Enteric Bacteria in Aerobically Digested Sludget - Applied and ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1984, p. 831-834

Vol. 47, No. 4

0099-2240/84/040831-04$02.00/0 Copyright C 1984, American Society for Microbiology

Enteric Bacteria in Aerobically Digested Sludget SAMUEL R. FARRAH* AND GABRIEL BITTON Department of Microbiology and Cell Science and Department of Environmental Engineering Sciences, University of Florida, Gainesville, Florida 32611 Received 1 July 1983/Accepted 27 January 1984

Indicator bacteria, Salmonella spp., and total aerobic bacteria were determined in samples of undigested sludge and sludge that had been treated by one or two stages of aerobic digestion. Aerobic sludge digestion reduced the level of indicator bacteria by 1 to 2 log10 per g. The level of Salmonella spp. was also reduced during aerobic treatment of sludge. In general, aerobic treatment of sludge reduced, but did not eliminate, indicator bacteria and Salmonella spp. Wastewater sludges may harbor a wide range of microbial pathogens and parasites which present potential health hazards to humans. The concentration of these pathogens in sludge is a result of their affinity for and subsequent association with wastewater solids and the failure of conventional wastewater treatment processes to completely inactivate these organisms (3-5, 11, 12). Much of the sludge produced in the United States is treated further by anaerobic digestion. Several studies on the fate of pathogens and indicator organisms during anaerobic digestion of sludge have been reported (2, 11-13, 15). In contrast to anaerobic digestion, aerobic sludge digestion is a less complex process that is used by many small communities. Aerobic digestion is one of the treatment processes considered by the Environmental Protection Agency to significantly reduce pathogens in wastewater sludge before land application (Federal Register, Vol. 44, no. 179, Sept. 13, 1979, p. 53463). The results of several studies indicate that indicator microorganisms, pathogenic bacteria, and viruses are inactivated during thermophilic aerobic digestion of sludge at temperatures ranging from 45 to 65°C (8-10). However, Pedersen (14) conducted a thorough review of the literature and concluded that little research had been conducted on the effect of mesophilic aerobic digestion on bacterial and viral pathogens. Kuchenrither and Benefield (10) and Farrah and Bitton (6) studied the impact of some environmental factors on the survival of indicator and pathogenic bacteria in aerobically digested sludge under controlled laboratory conditions. This paper presents the results of a field survey of indicator bacteria (total and fecal coliforms and fecal streptococci) and pathogens (Salmonella spp.) in undigested and aerobically digested sludge from wastewater treatment plants in Florida.

was aerobically digested in two stages in digestors with detention times of ca. 8.5 days for each tank. Wasted activated sludge at the Main Street plant was digested in two stages in digestors with 50-day detention times each. However, undigested primary sludge was added to the first aerobic digestor along with wasted activated sludge. It was difficult to know the proportions of these two sludges that were added to the digestors and to determine the numbers of bacteria added to this digestor. Therefore, calculations of bacterial removal were made by comparing bacteria in sludges from aerobic digestors 1 and 2 and considering the plant as having only one stage of aerobic sludge digestion. Sludge at the Tallahassee plant was digested in one stage with a detention time of 10 days. Sludge samples were obtained over two periods. From January to December 1981, 12 to 15 samples were taken from the Kanapaha and Main Street plants. From August to October 1983, monthly samples (in triplicate) were taken from the Main Street and Kanapaha plants and one set of triplicate samples was obtained from the Tallahassee plant. Bacteriological analyses. Sludge samples (45 ml) were mixed with 5 ml of a solution containing (per 100 ml of water): 9 g of sodium chloride, 0.1 g of Lubrol WX, and 0.1 g of sodium PP1 (7). Samples were homogenized for 1 min with a Tekmar homogenizer (Tekmar Co., Cincinnati, Ohio) and diluted in phosphate-buffered saline. The supernatant fraction remaining after centrifugation of sludge samples for 5 min at 500 x g was diluted in phosphate-buffered saline. Samples of 0.1 ml of homogenized sludge, supernatant fraction, or dilutions of these in phosphate-buffered saline were spread on agar plates or added to tubes of media. Media for bacterial analyses were obtained from Difco Laboratories, Detroit, Mich., or from BBL Microbiology Systems, Cockeysville, Md. The solid media employed and the bacteria enumerated were as follows: mEndo, total coliforms; mFC, fecal coliforms; KF, fecal streptococci; plate count agar, total aerobic bacteria. Total coliforms were also determined by using lactose broth and a most-probable number (MPN) procedure (1) with three tubes per dilution. Tubes of EC broth were inoculated with media from tubes of lactose broth that showed gas production to determine fecal coliforms. The mFC plates and EC tubes were incubated in a water bath at 44.5°C, and all other plates were incubated at 37°C. Salmonella spp. were recovered by the following procedures. (i) Pre-enrichment. In most cases, three tubes with 10 ml of double-strength nutrient broth were inoculated with 10 ml of sludge, three tubes containing 10 ml of singlestrength broth were inoculated with 1 ml of sludge, and a third set of three tubes with 10 ml of single-strength nutrient

