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 17 December 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 com-

position, 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). � 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 livestockmanure to make it easier to dispose of on land, and toproduce an inexpensive fertiliser. However, livestockmanure may contain zoonotic microbial pathogens,including Salmonella spp. and Escherichia coli O157.Composting as a means of treatment has the addedadvantage that if it is well managed, thermophilic tem-peratures may be attained, which will inactivate thosepathogens present in the manure, making it safe for landspreading. Problems may arise if pathogens have notbeen inactivated before land spreading, as it is knownthat some potentially serious microorganisms may sur-vive for a prolonged period in soil or on land. Salmo-nella is known to survive for several months in storedslurry; up to 6 months in cowpats and up to 100 days inslurry applied to grass (Mawdsley, 1993). Maule (1998)noted that E. coli O157 may survive for more than 56days in fresh cattle faeces, and in cattle slurry at 18 �Cfor up to 9 days. Kudva et al. (1998) also noted thelongevity of E. coli O157 in muck – it can survive for 21months in a manure pile. Another study (Himathongk-ham et al., 1999) found that survival times of E. coliO157:H7 and Salmonella typhimurium in cow manureand cow slurry was dependent on temperature, and

ranged from 6 days to 3 weeks in manure and 2 days to 5weeks in manure slurry.

The United States Environmental Protection Agen-cy’s publication (EPA, 1985) on control of pathogens inbiosolids, part 503, has a minimum time–temperaturerequirement for in-vessel and aerated static pile com-posting methods: the material must maintain a mini-mum temperature of 55 �C for at least three consecutivedays. For turned windrow composting, at least 55 �Cmust be maintained for 15 consecutive days with thematerial turned at least five times (Wu and Smith, 1999).These conditions are very stringent, and are designed toensure that the composted material will not containpathogenic organisms. However, thermal destruction ofbacterial pathogens (e.g. E. coli O157) may well dependon factors other than temperature, e.g. moisture con-tent, free ammonia concentration, duration of heat treat-ment and the presence of other microorganisms whichmay enhance or inhibit pathogen inactivation. For in-stance, in an industrial compost, Salmonella and E. coliwere found to survive for 59 days at about 60 �C, al-though the pathogens were destroyed during the cooler,curing process (Droffner and Brinton, 1995). In the samestudy, survival was different in different composts, whichdemonstrates that the mechanism for inactivation iscomplex, and not solely dependent on temperature andtime.

This study examines the inactivation of a non-toxicmarker E. coli strain in sterile straw, sterile pig farmyard

Bioresource Technology 84 (2002) 57–61

*Tel.: +44-1525-860-000; fax: +44-1525-861-735.

E-mail address: claire.turner@bbsrc.ac.uk (C. Turner).

0960-8524/02/$ - see front matter � 2002 Elsevier Science Ltd. All rights reserved.

PII: S0960-8524 (02 )00008-1

manure and sterile pig faeces at different temperaturesand different moisture contents. Although the markerstrain used was not conditioned for the temperaturesencountered during thermophilic composting, the aim ofthe study was to determine the minimum requirementsfor the inactivation of the marker strain under differentconditions, and infer the conditions that are thereforelikely to affect inactivation of similar pathogens duringcomposting. Data are also included in this study show-ing the growth of faecal coliforms throughout a compostheap of pig farmyard manure where the compostingtemperature was at mesophilic levels, demonstrating theimportance of ensuring that composting is carried out atsufficiently high temperatures. If a compost heap doesnot reach high enough temperatures, it is possible thatnot only will inactivation not occur, but pathogenicbacteria may in fact grow.

2. Methods

2.1. E. coli cultures

E. coli 11943 was cultivated at 37 �C and at 200 rpmin an orbital incubator for 16 h. Cultures were inocu-lated from freshly grown nutrient agar plates into six500 ml conical flasks containing 50 ml sterile nutrientbroth (Merck). After incubation and shaking for 16 h,the contents of all six flasks were pooled into a singleflask and the number of colonies per ml was measuredfrom the pooled flask. The volume of broth required foreach experiment was removed, and the flask containingthe remaining broth was kept unagitated at 20 �C for upto 72 h. This was to serve as a control for comparisonwith experiments where the broth had been added toeither straw, pig faeces or pig farmyard manure andincubated at different temperatures for up to 72 h (seebelow). The ammoniacal nitrogen content of the E. colibroth was 0.20 g/l.

2.2. Straw experiments

Wheat straw (WS) used in these experiments had a drymatter (DM) content of 91% (w/w) and an ammoniacalnitrogen content of 0 g/kg. One g WS was weighedout into each of several glass bottles and autoclaved at121 �C for 15 min.

2.3. Pig faeces experiments

Pig faeces (PF) used in these experiments was ob-tained from grower pigs fed on a proprietary grower pigfeed (from BOCM Pauls) containing copper, and pre-scription drug additive called Potencil. The faeces had adry matter (DM) content of 22% (w/w) and an ammo-niacal nitrogen content of 3.2 g/kg. Twenty g PF was

weighed out into each of several glass bottles and au-toclaved at 121 �C for 15 min.

