The thermal inactivation of E. coli in straw and pig manure
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Bioresource Technology 84 (2002) 57–61
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). Ó 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Composting; Pathogen inactivation; E. coli; Animal waste disinfection
1. Introduction ranged from 6 days to 3 weeks in manure and 2 days to 5
weeks in manure slurry.
Composting is a traditional way of treating livestock The United States Environmental Protection Agen-
manure to make it easier to dispose of on land, and to cy’s publication (EPA, 1985) on control of pathogens in
produce an inexpensive fertiliser. However, livestock biosolids, part 503, has a minimum time–temperature
manure may contain zoonotic microbial pathogens, requirement for in-vessel and aerated static pile com-
including Salmonella spp. and Escherichia coli O157. posting methods: the material must maintain a mini-
Composting as a means of treatment has the added mum temperature of 55 °C for at least three consecutive
advantage that if it is well managed, thermophilic tem- days. For turned windrow composting, at least 55 °C
peratures may be attained, which will inactivate those must be maintained for 15 consecutive days with the
pathogens present in the manure, making it safe for land material turned at least five times (Wu and Smith, 1999).
spreading. Problems may arise if pathogens have not These conditions are very stringent, and are designed to
been inactivated before land spreading, as it is known ensure that the composted material will not contain
that some potentially serious microorganisms may sur- pathogenic organisms. However, thermal destruction of
vive for a prolonged period in soil or on land. Salmo- bacterial pathogens (e.g. E. coli O157) may well depend
nella is known to survive for several months in stored on factors other than temperature, e.g. moisture con-
slurry; up to 6 months in cowpats and up to 100 days in tent, free ammonia concentration, duration of heat treat-
slurry applied to grass (Mawdsley, 1993). Maule (1998) ment and the presence of other microorganisms which
noted that E. coli O157 may survive for more than 56 may enhance or inhibit pathogen inactivation. For in-
days in fresh cattle faeces, and in cattle slurry at 18 °C stance, in an industrial compost, Salmonella and E. coli
for up to 9 days. Kudva et al. (1998) also noted the were found to survive for 59 days at about 60 °C, al-
longevity of E. coli O157 in muck – it can survive for 21 though the pathogens were destroyed during the cooler,
months in a manure pile. Another study (Himathongk- curing process (Droffner and Brinton, 1995). In the same
ham et al., 1999) found that survival times of E. coli study, survival was different in different composts, which
O157:H7 and Salmonella typhimurium in cow manure demonstrates that the mechanism for inactivation is
and cow slurry was dependent on temperature, and complex, and not solely dependent on temperature and
time.
*
Tel.: +44-1525-860-000; fax: +44-1525-861-735. This study examines the inactivation of a non-toxic
E-mail address: claire.turner@bbsrc.ac.uk (C. Turner). marker E. coli strain in sterile straw, sterile pig farmyard
0960-8524/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 6 0 - 8 5 2 4 ( 0 2 ) 0 0 0 0 8 - 158 C. Turner / Bioresource Technology 84 (2002) 57–61
manure and sterile pig faeces at different temperatures weighed out into each of several glass bottles and au-
and different moisture contents. Although the marker toclaved at 121 °C for 15 min.
strain used was not conditioned for the temperatures
encountered during thermophilic composting, the aim of 2.4. Farmyard manure experiments
the study was to determine the minimum requirements
for the inactivation of the marker strain under different Pig farmyard manure (FYM) composed of straw, pig
conditions, and infer the conditions that are therefore faeces and urine was obtained from sows fed a propri-
likely to affect inactivation of similar pathogens during etary brand of dry sow rations from BOCM Pauls, with
composting. Data are also included in this study show- no specific additives. The FYM used in these experi-
ing the growth of faecal coliforms throughout a compost ments had a dry matter (DM) content of 26% and an
heap of pig farmyard manure where the composting ammoniacal nitrogen content of 2.5 g/kg. Twenty g
temperature was at mesophilic levels, demonstrating the FYM was weighed out into each of several glass bottles
importance of ensuring that composting is carried out at and autoclaved at 121 °C for 15 min.
sufficiently high temperatures. If a compost heap does
not reach high enough temperatures, it is possible that 2.5. Experimental protocol for WS, PF and FYM
not only will inactivation not occur, but pathogenic experiments
bacteria may in fact grow.