MATERIALS AND METHODS Sources of sludge. Most of the results were obtained with undigested sludge (mixed liquor-suspended solids or wasted sludge) and aerobically digested sludge which were obtained from two wastewater treatment plants serving the city of Gainesville, Fla. (Main Street and Kanapaha plants). Samples of undigested and aerobically digested sludge were also obtained from a treatment plant serving the city of Tallahassee, Fla. Anaerobically digested sludge was obtained from the Tallahassee plant. At the Kanapaha plant, wasted sludge * Corresponding author. t Journal paper no. 4814 from the Florida Agriculture Experiment Station, Gainesville, FL 32611.

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FARRAH AND BITTON TABLE 1. Characteristics of Aerobically digested sludgesa

Treatment Treatment plant

Sludge

Total

source

Temp (C)

Dissolved

pH

solids

1 2 1 2

7-29 7-31 15-29 10-23

0.8-3.3 0.6-9.2 0.5-1.8 0.8-3.7

6.8-6.9 6.9-7.3 6.5-7.1 6.9-7.1

8.0-21.6 19.0-26.5 12.3-19.0 14.0-47.0

~~~~(digestor)

Kanapaha Main Street

a Measurements were made between January and

oxygen oxgn(glliter)

December 1981.

broth received 0.1 ml of sludge. Occasionally, 50 ml of sludge was added to 50 ml of double-strength nutrient broth. The samples were incubated for 18 to 24 h at 37°C. (ii) Enrichment. Pre-enrichment culture (1 ml) was transferred to a tube containing 10 ml of tetrathionate broth and incubated for 18 to 24 h. (iii) Inoculation. One loopful of the enrichment broth was streaked onto a plate containing one of the following media: bismuth sulfite, brilliant green, salmonella-shigella, XLD, and hektoen enteric. At least three different media were used for each sludge sample. The plates were incubated at 37°C for 24 to 48 h. (iv) Purification. Portions of colonies resembling Salmonella spp. were streaked onto plates of MacConkey or XLD agar for purification. (v) Characterization. Isolated colonies resembling Salmonella spp. were transferred to Kligler iron agar slants and incubated at 37°C for 24 h. Cultures giving reactions characteristic of Salmonella spp. were tested with polyvalent and group-specific antisera. Cultures that were agglutinated by Salmonella antisera were used to inoculate a series of biochemical tests in enterotubes (Roche Diagnostics, Nutley, N.J.). Physical and chemical measuretnents. The pH, temperature, and total solids of sludge samples were determined according to standard methods (1). The dissolved oxygen of sludge was measured with a Yellow Springs model 54Adissolved oxygen meter (Yellow Springs Instrument Co., Yellow Springs, Ohio). Statistical analyses. The numbers of bacteria in the different sludge samples were compared by using Duncan's new multiple range test (16). RESULTS The characteristics of the aerobically digested sludges used in most of the study are shown in Table 1. Values for the other sludges fell within the range of values shown in Table 1. In general, the temperature, dissolved oxygen, and total solids were relatively variable, whereas the pH was constant.

The levels of indicator and total aerobic bacteria declined during aerobic digestion at both the Kanapaha and Main Street plants (Table 2). The levels of total coliforms and fecal streptococci in undigested sludge were similar for trials 1 and 2. However, lower levels of these bacteria were generally found in the digested sludge during trial 2. The numbers shown in Table 2 are based on colony counts on solid media; similar trends were observed in results obtained with tube media (data not shown). The digestors were continuously mixed and had sludge added and removed at relatively constant rates. In a completely mixed system such as this, a portion of the influent sludge is removed along with the digested sludge. Bacteria in the influent sludge will therefore contaminate the digested sludge. The presence of these contaminating bacteria will place a limit on the amount of reduction in bacterial numbers that can be observed during the digestion process. Assuming that the bacterial numbers do not change appreciably