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Effect of organic acids and ensiling of cattle dung on the growth of Clostridium perfringens
Bioresource Technology 37 ( 1991 ) 115-119
Effect of Organic Acids and Ensiling of Cattle Dung on the Growth of Clostridium perfringens D. N. Kamra & S. K. Srivastava* Division of Animal Nutrition, Indian Veterinary Research Institute, Izatnagar 243 122, India (Received 5 September 1989; revised version received 27 August 1990; accepted 9 September 1990)
Abstract
The influence of organic acids at different pH values on the growth of Clostridium perfringens was examined: propionic acid was found to be the most inhibitory, followed by lactic, butyric and acetic acids. The inhibition of bacteria increased with decreasing pH and increasing concentration of organic acid at a given pH. C. perfringens was able to initiate growth at a pH as low as 4.6 in the absence of organic acids. The ensiling of cattle dung with wheat straw and sugarcane molasses had a detrimental effect on the growth of C. perfringens. When the above premix was inoculated with a mixture of Lactobacillus plantarum and Streptococcus faecalis, the pH of the wastelage after 20 days of fermentation at 37°C was 3"8 and C. perfringens was killed, but its number was also reduced considerably in a wastelage of pH 4.25, in which the lactic acid-producing bacteria had not been inoculated. Key words: Ensiling, cattle dung wastelage, Clostridium perfringens.
INTRODUCTION The excreta of ruminants fed on a high concentrate diet contain many nutrients (Lucas et aL, 1975; Jakhmola et al., 1986; Lall et al., 1989) which can be recycled for the feeding of livestock. But the bovine waste has been found to contain as high as 10 :° microbes/g dry weight, which included anaerobes, coliforms, spore formers, yeasts, fungi and streptomycetes (Rhodes & Hrubant, 1972). Citrobacter, Proteus and
Klebsiella were reported by Hrubant et al. (1972) in feedlot waste. The other pathogenic bacteria reported in bovine waste are clostridia, salmonellae, corynebacteria and mycobacteria (Miner etal., 1967; Benham, 1983). The presence of these pathogenic microbes and the smell makes the waste unfit for consumption as feed. In addition, the clostridia are responsible for the undesirable butyric acid fermentation which results in the production of a poor quality silage. Therefore, the elimination of such microbes is essential for the recycling of waste as feed. In the present study, the antagonistic effects of organic acids produced during ensiling againt Clostridium perfringens were studied, both in pure culture in a complex growth medium and in laboratory silos
METHODS
The culture of Clostridium perfringens was kindly supplied by the Head, Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, and cultures of Lactobacillus plantarum and Streptococcus faecalis were kindly supplied by the Head, Division of Dairy Bacteriology, National Dairy Research Institute, Karnal. C. perfringens was maintained on Robertson's cooked meat medium (Hi Media) and the lactic acid-producing bacteria were maintained on steri-lised skimmed milk. To study the effects of organic acids at different pH values on the growth of C. perfringens, lactic, acetic, propionic and butyric acids, at concentrations of 0.25%, 0.50% and 1.00% (v/v), were added to the Robertson's cooked meat medium and the pH of the medium was adjusted to between 4 and 7 with 20% sodium hydroxide. In a control, the medium was used without the addition of any organic acid and the pH was adjusted with hydrochloric acid. Six
*Present address: Division of Bacteriologyand Mycology, IVRI. Izatnagar243122. India. 115 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
D. N. Kamra, S. K. Srivastava
116
tubes of "each treatment were prepared, out of which one tube was used for the dilution of inoculum; the second for the measurement of pH after autoclaving the medium; three were inoculated with C. perfringens and the sixth tube of medium was used as a control blank for the measurement of optical density at 550 nm to assess the bacterial growth using a Spectronic-20 spectrophotometer. The inhibition at a given pH due to the presence of organic acids was calculated as described by Kamra (1989): AS
Inhibition ( % ) = - -
~
A,
At
three treatments were picked and grown in Robertson's cooked meat medium. The trypsinised supernatant of the culture (0.1 ml) was injected into the tail vein of albino mice of 18-22 g body weight to test for the production of epsilon toxin. The production of epsilon toxin in growth medium is indicative of the presence of Clostridium perfringens type D (Wilson, 1931). The wastelage saline extract was also used for estimation of pH, lactic acid (Barker & Summerson, 1941) and volatile fatty acids (Kroman et al., 1967).
