The thermal inactivation of E. coli in straw and pig manure

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1 Bioresource Technology 84 (2002) The thermal inactivation of E. coli in straw and pig manure Claire Turner * Silsoe Research Institute, Wrest Park, Silsoe, Bedford, MK45 4HS, UK Received 15 November 2001; received in revised form 15 December 2001; accepted 17December 2001 Abstract Livestock manure may contain pathogenic organisms which pose a risk to the health of animals or humans if the manure is not adequately treated or disposed of. One possible treatment method is composting. However to ensure that pathogen destruction occurs, temperatures need to be sufficiently high throughout the heap to ensure that pathogens are inactivated. The temperature required to inactivate a marker organism, Escherichia coli 11943, has been investigated, and found to depend on substrate composition, moisture content and duration of incubation. Results show that temperatures in excess of 55 C for 2 h are required for inactivation. Data are presented showing the levels of faecal coliforms in compost heaps where temperatures did not rise above mesophilic levels (35 C where samples were taken). Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Composting; Pathogen inactivation; E. coli; Animal waste disinfection 1. Introduction Composting is a traditional way of treating livestock manure to make it easier to dispose of on land, and to produce an inexpensive fertiliser. However, livestock manure may contain zoonotic microbial pathogens, including Salmonella spp. and Escherichia coli O157. Composting as a means of treatment has the added advantage that if it is well managed, thermophilic temperatures may be attained, which will inactivate those pathogens present in the manure, making it safe for land spreading. Problems may arise if pathogens have not been inactivated before land spreading, as it is known that some potentially serious microorganisms may survive for a prolonged period in soil or on land. Salmonella is known to survive for several months in stored slurry; up to 6 months in cowpats and up to 100 days in slurry applied to grass (Mawdsley, 1993). Maule (1998) noted that E. coli O157may survive for more than 56 days in fresh cattle faeces, and in cattle slurry at 18 C for up to 9 days. Kudva et al. (1998) also noted the longevity of E. coli O157in muck it can survive for 21 months in a manure pile. Another study (Himathongkham et al., 1999) found that survival times of E. coli O157:H7 and Salmonella typhimurium in cow manure and cow slurry was dependent on temperature, and * Tel.: ; fax: address: claire.turner@bbsrc.ac.uk (C. Turner). ranged from 6 days to 3 weeks in manure and 2 days to 5 weeks in manure slurry. The United States Environmental Protection Agency s publication (EPA, 1985) on control of pathogens in biosolids, part 503, has a minimum time temperature requirement for in-vessel and aerated static pile composting methods: the material must maintain a minimum temperature of 55 C for at least three consecutive days. For turned windrow composting, at least 55 C must be maintained for 15 consecutive days with the material turned at least five times (Wu and Smith, 1999). These conditions are very stringent, and are designed to ensure that the composted material will not contain pathogenic organisms. However, thermal destruction of bacterial pathogens (e.g. E. coli O157) may well depend on factors other than temperature, e.g. moisture content, free ammonia concentration, duration of heat treatment and the presence of other microorganisms which may enhance or inhibit pathogen inactivation. For instance, in an industrial compost, Salmonella and E. coli were found to survive for 59 days at about 60 C, although the pathogens were destroyed during the cooler, curing process (Droffner and Brinton, 1995). In the same study, survival was different in different composts, which demonstrates that the mechanism for inactivation is complex, and not solely dependent on temperature and time. This study examines the inactivation of a non-toxic marker E. coli strain in sterile straw, sterile pig farmyard /02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S (02)