during the mixing period, the fraction of the influent bacteria that will be found in the effluent sludge is related to the number of digestors used and to the detention time of the digestors. Calculations on the makimum detectable removal of bacteria at the three plants studied are presented in Table 3. Reductions in bacterial numbers were observed during aerobic digestion at the three plants studied (Table 4). The reductions in numbers of indicator bacteria varied between 0.77 and 2 logl0 per g of sludge and between 80 and 99%. Fecal streptococci were more stable than total or fecal coliforms at the Kanapaha and Main Street plants. Salmonella spp. were detected in sludge samples from all three treatment plants studied (Table 5). All of the Salmonella isolates were identified as Salmonella enteritidis. The serotypes of 22 isolates that were typed are as follows: B, 19%; C, 58%; E, 8%; not typed with available sera, 15%. S. enteritidis was isolated from 70% (7/10) of aerobically digested sludge samples that were between 7 and 11°C when collected. In contrast, S. enteritidis was isolated from 15% (4/26) of the samples that were between 12 and 31°C (Table 6).

TABLE 2. Bacteria in wastewater sludge

Treatment plant

Sludge source

1" 1.3 x 108 5.4 x 107 Trial

Undigested Aerobic digestor 1 Aerobic digestor 2 3.8 Main Street Aerobic digestor 1 2.0 1.9 Aerobic digestor 2 for the in same plant and the aNumbers b January through December 1981. ' August through October 1983.

Kanapaha

Bacteria per g of sludge' Fecal streptococci

Total coliforms Trial 2'

5.8 x 107 2.2 x 106 x 1o6 5.9 x 105 x 108 7.6 x 107 x 107 9.1 X 105 same column are significantly

Trial 1

1.6 6.9 1.5 2.3 3.9

x 106 x 105 x 105

x 106

Trial 2

1.8 1.9 4.2 1.5 2.9

x

106

x 105 x

104

x 106

Fecal

Total aerobic

coliforms:

bacteria:

trial 1

trial 1

1.5 3.6 4.5 2.8 2.3

x

107

x 106 x

105

x 107

x 106 x 105 x 104 different at the 95% confidence level.

1.8 8.8 2.0 2.0 3.2

x 109 x 108 x x x

108 109 108

VOL. 47, 1984

Treatment plant

ENTERIC BACTERIA IN SLUDGE

833

TABLE 3. Effect of sludge detention time on the maximum detectable removal of bacteriaa Maximum detectable removal Detention Fraction of undigested Stages of

Sagesbi

digestion

time per stage (days)

influent sludge expected in the final digested sample after dilution

Kanapaha 2 8.5 1/8.5 x 1/8.5 = 1/72.5 Main Street 1 50 1/50 Tallahassee 1 10 1/10 a Assuming that the sludge is added to and removed from the digestors at relatively constant rates and that change during the mixing period. b Percent = (1 - the value from column 4) x 100%. c Log10 (the value from column 4).

After low-speed centrifugation, ca. 22% of the total coliforms, fecal coliforms, and total aerobic bacteria were found in the supernatant fraction of anaerobically digested sludge, whereas the remainder were associated with the sludge flocs. However, greater than 95% of the bacteria in aerobically digested sludge were associated with the sludge solids after similar treatment, and only 5% could be found in the supernatant fraction.

DISCUSSION The possible transmission of pathogenic bacteria and viruses during disposal of wastewater sludge has led to several studies on the survival of pathogens during sludge digestion. Since anaerobic digestion of sludge is widely used, most workers have studied survival of enteric pathogens under anaerobic conditions (2, 12, 13, 15). In general, these studies have shown that anaerobic treatment can reduce the number of pathogens but usually does not completely eliminate such organisms. Since sludge digestors are usually operated as continuous systems, the digested sludge may be contaminated with freshly added undigested sludge (2). In contrast, relatively few studies on survival of indicator bacteria, bacterial pathogens, and viruses during aerobic digestion of sludge have been reported. Kabrick et al. (9) and Drnevich and Smith (48th Annual Water Pollut. Control Fed. Conf., Miami Beach, Fla., 1975) found that aerobic thermo-

%b

Loglo1

99 98 90

-1.86 -1.70 -1.00

