Download The effect of summer and winter seasons on the survival of

Journal of Applied Microbiology 2001, 91, 1036±1043
The effect of summer and winter seasons on the survival
of Salmonella typhimurium and indicator micro-organisms
during the storage of solid fraction of pig slurry
I. PlachaÂ, J. VenglovskyÂ, N. SasaÂkova and I.F. Svoboda1
Research Institute of Veterinary Medicine, KosÏice, Slovak Republic and 1SAC, Auchincruive, Ayr, Scotland, UK
813/5/01: received 11 April 2001, revised 21 May 2001 and accepted 1 June 2001
 , N . S A S A K O V A A N D I . F . S V O B O D A . 2001.
I . P L A C H AÂ , J . V E N G L O V S K Y
Aims: Investigations were carried out to observe the in¯uence of winter/spring and summer
periods on the survival of Salmonella typhimurium and indicator bacteria (psychrophilic,
mesophilic, coliform and faecal coliform bacteria and faecal streptococci) in the solid fraction of
pig slurry from agricultural wastewater treatment plant.
Methods and Results: Leather squares and PVC bottles with openings served as test carriers.
They were inoculated with broth culture of Salm. typhimurium and introduced directly into the
solid fraction. During the experiment, quantitative and qualitative examinations were carried
out to determine the presence of Salm. typhimurium and observe the dynamics of indicator
bacteria in the solid fraction.
Conclusions: Salmonella typhimurium survived for 26 d in summer and for 85 d in winter/
spring. The T90 values of indicator bacteria in summer ranged from 35á44 d (coliform bacteria)
up to 100á29 d (mesophilic bacteria). The winter T90 values of indicator bacteria ranged from
74á58 d (faecal coliform bacteria) to 233á07 d (coliform bacteria).
Signi®cance and Impact of the Study: The present study demonstrated that it is necessary
to pay increased attention to the manipulation of slurry solid fraction.
INTRODUCTION
The operation of large-capacity farms housing high number
of animals in one location creates problems with regard to
possible spreading of various diseases, some of them
transmissible also to humans, either directly through excrement or via animal products. Diseases that can be transmitted
include helminthoses, fungal diseases (e.g. trichophytosis),
salmonellosis, leptospirosis, tularaemia and colibacillosis.
Farms with litterless technology, producing liquid excrements, pose a serious risk. This technology of housing fails
to create conditions for biothermal processes in the manure
which, together with other factors, encourage destruction of
pathogens.
One way to solve these problems is the operation of
wastewater treatment plants (WTP) on livestock farms.
Wastewaters from large-capacity farms are processed by
technologies which separate the solid and liquid fractions on
Correspondence to: I. F. Svoboda, SAC, Auchincruive, Ayr KA6 5HW,
Scotland, UK.
vibrating screens or ®lter belts. The solid fraction is
disposed to ®eld manure storage or to land®lls which could
be a reservoir of many pathogens. On the other hand, it is
necessary to remember that excrements of farm animals are
an important source of organic substances which should be
returned to the soil. Therefore, all methods of manure
treatment should concentrate on the improvement of its
properties and a maximal utilization of its manuring value
(Tofant et al. 2000).
Venglovsky et al. (1994), NovaÂk (1994) and colleagues
concluded that the most suitable way of processing this
material was a biothermic composting in the thermophilic
temperature region which can ensure destruction of pathogens. Despite recommendations, insuf®cient attention has
been paid to a practical manipulation of slurry solid fraction
in practice. From this point of view we investigated survival
of Salmonella typhimurium and a series of indicator microorganisms in solid fraction of slurry. Salmonella typhimurium
was chosen as a model strain since it can cause serious
zoonoses disseminated via faecal contamination.
One of the most important factors affecting the survival of
micro-organisms in the environment is temperature.
ã 2001 The Society for Applied Microbiology
SURVIVAL OF SALM. TYPHIMURIUM IN PIG SLURRY
Therefore we investigated the in¯uence of winter and
summer conditions on survival of the above-mentioned
model strain and of indicator micro-organisms.
This study is intended to provide more information on
pathogen survival in solid fraction of pig slurry. Currently,
similar information is only available for slurry, sewage
sludge and manures.