x 100 RESULTS
where, As = absorbance of the culture at pH 7-0 without any addition of acid (standard conditions), At -- absorbance of the culture grown under test conditions. The 18-h old culture of C. perfringens (57.5 x 107 cfu/ml) was diluted 100 times and 1 ml of the diluted culture was inoculated into 9 ml of the test medium. In the second experiment, a mixture of cattle dung, wheat straw and sugarcane molasses, in the ratio of 70:20:10, was prepared and divided into three parts. The first part was used as a control without any microbial additive. In the second part, a 5% inoculum of C. perfringens was added in the premix, and in the third part, in addition to C. perfringens, 2"5% inocula of L. plantarum (2.75 x 106 cfU/ITd) and S. faecalis (23.95 x 107 cfu/ml) was also added. The premix was packed into 0-1-1itre test-tube silos and incubated at 37°C. Two silos from each treatment were opened after a fermentation period of 4, 14 and 20 days and a 10-g sample was added to 90 ml of sterilised physiological saline and placed in a rotary shaker for 1 h. The mixture was used for the estimation of lactic acid-producing bacteria (by pour-plate method) (Rogosa et al., 1951). The number of vegetative cells and spores of C. perfringens was determined on blood agar (Lapage & Shelton, 1970) containing neomycin sulphate (100/~g/ml of medium). After inoculation by spreading, the petri dishes were incubated in a carbon dioxide atmosphere in anaerobic jars. The jars were incubated at 37°C. The haemolytic colonies on blood agar were counted and 2-3 colonies from the premix of treatment 1 and the wastelage samples (after 20 days of fermentation) from all
The influence of pH on the growth of C perfringens in the presence of lactic acid is shown in Fig. l(a). When the pH was adjusted with hydrochloric acid and no lactic acid was included in the growth medium (control), the bacterium was able to initiate growth at pH as low as 4.6, but the growth was only 46% as compared with that in the control medium at pH 7"0. Increase in concentration of lactic acid enhanced inhibition of bacterial growth. High inhibition (78%) was observed in the presence of 0.25% acetic acid at pH 4.3 (Fig. l(b)). At a higher concentration of acetic acid (1% in growth medium), the growth was inhibited
CONTROL m-~..
~
~ N % ACITATt
N
~0°
i
/
~o.
2O
\ 5
°M
°
°L. S bN
T
6
T
(a/ ~*
ioo~
Gn % ~ATE , - - n OSG% OUTYOATE
o~~ o - - o ioo %lm~tO~MT|
ii
eoJ
"~i
'0'
°~
i.oo% IUTYRATE
!_
0 s
6
(c/
7
s
8
,
(d)
Fig. 1. Effect of pH on growth of Clostridium perfringens in the presence of (a) lactic acid, (b) acetic acid, (c) propionic acid and (d) butyric acid.
Wastelage and C. perfringens
completely at pH 5"6, but there was no inhibition at a pH of 6"5, irrespective of the concentration of acid used. Propionic acid seemed to be more inhibitory than acetic acid or lactic acid because it inhibited growth at pH 5.25 even at the lowest concentration of 0.25% (Fig. l(c)). The inhibition was reversed between pH 5.5 and 6.5, depending upon the concentration of acid in the medium. The effect of butyric acid on growth of C. perfringens is shown in Fig. l(d). The bacterium was not able to grow at a pH of 5.4, when the acid concentration in the medium was 0.5% or above. On lowering the concentration to 0.25%, the inhibition was reduced to 60% as compared to the control medium at pH 7.0. The inhibition was reversed at pH values higher than 6.0 even at the highest concentration of acid (1%) used in the present experiments. At the lowest concentration of acid used (0.25%), propionic acid was the most inhibitory, followed by lactic, butyric and acetic acids. The biochemical characteristics of cattle dung wastelage inoculated with C. perfringens and lactic acid-producing bacteria are shown in Table 1. The drop in pH was highest when the premix was inoculated with a mixture of L. plantarum and S. faecalis. The production of lactic acid was greatest and production of total volatile fatty acids was least in treatment III, and the differences from the other two treatments were statistically significant (P<0.01). The microbiological changes during fermentation are given in Table 2. In treatment, where neither of the two inocula was added, there were bacteria (16 x 1 0 2 cfu/g premix) which were able to grow on blood agar containing neomycin sulphate and produce haemolytic colonies. The number of such bacteria was reduced slightly on fermentation. In treatments II and III, where C.
