the first centrifugation of the primary and secondary sludges.

After comparison of the methods, method 2 was used to determine the levels of the different bacteriophages in the various sludge samples studied.

Survival of bacteriophages in de-watered, anaerobically, mesophilically-digested sludge

Sludge was placed in 1 litre glass bottles, which were wrapped with foil to avoid the effect of light, and placed at 4, 20 and 37 °C. A 25 g aliquot of each sample was removed at day 0, 10, 20 and 45, processed by method 2 and the bacteriophages assayed.

Statistical analysis

The Statistical Package for Social Science (SPSS V 6·01 Windows, Chicago, IL, USA) was used for the descriptive statistics, and the Mann–Whitney U non-parametric test was performed in order to determine the significance of the differences between the series of results.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences

Efficiency of the methods assayed for the elution of bacteriophages

The data shown in Table 2 allow comparisons to be made of the efficiency of the methods tested for the three groups of bacteriophages and different types of sludges. Methods 1 and 2 recovered phages of the three groups with similar efficiencies, in spite of the different physicochemical characteristics of the different types of sludges studied. However, method 3 recovered a significantly lower number of F-specific bacteriophages compared with methods 1 and 2 (Mann–Whitney U, P < 0·01). With regard to somatic coliphages and phages infecting B. fragilis, method 3 was as efficient as methods 1 and 2. The plaque assays performed to enumerate the bacteriophages presented the least problems with background bacterial flora in the phage suspension obtained by method 3. However, method 2 was selected to elute phages in subsequent experiments because it could be used for all three groups of bacteriophages.

Table 2.  Recovery of the different groups of bacteriophages from different sludge types using three different methods
SludgeMethodSomatic coliphagesF-specific coliphagesBacteroides fragilis phages
  • Geometric mean (standard deviation) of counts of the indicated micro-organisms 100 g−1 of sludge of six different experiments.

  • ND, not done

  • *

    De-watered, anaerobically, mesophilically-digested sludge.

  • Values significantly different (Mann–Whitney U, P < 0·01) from their pairs using the other methods.

  • Value that seems clearly lower than its pair using the other methods, but the difference is not significant (Mann–Whitney U, P < 0·01).

Primary12·6×106 (3·6×106)2·4×105 (7·5×106)2·4×104 (6·9×104)
Primary26·7×106 (1·0×107)3·1×106 (1·2×107)5·3×104 (9·6×104)
Primary32·1×106 (8·8×106)5·1×104 (3·1×105)3·5×104 (1·3×105)
Activated11·4×106 (2·6×106)9·2×104 (1·6×105)2·8×103 (9·5×103)
Activated21·5×106 (3·3×106)1·6×105 (1·4×105)4·1×103 (3·2×103)
Activated37·0×105 (3·5×106)1·6×104 (7·2×104)6·8×103 (3·2×103)
De-watered*1NDNDND
De-watered*21·5×106 (8·7×105)2·3×104 (1·3×104)9·3×103 (4·4×103)
De-watered*35·1×1052·5×103 (2·4×103)5·2×103 (8·5×102)

Levels of bacteriophages in different sludge types of plant A

The data from the sludges from treatment plant A are presented in Table 3. The primary sludge showed an accumulation of the different bacteriophages and indicator bacteria. None of the bacteriophages and bacteria seemed to accumulate more than the others, as the ratios between their concentrations were similar to those in sewage.

Table 3.  Geometric mean (standard deviation) of counts of the indicated micro-organisms 100 g−1 of the sludge types from plant A
 Primary sludge (without lime) (n = 9)Primary sludge (with lime) (n = 6)
  • *

    Significant differences (Mann–Whitney U, P < 0·01) in numbers in sludge with and without lime.

  • Significant differences (Mann–Whitney U, P < 0·05) in numbers in sludge with and without lime.

