Journal of Applied Bacteriology 19P2,53, 331-334 1067,/111/81

Inactivation of Salmonella during anaerobic digestion of sewage sludge

E.G. C A R R I N G T O N , S A R A H A. H A R M A N & E.B. P I K E Water Research Centre, Steuenuye Laboratory, Elder W a y , Steoenaye, Herts SG1 I TH, U K

Received 23 November 1981 and accepted 17 March 1982

C A R K I N G T O Y , E.G., H A K M A N , S A R A H A. & PIKE, E.B. 1982. Inactivation of Salmonella during anaerobic digestion of sewage sludge. Journal of Applied Bucte- rioloyy 53, 331-334.

The inactivation of Sulrnonella duesseldorf in sewage sludge during anaerobic di- gestion was investigated at 35 and 48'C with mean retention periods of between 10 and 20 days. Digesters were fed daily with raw sludge containing added Salm. duesseldorf after removal of digested sludge. During steady operation, the levels of Salm. duesseldorf in the digested and the feed sludge were determined and their specific rates of decay were estimated. The latter were: (i) greater at 48'C than at 35°C for the same retention time; (ii) similar for retention periods greater than I S d, but lower for 10 d ; (iii) greater when the level of salmonellas in the feed was lower. Gas production, a measure of steady state, was gradually lost when the mean retention period was reduced to 6.7 d. In experiments in which a single dose of Salm. duesddor f was added to digesting sludge. the inactivation appeared to follow first-order kinetics at 3S"C and the decimal decay rate, 1,6,'d, was similar to that in the daily feeding experiments (1.41d) with larger and similar inocula of Salm. dues- seldorf At 48-'C, however, the rate of inactivation declined with decreasing time from inoculation suggesting that the culture contained cells differing in thermal resistance. The degrees and rates of inactivation of salmonellas in those experiments were greater than in full-scale digesters, because the latter seldom operated under conditions ideal for inactivation or because indigenous salmonellas are more resist- ant

The use of sludge as a soil conditioner and ferti- liser is an attractive proposition to farmers and regional water authorities. Approximately 70% of sewage sludge produced in the UK is dis- posed of on land and about 42% of this is trea- ted by anaerobic digestion, (Anon. 1981a). Heated anaerobic digestion is recognized as a method of reducing certain pathogens which might be present in sewage sludge to a level where the risk of transmission of the disease to animals and man is low (Anon. 1981b). How- ever, the degree of inactivation of Salmonella spp. appears, in practice, to be incomplete and dependent upon the frequency of feeding and of withdrawal of sludge and upon the design and mixing characteristics of the digester (Pike &

0021-8847/82/1200-0331 $02.00 @ 1982 The Society for Applied Bacteriology

Carrington 1979). If the specific rates of decay of salmonellas in the sludge are known it should be possible to predict the proportion remaining in the sludge withdrawn from digesters oper- ating under specified conditions. This has been attempted experimentally by Ginnivan e t al. (1980) for pig slurry treated aerobically a t 55°C. It was the aim of these experiments to measure the rates and extent of inactivation of a strain of Salmonella duesseldorf in digesting sludge and to study the effects of temperatures, mean retention period and size of inoculum on inactivation.

Materials and Methods

D I G E S T E R S

Four anaerobic digesters were constructed using 5 1 Quickfit reaction vessels. These were initially

332 E. G . Carrington et al. filled with 4 1 of actively digesting sludge. The contents were maintained at a constant tem- perature by standing the digesters in water baths. The contents of a digester were mixed by a paddle rotating continuously at about 35 rev/min. The daily production of gas was meas- ured by a tipping-trough gas meter (Pearce 1975). Once a day a predetermined volume of sludge was withdrawn and then replaced with an equal volume of co-settled raw and activated sludge from a local sewage works. The retention time of the sludge in the digesters was varied by altering the volume of the sludge changed daily. Experimentation did not begin until the di- gesters had achieved a steady state of gas pro- duction and pH value.

S A L M O N E L L A S

Raw sewage sludge was obtained weekly from a local sewage works and was sieved before use to remove gross particles. The Salmonella content was low and variable, so that it was necessary to inoculate the sludge artificially before digestion with a strain of Salm. duesseldorf, originally iso- lated from activated sludge. This serotype is not normally encountered in sludge and could therefore be easily recognized during confirma- tion by serotyping. This strain could not be re- covered from sludge, when it had been added as an overnight broth culture, presumably because it was rapidly inactivated. In all experiments, the inoculum was an aged culture of Salm. duesseldorf, previously cultured in cooked meat medium (Oxoid CM439) and stored at 4'C for approximately 3 months before use.

E X P E R I M E N T A L P R O C E D U R E S

Sufficient aged culture of Salm. duesseldorf, di- luted in peptone water, was added to the accli- matized digesters a t the start of each experiment, to give the desired initial count of salmonellas. Thereafter, 1 ml of diluted culture was added daily to the raw sludge before feeding to give the same value of initial count in the raw sludge. The count of salmonellas in the diluted culture was determined by spread-plating on XLD agar (Oxoid CM469), incubated at 37°C for 18 h. Three times/week, the numbers of Salm. duesseldorf in samples withdrawn from the digester were determined using the most prob- able number (MPN) method of Carrington

(1980a). It was found that high concentration of sludge in the least dilute portions of sample often appeared, from the pattern of results in the MPN test, to inhibit detection of salmonellas. This has also been noted by Daniel & Lloyd (1980). In the calculation of MPN from the pat- terns of results, where this problem was en- countered, it was assumed that there were at least as many positive reactions at this lowest dilution of sample as at the next higher dilution.