MATERIALS AND METHODS
Solid fraction of slurry
The solid fraction of slurry used in our experiments was
obtained by slurry mechanical separation by vibrating
screens. This is the ®rst stage of the treatment process of
pig slurry from a farm in KosÏicka Polianka, Slovak Republic
with capacity of 23 000 fattening pigs. Slurry was treated by
an aerobic process in a WTP with mechanical, chemical and
biological treatment stages.
Storage
The solid fraction was placed into a 50-l container. This
container was located outdoors in a fenced space and covered
with a netting to prevent contamination with insects.
Experiments were carried out in the summer period in the
months of June to August and in winter/spring from January
to June. The temperature of the environment and in the solid
fraction was recorded during the experiments in 1-h intervals
by means of a programmable registration thermometer
Commeter (COMET System, RozÏnov p. R., Czech Republic).
Test carriers
Two test carriers were used. These were sterile leather
squares (4 ´ 4 cm) and sterile PVC bottles (100 ml, with
side openings to ensure the contact with the environment).
Sterile 20 cm long glass ampoules, 1 cm in diameter, were
used for controls.
Test bacterium and inoculation of test carriers
Â
A lyophilized strain of Salm. typhimurium SK 14/39 (SZU
Prague, Czech Republic) was used in our experiments.
Aliquots of 0á2 ml of 24-h broth cultures (Nutrient Broth
No. 2, Imuna, SÏarisÏske Michal'any, Slovakia) of Salm.
typhimurium were used to inoculate leather squares. Dried
leather pieces were transferred to a thermostat controlled at
37°C and incubated for 24 h to achieve good adhesion of
salmonellae to the carriers.
A 20-g sample of slurry solid fraction was transferred to
each PVC bottle, to which 0á5 ml aliquots of broth culture of
Salm. typhimurium were applied directly into the solid
1037
fraction. Additionally, 1 ml aliquots of broth culture of
Salm. typhimurium were transferred to control glass
ampoules which were sealed.
Leather pieces, PVC bottles and control glass ampoules
were inserted directly into the solid fraction placed in the
50-l container.
Quantitative and qualitative examinations
of Salm. typhimurium
Both carriers were examined by the method of MuÈller
(1973). Each leather carrier was transferred to 50 ml of
sterile 0á9% saline solution under sterile conditions and 20 g
of the material from the PVC bottle carrier was transferred
to 180 ml of sterile 0á9% saline solution, and both shaken for
30 min by mechanical shaker. After 30 min sedimentation,
each carrier was examined quantitatively and qualitatively
for presence of salmonellae. In quantitative examination we
prepared series of decimal dilutions up to 10±10 from both
carriers and the control ampoule and inoculated 0á1 ml on to
Xylose Lysine Deoxycholate Agar (XLD, Imuna) and on to
Salmonella Shigella Agar (SS, Imuna). Both plates were
incubated at 37°C for 24 and 48 h.
In qualitative examination we used for the purpose of
nonselective cultivation, Buffered Peptone Water (BPW,
Becton Dickinson, Cockeysville, MD, USA) as a preenrichment medium which was incubated at 37°C for 24 h.
The selective cultivation was then carried out in two
enrichment media: Selenite medium (Imuna) at 37°C for
48 h, and in a medium according to Rappaport and
Vassiliadis (Merck, Darmstadt, Germany) at 43°C for
48 h. A loopful from each of the selective broth was plated
on XLD and SS agars (Imuna) and both plates were
incubated at 37°C for 24 and 48 h.
The presumptive salmonella colonies were examined
biochemically using a system for identi®cation of bacteria
from the family of Enterobacteriaceae (BBL Enterotube,
Becton Dickinson). Serological examinations were carried
out at the State Veterinary Institute in KosÏice (Slovakia),
Department of Microbiology.
The carriers were examined in the time intervals presented in Table 1 (summer) and Table 4 (winter/spring). The
values obtained by quantitative examination, presented in
the tables, are arithmetic means of three parallel examinations. Qualitative tests were considered negative in those
cases in which all three parallel examinations for the
presence of Salm. typhimurium were negative.