the bacterial numbers do not

philic digestion of sludge significantly reduced the numbers of bacterial pathogens and indicator bacteria. The results of recent laboratory studies on survival of bacteria during mesophilic aerobic digestion of sludge (6, 10) have shown that the temperature of sludge digestion is a major factor influencing survival of bacteria. Inactivation rates were generally found to be higher at elevated temperatures. Lewin et al. (11) determined the level of Salmonella spp. in anaerobically digested sludge from four treatment plants and in aerobically digested sludge from one treatment plant. The range of Salmonella spp. was significantly lower in the aerobically digested sludge than in the anaerobically digested sludge samples (0.3 to 3 MPN per 100 ml compared with 171.4 to >2,400 MPN per 100 ml). However, these authors noted that the plant employing aerobic digestion was an experimental one. The levels of indicator bacteria in the undigested sludge samples were fairly constant during both trials of this study. The numbers of total coliforms and fecal streptococci varied by less than a factor of 3 for samples taken over three years. The levels of bacteria in the undigested sludge at the Kanapaha plant and in sludge from the first aerobic digestor from the Main Street plant were also similar during each trial. The addition of wasted primary sludge to the aerobic digestor at the Main Street plant increased the level of bacteria over that which might be expected for a digestor

TABLE 4. Reduction in bacterial numbers during aerobic digestion of sludge Treatment plant plant

% Reduction' ~~~~~~Bacteriad Bacterla Trial 1' Trial 2"

Total coliforms 97 99 Fecal coliforms 97 NDe 91 98 Fecal streptococci ND 88 Aerobic bacteria 99 99 Maximum possiblef Main Street 91 99 Total coliforms ND 92 Fecal coliforms 83 98 Fecal streptococci ND 84 Aerobic bacteria 98 98 Maximum possible! 88 ND Tallahassee Total coliforms ND 93 Fecal streptococci 90 ND Maximum possiblef (Bacteria in digested sludge/bacteria in undigested sludge) x 100% (data from Table 2).

Kanapaha

aLogl0

(bacteria in digested sludge/bacteria in undigested c January through December 1981. d August through October 1983. e ND, Not done. f From Table 3.

sludge); data from Table 2.

Reduction" (log1o) Trial 1

Trial 2

-1.53 -1.52 -1.03 -0.95 -1.86 -1.02 -1.09 -0.77 -0.80 -1.70 ND ND ND

-1.99 ND -1.63 ND -1.86 -1.92 ND -1.71 ND -1.70 -0.91 -1.10 -1.00

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FARRAH AND BITTON

TABLE 5. Detection of Salmonella spp. in wastewater sludge % of Treatment plant

Kanapaha

Main Street

Tallahassee

samples

No. of

source N.Sludge

Undigested Aerobic digestor 1 Aerobic digestor 2 Undigested Aerobic digestor 1 Aerobic digestor 2 Undigested Anaerobic digestor Aerobic digestor

Mean

positive for Salmonella spp.

MPN per g

11

27

4.6

11

36

2.3

10 7

40 29

0.9 7.5

7

14

0.4

8 5

25 40

0.8 30

3

33

25

3

100

33

s

receiving only wasted activated sludge. Significant reductions in bacterial numbers were observed during aerobic digestion of sludge at the three plants studied. The reduction at the Main Street and Kanapaha plants ranged between 1 and 2 log1o and was ca. 1 log1o at the Tallahassee plant. Except for samples obtained during trial 1 from the Main Street plant, the values for reduction in bacterial numbers were close to the maximum that could be detected for continuously fed reactors. In such reactors, a portion of the influent sludge would likely be withdrawn with the digested sludge because the reactors receive relatively constant additions of fresh sludge and are continuously mixed. S. enteritidis were isolated more frequently from sludge samples with relatively low temperatures. Although laboratory studies have shown that temperature affects the survival of bacteria in aerobic digestors (6, 10), the influence of temperature on the indicator and total aerobic bacteria studied was not clear. Supernatants from samples of anaerobically digested sludge had proportionally more bacteria than did supernatants from aerobically digested sludge. These results are consistent with previously reported laboratory data (6). In summary, it appears that aerobic digestion of sludge can reduce the levels of indicator and pathogenic bacteria. The reductions at mesophilic temperatures are less than those reported for thermophilic aerobic digestion (8, 9). Since the digestors are operated as continuous systems with relatively constant addition and removal of sludge, some

