Jorrrnol of Applied Bacteriology 1986,60, 93-96 21 40,'06,'85

A note on survival of salmonellas during anaerobic digestion of cattle dung

R . V . GADRE, D.R. R A N A D E & S.H. GODBOLE Department of Microbiology, M A C S Research Institute, Law College Road, Pune 411 004, India

Receiued 17 June 1985, revised 9 August 1985 and accepted 14 October 1985

G A u n t , R.V., R A N A D E , D.R. & GODBOLE, S . H . 1986. A note on survival of salmonellas during anaerobic digestion of cattle dung. Journal of Applied Bacte- riolog? 60, 93-96.

Anaerobic digestion of night soil with cattle dung slurry in biogas plants is advo- cated in Indian villages as a means of disposal of human excreta in the absence of conventional sanitary systems. Although intestinal pathogens are likely to be elimi- nated during anaerobic digestion, there is no conclusive evidence that this is so. Large numbers of saprophytic organisms in the fermenting mass make it impossible to detect the residual pathogens. Use of an antibiotic-resistant strain of Salmonella typhimurium as a test organism to study its survival during anaerobic digestion showed that the organism is totally eliminated in nine days.

Biogas technology has assumed importance in developing countries, particularly in rural areas. It has the advantages of producing fuel, supply- ing a well digested manure and disposing of human faeces in a safe manner. Biogas plant owners fear, however, that some human intesti- nal pathogens may survive and get redistributed through the slurry.

There is no conclusive evidence that intestinal pathogens are inactivated during anaerobic digestion although the number of these patho- gens is reduced considerably. Thus Carrington et al. (1982) showed that Salmonella duesseldorf was inactivated in sewage sludge anaerobic digesters run for a 10 to 20 day retention period. Dudley et al. (1980) on the other hand, showed that klebsiellas, shigellas, salmonellas, mycobac- teria, staphylococci and pseudomonads survived anaerobic digestion. Feachem et al. (1978) reported survival of salmonellas for several weeks in unheated anaerobic digesters and Findlay (1973) found that salmonellas may multiply in sterile sewage sludge in the absence of competition from other micro-organisms.

The major difficulty in studies on the survival of bacterial pathogens in anaerobic digesters is the recovery of pathogens from material that

contains many other micro-organisms. In spite of the use of several selective media and enrich- ment techniques, attempts to isolate these organisms often fail. Quantitative studies on inactivation of salmonellas made hitherto are by M P N technique of Carrington (1980). Studies were undertaken therefore, to determine the fate of a multiply-antibiotic resistant Salmonella typhimurium strain during anaerobic digestion.

Materials and Methods

0 R G A N I S M

Salmonella typhimurium, strain no. STMI3491 84, resistant to chloramphenicol and streptomy- cin was obtained from National Salmonella Phage Typing Centre, New Delhi.

M E D I A

Dehydrated Nutrient agar, MacConkey broth and MacConkey agar were obtained from Himedia Laboratories Pvt. Ltd, Bombay. Chlo- ramphenicol was obtained from Sigma Chemi- cals, Saint Louis, USA, and streptomycin sulphate from Sarabhai Chemicals, Baroda,

94 R. V . Gadre et al. India. Media were supplemented, after steriliza- tion, with 100 mg/l each of chloramphenicol and streptomycin which inhibited growth of all but Salm. typhimurium in fermenting cattle dung.

DIGESTERS

The anaerobic digestion of cattle dung was carried out in 1250 ml Corning glass digesters with different ports for the withdrawal of fer- menting mass and headspace gas samples.

S T A R T U P OF DIGESTERS

A mixture of 150 ml of fresh cattle dung slurry with 10% total solids and 150 ml fermented effluent of a 3 m3 Indian floating dome biogas plant (Dayal 1981), run on cattle dung, was placed in two digesters. The air present in the headspace of the digesters was replaced by oxygen-free nitrogen with a gassing manifold as described by Balch et al. (1979), to create anaer- obiosis. The ports of the digesters were closed with butyl rubber stoppers and aluminium seals. The temperature of the digesters was main- tained at 37°C and the volume of biogas pro- duced was measured daily by a gas flow meter with an electronic counter (Monelta & Albag- nac 1982). The gas phase in the headspace was analysed on alternate days by gas chromatog-

raphy (Ranade et al. 1980). After meth- anogenesis was established, one of the digesters was inoculated with the test organism.