Presence of Salmonella spp. in the solid
fraction of slurry
At the beginning of our experiments we examined the
original solid fraction for the presence of Salmonella spp.
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 1036±1043
1038 I . P L A C H AÂ ET AL.
Table 1 Detection time of Salmonella typhimurium in solid fraction ±
summer
Time
(d)
Leather carrier
(cfu ml)1)
PVC bottle
(cfu ml)1)
Control sample
(cfu ml)1)
0
1
5
26
48
60
72
Slope
T90
2á21 ´ 102
1á28 ´ 102
)
±
±
±
)0á42616
2á35
9á37 ´ 104
5á67 ´ 104
7á88 ´ 102
+
±
±
±
)0á42911
2á33
8á98 ´ 104
8á37 ´ 104
2á28 ´ 104
4á18 ´ 103
5á21 ´ 104
4á89 ´ 103
)0á01501
66á63
Psychrophilic and mesophilic bacteria. Plate counts of
psychrophilic and mesophilic bacteria were determined
using Nutrient agar No. 2 (Imuna). The psychrophilic
bacteria were incubated at 20°C for 72 h and the mesophilic
bacteria at 37°C for 24 h.
Faecal coliforms and coliform bacteria. Faecal coliforms
and coliform bacteria were determined using Endo Agar
(Imuna), incubated at 43°C for 48 h and at 37°C for 24 h,
respectively.
Faecal streptococci. Plate counts of faecal streptococci
were determined using Selective agar for isolation of faecal
streptococci (Imuna), incubated at 37°C for 24 h.
+, detected only qualitatively.
), not detected.
using the method of Philipp et al. (1990). For the nonselective cultivation we added 5 g of the solid fraction to 45 ml
of BPW (Becton Dickinson) and incubated the mixture at
37°C for 24 h. Selective cultivation was carried out in a
Selenite medium (Imuna) and Rappaport-Vassiliadis
medium (Merck). Aliquots from both selective media were
inoculated in parallel on XLD agar (Imuna) and Rambach
agar (Merck). The procedure used was the same as that
described above for the qualitative examination of carriers.
Physico-chemical parameters
The physico-chemical parameters ± pH, dry matter
content, ammonia nitrogen and total nitrogen ± were
determined according to Placha et al. (1997) and total
phosphorus according to the Standard Methods for Examination of Water and Wastewater (APHA 1985). The
samples for physico-chemical examinations were taken in
time intervals speci®ed in Table 3 (summer) and Table 6
(winter/spring).
Statistical analyses
Indicator bacteria ± media and bacteriological
procedures
Samples of the solid fraction for observation of dynamics of
indicator micro-organisms were examined in time intervals
speci®ed in Table 2 (summer) and Table 5 (winter/spring).
Twenty grams of the sampled material were transferred into
a 500-ml bottle, 180 ml of 0á9% saline solution were added
and the mixture was shaken for 5 min. Then, after 1 min of
sedimentation, the supernatant was examined to determine
the number of indicator micro-organisms. The samples were
diluted with 0á9% saline solution down to the concentration
of 10±6.
