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REDUCTION OF E-COLI, SULFITE-REDUCING ANAEROBIC BACTERIA AND F-SPECIFIC BACTERIOPHAGES IN THE PRODUCTION OF VACUUM FILTER PRESSED MUNICIPAL SLUDGE AT VEAS, Ø. Østensvik*, M. Myrmel*, A. Haarr** and P. Sagberg** * The Norwegian School of Veterinary Science, Dept. of Pharmacology, Microbiology and Food Hygiene, P.O.Box 8146-Dep., N-0033 Oslo, Norway. E-mail:[email protected] ** VEAS-Vestfjorden Wastewater Treatment Plant, Bjerkåsholmen 125, N-3470 Slemmestad, Norway. E.mail: [email protected] Steps of stabilization and hygienization are needed for safe utilization of sludge on farmland. At the VEAS wastewater treatment plant a thermal vacuum drier is introduced as the final step in the water removal and hygienization process of lime conditioned two stage anaerobic digested municipal sludge. This paper present the results from microbiological examination of sludge from six sites in the production line. The number of the indicator bacterium Escherichia coli per gram dry matter was reduced with at least 7 log10 units through the process. Spores of sulphite reducing anaerobic bacteria (Clostridia) and f-specific bacteriophages were reduced by at least 3 log10 units/g dry matter. In the final product, VEAS biosolids, E. coli and f-specific bacteriophages were not detected in 1 gram samples of dried sludge. The geometric mean concentration of spores of sulphite reducing anaerobic bacteria was 1000 per gram DS in final product. According to a working document for a proposed EU directive, the observed log10 reduction of E. Coli indicates a satisfactory hygienization being well above the demanded 5-6 log10 reduction. Based on the lower reduction of bacterial spores, the effect of vacuum drying on heat resistant pathogenic microorganisms is discussed. KEYWORDS Hygenization of sludge; vacuum drying; filterpressing; f-specific bacteriophages, e-coli; sulfite-reducing anaerobic bacteria. Sludge from sewage treatment plants contains high amounts of nutrients and organic matter. The use of sludge as fertilizer for farmland is recommended from a recycling point of view. However, sludge can contain significant amounts of pathogenic microorganisms. National and international regulations require that sludge shall be stabilized and hygienized before land application. Hygienization is a process intended to reduce the content of pathogenic microorganisms to a safe level. In the current national regulation the degree of hygienization is based on the level of Thermotolerant Coliform Bacteria, TCB (SHD/MD,1996), and hygienized sludge shall not contain more than 2500 TCB per gram dry matter. In a working document for a proposed directive from the European Union, Escherichia coli replaces TCB as a microbiological parameter. Through the processes in the treatment plant, the content of E. coli in sludge shall be reduced with more than 5 log10-units. E. coli is a vegetative bacterium and does not survive high temperatures. The D-value (decimal reduction time) for E. coli is 4-6 minutes at 55°C and 2 minutes at 60°C (Mossel et al., 1995). The use of E. coli as an indicator for the presence of pathogenic microorganisms in biosolids is limited to vegetative bacteria (i.e. Salmonella spp., Shigella spp.) with D-values in the same order of magnitude. In addition to vegetative bacterial pathogens, sewage may contain more heat resistant microorganisms (i.e. Cryptosporidium spp., Giardia spp., spores of aerobic and anaerobic bacteria). To indicate the presence of heat resistant microorganisms, spores of sulphite reducing anaerobic bacteria, SRC, (mostly Clostridium perfringens) may be used. The D-value for C. perfringens is 5-35 minutes at 90°C and 2.3-5.2 minutes at 110°C (Mossel et al., 1995). In contrast to bacteria, virus in general, are sensitive to heat and are inactivated by increased temperature. Studies on the inactivation of enteric viruses in human and animal waste and sludge indicate that temperature is a predominant factor (Spillmann et al., 1987 and Deng and Cliver, 1953). Bacteriophages are potential model organisms for determining the influence of sludge treatment on the enteric virus load (Lasobras et al., 1999). F-specific RNA coliphages also have a similar size and structure as enteric viruses and can be used as a processing parameter. The purpose of the present study was to describe the hygienization of sludge from the VEAS wastewater treatment plant using E. coli, spores of sulphite reducing anaerobic bacteria and f-specific bacteriophages as indicators. Special attention was directed to the recently introduced thermal vacuum drying process, described in (Sagberg, submitted). This step includes an increase in temperature above 80°C, and under ordinary operation the sludge is vacuum dried for 80-90 minutes. To describe the effect of increased heat treatment, vacuum drying for 60, 90, 120 and 150 minutes was performed. Sludge samples

Ordinary operation. Samples of sludge from 6 different sites in the treatment plant were collected in plastic
boxes with screw caps. From five sample sites a mixture of random samples were used. The samples taken
after thickening were 24 hours mixed samples. Three sampling series were performed: 6., 13. and 19.