At the start of each experiment, 1 or 10 ml of E. coli
culture broth previously incubated for 16 h was added to
2. Methods each of the sterile glass bottles containing either straw,
pig faeces or pig farmyard manure, and the bottles were
2.1. E. coli cultures shaken thoroughly. Two (i.e. duplicate bottles) were
assayed for E. coli titres immediately, two were kept at
E. coli 11943 was cultivated at 37 °C and at 200 rpm 20 °C for the duration of the experiment, and the re-
in an orbital incubator for 16 h. Cultures were inocu- maining bottles were put in an incubator at either 50 or
lated from freshly grown nutrient agar plates into six 55 °C. At 1, 2, 5, 24 and 48 h (and in some cases, 72 h),
500 ml conical flasks containing 50 ml sterile nutrient duplicate flasks were removed from the incubator and
broth (Merck). After incubation and shaking for 16 h, assayed for E. coli as described below.
the contents of all six flasks were pooled into a single
flask and the number of colonies per ml was measured 2.6. Extraction of E. coli from samples
from the pooled flask. The volume of broth required for
each experiment was removed, and the flask containing E. coli was extracted from each of the samples by
the remaining broth was kept unagitated at 20 °C for up adding 0.1 M sodium phosphate buffer, pH 7, to each
to 72 h. This was to serve as a control for comparison bottle, the volume added being dependent upon how dry
with experiments where the broth had been added to the material was (i.e. whether it was WS, PF or FYM,
either straw, pig faeces or pig farmyard manure and and how much culture broth had been added; in the case
incubated at different temperatures for up to 72 h (see of straw with 10 ml E. coli broth added, no sodium
below). The ammoniacal nitrogen content of the E. coli phosphate was needed, but in the case of FYM with
broth was 0.20 g/l. only 1 ml broth, 30 ml was required). The contents of
the bottles were mixed thoroughly, and the contents
2.2. Straw experiments decanted and, except in the case of WS samples, were
centrifuged at 1700g in an IEC Centra 3E centrifuge
Wheat straw (WS) used in these experiments had a dry (IEC, Dunstable, Bedfordshire, UK) for 10 min. The
matter (DM) content of 91% (w/w) and an ammoniacal supernatants were then assayed for E. coli counts.
nitrogen content of 0 g/kg. One g WS was weighed
out into each of several glass bottles and autoclaved at 2.7. E. coli counts
121 °C for 15 min.
E. coli titres or counts (cfu) were measured as follows:
2.3. Pig faeces experiments samples (sodium phosphate extracted for WS; extracted
and centrifuged for PF and FYM, and neat for E. coli
Pig faeces (PF) used in these experiments was ob- broth samples) were serially diluted by adding 1 ml to
tained from grower pigs fed on a proprietary grower pig glass bottles containing 9 ml 0.7% NaCl. 100 ll of each
feed (from BOCM Pauls) containing copper, and pre- dilution was then added to fresh nutrient agar plates,
scription drug additive called Potencil. The faeces had a and spread with an alcohol sterilised glass spreader. The
dry matter (DM) content of 22% (w/w) and an ammo- plates were incubated at 37 °C for 24 h, after which
niacal nitrogen content of 3.2 g/kg. Twenty g PF was colonies were counted and titres obtained in cfu. In eachC. Turner / Bioresource Technology 84 (2002) 57–61 59
case, the titres were calculated back to per ml of culture 3. Results and discussion
broth to allow direct comparisons to be made between
WS, PF and FYM experiments at different added E. coli 3.1. Heated wheat straw samples
levels.