117
perfringens was inoculated in the premix, the number of neomycin resistant and haemolytic bacteria reduced gradually in the first 14 days of fermentation, but there was not much change in the number of such bacteria between 14 and 20 days after initiation of fermentation. Representative colonies on blood agar were picked from the premix of treatment I and wastelage (after 20 days of fermentation) of all the three treatments. The isolates were tested for the production of epsilon toxin in albino mice of 18-22 g body weight. The results showed that the haemolytic bacteria present in the waste were not C perfringens. Only in wastelage of treatment II, in which C. perfringens was inoculated in the premix, were there some bacteria which survived the fermentation for up to 20 days at 37°C but their number was considerably reduced. In treatment III, where lactic Table 2. Microbiological changes on fermentation of cattle dung wastelage inoculated with Clostridium perfringens and lactic acid producing bacteria
Treatment"
Fermentation period (days) (LogNo. ofcells/g) b
Lactic acid bacteria (Log No. of cells/g)'
0
0
C. peffringens
I II III
4
14
20
4
14
20
3.20 2.88 2-40 2.40 5"20 4.81 4.78 5.48 8.00 7-35 2-85 2.48 4.86 4.73 5.05 5.59 7.86 4.95 2.78 2.30 7.05 6.88 7-43 7.86
a I - P r e m i x consisted of cattle dung, wheat straw and molasses in the ratio of 7 0 : 2 0 : 1 0 , respectively. II -- Premix I + inoculum of C perfringens. I I I = Premix I +inoculum of C perfringens, Lactobacillus
plantarum and Streptococcus faecalis. bC perfringens and haemolytic cfu on blood agar containing neomycin sulphate; see text. 'cfu on Rogosa medium. Results represent the averages of two silages.
Table 1. Biochemical changes on fermentation of cattle dung wastelage inoculated with Clostridium perfringens and lactic acid producing bacteria
Treatment"
Fermentation period (days) Lactic acid (% DM)
pH
I II III
Total volatile fatty acids (% DM)
Lactic acid (% total acids)
0
4
14
20
4
14
20
4
14
20
20
7.40 7.25 7.05
4.30 4.25 3'85
4.25 4.30 3'75
4"30 4.25 3.80
3-78 4-10 5-52
5.24 5"30 6"52
6.00 6.02 7"32
3.48 3.37 2.64
3.94 3"53 2'89
4.85 3.69 2"73
55"30 62.00 72.84
" I = Premix of cattle dung, wheat straw and molasses in the ratio of 70 : 20: 10. II = Premix I + inoculum of C. perfringens, Ill = Premix I + inoculum of C perfringens, Lactobacillus plantarum and Streptococcusfaecalis. Results represent the averages of two silages.
118
D. N. Kamra, S. K. Srivastava
acid producing-bacteria were also inoculated in the premix and the pH of the final product was 3"8, C. perfringens was almost completely removed.
DISCUSSION A silage temperature of 37°C was chosen as it is the optimum for C perfringens and the ambient temperature in many parts of India is above 30°C for most of the year. The optimum pH for growth of C perfringens was found in the range 6"5-7"0, but the bacterium was able to initiate growth at a pH of as low as 4.6 in the absence of any organic acid. The growth inhibition increased with decreasing p H and increasing concentration of organic acids. The inhibitory effect of the organic acids might be similar to that observed with yeasts and weak acids (Warth, 1977). The acids pass through the cell wall and accumulate within the cells. The p K a values for the present acids vary between pH 3.7 and 4.8 (Woolford, 1975), at which the organic acids are half dissociated and half undissociated. At a pH lower than the p K a value of an acid, the concentration of undissociated acid (free acid) increases, while at a pH higher than this, the concentration of anions increases. Therefore, at a lower pH, the free acids enter the cells; but the pH inside the cell is near neutrality and the acids dissociate and cannot diffuse out as the cell wall is impermeable to the anions (Warth, 1977). Therefore, the toxic anions accumulate within the cell at a lower pH and ultimately kill the cell. A similar mechanism for the toxic effect of organic acids was proposed by Levine and Fellers (1940). The inhibition of C. perfringens has been observed at a pH below 5.0 at low concentrations of organic acids (below 1%). Similarly, Woolford (1975) also observed that very low concentrations of acetic acid (47 mmoles/litre), propionic acid (8 mmoles/litre) and lactic acid (8 mmoles/litre) at pH 5"0 were inhibitory for the growth of Clostridia. But in a good quality wastelage of pH values lower than 4.3, the concentrations of lactic acid and volatile fatty acids have been found to be 7.5% and 3.4% (Kamra et al., 1989), which is much higher than the concentration of acids required to inhibit C perfringens, indicating that C. perfringens should not survive during the fermentation of cattle waste with other feed ingredients. In the second experiment, the pathogenic bacteria were killed in 20 days of fer-
mentation. Relatively faster killing of C. perfringens was observed by Talkington et al. ( 1981 ) in fermented edible wastes at 30°C. In another study only a decline in the number of C sporogenes was observed in a bovine waste and corn forage feed ensiled for 60 days (McCaskey & Anthony, 1978). The results of these experiments indicate that C perfringens was inhibited by the organic acids which are usually formed in wastelage and the inhibition increased at a lower pH. The ensiling of cattle dung with other feeds resulted in the abolition of C. perfringens within 20 days at pH 3.8. when fermented at 73°C. The chances of clostridial infection would be minimal if the cattle dung were fermented properly.