2.4. Farmyard manure experiments

Pig farmyard manure (FYM) composed of straw, pigfaeces and urine was obtained from sows fed a propri-etary brand of dry sow rations from BOCM Pauls, withno specific additives. The FYM used in these experi-ments had a dry matter (DM) content of 26% and anammoniacal nitrogen content of 2.5 g/kg. Twenty gFYM was weighed out into each of several glass bottlesand autoclaved at 121 �C for 15 min.

2.5. Experimental protocol for WS, PF and FYMexperiments

At the start of each experiment, 1 or 10 ml of E. coliculture broth previously incubated for 16 h was added toeach of the sterile glass bottles containing either straw,pig faeces or pig farmyard manure, and the bottles wereshaken thoroughly. Two (i.e. duplicate bottles) wereassayed for E. coli titres immediately, two were kept at20 �C for the duration of the experiment, and the re-maining bottles were put in an incubator at either 50 or55 �C. At 1, 2, 5, 24 and 48 h (and in some cases, 72 h),duplicate flasks were removed from the incubator andassayed for E. coli as described below.

2.6. Extraction of E. coli from samples

E. coli was extracted from each of the samples byadding 0.1 M sodium phosphate buffer, pH 7, to eachbottle, the volume added being dependent upon how drythe material was (i.e. whether it was WS, PF or FYM,and how much culture broth had been added; in the caseof straw with 10 ml E. coli broth added, no sodiumphosphate was needed, but in the case of FYM withonly 1 ml broth, 30 ml was required). The contents ofthe bottles were mixed thoroughly, and the contentsdecanted and, except in the case of WS samples, werecentrifuged at 1700g in an IEC Centra 3E centrifuge(IEC, Dunstable, Bedfordshire, UK) for 10 min. Thesupernatants were then assayed for E. coli counts.

2.7. E. coli counts

E. coli titres or counts (cfu) were measured as follows:samples (sodium phosphate extracted for WS; extractedand centrifuged for PF and FYM, and neat for E. colibroth samples) were serially diluted by adding 1 ml toglass bottles containing 9 ml 0.7% NaCl. 100 ll of eachdilution was then added to fresh nutrient agar plates,and spread with an alcohol sterilised glass spreader. Theplates were incubated at 37 �C for 24 h, after whichcolonies were counted and titres obtained in cfu. In each

58 C. Turner / Bioresource Technology 84 (2002) 57–61

case, the titres were calculated back to per ml of culturebroth to allow direct comparisons to be made betweenWS, PF and FYM experiments at different added E. colilevels.

2.8. Compost heap experiment

One tonne of pig farmyard manure (from the samesource as that used in the small scale experiments) wasplaced in a specially constructed rig and forcibly aer-ated. The temperature at various points in the materialwas monitored, and kept at mesophilic temperaturesthrough the cooling effects of aeration. At time intervalsover 3 weeks, samples were taken from a particular partof the heap and cfu counts on petri dishes containingnutrient agar and MacConkey agar were taken byadding 20 or 30 ml 0.1 M sodium phosphate buffer, pH7, shaking, centrifuging, serially diluting in 0.7% NaCland plating as described above. The temperature of thecompost heap from where the samples were taken wasmonitored.

3. Results and discussion

3.1. Heated wheat straw samples

Results of heating E. coli in WS at 50 and 55 �C withlow or high moisture content (i.e. either 1 or 10 ml E.coli culture broth added) are given in Table 1. E. coli wasinactivated within 2 h at 55 �C; however, at 50 �C, it wasstill viable after 72 h. The moisture content of thesamples also played a role in inactivation. When 10 mlE. coli broth was added to the WS, inactivation occurredless rapidly than when 1 ml was added at both 50 and 55�C. In the case where 1 ml was added, the broth soakedinto the straw, with no residual liquid, whereas when 10ml was added, it was a liquid culture.

3.2. Heated pig faeces samples

Results of heating E. coli in PF at 50 and 55 �C withlower or higher moisture content (i.e. either 1 or 10 mlE. 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

Time 50 �C (as log10 cfu/ml) 55 �C (as log10 cfu/ml)

10 ml broth 1 ml broth 10 ml broth 1 ml broth

0 h 10 9.2 9.3 9.4

1 h 8.7 5.3 6.8 0

2 h 8.3 2.4 0 0

5 h 3.5 2.3 0 0

24 h 3.1 2.0 0 0

48 h nd 2.4 0 0

72 h nd 3.3 0 0

Control 0 h 10.0 9.2 9.1 9.4

Control 24 h 9.2 8.3 8.9 8.9

Control 48 h nd 9.0 8.5 8.5

Control 72 h nd 8.2 nd 8.7

Results for each experiment are averages of two samples; nd¼ not done; ‘‘control’’ refers to E. coli broth kept unagitated at 20 �C.

