1. Factors Affecting E. coli Growth in Limed Versus Anaerobically Digested Sludge Justin Hayes and Dr. Carol Bair, Dept. of Biological Sciences, York College of PA ABSTRACT A useful method for disposing of stabilized sewage sludge (“biosolids”) is field application, particularly agricultural fields, where it can be used as a fertilizer. It has been demonstrated that pathogens such as Salmonella or E. coli can grow in biosolids, especially when inoculated by animals (Sidhu 2001). Alkali (lime) addition and anaerobic digestion are two different techniques used to stabilize Class B sludge before field application, which suggests that growth of inoculated bacteria could differ substantially between samples. The objective of this study was to analyze bacterial growth in limed and anaerobically digested biosolids, in relation to nutrients, pH and indigenous microflora. The pH of the limed samples was found to be significantly higher (p=0.029) compared to the non-limed (anaerobically digested) samples for the 3 trials. Indigenous populations, on the other hand, were generally steady during the 3 trials, with no significant differences found between limed and non- limed samples. Nutrient analysis is planned for future studies. While seeded E. coli R grew well (10 7 -10 10 /g dry wt) in non-limed samples over six-hours, its growth in limed samples was minimal (10 5 max/g dry wt), and mostly in the first hour of seeding. Due to the fact that pre-limed (samples from the alkali plant not containing lime) control samples allowed extensive growth (10 8 -10 11 /g dry wt) of seeded E. coli R, it was concluded that the pH level is likely the main factor in affecting bacterial growth in these types of biosolids, no matter what the nutrient content may have been. The results suggest that bacteria could potentially grow to notably higher levels in anaerobically digested biosolids compared to limed biosolids, which may raise some safety concerns. INTRODUCTION Sewage sludge is a solid, semi-solid, or liquid residue generated from the treatment of municipal sewage, and can be classified as Class A or B following treatment. Class B biosolids, used in this study, may contain some pathogens, and are therefore applied only to farmland, reclamation sites, forests, or public contact sites where crop harvesting, animal grazing, and public contact is restricted for a certain period of time. Alkali addition and anaerobic digestion are two common methods used to stabilize sewage sludge. Anaerobic digestion breaks down most of the biodegradable material, which helps to reduce the mass of volatile solids in sewage sludge by at least 38 percent. Alkali (lime) addition raises the pH of sludge to 12 or higher, which is maintained for 2 hours, along with a pH of 11.5 or higher for an additional 22 hours. These processes also help to reduce the attraction of vectors (animals that may carry pathogens) to biosolids by significantly reducing bacterial levels. If indigenous bacterial levels are not reduced, organic matter could be broken down and vector-attracting odors subsequently released. Vectors could inoculate biosolids with potentially pathogenic organisms after land application, or could transmit pathogens from sewage sludge to humans (PADEP 2000). In past studies, bacteria such as Salmonella were used to test growth potential in biosolids that were composted following anaerobic digestion. A Salmonella typhimurium strain (Sidhu 1999) was found to grow as high as 10 6 /g, and the indigenous microbial population played a significant role in controlling Salmonella growth (Sidhu 2001). For this study, it was expected that existing microbes, pH levels and nutrient levels would affect bacterial growth in both samples. It was expected that the limed sample would support less growth predominantly because of pH, making it extremely difficult for survival or growth of any organism to occur. METHODS Sample Collection: Samples were obtained from two different wastewater treatment plants, one that uses anaerobic digestion and the other using alkali addition for stabilization. For the 2 nd and 3 rd trials, additional samples containing no lime (“pre-limed”) were obtained from the alkali addition plant. Samples were transported to the lab on ice and large materials were removed by sieving. Moisture Content: Dry weight was determined for each sample. LITERATURE CITED APHA, AWWA, and WEF. 1995. Standard methods for the examination of water and wastewater. 19 th ed. American Public Health Association Washington, DC. Pennsylvania Department of Environmental Protection [PADEP]. 