February 2001. The samples were transported to the laboratory the same day they were collected.

Increased length of vacuum drying. Sludge samples were collected after increasing lengths of heat
treatment. Three samples without heat treatment were used as a reference. Three parallel sludge-samples
were collected after respectively 60, 90, 120 and 150 minutes of vacuum drying.

Methods

Sample pretreatment, homogenization and dilution . The analyses were started the day the samples arrived
the laboratory. Initial suspension and decimal dilutions were made according to ISO 6887-1 (ISO, 1999).
Ten grams of sludge were mixed with 90 ml of sterile peptone saline solution in sterile stomacher plastic-
bags, and homogenized in an IUL masticator for 30 seconds. To make a proper suspension of the final
product, 53-68% dry matter, these samples were first crushed mechanically and the plastic bags were stored
for about one hour before stomaching. From the initial dilution (10-1) further decimal dilutions were made in
glass tubes containing 9 ml of sterile peptone saline solution.

Quantification of Escherichia coli . E. coli was quantified using a multiple tube fermentation (MTF)
technique, ISO 7218 (ISO, 1996). Identification of E. coli requires additional steps as compared to
Thermotolerant Coliform Bacteria, TCB. Methods for quantification of TCB are based on fermentation of
lactose and formation of indole from tryptophane at 44.0 – 44.5°C. There is no international standard
method for detection and enumeration of E. coli in sludge. However, methods based on the activity of the
enzyme beta-glucuronidase are proposed for both foodstuff and water (Frampton and Restaino, 1993).
Activity of the specific enzyme is visualized using chromogenic or fluorogenic substrates (Manafi et al., 1991). In this study E. coli was defined as a bacterium producing the enzyme beta-glucuronidase which gives a positive MUG-reaction in EC-MUG broth (NN AmPubHealthAss, 1995). From appropriate dilutions, tubes containing MacConkey broth were inoculated with 1 ml of diluted samples. Three tubes were inoculated from each dilution. In addition, from the sample of the final product 10 MacConkey tubes were inoculated with 1 ml from the 10-1 dilution, corresponding to 1 gram sludge. MacConkey-tubes were incubated at 37 ± 1°C for 18-24 hours. From positive MacConkey-tubes (growth of bacteria with acid and gas formation) material was transferred to EC-broth containing 4-methylumbelliferyl-β-D-glucuronide (MUG) (Manafi et al., 1991) and Tryptone water. The EC and Tryptone tubes were incubated at 44 ± 0,5°C for 18-24 hours in water-bath. After incubation, the production of gas and fluorescence in EC-MUG broth and indole reaction in Tryptone water was recorded. The Most Probable Number (MPN) of E. coli per gram sludge was determined using an appropriate MPN-table, ISO 8199 (ISO, 1988). The MPN-values are calculated from the combinations of MUG-positive and MUG-negative tubes in three different dilutions. The limit of detection using the standard MTF-method is a MPN value of 3 bacteria per gram. The 10-tube examination of the final product has a detection limit of 1 bacterium per gram. Quantification of spores of sulphite reducing anaerobic bacteria. Before inoculation, dilutions of sludge samples were heated in water-bath at 75°C for 15 min to kill vegetative bacteria. From appropriate dilutions, 1 ml samples were transferred to sterile 9 cm Petri-dishes and mixed with molten and tempered TSC-agar (SFP Agar Base, Difco, with 0.04 % D-cycloserine). When the plates had solidified, they were incubated in anaerobic jars at 37±1°C for 18-24 hours. Anaerobic atmosphere was produced using Anaerocult anaerobic system (Merck). Black colonies were counted as sulphite-reducing anaerobic bacteria. The unit is named CFU-colony forming units. Quantification of f-specific bacteriophages. ISO 10705-1 was used for the quantification of f-specific bacteriophages (ISO, 1995). One ml of the sample was mixed with nalidixic acid (100 µg/ml) and liquid semi solid agar at 45 ± 1°C prior to the addition of 1 ml of host bacteria (modified S. typhimurium, WG49). The mixture was poured onto solid agar and incubated at 37 ± 1°C for 18 ± 2 hours. Plaques were counted and the number of f-specific bacteriophages was given as plaque forming units (PFU). Presentation of results. All quantitative results are referred to dry weight of the samples unless otherwise stated. E. coli. The MPN-values from individual analysis were transformed to log10-values, corrected for dry solids