The in¯uence of physico-chemical parameters on the
survival of Salm. typhimurium and on the dynamics of
indicator bacteria was evaluated by means of correlation
analysis of logarithmically transformed data. The time of
survival of the observed micro-organisms was expressed as
T90 values. The decimation time (T90), as de®ned by
Schlundt (1984), is the time taken for viable counts of a
population to decrease by one logarithmic unit (log10) which
is equivalent to a 90% reduction. The decimation time T90
was calculated according to the formula:
T90 ˆ ÿ1=a
Time
(d)
Psychrophilic
bacteria
(cfu ml)1)
Mesophilic
bacteria
(cfu ml)1)
Coliforms
(cfu ml)1)
Faecal
coliforms
(cfu ml)1)
0
5
26
48
60
72
Slope
T90
6á67 ´ 107
4á88 ´ 105
2á18 ´ 107
2á71 ´ 105
3á51 ´ 105
1á1 ´ 105
)0á02803
35á68
3á38 ´ 107
1á64 ´ 107
1á57 ´ 106
3á12 ´ 106
2á01 ´ 106
9á87 ´ 106
)0á00997
100á29
6á92 ´ 105
6á20 ´ 106
4á52 ´ 106
6á11 ´ 104
3á02 ´ 104
6á78 ´ 104
)0á02821
35á44
9á35 ´
9á76 ´
8á51 ´
8á53 ´
7á02 ´
8á76 ´
)0á0103
97á09
104
104
105
104
104
104
Faecal
streptococci
(cfu ml)1)
Table 2 Dynamics of indicator microorganisms in solid fraction ± summer
2á37 ´ 104
1á93 ´ 104
4á63 ´ 103
5á00 ´ 103
3á01 ´ 102
2á98 ´ 102
)0á02711
36á89
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 1036±1043
SURVIVAL OF SALM. TYPHIMURIUM IN PIG SLURRY
Table 3 Physico-chemical parameters in
solid fraction ± summer
1039
Time
(d)
pH
Dry matter
(%)
N-total
(g kg)1)
N-NH4
(g kg)1)
Volatile
solids (%)
P-total
(g kg)1)
Temperature
(°C)
0
5
26
48
60
72
8á08
7á28
6á50
5á75
5á80
5á91
21á37
20á02
20á66
14á96
13á81
13á43
39á56
42á99
20á20
28á21
31á20
32á10
0á78
0á84
0á54
0á48
0á52
0á50
93á20
92á74
91á80
90á88
88á02
81á21
12á24
12á19
11á92
9á34
9á81
9á57
21á96
22á21
22á57
20á87
21á82
21á24
where a is a decimation constant corresponding to the line
slope compared to the axis x. The T90 values were determined
using the method of minimum squares. In addition to the
values of T90, the tables also contain the gradient values of
regression lines ®tted through the given points.
RESULTS
Summer period
Survival of Salm. typhimurium. During the summer
period, Salm. typhimurium survived the longest period of
26 d in the PVC bottle carrier (Table 1). During this time a
change in the pH value from alkaline to acidic was recorded
(from the original value of 8á08±6á50 ± Table 3). The counts
of Salm. typhimurium bacteria in the control glass ampoules
decreased only by one order of magnitude at the end of the
experiment. The values of T90 for Salm. typhimurium
bacteria were 2á35 d on the leather carrier, 2á33 d in the
PVC bottle and 66á63 d in the control ampoule (Table 1).
Indicator bacteria. The numbers of indicator and other
bacteria in the solid fraction, ranged from 103 to 107 cfu ml±1
at the beginning of the experiment (Table 2). The most
pronounced decrease (by two orders of magnitude) was
observed in psychrophilic bacteria and faecal streptococci,
and a lesser decrease (by one order) was observed in coliforms
and mesophilic bacteria. Faecal coliforms persisted on the
same level during the entire experiment with the exception of
d 26, when an increase by one order was recorded followed by
a subsequent decrease to the original value on d 48. The
decimation time values ranged from 35á44 d (coliform
bacteria) to 100á29 d (mesophilic bacteria, Table 2).
Physico-chemical parameters. Of the physicochemical
parameters observed, the most marked decrease was detected in the values of pH, which declined from the starting
value of 8á08 to the ®nal value of 5á91 at the end of the
experiment. The values of NH4+-N showed a decreasing
tendency with the exception of d 5 of the experiment when a
slight increase with regard to the starting value was
observed. Other values showed slightly decreasing tendency
(Table 3).
The temperature of the outer environment ranged from
10°C to 33°C and the temperature in the solid fraction
varied between 17°C and 26°C.
Winter/spring period
Survival of Salm. typhimurium. In the winter period,
Salm. typhimurium was detected qualitatively until d 85 on
both carriers (Table 4). Quantitative determinations provided positive results until d 4 on the leather carrier and until
d 54 in the PVC bottle. The values of decimation times T90
reached 0á96 d on the leather carrier and 12á91 d in the PVC
bottles (Table 4).