2000 December. Sampling manual for pollutant limits, pathogen and vector attraction reductions in sewage sludge. Available from: http://www.dep.state.pa.us/. Accessed 2001 July 2. Sidhu, J., Gibbs, R.A., Ho, G.E. and Unkovich, I. 1999. Selection of Salmonella typhimurium as an indicator for pathogen regrowth potential in composted biosolids. Letters in Applied Microbiology 29:303-307. Sidhu, J., Gibbs, R.A., Ho, G.E. and Unkovich, I. 2001. The role of indigenous microorganisms in suppression of Salmonella regrowth in composted biosolids. Water Research 35:913-920. ACKNOWLEDMENTS Thanks to Dr. Carol Bair for her guidance and to the wastewater treatment plants for allowing me to obtain samples. Thanks to Mrs.Taylor for lab assistance. DISCUSSION We hypothesized that limed biosolids would encourage less bacterial growth than anaerobically digested biosolids, predominantly because of pH, and this was supported. These preliminary results are based on 3 trials conducted within a small period of time. Considering the extreme pH differences, some conclusions can be made: Although it is unlikely that nutrient content (Carbon, Nitrogen) would have been a factor, these studies remain to be completed. Since the limed and non-limed indigenous populations were generally steady throughout the trials, they would probably not impact growth to the same degree as the pH differences. The fact that pre-limed control samples allowed extensive growth of seeded E. coli R suggested that the addition of lime, which raises the pH, provided a hostile environment for bacterial growth and was likely the reason why seeded E. coli R levels were more inhibited in limed samples. pH analysis: Each sample was diluted 1:10 and the pH was recorded. Enumeration of indigenous bacteria, fungi, and actinomycetes: The standard spread plate count procedure was used as described in APHA (1995). Serial ten-fold dilutions were prepared in phosphate buffer (0.25M) for all 3 types of indigenous organisms. All plating was in duplicate. Bacteria were isolated on R2A agar (Difco). Fungi were isolated on streptomycin-terramycin-malt extract agar (STMEA). Actinomycetes were isolated on double-layer plates of starch-casein-cycloheximide agar (SCA). Viability plating: A standard E. coli lab strain was selected for Rifampicin resistance, maintained at –90°C, and grown in peptone water. Fifty grams of autoclaved biosolids were mixed with 45 mL of H 2 O and 5 mL (~1.5 x 10 9 cells) of an overnight E. coli R culture, followed by incubation in a 37°C shaking water bath. Using timed intervals, the samples were plated on nutrient agar containing Rifampicin (50  g/mL), followed by incubation at 37°C for 24 hours and colony counting (Sidhu 2001). Statistical Analyses: To control for variability of different moisture levels on different days, data were compared as to dry weight (per gram). All statistical analyses were calculated at the 0.05 level using non-parametric Mann-Whitney tests (Instat). RESULTS The median pH value for the limed biosolids was 12.19, while the non-limed and pre-limed values were 8.53 and 7.93, respectively (Figure 1). The differences in pH were significant between limed biosolids and both non-limed and pre-limed biosolids. Figure 1. pH values for the 3 trials. Variation among column medians was significant between limed (*) and pre-limed biosolids (p=0.016), and between limed and non-limed biosolids (p=0.029). The 95% confidence interval is represented by the bars. Figure 2 displays results of the indigenous microflora counts. No significant differences were found among median population levels of limed and non- limed samples. Figure 3. Comparison of E. coli R viability in limed, non-limed and pre-limed biosolids. During viability plating, seeded E. coli R grew well in non-limed samples, with values ranging from 3.1 x 10 7 – 8.5 x 10 10 /g dry weight. Similar growth values (1.5 x 10 8 – 1.4 x 10 11 /g dry weight) were found for the pre-limed samples. However, the limed samples allowed minimal growth (1.6 x 10 5 maximum/g dry weight), occurring mostly within the first hour, with almost all of the E. coli R falling below detectable levels afterwards (Figure 3). It would seem that additional treatment should be performed on the anaerobically digested sludge to prevent the possibility of potentially high bacterial growth, and subsequent possible danger to the public. In actual practice, pH of soil is considered in choice of fields where sludge is applied. Although PADEP has not established fecal coliform limits in Class B limed and anaerobically digested biosolids, it does mandate that fields receiving Class B biosolids must not be open to the public or cultivated for an extensive period of time, which also lessens any safety risks. It is also important to point out that there is variability among samples from each plant, and from the same plant. The fact that each treatment facility has different industrial sources implies that each sample may have contained different varieties and concentrations of metals. Therefore, heavy metals could have played a significant role in affecting bacterial growth in either sample. Each treatment plant also has different domestic and commercial sources that would likely cause variability in organic matter composition of each set of samples. Neither of these variables has been addressed. Future studies will address the following: 1.Investigate the role of nutrient content in relation to pH levels 2.Conduct heavy metal analysis of each sample as an additional factor affecting growth. 3.Compare E. coli R growth in non-sterilized biosolids to that of sterilized biosolids used in this study. Figure 2. Indigenous microflora counts for the 3 trials. The results of the Mann- Whitney tests are that variation in median population levels was not significant between limed and non-limed samples (p values of 0.400, 0.826, and 0.700 for bacteria, fungi and actinomycetes, respectively). The 95% confidence interval is represented by the bars. Limed Anaerobically Digested