REFERENCES Barker, S. B. & Summerson, W. H. (1941). The colorimetric determination of lactic acid in biological materials. J. Biol. Chem., 138, 535-54. Benham, C. L. (1983). A review of the possible microbiological and chemical risks associated with the feeding of animal manure to livestock. In Animals as Waste Converters, ed. E. H. Ketelaars & S. Boer Iwena. Proc. International Symp., Pudoc, Wageningen, Netherlands, 30 November-2 December 1983, pp. 22-7. Hrubant, G. R., Dougherty, R. V. & Rhodes, R. A. (1972). Enterobacteria in feedlot waste and run off. Appl. Microbiol., 24, 378-83. Jakhmola, R. C., Singh, R., Jindal, S. K. & Kamra, D. N. (1986). Buffalo dung wastelage as a sole or partial source of nutrients to sheep. Agric. Wastes, 17, 91-8. Kamra, D. N. (1989). Interaction between pathogenic and lactic acid producing bacteria during ensiling of animal wastes. PhD thesis, Indian Veterinary Research Institute Deemed University, Izatnagar. Kamra, D. N., Lall, D., Kewalramani, N. & Pathak, N. N. (1989). Ensiling characteristics of cattle waste fermented with wheat straw and green berseem ( Trifolium alexandrinum). Anita. Feed Sci. Technol., 25, 149-55. Kroman, R. E, Meyer, J. H. & Stielau, W. J. (1967). Steam distillation of volatile fatty acids in rumen digesta. J. Dairy Sci., 50, 73-6. Lall, D., Pathak, N. N., Kewalramani, N. & Kamra, D. N. (1989). Nutritional contribution of cattle dung in wastelage feeding in buffalo. Biol. Wastes, 28, 175-9. Lapage, S. D. & Shelton, J. E. (1970). Media for maintenance and preservation of bacteria. In Methods in Microbiology, Vol. 3A, ed. J. R. Norris & D. W. Ribbons. Academic Press, London, p. 94. Levine, A. S. & Fellers, C. R. (1940). Action of acetic acid on food spoilage microorganisms. J. Bacteriol., 39,499-515. Lucas, D. M., Fontenot, J. E & Webb, K. E. Jr (1975). Composition and digestibility of cattle faecal waste. J. Anita. Sci., 41, 1480-6. McCaskey, T. A. & Anthony, W. B. (1978). Evaluation of the health significance of clostridia in wastelage and corn silage. Amer. Dairy Sci. Assoc. Meeting, 9-13 July, East Lansing, Michigan. Miner, J. R., Fina, L. R. & Piatt, C. (1967). Salmonella
Wastelage and C. perfringens infant& in cattle feedlot run off. Appl. Microbiol., 15, 627-8. Rhodes, R. A. & Hrubant, G. R. (1972). Microbial population of feediot waste and associated sites. Appl. Microbiol., 24,369-77. Rogosa, M., Mitchell, J. A. & Wiseman, R. E ( 1951 ). A selective medium for isolation and enumeration of oral and faecal lactobacilli. J. Bacteriol., 62, 136. Talkington, F. D., Shotts, E. B., Wooley, R. E., Whitehead, W. K. & Dobbins, C. N. (1981 ). Introduction and reisolation of gram positive bacteria from fermented edible wastes. Amer. J. Vet. Res., 42, 1302-5.
1 19
Warth, A. D. (1977). Mechanism of resistance of Saccharomyces bailii to benzoic, sorbic and other weak acids used as food preservatives. J. Appl. Bacteriol., 43, 215-30. Wilson, A. J. (1931). Observations on the classification of Bacillus welchii. Univ. Cambridge, Inst. Ann. Path. 2nd Report 53. Cited by Whitiock, J. H. & Fabricant, J. 1947. The use of Clostridium welchii type D anacuiture in the prevention of over eating disease (Enterotoxaemia) in sheep. Cornell Vet., 37, 211-30. Woolford, M. K. (1975). Microbiological screening of the straight chain fatty acids (Cz-Ct2) as potential silage additives. J. Sci. Food Agric., 26, 219-28.