Indicator bacteria. The counts of indicator bacteria in the
solid fraction during winter ranged from 102 to 108 cfu ml±1
of the sample (Table 5). During the storage of the solid
fraction the numbers of psychrophilic and mesophilic
bacteria decreased by one order of magnitude. The numbers
of faecal streptococci and faecal coliforms decreased by two
orders and the numbers of coliforms decreased by one order
by d 54 of the experiment. However, on d 120 an increase to
original numbers was observed. The values of T90 ranged
from 74á58 d (faecal coliforms) to 233á07 d (coliform
microorganisms) (Table 5).
Physico-chemical parameters. The physicochemical
examination showed considerable decrease in ammonia
nitrogen, which declined to 38% of the initial concentration
(Table 6). The values of total nitrogen and total phosphorus
showed a slightly increasing tendency. The value of pH
decreased from the starting 8á52 to 7á51 and the values of
volatile solids also decreased. Dry matter content was
decreasing till d 40 of the experiment and then it increased
to the starting value (Table 3).
The ambient temperatures ¯uctuated in the range
between )11°C and +34°C and the temperature in the
solid fraction ranged from )1°C to 30°C.
Statistical evaluation
In the summer period a statistically signi®cant correlation
was observed between survival of Salm. typhimurium and pH
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 1036±1043
1040 I . P L A C H AÂ ET AL.
Table 4 Detection time of Salmonella typhimurium in solid fraction ±
winter/spring
Time
(d)
Leather carrier
(cfu ml)1)
PVC bottle
(cfu ml)1)
Control sample
(cfu ml)1)
0
1
4
40
54
76
85
120
137
152
Slope
T90
2á43 ´ 104
3á35 ´ 102
+
+
+
+
)
)
)
)1á03643
0á96
1á48 ´ 107
4á32 ´ 107
5á91 ´ 107
8á43 ´ 104
1á60 ´ 103
+
+
)
)
)
)0á07744
12á91
1á54
5á50
4á69
3á23
2á99
2á39
3á39
7á12
5á67
NR
´
´
´
´
´
´
´
´
´
107
107
108
107
107
108
108
107
107
NR, no reduction, the slope of survival curves was zero.
+, detected only qualitatively.
), not detected.
on both leather and PVC carrier (r ˆ 0á904, r ˆ 0á907,
respectively). Signi®cantly positive correlations were also
observed between survival of salmonellae and the level of
N-NH3 (r ˆ 0á959), N-total (r ˆ 0á972), volatile solids
(r ˆ 0á973), P-total (r ˆ 0á999), and a negative correlation
between temperature and survival of salmonellae on PVC
carrier (r ˆ 0á936). Temperature and the survival of
salmonellae on the leather carrier exhibited a tendency to
negative correlation (r ˆ 0á869) while dry matter and
survival of the tested salmonellae correlated positively
(r ˆ 0á842). The in¯uence of other physico-chemical
parameters on the survival of salmonellae was not signi®cant.
In this period a signi®cantly positive correlation was
observed between survival of coliform bacteria and P-total
(r ˆ 0á908). Positive relationships were detected between
faecal streptococci and volatile solids and dry matter
(r ˆ 0á865, r ˆ 0á859, respectively). A tendency to positive
Time
(d)
Psychrophilic
bacteria
(cfu ml)1)
Mesophilic
bacteria
(cfu ml)1)
0
4
40
54
76
85
120
137
152
Slope
T90
2á59 ´ 107
2á80 ´ 108
1á87 ´ 108
7á12 ´ 107
3á44 ´ 107
9á64 ´ 106
9á00 ´ 106
6á41 ´ 106
7á34 ´ 106
)0á00896
111á67
3á01 ´ 107
5á37 ´ 107
4á21 ´ 106
1á73 ´ 105
6á50 ´ 106
9á17 ´ 106
6á32 ´ 105
3á03 ´ 106
2á13 ´ 106
)0á00738
135á55
correlations was also observed between coliform and
psychrophilic bacteria and dry matter (r ˆ 0á891,
r ˆ 0á837, respectively). No signi®cant relationship was
observed between other investigated parameters.