content and multiplied with the dilution factor to obtain the log10-MPN per gram dry matter. Spores of sulphite reducing anaerobic bacteria. Arithmetic values (CFU/gram) were transformed to log10 values, corrected for dry solids content and multiplied with the dilution factor to obtain the log10 CFU per gram dry matter. F-specific bacteriophages. Arithmetic values (PFU/gram) were transformed to log10 values, corrected for dry solids content and multiplied with the dilution factor to obtain the log10 PFU per gram dry matter. Ordinary operation. Through the sludge treatment process at VEAS the reduction of E. coli and spores of sulphite reducing anaerobic bacteria (SRC) showed notable differences (Table 1). Table 1. Escherichia coli, spores of sulphite reducing anaerobic bacteria and f-specific bacteriophages in sludge samples from six different sample sites at VEAS wastewater treatment plant. The results from three sample series (two sample series for bacteriophages) and the geometric mean are presented. __________________________________________________________________________________________ ______________________________________________________________________________ Sample site ____________________________________________________________________________________ 1. Raw sludge, 3.5% 6.94 7.64 7.34 7.31 5.89 6.52 6.26 6.22 nd 4.23 4.23 2. After thickening, 7.92 7.64 6.89 7.48 6.30 6.46 6.27 6.37 5.26 5.78 5.52 5.5% 3. After biological hydrolysis, 4.8% 7.36 7.36 6.70 7.14 6.66 6.89 6.4 6.62 5.02 3.32 4.17 4. After anaerobic digestion, 3.6% 4.40 4.62 4.30 4.44 6.27 6.56 6.49 6.28 <2.44 2.77 <2.59 5. After addition of slaked lime, 4.5% 2.83 1.83 1.83 2.16 5.55 5.55 5.29 5.49 <2.3 <2.3 < 2.3 5b. After mechanical pressing ex. vacuum and heating, 38% 3.35 4.67 4.75 4.26 n.a. n.a. n.a. 6.After vacuum drying, final product 58%, <0.23 <0.23 <0.23 <0.23 LW-1.84 HW 3.99 2.92 <1.23 <1.23 < 1.23 ____________________________________________________________________________________ The reduction of E. coli was more than 7 log10-units, and the reduction of spores of SRC was 3 log10 units. In raw sewage the mean log10 MPN of E. coli was 7.3/g DS, the highest value was 7.6 and the lowest was 6.9. The highest log10 MPN for E. coli during the process was observed after thickening, 7.9. In the final product no test tubes gave a positive reaction for E. coli, corresponding to a MPN-value of <1 per gram sample. The mean log10 CFU of SRC in raw sludge was 6.2; the highest value was 6.5 and the lowest 5.9. The highest value for SRC was observed after biological hydrolysis, 6.9. The final product showed the lowest count, with a mean value for 12 samples from the four different drying times of 2.9 log10 CFU/gram DS, ranging from 1.8 to 3.5. F-specific bacteriophages showed a lower log10 value, 4.2, in raw sludge than E. coli and SRC. The highest value was found after thickening (5.8 log10 units) and no bacteriophages were detected in the final product (1:10 dilution) indicating a value of <10 PFU per gram sludge at approx 58% dry matter. The reduction of E. coli and SRC expressed as the geometric mean per gram dry matter are presented graphically in Figure 1. Sample site
Figure 1. Concentration of E. coli (MPN/g dry matter), spores of sulphite reducing anaerobic bacteria (CFU/g dry matter) (Clostridia) and f-specific bacteriophages (PFU/g dry matter) at 6 sample sites at the VEAS wastewater treatment plant. The sample sites correspond with the explanation given in Table 1. The steps with the highest reduction of E. coli seem to be the anaerobic digestion, the treatment with slaked lime and the thermal vacuum drying. During these three steps the mean log10 reduction of E. coli were 2.7, 2.3 and >2, respectively. E. coli was not detected in samples of the final product. The reduction of SRC, after addition of slaked lime, was 0.8 log10-units. Through the thermal vacuum drying the reduction of SRC was 2.6 log10 units. The total effect of the sludge treatment on the f-specific bacteriophages seems to parallel the effect on E. coli. As f-specific bacteriophages only was detected in one sample after anaerobic digestion and was undetected after lime addition and pressing, this study could not reveal the specific effect of lime addition and vacuum drying on the destruction of the bacteriophages. The variation of E. coli concentration from three different sample series is presented in Figure 2. The results from the three series showed a similar pattern. Increased time of vacuum drying. At normal operation the vacuum drying lasts for 90 minutes. The varied time of vacuum drying, up to 150 minutes, did not have obvious effects on the content of SRC-spores in VEAS-biosolids (Table 2). Sample site