Effect of Organic Acids and Ensiling of Cattle Dung on the Growth of Clostridium perfringens D. N. Kamra & S. K. Srivastava* Division of Animal Nutrition, Indian Veterinary Research Institute, Izatnagar 243 122, India (Received 5 September 1989; revised version received 27 August 1990; accepted 9 September 1990)
Abstract
The influence of organic acids at different pH values on the growth of Clostridium perfringens was examined: propionic acid was found to be the most inhibitory, followed by lactic, butyric and acetic acids. The inhibition of bacteria increased with decreasing pH and increasing concentration of organic acid at a given pH. C. perfringens was able to initiate growth at a pH as low as 4.6 in the absence of organic acids. The ensiling of cattle dung with wheat straw and sugarcane molasses had a detrimental effect on the growth of C. perfringens. When the above premix was inoculated with a mixture of Lactobacillus plantarum and Streptococcus faecalis, the pH of the wastelage after 20 days of fermentation at 37°C was 3"8 and C. perfringens was killed, but its number was also reduced considerably in a wastelage of pH 4.25, in which the lactic acid-producing bacteria had not been inoculated. Key words: Ensiling, cattle dung wastelage, Clostridium perfringens.
INTRODUCTION The excreta of ruminants fed on a high concentrate diet contain many nutrients (Lucas et aL, 1975; Jakhmola et al., 1986; Lall et al., 1989) which can be recycled for the feeding of livestock. But the bovine waste has been found to contain as high as 10 :° microbes/g dry weight, which included anaerobes, coliforms, spore formers, yeasts, fungi and streptomycetes (Rhodes & Hrubant, 1972). Citrobacter, Proteus and
Klebsiella were reported by Hrubant et al. (1972) in feedlot waste. The other pathogenic bacteria reported in bovine waste are clostridia, salmonellae, corynebacteria and mycobacteria (Miner etal., 1967; Benham, 1983). The presence of these pathogenic microbes and the smell makes the waste unfit for consumption as feed. In addition, the clostridia are responsible for the undesirable butyric acid fermentation which results in the production of a poor quality silage. Therefore, the elimination of such microbes is essential for the recycling of waste as feed. In the present study, the antagonistic effects of organic acids produced during ensiling againt Clostridium perfringens were studied, both in pure culture in a complex growth medium and in laboratory silos
METHODS
The culture of Clostridium perfringens was kindly supplied by the Head, Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, and cultures of Lactobacillus plantarum and Streptococcus faecalis were kindly supplied by the Head, Division of Dairy Bacteriology, National Dairy Research Institute, Karnal. C. perfringens was maintained on Robertson's cooked meat medium (Hi Media) and the lactic acid-producing bacteria were maintained on steri-lised skimmed milk. To study the effects of organic acids at different pH values on the growth of C. perfringens, lactic, acetic, propionic and butyric acids, at concentrations of 0.25%, 0.50% and 1.00% (v/v), were added to the Robertson's cooked meat medium and the pH of the medium was adjusted to between 4 and 7 with 20% sodium hydroxide. In a control, the medium was used without the addition of any organic acid and the pH was adjusted with hydrochloric acid. Six
*Present address: Division of Bacteriologyand Mycology, IVRI. Izatnagar243122. India. 115 Bioresource Technology 0960-8524/91/S03.50 © 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
D. N. Kamra, S. K. Srivastava
116
tubes of "each treatment were prepared, out of which one tube was used for the dilution of inoculum; the second for the measurement of pH after autoclaving the medium; three were inoculated with C. perfringens and the sixth tube of medium was used as a control blank for the measurement of optical density at 550 nm to assess the bacterial growth using a Spectronic-20 spectrophotometer. The inhibition at a given pH due to the presence of organic acids was calculated as described by Kamra (1989): AS
Inhibition ( % ) = - -
~
A,
At
three treatments were picked and grown in Robertson's cooked meat medium. The trypsinised supernatant of the culture (0.1 ml) was injected into the tail vein of albino mice of 18-22 g body weight to test for the production of epsilon toxin. The production of epsilon toxin in growth medium is indicative of the presence of Clostridium perfringens type D (Wilson, 1931). The wastelage saline extract was also used for estimation of pH, lactic acid (Barker & Summerson, 1941) and volatile fatty acids (Kroman et al., 1967).
