Journal of Applied Microbiology 1998, 84, 138–142

Inactivation of Salmonella typhi by high levels of volatile fattyacids during anaerobic digestion

D.P. Kunte 1, T.Y. Yeole1, S.A. Chiplonkar 2 and D.R. Ranade 1

1Division of Microbial Sciences, and 2Biometry Group, Agharkar Research Institute, Pune, India

6056/01/97: received 10 January 1997, revised 7 May 1997 and accepted 9 May 1997

D.P. KUNTE, T.Y. YEOLE, S.A. CHIPLONKAR AND D.R. RANADE. 1998. Survival of Salmonellatyphi was investigated in an anaerobic digester for cattle dung with volatile fatty acid (VFA)levels of 5000 mg l−1 and pH 6·0. The organism was added to the digester only oncein the first experiment and daily in the other. Survival was monitored on alternatedays. In the single dose experiment, the counts of Salm. typhi declined rapidly and thepathogen was completely eliminated within 12 d in the experimental digester (VFA ca 5000mg l−1 and pH 6·0), whereas 26 d were required in the control digester (VFA ca 100mg l−1 and pH 6·8). T90 values for the experimental and control digesters were 2·44 d and4·80 d, respectively. In the daily dose experiment, a four log reduction in the pathogencount was observed in the experimental digester, but only a two log reduction inthe control digester at the end of the experimental period. The mean T90 values for theexperimental and the control digester were 4·22 d and 18·63 d, respectively. In both theexperiments, statistical analysis of the data showed significant differences in the survivalpattern of Salm. typhi in the two digesters.

INTRODUCTION survive in the animal slurries for a considerable time (Jones1980; Larsen and Munch 1982). Thus, there is a possibility

Disposal of animal and human wastes is one of the majorof spread of infectious diseases during land application of the

sanitation problems in rural parts, particularly in developing digested slurry (Jones and Matthews 1975; Ginnivan et al.countries. Anaerobic digestion offers a good alternative for 1980). It therefore becomes essential to determine the fate ofthe disposal of these wastes. It has selective advantages over bacterial pathogens in anaerobic digestion, and to improveother methods of waste treatment because it generates biofuel the existing design and operational parameters of the bio-in the form of methane and organic manure as digested sludge gas plants for achieving complete elimination of bacterial(Dahiya and Vasudevan 1986; Gadre et al. 1986). Anaerobic pathogens.digesters have become popular in rural parts of India as ‘gobar Carrington et al. (1982) reported that Salm. dusseldorf wasgas plants’ or ‘biogas plants’ in the past 20 years. At present, inactivated during anaerobic digestion of sewage sludgethere are over 1·9 million biogas plants in the country and within 10–20 d of retention period. Abdul and Lloyd (1985a)most of these are family-size plants run on cattle dung. have shown that counts of E. coli declined rapidly duringFurthermore, the number of families intending to connect the anaerobic digestion of pig slurry, and that the antibioticlatrines to biogas plants is increasing every year. However, resistant strains persisted longer than the sensitive strains.because of the lack of conclusive evidence for the inactivation Dudley et al. (1980) have shown that bacterial pathogens,of bacterial pathogens during anaerobic digestion, there is such as salmonellas, shigellas, klebsiellas, mycobacteria,confusion in the minds of end-users about the safe disposal pseudomonads, etc. survive the process of anaerobic diges-of these wastes. tion. The finding by Gadre et al. (1986) that Salm. typhi-

It has been reported that animal wastes show the presence murium was completely inactivated within 9 d in batch anaer-of pathogenic bacteria, including Salmonella spp., Escherichia obic digestion of cattle dung should be viewed vis-a-vis con-coli, Campylobacter spp., Listeria monocytogenes, etc., which ventional practice of semi-continuous anaerobic digestion for

running the biogas plants in India. Kearney et al. (1993a)Correspondence to: Dr D.R. Ranade, Division of Microbial Sciences,have reported that counts of Salm. typhimurium, E. coli andAgharkar Research Institute, G.G. Agarkar Road, Pune-411 004, India (e-

mail: drr@aripune.ernet.in). Yersinia enterolytica declined during the process of anaerobic