Figure 2. Reduction of Escherichia coli in three sample series through the sludge process at VEAS wastewater treatment plant. The sample sites correspond to the explanation given in Table 1. Table 2. Reduction of spores of sulphite reducing anaerobic bacteria in sludge during different times of thermal vacuum drying. The results are presented as log10 CFU per gram. _____________________________________________________________________________ _______________________________________________________________________ ___________________________________________________________________________ In this study Escherichia coli, spores of sulphite reducing anaerobic bacteria (SRC) and f-specific bacteriophages were used to describe the hygienization of sludge. The results from ordinary operation of the treatment plant, 80-90 minutes of vacuum drying, showed a more than 7-log10 reduction of E. coli per gram dry matter and a 3-log10 reduction of bacteriophages and spores of anaerobic sulphite reducing bacteria. Extending the time of heat treatment up to 150 minutes did not demonstrate a significant reduction of SRC. The final product contained about 1000 CFU per gram dry matter. The VEAS-biosolids can not be declared free for heat resistant pathogenic microorganisms, Cryptosporidium oocysts, Giardia cysts and spore forming bacteria, but a 99,9% reduction of SRC has been demonstrated. Three steps in the production of VEAS-biosolids, anaerobic digestion, addition of slaked lime and thermal vacuum drying, showed significant reduction in the concentration of E. coli and f-specific bacteriophages. In the present study the reduction of SRC, E. coli and f-specific bacteriophages during anaerobic digestion was nil, 3.0 and 1.6 log10-units per gram dry matter , respectively. In a Canadian study, the reduction of Clostridium perfringens, Enterococcus spp., Cryptosporidium oocysts and Giardia cysts was not significant during anaerobic digestion (Chauret et al., 1994), but the reduction of fecal coliform bacteria was 1.56 log10-units. These results may indicate differences in the sludge composition and/or in the conditions during anaerobic digestion, but in both cases the reduction of SRC seemed to be limited. The addition of slaked lime reduced the number of SRC and E. coli with 0.8 and 2.3 log10-units per gram dry matter, respectively. This step has a more pronounced effect on vegetative bacteria than spores. The results indicate that the f-specific bacteriophages were sensitive to the anaerobic digestion, a reduction of >1.6 log10-units per gram dry matter was found. During the anaerobic digestion the sludge temperature was stable at 37°C for about 22 days. The inactivation is probably due to chemical and microbial activity (Deng and Cliver, 1995). The effect of slaked lime on f-specific bacteriophages was not demonstrated as the initial level was close to the detection limit. F-specific bacteriophages could not be detected after this step. The final thermal vacuum drying would probably inactivate residual enteric viruses as anaerobic digestion at 54-56°C reduced the titer of a human rotavirus in sludge with more than 8.5 log10-units per hour (Spillmann et al., 1987). The thermal resistant hepatitis A virus has a D-value < 10 minutes at temperatures above 55 °C (Lemon et al., 1994). Thermal vacuum drying reduced the concentration of E. coli from 2.2 log10-units to below the detection limit, <1 CFU per gram or less than 0 log10-units. The reduction of SRC was 1.3 log10-units compared with drying without heating and vacuum and 2.6 log10-units compared with lime slaked sludge. Since E. coli was not detected in the final product, the observed reduction may represent an underestimation. D-values given for E. coli are 4-6 minutes at 55°C, and 2 minutes at 60°C (Mossel et al., 1995). During thermal vacuum drying the temperature rises to about 80°C, and the ordinary treatment time is 90 minutes. In addition flash evaporation occurs when the vacuum is connected, resulting in cell wall ruptures. From these data it should assumed that the hygienization of VEAS-biosolids, based on E. coli, exceeds the proposed EU-regulations, which has been demonstrated in this study. However, hygienization of sludge expressed as the reduction of the vegetative indicator bacterium E. coli may indicate a false safety regarding the possible presence of more heat resistant pathogenic microorganisms. The