EXPERIMENTAL P R O C E D U R E

Five ml of saline suspension (2.9 x lo5 cells/ml by viable count) of a 24 h growth of the test organism on nutrient agar was added to one digester. The other digester, run as control, received 5 ml of sterile saline. The contents of the digesters were mixed thoroughly. Five ml amounts of the contents of both digesters were withdrawn immediately and at 24 h intervals thereafter. Samples were homogenized on a Cyclomixer (Remi-CM 101 Bombay) and a I ml sample was diluted serially 10-fold in sterile saline. One ml of each dilution was used to prepare duplicate pour plates in MacConkey agar for viable counts of Salrn. typhirnurium.

To determine the minimum detectable level of the organism, enrichments of dilutions were made by adding 1 ml amounts of each dilution into 9 ml MacConkey broth. One tube was used for each dilution. The tubes were incubated at 37°C for 48 h and a loopful from each was streaked onto MacConkey agar. The plates were incubated at 37°C for 24 h. Representative colonies from the viable count plates and after enrichment were identified biochemically to

Table 1. Inactivation of Salmonella typhimurium during anaerobic digestion

Minimum detectable level

Maximum Viable count dilution Minimum

showing detectable Day of per Death positive number

incubation ml ( % I ) results per ml 0 4606 - 10-3 4.6 1 3786 17.8 10-3 3.7 2 3630 21.2 10-3 3.6 3 3043 33.9 10-3 3.0 4 2560 44.4 10-3 2.5 5 2256 51.0 1 0 - 3 2.2 6 2073 54.9 10-3 2.0 7 675 85.3 10-2 6.7 8 11 99.7 lo-' 1.1 9 1 99.9 Original 1 .o

10 0 100.0 1 1 0 100.0 - -

12 0 100.0 13 0 100~0

sample - -

- - - -

Survival of salmonellas Table 2. Performance of digesters with respect to methanogenesis

Test digester Control digester

Day of Volume of biogas CH, Volume of biogas CH, incubation (mlid) (%I) (mlid) (%I)

- - - - 0 1 105 42 114 44 2 81 50 78 49 3 70 4 58 49 65 60 5 58 6 58 55 63 50 7 58 8 58 55 60 56 9 46

10 46 55 48 57 11 46 12 35 5 1 36 56 13 35

Average 58 51 62 53

- 76

64

61

59

47

36

-

- -

- -

- -

- -

- -

95

genus level. The minimum detectable number of Salm. typhimurium was calculated from viable count and the maximum dilution showing posi- tive results.

Results and Discussion

The use of an antibiotic-resistant strain of Sal- monella typhimurium made it possible to detect it in the fermenting slurry by suppressing the growth of the large number of other micro- organisms which were completely inhibited by the streptomycin and chloramphenicol. It can be seen (Table 1) that there is a uniform decrease in the number of Salm. typhimurium during the course of anaerobic digestion. Studies by enrichment substantiated these find- ings. The enrichment procedure was sensitive enough to detect as few as 1 cell/ml on the 9th day. The viable count results corroborate this finding.

The comparative results on the biogas and its methane content in the test and control digester (Table 2) indicate satisfactory methanogenic activity in both the digesters.

Kabrick & Jewel1 (1982) reported that meso- philic anaerobic digestion does not completely inactivate salmonellas. The present studies show that anaerobic mesophilic digestion results in complete inactivation of Salm. typhimurium in 10 days. In the Indian design of biogas plants,

animal dung is digested along with night soil with a detention time of more than 30 days. Thus, there is no possibility that Salm. typhimu- rium will survive in the digested slurry of biogas plant. It is likely that other members of the Enterobacteriaceae would also be eliminated in the mesophilic anaerobic digestion.

The authors are thankful to the Department of Non-Conventional Energy Sources, Govern- ment of India for the financial support.

References

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CARRINGTON, E.G. 1980 The isolation and identifica- tion of Salmonella sp. in sewage sludges, a compari- son of methods and recommendations for a standard technique. Technical Report TR 129. Ste- venage and Medmenham: Water Research Centre, UK.

CARRINGTON, E.G., HARMAN, S.A. & PIKE, E.B. 1982 Inactivation of Salmonella during anaerobic digestion of sewage sludge. Journal of Applied Bac- teriology 53, 331-334.

DAYAL, M. 1981 Biogas Technology and Utilization. A Status Report. New Delhi: Department of Science and Technology, Government of India.

DUDLEY, D.J., GUENTZEL, M.N., IBARRA, M.J., MOORE, B.E. & SAGIK, B.P. 1980 Enumeration of potentially pathogenic bacteria from sewage sludge. Applied and Environmental Microbiology 39, 118-126.

96 R. I/. Gadre et al. FEACHEM, R.G., BRADLEY, D.J., GARELICK, H. &

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RANADE, D.R., GORE, J.A. & GODBOLE, S.H. 1980 Methanogenic organisms from fermenting slurry of gobar gas plant. Current Science 49, 395-397.