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

Time 50 �C (as log10 cfu/ml) 55 �C (as log10 cfu/ml)

10 ml broth 1 ml broth 10 ml broth 1 ml broth

0 h 9.4 8.7 8.9 9.0

1 h 9.0 8.1 0 4.2

2 h 7.6 5.0 0 0

5 h 2.3 3.1 0 0

24 h 0 2.9 0 0

48 h 1.8 2.3 0 0

72 h 3.0 2.8 0 0

Control 0 h 9.4 9.0 9.7 9.1

Control 24 h nd 8.7 nd 8.7

Control 48 h nd 8.3 nd nd

Control 72 h 9.7 8.4 8.4 8.5

Results for each experiment are averages of two samples; nd¼ not done; ‘‘control’’ refers to E. coli broth kept unagitated at 20 �C.

C. Turner / Bioresource Technology 84 (2002) 57–61 59

results show that E. coli was not destroyed at 50 �C inPF, and where 10 ml broth was added, although theE. coli counts dropped to below detectable levels after24 h, the bacteria recovered and were able to grow, pre-sumably when conditions selected for more temperatureresistant individuals. There were no significant differ-ences in E. coli survival in the 1 and 10 ml broth samplesat 50 �C. At 55 �C, E. coli was not detected in eitherexperiment after 1 h at that temperature, although it didsurvive for at least 1 h in the drier sample.

3.3. Heated pig farmyard manure samples

Results of heating E. coli in FYM at 50 and 55 �Cwith lower or higher moisture content (i.e. either 1 or 10ml E. coli culture broth added) are given in Table 3. At50 �C, the E. coli did not appear to survive for as long inthe FYM as in PF with both the 1 and 10 ml brothexperiments. The differences in dry matter and ammo-niacal nitrogen levels in the samples did not seem toaccount for these differences, although there did not

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

Time 50 �C (as log10 cfu/ml) 55 �C (as log10 cfu/ml)

10 ml broth 1 ml broth 10 ml broth 1 ml broth

0 h 8.3 8.0 9.6 8.9

1 h <4.3 6.6 1.8 2.4

2 h <4.3 3.8 0 <2.2

5 h <4.3 <2.2 0 <2.2

24 h 0 <2.2 0 <2.2

48 h 0 <2.2 0 <2.2

72 h 0 <2.2 nd nd

Control 0 h 8.8 8.8 10.49 9.6

Control 24 h nd nd nd nd

Control 48 h nd nd 9.4 8.9

Control 72 h 8.3 8.3 nd nd

Results for each experiment are averages of two samples; nd¼not done; ‘‘control’’ refers to E. coli broth kept unagitated at 20 �C.

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 8.7 nd 8.9 8.3

72 8.8 nd 9.3 7.9

Results given as log10 cfu/ml.

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.

60 C. Turner / Bioresource Technology 84 (2002) 57–61

appear to be differences in survival of E. coli at 55 �C,where E. coli was not detected after 1 h, both when 1 or10 ml E. coli broth was added.

3.4. Pig faeces and farmyard manure at 22 �C

In experiments where either 1 or 10 ml E. coli brothwas added to PF or FYM and maintained at 22 �C for72 h, there was no significant loss of titre of E. coli overthat time, and this is shown in Table 4. E. coli broth leftunagitated at 22 �C for 72 h also did not show anyappreciable loss of titre. This can be seen in Tables 2 and3, where values given for ‘‘controls’’ were of E. colibroth left at 20 �C.

3.5. Compost experiment

The above experiments demonstrate that E. coliwould be inactivated in farmyard manure, pig faecesand straw (the components of muck heaps) if kept at 55�C for more than 2 h. Fig. 1 shows the results of bac-terial counts from samples taken from a compost heapat mesophilic temperatures of pig farmyard manuregrown on both MacConkey agar (for faecal coliforms)and nutrient agar. The trend of bacterial counts duringthe course of the compost experiment seemed to followthe temperature trend of the area in the compost heapfrom which the sample was taken. The temperature inthe sampled area of the heap reached 35 �C, and thiscoincided with the highest total bacterial and faecalcoliform counts. It therefore shows that the compostingprocess will not inactivate coliforms (and thus patho-gens such as E. coli O157 and Salmonella) at mesophilictemperatures.

4. Conclusions

This work has shown the conditions required for theinactivation of a lab strain of E. coli, acting as a markerfor common pathogenic organisms that may be presentin animal manures. Results demonstrated that at 50 �C,

inactivation of E. coli may depend on the moisturecontent and the nature of the material. However, whenthe temperature is increased to 55 �C, inactivation pro-ceeds rapidly, and in all cases, E. coli was inactivated tobelow detectable levels within 2 h. Although tempera-tures required for inactivation of the lab strain maynot be as high as those required for the destruction of‘‘conditioned’’ pathogens, the results indicate that theinactivation is not merely temperature dependent, but isaffected by the moisture content and the nature of thematerial. If incomplete inactivation has taken place dueto insufficiently high temperature, recovery and growthof the damaged population may be possible.

Results from a composting experiment demonstratedthat coliforms grew in the compost heap if the compo-sting was conducted at mesophilic temperatures, so careshould be taken in maintaining adequate composting ofmaterial, rather than just leaving a heap unmonitored.

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