The decimal decay rate, k , , was calculated using the formula of Ginnivan et al. (1980):

P/P , = R/( 10RBka - 1 + R) ,

where P = the count of Salm. duesseldorfin the withdrawn sludge, P, = the count in raw sludge, R = the fractional volume replaced at each feed and 0 = the mean retention period.

In experiments to determine survival curves, digesters were given single doses of raw sludge containing the desired count of salmonellas and samples of sludge were withdrawn at intervals for analysis. The decimal decay rate was deter- mined as the slope k , , of the linear regression of log,, fraction surviving against time from in- oculation.

Results

Measurements of the salinity of sludge, drawn off from a digester a t 2 min intervals after the addition of a feed of sludge containing added sodium chloride as a tracer, showed that the feed sludge was homogeneously mixed with the digester contents within 6 min.

Table 1 indicates that the decimal decay rate for daily feeding was greater at 48°C than at 35°C for the same retention period, equivalent with retention periods of 16 and 20 d at each temperature, but less when the retention period was 10 d and greater when the level of salmon- ellas in the feed was lower.

In the experiments to determine survival curves, inactivation at 35°C (Fig. la) appeared to follow first-order kinetics and the value of k , from four experiments was 1.6/d. In the three experiments a t 48"C, the rate of inactivation de- creased with increasing time from inoculation (Fig. lb).

When the mean retention period of the diges- ters at 35°C was increased to 6.7 d, the rate of production of gas declined progressively.

Salmonellus in unaerobic digestion Table 1. Decimal decay rates of Sulmonellu diwsseldurf in anaerobic digesters fed daily

and operating under various conditions

333

48

~

Mean retention period W) '0, 20 16 I6 10 20 10

-

Sulmonelluj100 ml* . ~~ ~~

In feed Withdrawn P

500 1 1.3 1 0 5 920

350 1 1.3 ; 10' 9 20 7.8 I( 103 700 5.5 Y 10' 1 9.1 % 1 0 5 40

~~~ ~~~

Number of observations

30 52 12 49 28 28 24

~- ~-

Decimal decay rate,

k , ( d - ' )

I .4 0.9 I .4 I .o 0.3 4.4 3-4

-. ~ ~~~

* Median for duplicate experiments with each set of conditions.

Discussion

Findlay (1973) has shown that salmonellas may multiply in sterile sludge in the absence of com- petition from other micro-organisms. However, a considerable amount of literature reviewed by Carrington (1978, 1980b) and data from a nat- ional survey (Pike 1981) have shown that Sal- monella counts are usually reduced during anaerobic digestion but these papers do not give sufficient details for death rates to be calculated.

Whilst this work confirms that salmonellas

(

0-

0.0

0.oc

0. ooc

are inactivated in sludge treated in heated anaerobic digesters the rates of inactivation in the field remain to be determined. Studies with tracers at the authors' laboratory and elsewhere (Monteith & Stephenson 1981) show that anaer- obic digesters depart seriously from the ideal model of the continuously stirred-tank reactor required for efficient digestion and gas pro- duction, because of short-circuiting of flow, dead spaces in the digester and ineflicient mixing. In practice, they are fed discontinuously, whereas

- I 2 0

1 n A 1

Time from inoculation (h)

2 4 6 a Time frorn inoculation (d)

Fig. 1. Fraction of Salmonella duesseldorf added to digesting sewage sludge, remaining after various intervals from addition. (a) Four experiments at 35'C, points are for geometric mean counts of number of determinations shown. For (a) log,, y = -1,603 x +0.1120; r = -0.864; n = 23. (b) Three experiments at 48°C. Salmonella duesseldorf'added to give an initial count of approximately 104/100 ml in sludge.

E . G. Carrington et al. plug-flow or batch conditions are required to obtain the greatest degree of inactivation. It is probably for these reasons, and because of dif- ferences in sensitivity of the culture of Salrn. duesseldorf used here, compared with those of indigenous salmonellas in sludge, that the frac- tion remaining in the treated sludge was much smaller (Table 1) than reported from sewage works (Carrington 1978, 1980b; Pike 1981).

The differences in the kinetics of inactivation for digestion at 35 and 48°C (Fig. 1 ) are prob- ably fundamental since they suggest that the factors responsible operated constantly over the whole population of salmonellas at 35°C but not at 48°C. The supposition is that thermo- tolerance is a factor at 48°C and that the popu- lation of Salrn. duesseldorf contained a small fraction of heat-resistant cells. It will be noted that the value of k , obtained at 35°C in these inactivation curve experiments (16/d) was com- parable with those values (1.4/d) obtained in the daily feeding experiments with the higher count in feed, showing that the two methods were equivalent, as expected if inactivation followed first-order kinetics.

References

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ANON. 1981b The risk to health of microbes in sewage sludge applied to land. EURO Reports and Studies 54. Copenhagen: Ofice for Europe, World Health Organization.

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MONTEITH, H.D. & STEPHENSON, J.P. 1981 Mixing efi- ciencies in full scale anaerobic digesters by tracer methods. Journal of Water Pollution Control Feder- ation 53, 78-84.

PEARTE, D. 1975 Equipment for measurement of gas production at low rate of flow. Technical Memor- andum TM104 Stevenage and Medmenham: Water Research Centre.

PIKE, E.B. 1981 Salmonellae in sewage sludges: a survey of sewage works in England and Wales. In Proceedings of First European Symposium on Treat- ment and Use of Sewage Sludge, Cadarache, France, 13-15 February, 1979 ed. Alexandre, D. & Ott, H. pp. 189-200. Brussels: Commission of the Euro- pean Communities.

PIKE, E.B. & CARRINGTON, E.G. 1979 The effects of conventional sludge treatment processes on patho- gens. In Proceedings of Conference on Utilization of Sewage Sludye on Land, Oxford, 10-13 April 1978 pp. 198-218. Stevenage: Water Research Centre.