United States Environmental Protection Agency Water Engineering Research Laboratory Cincinnati OH 45268 Research and Development EPA/600/S2-86/047 June 1986 Project Summary Inactivation of Enteric Pathogens During Aerobic Digestion of Wastewater Sludge Samuel R. Farrah, Gabriel Bitton, and Stephen G. Zam A study was conducted to provide data on the ability of aerobic digestion to re- duce pathogens and to determine the ef- fect of important variables. Laboratory and field studies investigated the effects of aerobic and anaerobic digestion on enteric viruses, enteric bacteria, total aerobic bacteria, and intestinal parasites. Under laboratory conditions, the temperature of the sludge digestion was the major factor influencing survival of bacteria and vi- ruses. The survival of both bacteria and viruses was increased substantially by decreasing the temperature of sludge di- gestion from 28° to 7°C. Lowering the temperature or dissolved oxygen level reduced the percentage of solids-asso- ciated organisms for bacteria but not for viruses. Bacteria were inactivated at different rates during aerobic sludge digestion. Streptococcus faecalis was more stable than Salmonella typhimurium or Escheri- chia coll. Varying detention time or source of sludge did not affect the rate of inacti- vation of viruses or bacteria. Aeration of stock Ascaris suum ova in 0.1 N H2SO4 resulted in 91% embryon- ation. However, aeration in sludge resulted in only 19% to 50% embryonation after 30 days. Most of the ova embryonated in 0.1 N H2SO4 (79% to 93%) were infec- tive for rats. In contrast, only 9% to 12% of the ova embryonated in aerobically digesting sludge were infective for rats. Full-scale aerobic digestion of sludge re- duced densities of bacteria and entero- viruses. In most cases, the reductions were close to the maxima predicted for completely mixed digesters that contin- ually receive undigested sludge. The number of parasitic ova varied greatly with the community served by the treatment plant. Parasitic ova were re- covered from most of the samples of di- gested and undigested sludge from certain treatment plants but not from others. The relatively low numbers of ova recovered from the sludge samples make it difficult to evaluate the effects of sludge digestion on these agents. The inactivation rate of laboratory-grown bacteria in aerobically digested sludge was higher than that for indigenous bacteria. The indigenous bac- teria were probably better protected from predation by protozoans and other an- imals. Predation was the major factor in- fluencing survival of both indigenous and laboratory-grown bacteria during aerobic digestion of sludge under laboratory con- ditions. Predation had little effect on sur- vival of viruses, however. This Project Summary was developed by EPA's Water Engineering Research Laboratory, Cincinnati, OH, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report order- ing information at back). Introduction A large number of enteric bacteria, viral pathogens, and parasitic ova may be ex- creted by infected individuals and may therefore be present in untreated sewage. Since a large number of these pathogens become associated with wastewater so- ------- lids, many are not completely inactivated during sewage treatment processes and are merely transferred to wastewater slud- ges. These sludges are processed further, generally by aerobic or anaerobic diges- tion, to improve their dewaterability, reduce their unpleasant odor, and reduce their pathogen content. The ability of digestion processes to reduce sludge pathogens has received considerable investigation, though num- erous data gaps exist. The information is particularly sparse for aerobic digestion. This investigation was undertaken to pro- vide more definitive information on the ability of aerobic digestion to reduce pathogens and to determine the effects of important variables. The investigation studied the effects of aerobic digestion on pathogenic bacteria, bacterial indicator organisms, parasitic ova, and animal viruses. Experimental Procedures In laboratory experiments (which em- ployed temperature-controlled, laboratory, aerobic and anaerobic digesters), cultured bacteria and viruses were generally used. In field studies, conducted at existing plants in the vicinity of Gainesville, Florida, indigenous microbial species were used. Laboratory Experiments Aerobic digestion was conducted in temperature-controlled vessels holding 15 L of sludge. Sludge was mechanically agitated, and humidified air was intro- duced through spargers. Anaerobic diges- tion was conducted in externally heated carboys holding 8 L of sludge. Bacteria and viruses charged to the digesters were grown from type cultures. Ascaris suum ova were obtained from the feces of naturally infected hogs. Detailed proced- ures for growing organisms and measur- ing densities are presented in the report. Survival of bacteria and viruses and the association of these agents with sludge floes were studied in 11 individual trials. During each trial, two to four digesters were operated under different conditions of temperature, dissolved oxygen, and de- tention time. Aerobically digested sludge was obtained from one of three local treat- ment plants and aerated for 2 to 4 days at the desired temperature before the exper- iments were started, thus stabilizing the sludge at the desired conditions before an experimental trial was started. An anaero- bic sludge digester was