In the winter/spring period, comparison of survival of
Salm. typhimurium and physico-chemical parameters showed
signi®cant correlations between survival and temperature on
PVC and leather carriers (r ˆ 0á997, r ˆ 0á995, respectively). Salmonellae on the leather carrier were affected
positively by dry matter and volatile solids (r ˆ 0á822,
r ˆ 0á886, respectively) and negatively by N-total
(r ˆ 0á888). A tendency to positive correlations between
survival of salmonellae and pH and N-NH3 was observed on
the PVC carrier (r ˆ 0á862, r ˆ 0á847, respectively). A
tendency to a positive correlation was also recorded between
pH and faecal streptococci (r ˆ 0á802) and of negative
correlations between temperature and faecal coliform
bacteria and faecal streptococci (r ˆ 0á800, r ˆ 0á876,
respectively).
DISCUSSION
The results indicate that the survival of Salm. typhimurium
and indicator bacteria was considerably affected by temperature. Our results point to the differences in the survival
of salmonellae during summer (26 d) and winter/spring
(85 d) and therefore con®rm the ®ndings of a number of
authors (Dean and Lund 1981; Strauch 1991; Ahmed and
Sorensen 1995; Cabadaj et al. 1995). This observation is also
supported by MuÈller (1973) who stated that different strains
of salmonellae survived in slurry for 4±97 d in summer and
up to 87 d in winter/spring.
In our study we refer to a winter/spring period because
the experiment began in January and terminated in May.
The tested strain was recovered qualitatively up to March
but to terminate the experiment three subsequent negative
analyses were needed. Shorter time of survival of Salm.
Coliforms
(cfu ml)1)
Faecal
coliforms
(cfu ml)1)
Faecal
streptococci
(cfu ml)1)
3á48 ´ 105
1á58 ´ 105
2á31 ´ 105
5á03 ´ 104
2á55 ´ 104
3á90 ´ 104
5á05 ´ 105
5á19 ´ 104
1á34 ´ 104
)0.00429
233á07
5á32 ´ 105
7á04 ´ 104
3á51 ´ 104
1á29 ´ 104
1á69 ´ 104
1á23 ´ 104
3á00 ´ 102
5á15 ´ 103
4á13 ´ 103
)0á01341
74á58
1á38 ´ 105
1á80 ´ 105
1á49 ´ 104
3á72 ´ 104
1á50 ´ 104
2á93 ´ 104
7á30 ´ 103
6á85 ´ 103
2á11 ´ 103
)0á01048
95á38
Table 5 Dynamics of indicator microorganisms in solid fraction ± winter/spring
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 1036±1043
SURVIVAL OF SALM. TYPHIMURIUM IN PIG SLURRY
Table 6 Physico-chemical parameters in
solid fraction )winter/spring
1041
Time
(d)
pH
Dry matter
(%)
N-total
(g kg)1)
N-NH4
(g kg)1)
Volatile
solids (%)
P-total
(g kg)1)
Temperature
(°C)
0
4
40
54
76
85
120
137
152
8á52
8á14
8á26
8á60
8á31
8á10
7á01
7á28
7á51
16á06
15á95
13á09
15á48
15á55
15á13
15á09
16á68
16á21
3á49
3á67
4á54
4á97
4á11
4á41
4á75
4á49
4á48
1á37
0á99
1á12
0á70
0á84
0á91
0á94
0á49
0á52
91á07
89á33
89á47
91á66
89á44
87á71
87á91
88á88
87á71
1á68
2á09
1á27
1á16
1á20
1á06
2á01
1á88
1á57
1á75
)0á30
5á56
9á53
3á73
5á28
17á05
16á18
20á62
typhimurium in the summer period in comparison with
winter/spring period may be explained, besides other
physicochemical factors, also by higher temperature in the
summer. The increase in temperature in the spring was most
likely one of the important factors contributing to devitalization of salmonellae in the winter/spring period. This is
supported also by our statistical analyses (signi®cant correlation between survival and temperature on PVC and leather
carriers r ˆ 0á997, r ˆ 0á995, respectively).
In addition to the temperature effect, the survival of
salmonellae is also affected signi®cantly by the dry matter
content. Mitscherlich and Marth (1984) compared the
survival of Salm. typhimurium in three types of manure
differing by dry matter content and concluded that
Salm. typhimurium micro-organisms survived for 84 d in
the manure with dry matter content of 17á3%, comparable
with dry matter percentage in the solid fraction used in our
experiments (Table 6), at a temperature of 10°C. This result
is comparable with our results, which showed that Salm.
typhimurium micro-organisms survived for 85 d in the solid
fraction during the winter period (Table 4).