© 1998 The Society for Applied Microbiology

PATHOGEN SURVIVAL IN ANAEROBIC DIGESTION 139

digestion of cattle dung. However, complete elimination of the digesters were fed daily with fresh cattle dung slurry(6% TS). In order to achieve high levels of VFA in thethe pathogens was not achieved, even after a digestion period

of 20 d. Thus, there is no conclusive evidence that the intes- experimental digester, the HRT was maintained at 15 d andfurther, the daily feed was supplemented with 1% (w/v)tinal pathogens are totally eliminated during anaerobic

digestion, although their numbers reduce considerably. glucose. The HRT for the control digester was maintainedat 30 d. Performance of the digesters was determined in termsOperational parameters of the anaerobic digestion process

such as temperature, pH, hydraulic retention time (HRT), of total biogas produced and its methane content, pH andVFA. The studies on survival of Salm. typhi were carried outtotal solids (TS) and volatile fatty acids (VFA) are likely to

affect the survival of bacterial pathogens (Henry et al. 1983). only when the experimental digester attained a constant VFAlevel of around 5000 mg l−1.The in vitro inhibitory effects of VFA on bacterial pathogens

have been reported earlier (Goepfert and Hicks 1969; Freeseet al. 1973; Fay and Farias 1975; Cherrington et al. 1990;

Survival studiesAbdul and Lloyd 1985b). It is therefore logically expectedthat high levels of VFA in cattle dung- or human night soil- Single dose experiment. Overnight culture of Salm. typhi,based anaerobic digesters, would lead to greater inhibition of grown in Brain Heart Infusion Broth at 37 °C, was added topathogenic bacteria. Very little information is available on the both the control and the experimental digester so as to achievefate of bacterial pathogens under such conditions. an initial count of 106–107 cells ml−1 of the digesting slurry.

The present investigation was undertaken to study the Samples from both the digesters were serially diluted ineffect of high levels of VFA on the survival of Salm. typhi normal saline and 0·1 ml of each dilution was spread onduring anaerobic digestion of cattle dung. the selective medium to determine the initial count of the

pathogen. Viable counts of the pathogen in both the digesterswere determined after an inverval of 2 d.MATERIALS AND METHODS

Bacterial strain Daily dose experiment. Initially, both the control and exper-imental digesters were loaded with overnight-grown cultureSalmonella typhi MCM B-323 was a clinical isolate obtainedof Salm. typhi so as to achieve an initial count of 106–107 cellsfrom the MACS Collection of Micro-organisms (MCM).ml−1 and mixed well. Samples from both the digesters wereThe culture was maintained on Brain Heart Infusion Agarcollected and the initial count of the pathogen was deter-(Hi-Media, Mumbai, India) and was stored at 4 °C. Themined. From the next day onwards, along with the daily feed,culture showed high levels of resistance to the commonly useda dose of Salm. typhi (106 cells ml−1) was given to both theantibiotics, penicillin, ampicillin, chloramphenicol, rifampi-digesters. The survival of the pathogen was monitored aftercin, cloxacillin, trimethoprim, streptomycin and tetracycline.every 2 d.

Growth and selective mediumAnalytical methods

Salmonella typhi was grown in Brain Heart Infusion Broth atTotal biogas produced daily was measured using a wet gas37 °C. Wilson-Blair Agar (Hi-Media), supplemented withflow meter (Toshniwal Instruments Ltd, Chennai, India) andstreptomycin, chloramphenicol, ampicillin, trimethoprimanalysed daily for methane on a gas chromatograph (Chemitoand tetracycline at final concentrations of 10, 30, 10, 20 and3800, Toshniwal Instruments Ltd, Mumbai, India), equipped10 mg ml−1, respectively, was used for the selective isolationwith a thermal conductivity detector (Ranade et al. 1987).of Salm. typhi from the digester slurry without supportingOn every alternate day, VFA concentration in the digesterthe growth of undesirable indigenous flora. The temperaturesamples was determined on a gas chromatograph equippedfor incubation during the enumeration of the pathogen waswith a flame ionization detector (Yeole et al. 1989). Data37 °C.computation was done using an integrator (SpectraphysicsSP 4270, San Jose, CA, USA). Total solids and pH of the

Anaerobic digesters cattle dung slurry were determined according to Greenberget al. (1992).The anaerobic digesters used in the study were KVIC model

floating dome digesters (Dayal 1981) with a working capacityof 9·5 l. Initially, two digesters were filled with cattle dung

Statistical methodsslurry (6% TS) and kept for stabilization. After the estab-lishment of methanogenesis, as evidenced by satisfactory In the single dose experiment, regression analysis was used

to obtain the best fit line for the survival curves of Salm.biogas production containing a minimum of 50% methane,

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 138–142

140 D.P. KUNTE ET AL.

typhi in the control and experimental digesters. The decimal was completely eliminated in the experimental digester in 12d whereas 26 d were required in the control digester. Thereduction time (T90), which is the time taken for a 90%

reduction in the viable population, was determined from the absence of pathogen in the digester slurry was further con-firmed by enrichment in Selenite F broth. The digester per-regression equation according to the method described by