2 58 C. Turner / Bioresource Technology 84 (2002) manure and sterile pig faeces at different temperatures and different moisture contents. Although the marker strain used was not conditioned for the temperatures encountered during thermophilic composting, the aim of the study was to determine the minimum requirements for the inactivation of the marker strain under different conditions, and infer the conditions that are therefore likely to affect inactivation of similar pathogens during composting. Data are also included in this study showing the growth of faecal coliforms throughout a compost heap of pig farmyard manure where the composting temperature was at mesophilic levels, demonstrating the importance of ensuring that composting is carried out at sufficiently high temperatures. If a compost heap does not reach high enough temperatures, it is possible that not only will inactivation not occur, but pathogenic bacteria may in fact grow. 2. Methods 2.1. E. coli cultures E. coli was cultivated at 37 C and at 200 rpm in an orbital incubator for 16 h. Cultures were inoculated from freshly grown nutrient agar plates into six 500 ml conical flasks containing 50 ml sterile nutrient broth (Merck). After incubation and shaking for 16 h, the contents of all six flasks were pooled into a single flask and the number of colonies per ml was measured from the pooled flask. The volume of broth required for each experiment was removed, and the flask containing the remaining broth was kept unagitated at 20 C for up to 72 h. This was to serve as a control for comparison with experiments where the broth had been added to either straw, pig faeces or pig farmyard manure and incubated at different temperatures for up to 72 h (see below). The ammoniacal nitrogen content of the E. coli broth was 0.20 g/l Strawexperiments Wheat straw (WS) used in these experiments had a dry matter (DM) content of 91% (w/w) and an ammoniacal nitrogen content of 0 g/kg. One g WS was weighed out into each of several glass bottles and autoclaved at 121 C for 15 min Pig faeces experiments Pig faeces (PF) used in these experiments was obtained from grower pigs fed on a proprietary grower pig feed (from BOCM Pauls) containing copper, and prescription drug additive called Potencil. The faeces had a dry matter (DM) content of 22% (w/w) and an ammoniacal nitrogen content of 3.2 g/kg. Twenty g PF was weighed out into each of several glass bottles and autoclaved at 121 C for 15 min Farmyard manure experiments Pig farmyard manure (FYM) composed of straw, pig faeces and urine was obtained from sows fed a proprietary brand of dry sow rations from BOCM Pauls, with no specific additives. The FYM used in these experiments had a dry matter (DM) content of 26% and an ammoniacal nitrogen content of 2.5 g/kg. Twenty g FYM was weighed out into each of several glass bottles and autoclaved at 121 C for 15 min Experimental protocol for WS, PF and FYM experiments At the start of each experiment, 1 or 10 ml of E. coli culture broth previously incubated for 16 h was added to each of the sterile glass bottles containing either straw, pig faeces or pig farmyard manure, and the bottles were shaken thoroughly. Two (i.e. duplicate bottles) were assayed for E. coli titres immediately, two were kept at 20 C for the duration of the experiment, and the remaining bottles were put in an incubator at either 50 or 55 C. At 1, 2, 5, 24 and 48 h (and in some cases, 72 h), duplicate flasks were removed from the incubator and assayed for E. coli as described below Extraction of E. coli from samples E. coli was extracted from each of the samples by adding 0.1 M sodium phosphate buffer, ph 7, to each bottle, the volume added being dependent upon how dry the material was (i.e. whether it was WS, PF or FYM, and how much culture broth had been added; in the case of straw with 10 ml E. coli broth added, no sodium phosphate was needed, but in the case of FYM with only 1 ml broth, 30 ml was required). The contents of the bottles were mixed thoroughly, and the contents decanted and, except in the case of WS samples, were centrifuged at 1700g in an IEC Centra 3E centrifuge (IEC, Dunstable, Bedfordshire, UK) for 10 min. The supernatants were then assayed for E. coli counts E. coli counts E. coli titres or counts (cfu) were measured as follows: samples (sodium phosphate extracted for WS; extracted and centrifuged for PF and FYM, and neat for E. coli broth samples) were serially diluted by adding 1 ml to glass bottles containing 9 ml 0.7% NaCl. 100 ll of each dilution was then added to fresh nutrient agar plates, and spread with an alcohol sterilised glass spreader. The plates were incubated at 37 C for 24 h, after which colonies were counted and titres obtained in cfu. In each

3 C. Turner / Bioresource Technology 84 (2002) case, the titres were calculated back to per ml of culture broth to allow direct comparisons to be made between WS, PF and FYM experiments at different added E. coli levels Compost heap experiment One tonne of pig farmyard manure (from the same source as that used in the small scale experiments) was placed in a specially constructed rig and forcibly aerated. The temperature at various points in the material was monitored, and kept at mesophilic temperatures through the cooling effects of aeration. At time intervals over 3 weeks, samples were taken from a particular part of the heap and cfu counts on petri dishes containing nutrient agar and MacConkey agar were taken by adding 20 or 30 ml 0.1 M sodium phosphate buffer, ph 7, shaking, centrifuging, serially diluting in 0.7% NaCl and plating as described above. The temperature of the compost heap from where the samples were taken was monitored. 3. Results and discussion 3.1. Heated wheat straw samples Results of heating E. coli in WS at 50 and 55 C with low or high moisture content (i.e. either 1 or 10 ml E. coli culture broth added) are given in Table 1. E. coli was inactivated within 2 h at 55 C; however, at 50 C, it was still viable after 72 h. The moisture content of the samples also played a role in inactivation. When 10 ml E. coli broth was added to the WS, inactivation occurred less rapidly than when 1 ml was added at both 50 and 55 C. In the case where 1 ml was added, the broth soaked into the straw, with no residual liquid, whereas when 10 ml was added, it was a liquid culture Heated pig faeces samples Results of heating E. coli in PF at 50 and 55 C with lower or higher moisture content (i.e. either 1 or 10 ml E. coli culture broth added) are given in Table 2. These Table 1 Results of E. coli culture (either 1 or 10 ml) added to 1 g straw and incubated at 50 or 55 C for 72 h 0 h h h h h h nd h nd Control 0 h Control 24 h Control 48 h nd Control 72 h nd 8.2 nd 8.7 Table 2 Results of E. coli culture (either 1 or 10 ml) added to 20 g pig faeces and incubated at 50 or 55 C for 72 h 0 h h h h h h h Control 0 h Control 24 h nd 8.7nd 8.7 Control 48 h nd 8.3 nd nd Control 72 h