x 100 RESULTS
where, As = absorbance of the culture at pH 7-0 without any addition of acid (standard conditions), At -- absorbance of the culture grown under test conditions. The 18-h old culture of C. perfringens (57.5 x 107 cfu/ml) was diluted 100 times and 1 ml of the diluted culture was inoculated into 9 ml of the test medium. In the second experiment, a mixture of cattle dung, wheat straw and sugarcane molasses, in the ratio of 70:20:10, was prepared and divided into three parts. The first part was used as a control without any microbial additive. In the second part, a 5% inoculum of C. perfringens was added in the premix, and in the third part, in addition to C. perfringens, 2"5% inocula of L. plantarum (2.75 x 106 cfU/ITd) and S. faecalis (23.95 x 107 cfu/ml) was also added. The premix was packed into 0-1-1itre test-tube silos and incubated at 37°C. Two silos from each treatment were opened after a fermentation period of 4, 14 and 20 days and a 10-g sample was added to 90 ml of sterilised physiological saline and placed in a rotary shaker for 1 h. The mixture was used for the estimation of lactic acid-producing bacteria (by pour-plate method) (Rogosa et al., 1951). The number of vegetative cells and spores of C. perfringens was determined on blood agar (Lapage & Shelton, 1970) containing neomycin sulphate (100/~g/ml of medium). After inoculation by spreading, the petri dishes were incubated in a carbon dioxide atmosphere in anaerobic jars. The jars were incubated at 37°C. The haemolytic colonies on blood agar were counted and 2-3 colonies from the premix of treatment 1 and the wastelage samples (after 20 days of fermentation) from all
The influence of pH on the growth of C perfringens in the presence of lactic acid is shown in Fig. l(a). When the pH was adjusted with hydrochloric acid and no lactic acid was included in the growth medium (control), the bacterium was able to initiate growth at pH as low as 4.6, but the growth was only 46% as compared with that in the control medium at pH 7"0. Increase in concentration of lactic acid enhanced inhibition of bacterial growth. High inhibition (78%) was observed in the presence of 0.25% acetic acid at pH 4.3 (Fig. l(b)). At a higher concentration of acetic acid (1% in growth medium), the growth was inhibited
CONTROL m-~..
~
~ N % ACITATt
N
~0°
i
/
~o.
2O
\ 5
°M
°
°L. S bN
T
6
T
(a/ ~*
ioo~
Gn % ~ATE , - - n OSG% OUTYOATE
o~~ o - - o ioo %lm~tO~MT|
ii
eoJ
"~i
'0'
°~
i.oo% IUTYRATE
!_
0 s
6
(c/
7
s
8
,
(d)
Fig. 1. Effect of pH on growth of Clostridium perfringens in the presence of (a) lactic acid, (b) acetic acid, (c) propionic acid and (d) butyric acid.
Wastelage and C. perfringens
completely at pH 5"6, but there was no inhibition at a pH of 6"5, irrespective of the concentration of acid used. Propionic acid seemed to be more inhibitory than acetic acid or lactic acid because it inhibited growth at pH 5.25 even at the lowest concentration of 0.25% (Fig. l(c)). The inhibition was reversed between pH 5.5 and 6.5, depending upon the concentration of acid in the medium. The effect of butyric acid on growth of C. perfringens is shown in Fig. l(d). The bacterium was not able to grow at a pH of 5.4, when the acid concentration in the medium was 0.5% or above. On lowering the concentration to 0.25%, the inhibition was reduced to 60% as compared to the control medium at pH 7.0. The inhibition was reversed at pH values higher than 6.0 even at the highest concentration of acid (1%) used in the present experiments. At the lowest concentration of acid used (0.25%), propionic acid was the most inhibitory, followed by lactic, butyric and acetic acids. The biochemical characteristics of cattle dung wastelage inoculated with C. perfringens and lactic acid-producing bacteria are shown in Table 1. The drop in pH was highest when the premix was inoculated with a mixture of L. plantarum and S. faecalis. The production of lactic acid was greatest and production of total volatile fatty acids was least in treatment III, and the differences from the other two treatments were statistically significant (P<0.01). The microbiological changes during fermentation are given in Table 2. In treatment, where neither of the two inocula was added, there were bacteria (16 x 1 0 2 cfu/g premix) which were able to grow on blood agar containing neomycin sulphate and produce haemolytic colonies. The number of such bacteria was reduced slightly on fermentation. In treatments II and III, where C.