Olsen and Larsen (1987). The difference between the formance, in terms of pH, TS and VFA of the digestingslurry, and total biogas produced along with its methaneregression coefficients was assessed by using the F-test

(Snedecor and Cochran 1967). The two survival curves were content, remained relatively constant during the course ofthe experiment. The statistical analysis (F-test) of the survivalconsidered significantly different if P³ 0·05. In the daily

dose experiment, after every two days, T90 was calculated by curves of Salm. typhi in the control and experimental digestersshowed significant difference in their regression coefficients.the formula described by Kearney et al. (1993b) and the mean

T90 was calculated. The difference in the survival curves was In the daily dose experiment, the counts of Salm. typhideclined rapidly for the initial period of 8 d. During thiscalculated by comparing the population medians using the

Mann-Whitney Confidence Interval Test (Snedecor and period, a four log-reduction in the pathogen count wasobserved in the experimental digester compared to a threeCochran 1967). The computations were done using the

Mintab Statistical Package (Rayan et al. 1985). log-reduction in the control digester. Later, the pathogencount remained stable at 102 and 104 cells ml−1 in the exper-imental and control digesters, respectively (Fig. 2). The mean

RESULTST90 values for the experimental and control digesters were 4·2and 18·6 d, respectively (Table 2). The data, when subjectedThe performance of the digesters is described in Table 1. It

can be seen that the addition of 1% glucose in the exper- to statistical analysis using the Mann-Whitney ConfidenceInterval Test, showed a significant difference in the twoimental digester led to an increase in VFA (4500–5700 mg

l−1) compared to the control digester which showed a VFA population medians.level of 90–140 mg l−1. The treatment also resulted in slightlyhigher total gas production but with less methane content

DISCUSSION(38–45%) than in the control digester (55–60%). Further,the pH of the digesting slurry in the experimental digester The experimental digester was supplemented with an easily

degradable substrate, ie. glucose (1% w/v); this resulted inwas slightly acidic (pH 5·8–6·2) compared to that of thecontrol digester (pH 6·8–7·1). an increased VFA level (5000 mg l−1) because cattle dung

contains low amounts of easily degradable substrates.The survival pattern of Salm. typhi in the experimentaland control digesters during the single dose and daily dose The T90 value indicates the degree of inactivation of bac-

terial pathogens in anaerobic digestion. Various factors suchexperiments is shown in Figs 1 and 2, respectively. In thesingle dose experiment, viable counts of Salm. typhi, in both as temperature, pH, TS, VFA etc., are known to influence

the values of T90 (Henry et al. 1983). In the present study,the experimental and control digester, declined rapidly foran initial period of 6 d. This was followed by a phase in which the temperature and TS were identical for both the digesters.

Therefore, the difference in the survival of Salm. typhi in thethe decline was gradual. The rate of decline in the counts ofSalm. typhi was significantly greater in the experimental two digesters could be attributed to pH and high VFA. The

in vitro toxic effects of VFA on bacterial pathogens have beendigester than in the control (Fig. 1). This is reflected in thesmaller T90 value for Salm. typhi in the experimental digester reported earlier (Freese et al. 1973; Abdul et al. 1985a). It has

also been reported that the toxicity of VFA is greater at lower(2·44 d) than the control (4·80 d) (Table 2). The pathogen

Table 1 Performance of anaerobicdigesters

—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Single dose experiment Daily dose experiment—–––––––––––––––––––––––––– —––––––––––––––––––––––––––Control Experimental Control Experimental

Parameter digester digester digester digester—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––HRT (d) 30 15 30 15pH 6·7–6·9 5·9–6·1 6·7–7·1 5·8–6·2Methane (%) 55–59 40–45 55–60 38–41Total biogas (ml d−1) 2700–3000 3600–5400 3000–3200 3400–3900VFA (mg l−1) 90–140 4500–5000 100–125 4800–5700—–––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 138–142

PATHOGEN SURVIVAL IN ANAEROBIC DIGESTION 141

Fig. 1 Survival of Salmonella typhi in thesingle dose experiment. (Ž), Experimentaldigester; (�), VFA in experimental digester;(R), Control digester; (r), VFA in controldigester

30

8

00

Time (d)

Lo

g n

o. o

f cells m

l–1

5

10

VFA

(m

g l

–1)

7000

0

5000

7

6

4

3

2

1

12 14 162 4 6 8

6000

4000

3000

2000

100

18 20 24 26 2822

Fig. 2 Survival of Salmonella typhi in the dailydose experiment. (Ž), Experimental digester;(�), VFA in experimental digester; (R),Control digester; (r), VFA in controldigester