Faecal coliforms1·2×108 (3·5×108)*4·2×105 (5·1×106)*
Faecal streptococci8·6×106 (1·9×106)*8·6×105 (2·7×106)*
Somatic coliphages2·9×106 (4·1×106)1·2×106 (4·8×106)
F-specific phages6·5×105 (7·2×105)8·3×104 (2·6×105)
Bacteroides fragilis phages5·2×104 (1·6×105)8·1×103 (6·7×104)

The sludge obtained after lime-aided sedimentation showed a different pattern. Although the numbers of somatic coliphages and phages of B. fragilis were lower in the sludge obtained with lime, there was no significant difference between the two sludge types. The numbers of F-specific bacteriophages were lower in the sludge obtained with lime but with a low level of significance (Mann-Withney U, P < 0·05). In contrast, the numbers of indicator bacteria, both faecal coliforms and faecal streptococci, were significantly lower (Mann-Withney U, P < 0·01) than in the primary sludge.

Levels of bacteriophages in different sludge types from plant B

The data for the sludge from treatment plant B are presented in Table 4. The primary sludge showed an accumulation of the different bacteriophages and indicator bacteria, and as in plant A, all the micro-organisms studied appeared to accumulate in a similar fashion, as the ratios between their concentrations were similar to those in sewage.

Table 4.  Geometric mean (standard deviation) of counts of the indicated micro-organisms 100 g−1 of the different sludge types produced by plant B
 Primary sludge (n = 10)Activated sludge (n = 10)Raw sludge* (n = 9)Dewatered sludge (n = 9)
  • *

    Mixture of primary (two thirds) and secondary (one third) sludges.

  • De-watered, anaerobically, mesophilically-digested sludge.

  • Results not available.

Faecal coliforms5·3×108 (2·3×109)8·6×106 (9·8×106)3·4×108 (9·5×108)NA
Faecal streptococci2·4×107 (3·7×107)1·6×106 (4·2×106)1·5×107 (2·0×1075·0×106 (7·2×106)
Somatic coliphages1·1×107 (9·8×106)1·1×106 (2·7×106)7·0×106 (7·5×106)8·1×105 (7·0×105)
F-specific phages4·4×106 (7·2×105)1·6×105 (1·5×105)2·9×106 (2·1×106)3·4×104 (3·0×104)
Bacteroides fragilis phages8·4×104 (9·7×104)3·4×103 (3·9×103)5·6×104 (7·1×104)1·1×104 (7·7×103)

Phages and bacteria also accumulated in activated sludge, but the numbers were significantly lower (Mann-Withney U, P < 0·01) than in the primary sludge. However, all the micro-organisms studied seemed to accumulate in a similar fashion, as the ratios between their concentrations were similar to those in sewage and in the primary sludge. It is possible that the numbers of micro-organisms in these samples may have been underestimated, as no special treatment to disperse flocs was applied.

With regard to de-watered, anaerobically, mesophilically-digested sludge, the numbers of F-specific bacteriophages were significantly lower (Mann-Withney, P < 0·01) than in the primary sludge, whereas neither faecal streptococci nor the other groups of phages differed significantly. Unfortunately, the analysis of faecal coliforms in this sludge gave highly variable results which did not allow comparisons with the other parameters.

Persistence of bacteriophages in de-watered, anaerobically, mesophilically-digested sludge

The inactivation of somatic coliphages and bacteriophages infecting B. fragilis is very similar, both in kinetics and in the overall reduction in numbers (Fig. 1). Their inactivation depends on temperature, with clear differences between 4, 20 and 37 °C.

image

Figure 1. Inactivation of the different groups of bacteriophages after storage of dewatered sludge at (a) 4 °C, (b) 20 °C, (c) 37 °C

Download figure to PowerPoint

The inactivation of F-specific bacteriophages clearly differs from inactivation of the other groups of phages, both in kinetics and overall reduction in numbers. Their inactivation also depends on temperature, but the kinetics of inactivation at 20 and 37 °C are very similar, and only very minor differences in the reduction of numbers can be observed after 45 d.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences

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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences

This work was supported by the Generalitat de Catalunya 1997 – SGR 00069. The authors thank EMSSA for co-operation in obtaining the samples.

ReferencesReferences

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. ReferencesReferences
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