TABLE 6. Influence of sludge temperature on detection of S. enteritidis Temp of aerobically digested sludge (°C)

No. of samples

% of samples positive for S. enteritidis

Range of values (MPN per 100 ml)

7-11 12-31

10 26

70 15

1.4-4 3.0-16

contamination of digested sludge with fresh undigested sludge can be expected. Therefore, the digested sludge will likely have reduced levels of bacterial pathogens but would not be expected to be free of such organisms. ACKNOWLEDGMENTS This work was supported by grant no. R806290 from the U.S. Environmental Protection Agency. The technical assistance of Orlando Lanni is gratefully acknowledged. LITERATURE CITED 1. American Public Health Association. 1975. Standard methods for the examination of water and wastewater, 14th ed. American Public Health Association, Inc., New York. 2. Berg, G., and D. Berman. 1980. Destruction by anaerobic mesophilic and thermophilic digestion of viruses and indicator bacteria indigenous to domestic sludges. Appl. Environ. Microbiol. 39:361-368. 3. Bitton, G. 1980. Adsorption of viruses to surfaces: technological and ecological implications, p. 439. In G. Bitton and K. C. Marshall (ed.), Adsorption of microorganisms to surfaces. John Wiley & Sons, Inc., New York. 4. Cliver, D. 0. 1975. Virus associated with wastewater solids. Environ. Lett. 10:215-223. 5. Dudley, D. J., M. N. Guentzel, M. J. Ibarra, B. E. Moore, and B. P. Sagik. 1980. Enumeration of potentially pathogenic bacteria from sewage sludge. Appl. Environ. Microbiol. 39:118-126. 6. Farrah, S. R., and G. Bitton. 1983. Bacterial survival and association with sludge flocs during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions. Appl. Environ. Microbiol. 45:174-181. 7. Gayford, C. G., and J. P. Richards. 1970. Isolation and enumeration of aerobic heterotrophic bacteria in activated sludge. J. Appl. Bacteriol. 33:342-350. 8. Jewell, W. J., R. M. Kabrick, and J. A. Spada. 1982. Autoheated aerobic thermophilic digestion with air aeration. Research report no. EPA-600/S2-82-023, Environmental Protection Agency, Cincinnati, Ohio. 9. Kabrick, R. M., W. J. Jewell, B. V. Salotto, and D. Berman. 1979. Inactivation of viruses, pathogenic bacteria, and parasites in the autoheated thermophilic digestion of sewage sludges. Proc. 34th Ind. Waste Conf. Purdue University, Lafayette, Ind. 10. Kuchenrither, R. D., and L. D. Benefield. 1983. Mortality patterns of indicator organisms during aerobic digestion. J. Water Pollut. Control Fed. 55:76-80. 11. Lewin, V. H., P. W. Jones, and D. L. Redhead. 1981. The fate of bacterial pathogens in sewage treatment processes. Water Pollut. Control. 80:42-50. 12. Lund, E. 1970. Observations of virus binding capacity of sludge, p. 1-24. In S. H. Jenkins (ed.), Fifth International Conference on Water Pollution Research. Pergamon Press, Inc., San Fran-

cisco, Calif. 13. McKinney, R. E., H. E. Langley, and H. D. Tomlinsom. 1958. Survival of Salmonella typhosa during anaerobic digestion. I. Experimental methods and high rate digester studies. Sewage Ind. Wastes 30:1467-1477. 14. Pedersen, D. C. 1981. Density levels of pathogenic organisms in municipal wastewater sludge: a literature review. Research report no. EPA-600/S2-81-170, Environmental Protection Agency, Cincinnati, Ohio. 15. Pramer, D., H. Heukelekian, and R. A. Ragotskie. 1950. Survival of tubercule bacilli in various sewage treatment processes. I. Development of a method for the quantitative recovery of mycobacteria from sewage. Public Health Rep. 65:851-859. 16. Steele, R. G. D., and J. H. Torrie. 1960. Principles and procedures of statistics, p. 107. McGraw-Hill Book Co., New York.