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.
2.8. Compost heap experiment
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
One tonne of pig farmyard manure (from the same
still viable after 72 h. The moisture content of the
source as that used in the small scale experiments) was
samples also played a role in inactivation. When 10 ml
placed in a specially constructed rig and forcibly aer-
E. coli broth was added to the WS, inactivation occurred
ated. The temperature at various points in the material
less rapidly than when 1 ml was added at both 50 and 55
was monitored, and kept at mesophilic temperatures
°C. In the case where 1 ml was added, the broth soaked
through the cooling effects of aeration. At time intervals
into the straw, with no residual liquid, whereas when 10
over 3 weeks, samples were taken from a particular part
ml was added, it was a liquid culture.
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 3.2. Heated pig faeces samples
7, shaking, centrifuging, serially diluting in 0.7% NaCl
and plating as described above. The temperature of the Results of heating E. coli in PF at 50 and 55 °C with
compost heap from where the samples were taken was lower or higher moisture content (i.e. either 1 or 10 ml
monitored. 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
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
0h 10 9.2 9.3 9.4
1h 8.7 5.3 6.8 0
2h 8.3 2.4 0 0
5h 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 0h 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; nd ¼ 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
0h 9.4 8.7 8.9 9.0
1h 9.0 8.1 0 4.2
2h 7.6 5.0 0 0
5h 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 0h 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; nd ¼ not done; ‘‘control’’ refers to E. coli broth kept unagitated at 20 °C.60 C. Turner / Bioresource Technology 84 (2002) 57–61
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
0h 8.3 8.0 9.6 8.9
1hC. Turner / Bioresource Technology 84 (2002) 57–61 61
appear to be differences in survival of E. coli at 55 °C, inactivation of E. coli may depend on the moisture
where E. coli was not detected after 1 h, both when 1 or content and the nature of the material. However, when
10 ml E. coli broth was added. the temperature is increased to 55 °C, inactivation pro-
ceeds rapidly, and in all cases, E. coli was inactivated to
3.4. Pig faeces and farmyard manure at 22 °C below detectable levels within 2 h. Although tempera-
tures required for inactivation of the lab strain may
In experiments where either 1 or 10 ml E. coli broth not be as high as those required for the destruction of
was added to PF or FYM and maintained at 22 °C for ‘‘conditioned’’ pathogens, the results indicate that the
72 h, there was no significant loss of titre of E. coli over inactivation is not merely temperature dependent, but is
that time, and this is shown in Table 4. E. coli broth left affected by the moisture content and the nature of the
unagitated at 22 °C for 72 h also did not show any material. If incomplete inactivation has taken place due
appreciable loss of titre. This can be seen in Tables 2 and to insufficiently high temperature, recovery and growth
3, where values given for ‘‘controls’’ were of E. coli of the damaged population may be possible.
broth left at 20 °C. Results from a composting experiment demonstrated
that coliforms grew in the compost heap if the compo-
3.5. Compost experiment sting was conducted at mesophilic temperatures, so care
should be taken in maintaining adequate composting of
The above experiments demonstrate that E. coli material, rather than just leaving a heap unmonitored.
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 bac- References
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4. Conclusions Mawdsley, 1993. Pathogenic microorganisms in livestock waste and
factors influencing their transport to the aqueous environment.
Appendix 1 of: Protozoan, bacterial and virus pathogens, farm
This work has shown the conditions required for the
wastes and water quality protection. Final Report for Ministry of
inactivation of a lab strain of E. coli, acting as a marker Agriculture, Fisheries and Food Open Contract CSA2064.
for common pathogenic organisms that may be present Wu, N., Smith, J.E., 1999. Reducing pathogen and vector attraction
in animal manures. Results demonstrated that at 50 °C, for biosolids. Biocycle (November), 59–61.You can also read