117
perfringens was inoculated in the premix, the number of neomycin resistant and haemolytic bacteria reduced gradually in the first 14 days of fermentation, but there was not much change in the number of such bacteria between 14 and 20 days after initiation of fermentation. Representative colonies on blood agar were picked from the premix of treatment I and wastelage (after 20 days of fermentation) of all the three treatments. The isolates were tested for the production of epsilon toxin in albino mice of 18-22 g body weight. The results showed that the haemolytic bacteria present in the waste were not C perfringens. Only in wastelage of treatment II, in which C. perfringens was inoculated in the premix, were there some bacteria which survived the fermentation for up to 20 days at 37°C but their number was considerably reduced. In treatment III, where lactic Table 2. Microbiological changes on fermentation of cattle dung wastelage inoculated with Clostridium perfringens and lactic acid producing bacteria
Treatment"
Fermentation period (days) (LogNo. ofcells/g) b
Lactic acid bacteria (Log No. of cells/g)'
0
0
C. peffringens
I II III
4
14
20
4
14
20
3.20 2.88 2-40 2.40 5"20 4.81 4.78 5.48 8.00 7-35 2-85 2.48 4.86 4.73 5.05 5.59 7.86 4.95 2.78 2.30 7.05 6.88 7-43 7.86
a I - P r e m i x consisted of cattle dung, wheat straw and molasses in the ratio of 7 0 : 2 0 : 1 0 , respectively. II -- Premix I + inoculum of C perfringens. I I I = Premix I +inoculum of C perfringens, Lactobacillus
plantarum and Streptococcus faecalis. bC perfringens and haemolytic cfu on blood agar containing neomycin sulphate; see text. 'cfu on Rogosa medium. Results represent the averages of two silages.
Table 1. Biochemical changes on fermentation of cattle dung wastelage inoculated with Clostridium perfringens and lactic acid producing bacteria
Treatment"
Fermentation period (days) Lactic acid (% DM)
pH
I II III
Total volatile fatty acids (% DM)
Lactic acid (% total acids)
0
4
14
20
4
14
20
4
14
20
20
7.40 7.25 7.05
4.30 4.25 3'85
4.25 4.30 3'75
4"30 4.25 3.80
3-78 4-10 5-52
5.24 5"30 6"52
6.00 6.02 7"32
3.48 3.37 2.64
3.94 3"53 2'89
4.85 3.69 2"73
55"30 62.00 72.84
" I = Premix of cattle dung, wheat straw and molasses in the ratio of 70 : 20: 10. II = Premix I + inoculum of C. perfringens, Ill = Premix I + inoculum of C perfringens, Lactobacillus plantarum and Streptococcusfaecalis. Results represent the averages of two silages.
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D. N. Kamra, S. K. Srivastava
acid producing-bacteria were also inoculated in the premix and the pH of the final product was 3"8, C. perfringens was almost completely removed.
DISCUSSION A silage temperature of 37°C was chosen as it is the optimum for C perfringens and the ambient temperature in many parts of India is above 30°C for most of the year. The optimum pH for growth of C perfringens was found in the range 6"5-7"0, but the bacterium was able to initiate growth at a pH of as low as 4.6 in the absence of any organic acid. The growth inhibition increased with decreasing p H and increasing concentration of organic acids. The inhibitory effect of the organic acids might be similar to that observed with yeasts and weak acids (Warth, 1977). The acids pass through the cell wall and accumulate within the cells. The p K a values for the present acids vary between pH 3.7 and 4.8 (Woolford, 1975), at which the organic acids are half dissociated and half undissociated. At a pH lower than the p K a value of an acid, the concentration of undissociated acid (free acid) increases, while at a pH higher than this, the concentration of anions increases. Therefore, at a lower pH, the free acids enter the cells; but the pH inside the cell is near neutrality and the acids dissociate and cannot diffuse out as the cell wall is impermeable to the anions (Warth, 1977). Therefore, the toxic anions accumulate within the cell at a lower pH and ultimately kill the cell. A similar mechanism for the toxic effect of organic acids was proposed by Levine and Fellers (1940). The inhibition of C. perfringens has been observed at a pH below 5.0 at low concentrations of organic acids (below 1%). Similarly, Woolford (1975) also observed that very low concentrations of acetic acid (47 mmoles/litre), propionic acid (8 mmoles/litre) and lactic acid (8 mmoles/litre) at pH 5"0 were inhibitory for the growth of Clostridia. But in a good quality wastelage of pH values lower than 4.3, the concentrations of lactic acid and volatile fatty acids have been found to be 7.5% and 3.4% (Kamra et al., 1989), which is much higher than the concentration of acids required to inhibit C perfringens, indicating that C. perfringens should not survive during the fermentation of cattle waste with other feed ingredients. In the second experiment, the pathogenic bacteria were killed in 20 days of fer-
mentation. Relatively faster killing of C. perfringens was observed by Talkington et al. ( 1981 ) in fermented edible wastes at 30°C. In another study only a decline in the number of C sporogenes was observed in a bovine waste and corn forage feed ensiled for 60 days (McCaskey & Anthony, 1978). The results of these experiments indicate that C perfringens was inhibited by the organic acids which are usually formed in wastelage and the inhibition increased at a lower pH. The ensiling of cattle dung with other feeds resulted in the abolition of C. perfringens within 20 days at pH 3.8. when fermented at 73°C. The chances of clostridial infection would be minimal if the cattle dung were fermented properly.