present results indicate that the sludge treatment processes at VEAS also have reasonably good removal effects on SRC and very good removal effects on f-specific bacteriophages. Prolonged time of thermal vacuum drying indicated a limited additional reduction of SRC. The results after 90 minutes were lower than 150 minutes of drying. However, the number of analyses was limited. In the present study three parallel samples from each treatment time were examined. These observations may illustrate the variation of microbial concentration in sludge samples. To eliminate this variation, Chauret et al. (1999) underlines the importance of collecting a large number of samples over a long period of time. The content of specific pathogenic microorganisms at VEAS wastewater treatment plant was not examined. VEAS receive sewage from the main parts of Oslo, and may contain all the pathogens excreted from a large human population. Heat resistant cysts from various kinds of parasites, spores of bacilli and clostridia and different kinds of virus may be present, and it might be advantageous to carry out a further evaluation of the the effect of the sludge treatment on these microorganisms. The results from this study showed a reduction of E. Coli, SRC and f-specific bacteriophages in sludge through the VEAS wastewater treatment plant in the order of >7, 3 and >3 log10-units/ g dry matter, respectively. Important steps in the hygienization process were anaerobic digestion, addition of slaked lime and thermal vacuum drying. From the final product, VEAS-biosolids, no E. coli and f-specific bacteriophages were detected per gram dried material. With reference to the working document for a proposed EU directive, the reduction of E. coli through the sludge processing are well in accordance with the proposed limits. Acknowledgements: The authors will thank Brit Heidenreich, Lone Larsen and Indira Secic for technical assistance. Chauret, C., Springthorpe, S., Sattar, S. (1999) Fate of Cryptosporidium oocysts, Giardia cysts, and microbial indicators during wastewater treatment and anaerobic sludge digestion. Canadian Journal of Microbiology , 45, 257-262. Deng, M.Y.and Cliver, D.O. (1995) Persistence of inoculated hepatitis A virus in mixed human and animal wastes. Appl. Env. Microbiol., 61, 87-91. Frampton E.W., Restaino L. (1999) Methods for Escherichia coli identification in food, water and clinical samples based on beta-glucuronidase detection. Journal of Applied Bacteriology 1993; 74: 223-233. ISO 6887-1: Microbiology of food and animal feeding stuffs – Preparation of test samples, initial suspension and decimal dilutions for microbiological examination – Part 1: General rules for the initial suspension and decimal dilutions. International Organization for Standardization, Genève, Switzerland. ISO 7218. (1996) Microbiology of food and animal feeding stuffs – General rules for microbiological examinations. International Organization for Standardization, Genève, Switzerland. ISO 8199. (1988) Water quality – General guide to the enumeration of micro-organisms by culture. International Organization for Standardization, Genève, Switzerland. ISO 10705-1 (1995) Water quality – Detection and enumeration of bacteriophages. Part 1: Enumeration of F-specific RNA bacteriophages. International Organization for Standardization, Genève, Switzerland. Lasobras, J., Dellunde, J., Jofre, J. and Lucena, F. (1999) Occurrence and levels of phages proposed as surrogate indicators of enteric viruses in different types of sludges. J. Appl. Microbiol., 86, 723-729. Lemon, S.M. (1994) Hepatitis A virus. In Webster, R.G. and Granoff, A. (eds.), Encyclopedia of Virology, Manafi M., Kneifel W., Bascomb S. (1991) Fluorogenic and Chromogenic Substrates Used in Bacterial Diagnostics. Microbiological Reviews, 55, 335-348. Mossel, D.A.A., Corry, J.E.L., Struijk, C.B., Baird, R.M. (1995) Essentials of the microbiology of foods – A textbook for advanced studies. John Wiley & Sons, UK Chichester. NN AmPubHealthAss.(1995) Standard Methods for the Examination of Water and Wastewater. 19th ed. American Public Health Association, Washington D.C. Sagberg, P. (2003) Hygienization of Municipal Sludge in Automatically Operated Chamber Filter Presses SHD/MD (1996) Forskrift om avløpslam. Sosial- og helsedepartementet og Miljøverndepartementet, ISBN Spillmann, S.K., Traub, F., Schwyzer, M. and Wyler, R. (1987) Inactivation of animal viruses during sewage sludge treatment. Appl. Environ. Microbiol., 53, 2077-2081.

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