4 60 C. Turner / Bioresource Technology 84 (2002) Table 3 Results of E. coli culture (either 1 or 10 ml) added to 20 g pig farmyard manure and incubated at 50 or 55 C for 72 h 0 h h < h < <2.2 5h <4.3 <2.2 0 < h 0 <2.2 0 < h 0 <2.2 0 <2.2 72h 0 <2.2 nd nd Control 0 h Control 24 h nd nd nd nd Control 48 h nd nd Control 72 h nd nd Table 4 Results of E. coli culture (either 1 or 10 ml) added to 20 g pig faeces (PF) or pig farmyard manure (FYM) and incubated at 22 C for 72 h Time (h) 1 ml broth to PF 10 ml broth to PF 1 ml broth to FYM 10 ml broth to FYM 0 8.7nd nd Results given as log 10 cfu/ml. results show that E. coli was not destroyed at 50 C in PF, and where 10 ml broth was added, although the E. coli counts dropped to below detectable levels after 24 h, the bacteria recovered and were able to grow, presumably when conditions selected for more temperature resistant individuals. There were no significant differences in E. coli survival in the 1 and 10 ml broth samples at 50 C. At 55 C, E. coli was not detected in either experiment after 1 h at that temperature, although it did survive for at least 1 h in the drier sample Heated pig farmyard manure samples Results of heating E. coli in FYM at 50 and 55 C with lower or higher moisture content (i.e. either 1 or 10 ml E. coli culture broth added) are given in Table 3. At 50 C, the E. coli did not appear to survive for as long in the FYM as in PF with both the 1 and 10 ml broth experiments. The differences in dry matter and ammoniacal nitrogen levels in the samples did not seem to account for these differences, although there did not Fig. 1. Bacterial counts measured on nutrient agar and MacConkey agar for muck samples taken during experiment where pig farmyard manure was composted at mesophilic temperatures for 3 weeks.

5 C. Turner / Bioresource Technology 84 (2002) appear to be differences in survival of E. coli at 55 C, where E. coli was not detected after 1 h, both when 1 or 10 ml E. coli broth was added Pig faeces and farmyard manure at 22 C In experiments where either 1 or 10 ml E. coli broth was added to PF or FYM and maintained at 22 C for 72 h, there was no significant loss of titre of E. coli over that time, and this is shown in Table 4. E. coli broth left unagitated at 22 C for 72 h also did not show any appreciable loss of titre. This can be seen in Tables 2 and 3, where values given for controls were of E. coli broth left at 20 C Compost experiment The above experiments demonstrate that E. coli would be inactivated in farmyard manure, pig faeces and straw (the components of muck heaps) if kept at 55 C for more than 2 h. Fig. 1 shows the results of bacterial counts from samples taken from a compost heap at mesophilic temperatures of pig farmyard manure grown on both MacConkey agar (for faecal coliforms) and nutrient agar. The trend of bacterial counts during the course of the compost experiment seemed to follow the temperature trend of the area in the compost heap from which the sample was taken. The temperature in the sampled area of the heap reached 35 C, and this coincided with the highest total bacterial and faecal coliform counts. It therefore shows that the composting process will not inactivate coliforms (and thus pathogens such as E. coli O157and Salmonella) at mesophilic temperatures. 4. Conclusions This work has shown the conditions required for the inactivation of a lab strain of E. coli, acting as a marker for common pathogenic organisms that may be present in animal manures. Results demonstrated that at 50 C, inactivation of E. coli may depend on the moisture content and the nature of the material. However, when the temperature is increased to 55 C, inactivation proceeds rapidly, and in all cases, E. coli was inactivated to below detectable levels within 2 h. Although temperatures required for inactivation of the lab strain may not be as high as those required for the destruction of conditioned pathogens, the results indicate that the inactivation is not merely temperature dependent, but is affected by the moisture content and the nature of the material. If incomplete inactivation has taken place due to insufficiently high temperature, recovery and growth of the damaged population may be possible. Results from a composting experiment demonstrated that coliforms grew in the compost heap if the composting was conducted at mesophilic temperatures, so care should be taken in maintaining adequate composting of material, rather than just leaving a heap unmonitored. References EPA: Composting municipal wastewater sludges, 1985, EPA/625/4-85/ 014, 66 p. Droffner, M.L., Brinton, W.F., Survival of E. coli and Salmonella populations in aerobic thermophilic composts as measured with DNA gene probes. Zbl. Hyg. 197, Himathongkham, S., Bahari, S., Riemann, H., Cliver, D., Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure ad cow manure slurry. FEMS Microbiol. Lett. 178, Kudva, I.T., Blanch, K., Hovde, C.J., Analysis of Escherichia coli O157:H7 survival in ovine or bovine manure and manure slurry. Appl. Environ. Microbiol. 64 (9), Maule, A., Environmental survival of Escherichia coli O157: implications for spread of disease. Paper presented at International Food Hygiene Conference on E. coli O157, October 1998, Paris, France. Mawdsley, Pathogenic microorganisms in livestock waste and factors influencing their transport to the aqueous environment. Appendix 1 of: Protozoan, bacterial and virus pathogens, farm wastes and water quality protection. Final Report for Ministry of Agriculture, Fisheries and Food Open Contract CSA2064. Wu, N., Smith, J.E., Reducing pathogen and vector attraction for biosolids. Biocycle (November),