REFERENCES Barker, S. B. & Summerson, W. H. (1941). The colorimetric determination of lactic acid in biological materials. J. Biol. Chem., 138, 535-54. Benham, C. L. (1983). A review of the possible microbiological and chemical risks associated with the feeding of animal manure to livestock. In Animals as Waste Converters, ed. E. H. Ketelaars & S. Boer Iwena. Proc. International Symp., Pudoc, Wageningen, Netherlands, 30 November-2 December 1983, pp. 22-7. Hrubant, G. R., Dougherty, R. V. & Rhodes, R. A. (1972). Enterobacteria in feedlot waste and run off. Appl. Microbiol., 24, 378-83. Jakhmola, R. C., Singh, R., Jindal, S. K. & Kamra, D. N. (1986). Buffalo dung wastelage as a sole or partial source of nutrients to sheep. Agric. Wastes, 17, 91-8. Kamra, D. N. (1989). Interaction between pathogenic and lactic acid producing bacteria during ensiling of animal wastes. PhD thesis, Indian Veterinary Research Institute Deemed University, Izatnagar. Kamra, D. N., Lall, D., Kewalramani, N. & Pathak, N. N. (1989). Ensiling characteristics of cattle waste fermented with wheat straw and green berseem ( Trifolium alexandrinum). Anita. Feed Sci. Technol., 25, 149-55. Kroman, R. E, Meyer, J. H. & Stielau, W. J. (1967). Steam distillation of volatile fatty acids in rumen digesta. J. Dairy Sci., 50, 73-6. Lall, D., Pathak, N. N., Kewalramani, N. & Kamra, D. N. (1989). Nutritional contribution of cattle dung in wastelage feeding in buffalo. Biol. Wastes, 28, 175-9. Lapage, S. D. & Shelton, J. E. (1970). Media for maintenance and preservation of bacteria. In Methods in Microbiology, Vol. 3A, ed. J. R. Norris & D. W. Ribbons. Academic Press, London, p. 94. Levine, A. S. & Fellers, C. R. (1940). Action of acetic acid on food spoilage microorganisms. J. Bacteriol., 39,499-515. Lucas, D. M., Fontenot, J. E & Webb, K. E. Jr (1975). Composition and digestibility of cattle faecal waste. J. Anita. Sci., 41, 1480-6. McCaskey, T. A. & Anthony, W. B. (1978). Evaluation of the health significance of clostridia in wastelage and corn silage. Amer. Dairy Sci. Assoc. Meeting, 9-13 July, East Lansing, Michigan. Miner, J. R., Fina, L. R. & Piatt, C. (1967). Salmonella
Wastelage and C. perfringens infant& in cattle feedlot run off. Appl. Microbiol., 15, 627-8. Rhodes, R. A. & Hrubant, G. R. (1972). Microbial population of feediot waste and associated sites. Appl. Microbiol., 24,369-77. Rogosa, M., Mitchell, J. A. & Wiseman, R. E ( 1951 ). A selective medium for isolation and enumeration of oral and faecal lactobacilli. J. Bacteriol., 62, 136. Talkington, F. D., Shotts, E. B., Wooley, R. E., Whitehead, W. K. & Dobbins, C. N. (1981 ). Introduction and reisolation of gram positive bacteria from fermented edible wastes. Amer. J. Vet. Res., 42, 1302-5.
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Warth, A. D. (1977). Mechanism of resistance of Saccharomyces bailii to benzoic, sorbic and other weak acids used as food preservatives. J. Appl. Bacteriol., 43, 215-30. Wilson, A. J. (1931). Observations on the classification of Bacillus welchii. Univ. Cambridge, Inst. Ann. Path. 2nd Report 53. Cited by Whitiock, J. H. & Fabricant, J. 1947. The use of Clostridium welchii type D anacuiture in the prevention of over eating disease (Enterotoxaemia) in sheep. Cornell Vet., 37, 211-30. Woolford, M. K. (1975). Microbiological screening of the straight chain fatty acids (Cz-Ct2) as potential silage additives. J. Sci. Food Agric., 26, 219-28.