The longer survival of salmonellae at lower temperatures
is also indicated by decimation time values, T90, determined
in our experiment. The T90 values for the tested strain Salm.
typhimurium on leather carriers and in the PVC bottles were
0á96 d and 12á91 d, respectively, in winter and 2á35 d and
2á33 d, respectively, in summer.
The in¯uence of temperature on the survival of indicator
micro-organisms is evident also in our experiments. The
counts of indicator bacteria during summer and winter are
comparable with the counts obtained by OndrasÏovicÏovaÂ
et al. (1994) during the storage of slurry, with the difference
that the results show considerable decrease in the number of
coliform and faecal coliform micro-organisms during 6-week
storage of slurry while in our experiment only psychrophilic
micro-organisms in summer and faecal streptococci in
winter exhibited pronounced decreases.
Our results are also comparable with the ®ndings of
Jepsen et al. (1997) from the storage of sludges in which a
signi®cant reduction of salmonellae was observed in summer
(average temperature 20°C) and a slower reduction in winter
(average temperature below 10°C). These authors did not
10
9
8
cfu ml–1, log10
7
6
5
4
3
2
1
Fig. 1 Indicator bacteria in an in¯uent (h)
and in a solid fraction (j)
0
Psychrophilic bacteria
Mesophilic bacteria
ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 91, 1036±1043
Coliforms
Faecal coliforms
1042 I . P L A C H AÂ ET AL.
observe systematic reduction in faecal streptococci during
the storage and did not recommend to use them as indicator
micro-organisms in the process of improving the hygienic
quality of sludges by storage.
During our experiments we detected an increase of total
nitrogen. Nitrogen is the major nutrient required by
micro-organisms in the assimilation of the carbon substrates
in organic wastes. Golueke (1977) postulated that 2/3 of the
carbon in the material consumed is given off as CO2 and the
other 1/3 is combined with nitrogen in the living cell
leading to an increase in total N.
Our results proved that the decrease of the pH value has a
devitalization effect on micro-organisms. This was also
con®rmed by statistical evaluation which showed that the
more apparent pH change from d 85 in winter and from d
48 in summer resulted in reduction in the counts of
indicator micro-organisms. Our statistical evaluations point
to the fact that the pH changes affect essentially the vitality
of micro-organisms. The observations by Strauch (1987),
according to whom 90% of salmonellae reduction is
connected with pH decrease in the substrate, could be
con®rmed by our results. According to Strauch (1987) the
decrease of the pH value during storage is in¯uenced by the
natural bacterial ¯ora producing fatty acids which have toxic
effects upon salmonellae. The latter, in contrast to natural
bacterial ¯ora, are not able to secure nutrients and this
probably causes their die-off.
Numbers of indicator bacteria in the solid fraction
detected by Placha et al. (1997) were higher than in an
in¯uent to a WTP found by Venglovsky et al. (1994), Fig. 1.
Our results, well as those of other authors (VenglovskyÂ
et al. 1994; PacÏajova and Venglovsky 1997), indicate that the
solid fraction of pig slurry is highly contaminated with
pathogenic micro-organisms and therefore its treatment
deserves increased attention.
Niewolak (1994) stated that micro-organisms (Escherichia,
Salmonella) that reach the soil by means of application of
contaminated pig slurry can penetrate to the depth of
160±180 cm. Henry et al. (1995) isolated salmonellae from
pasture 2 months, and from soil 8 months, after the
application of contaminated pig slurry. This is the reason
why it is necessary to ensure proper sanitation of
contaminated slurry. The most suitable way of processing
of the solid fraction of slurry is composting. Several
authors, including Niewolak and Szelagiewicz (1997),
NovaÂk (1994) and Plachy (1995), have reported that
composting results in signi®cant decreases in pathogenic
bacteria, fungi and helminth eggs, and resulting high
quality organic manure with a considerable portion of
humic substances.
The present study demonstrated that it is necessary to pay
increased attention to the manipulation of slurry solid
fraction.
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