24

7

00

Time (d)

Lo

g n

o. o

f cells m

l–1

5

10

VFA

(m

g l

–1)

7000

0

5000

6

4

3

2

1

12 14 162 4 6 8

6000

4000

3000

2000

100

18 20 22

Table 2 Decimation times (T90) for Salmonella typhi in control It should be noted here that the magnitude of differenceand experimental digesters in the T90 values for the control and the experimental digester—––––––––––––––––––––––––––––––––––––––––––––––––––––– was greater in the daily dose experiment than the single

T90 (d) dose experiment. Complete elimination of the pathogen was—–––––––––––––––––––––––––––––––––––––

achieved in the single dose experiment. However, the sameExperiment Control digester Experimental digestercould not be achieved in the daily dose experiment, although—–––––––––––––––––––––––––––––––––––––––––––––––––––––a four log-reduction in the pathogen count was observed. ThisSingle dose 4·80 2·44might be due to the fact that in the daily dose experiment,Daily dose* 18·63 4·22

—––––––––––––––––––––––––––––––––––––––––––––––––––––– the digesters were fed daily with fresh cattle dung slurry*In the daily dose experiment, T90 was calculated after every two supplemented with a culture of Salm. tyhpi (106 cells ml−1).days and the value in the table is the arithmetic mean. Such a high cell density of the pathogen is not normally likely

to be encountered in an anaerobic digester running on cattledung or human night soil. It is, therefore, expected thatlower pathogen densities (less than 104 cells ml−1), high VFApH (Henry et al. 1983). In the present investigation, the effect(approximately 5000 mg l−1) and slightly acidic pH (5·5–6·0)of high VFA on survival of Salm. typhi was studied in ain the digester, would result in complete elimination of thecomplex substrate, cattle dung. High VFA and slightly acidicpathogens, even with the daily dose.pH in the experimental digester caused greater inactivation

In India, cattle dung is anaerobically digested alone or inof the pathogen than in the control digester with low VFAand neutral pH. combination with human night soil. The latter contains easily

© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 138–142

142 D.P. KUNTE ET AL.

acids on the growth of Escherichia coli. Journal of General Micro-degradable substrates and therefore it is possible to achievebiology 91, 233–244.high VFA levels by lowering the HRT. On the basis of the

Freese, E., Sheu, C. W. and Gallier, E. (1973) Function of lipophilicpresent study, it is proposed that the acidogenic andacids as antimicrobial food additives. Nature 241, 321–325.methanogenic phases should be separated for anaerobic diges-

Gadre, R.V., Ranade, D.R. and Godbole, S.H. (1986) A note ontion of human night soil. During the acidogenic phase, there survival of salmonellas during anaerobic digestion of cattle dung.would be almost complete elimination of the bacterial patho- Journal of Applied Bacteriology 60, 93–96.gens because of high VFA and low pH. The chance survivors Ginnivan, M.J., Woods, J.L. and O’Callaghan, J.R. (1980) Survivalwould then be further exposed to the methanogenic phase of Salmonella dublin in pig slurry during aerobic thermophilic

treatment in batch, cyclic and continuous systems. Journal offor at least 10–15 d. This would certainly result in theirApplied Bacteriology 49, 13–18.complete elimination. Thus, the two-stage digestion process

Geopfert, J.M. and Hicks, R. (1969) Effect of volatile fatty acids onwould effectively overcome the problems of pathogen survivalSalmonella typhimurium. Journal of Bacteriology 97, 956–958.during anaerobic digestion.

Greenberg, E.A., Clesceri, L.S. and Eaton, A.D. (ed.) (1992) Stan-dard Methods for the Examination of Water and Wastewater, 18thedn. Washington DC: APHA, AWWA and WPCF.

Henry, D.P., Frost, A.J., Samuel, J.L., O’Boyle, D.A. and Thom-ACKNOWLEDGEMENT son, R.H. (1983) Factors affecting the survival of Salmonella and

Escherichia coli in anaerobically fermented pig waste. Journal ofThe authors wish to thank the Department of Science andApplied Bacteriology 55, 89–95.Technology, Government of India, New Delhi, for financial

Jones, P.W. (1980) Health hazards associated with the handling ofsupport during this study. animal wastes. The Veterinary Record 106, 4–7.

Jones, P.W. and Matthews, P.R. (1975) Examination of slurry fromcattle for pathogenic bacteria. Journal of Hygiene Cambridge 74,57–64.

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© 1998 The Society for Applied Microbiology, Journal of Applied Microbiology 84, 138–142