started using anaerobically digested sludge obtained from Tallahassee. At the beginning of each trial, bacteria and viruses were added to each of the digesters to obtain an initial concentration of approximately 105/mL On each subsequent day, a portion of sludge was removed from each digester for analysis, and a portion of wasted sludge from the plant that was the source of the aerobically digested sludge was seeded with bacteria and viruses and add- ed to the digesters. The wasted sludge that was used for the daily additions to the digesters was obtained at the beginning of each trial and was kept at 4 °C without aeration. The volume of digested sludge removed and replaced with wasted sludge was determined by the detention time de- sired: 1/15 to 1/40 the volume for 15- or 40-day detention times, respectively. Laboratory studies were also conducted on the survival of seeded Ascaris suum in aerobically digested sludge. Three aeration procedures were used that produced dif- ferent degrees of mechanical abrasion on the ova. Aeration was provided by shak- ing 200 ml of sludge in 500-mL flasks on a reciprocal shaker, by mixing 200 ml of sludge in 1000-mL beakers using a mag- netic stirrer, or by aerating 200-mL samples in 500-mL flasks with air dif- fusers connected to a small air pump. All three methods provided approximately 5 mg/L of dissolved oxygen. The aerobic digestion was run under two conditions: batch operation and daily feeding to sim- ulate operation with 40-day residence. Samples of the sludge were examined for total recovered ova, ova embryonation, and ova infectivity in rats. Field Studies Sludge samples from nearby waste- water treatment plants were obtained over two periods: From January to December 1981, and from August to October 1983. The sludge samples were examined for the presence of enteric bacteria, enteric viruses, and parasitic ova. The dissolved oxygen, temperature, pH, and total solids of the sludge samples were also deter- mined. Unlike the laboratory experiments, no organisms were seeded into the slud- ges; only densities of indigenous micro- organisms were measured. Results and Discussion Bacteriological Investigations Laboratory Studies The influence of temperature and dis- solved oxygen level on inactivation of bacteria during aerobic and anaerobic sludge digestion was studied in 11 trials, each lasting approximately 9 days. The re- sults from one trial appear in Table 1, which presents the log daily change in bacterial densities. The log daily change is the difference between the logs of the bacterial densities of the sludge in the digester shortly after introduction of the feed sludge containing the inoculum and the sludge withdrawn from the digester 24 hr later (before addition of the next batch of feed). Table 1 shows the effects of temperature (28.3° and 6.2°C) and dissolved oxygen concentration on the bacteria in one of the trials. These results are typical of those obtained in other trials. Wasted, aerobically digested sludge from a nearby plant was added daily to the digesters to provide a detention time of 15 days. Results show much greater reduc- tions for S. typhimurium, S. faecalis, and E. coli than for P. aeruginosa and total aerobic bacteria. For all bacterial types, the log daily change was much lower at 6.2 °C than at 28.3°C (compare Conditions I and II in Table 1). The presence or absence of air (compare Conditions I and III in Table 1) produced a small effect that was usually not significant. The bacteria in the digesting sludge were either in the supernatant or were oc- cluded to solids. Operation at 28.3°C in the aerobic state showed a markedly dif- ferent distribution of bacteria from the other conditions, averaging about 8% in the supernatant compared with 36% for the other conditions. Subsequent experi- ments confirmed that grazing by protozoa accounted for the lower proportion of bac- teria in the supernatant at 28.3°C and contributed to the greater daily reduction in bacteria obtained at these conditions. Subsequent trials with sludges from three different wastewater treatment plants sludges showed that the source of sludge did not significantly affect the results. In all of the previously described ex- periments, bacteria were added to the di- gesters daily along with fresh sludge. The values for change in total bacteria there- fore reflect changes that occurred in the first 24 hr following bacterial addition. To determine whether the rate of daily change in bacteria would fall off with time, in- dicating the presence of a resistant frac- tion of added bacteria, digesters were operated under aerobic conditions without the addition of bacteria or sludge. Two digesters were operated at 28 °C and one at 6°C. All digesters were operated with greater than 4 mg/L dissolved oxygen. Re- sults with S. typhimurium, S. faecalis, and total aerobic count showed that curves of log density versus time were approximate ly linear (with negative slopes) over the ------- Table 1. Bacterial Survival and Association with Sludge Floes During Aerobic and Anaerobic Digestion of Sludge: The Influence of Sludge Digestion Conditions on Individual Bacteria Sludge Dissolved Total Daily Change Bacteria in Digestion Temperature Oxygen Solids in Total Supernatant Bacteria Condition* (°C) (mg/L) pH (g/L) Log10 (%) Salmonella typhimurium Streptococcus faecalis Escherichia coli Pseudomonas aeruginosa Total aerobic bacteria 1 II III 1 II III 1 II III 1 II III 1 II III 28.3 6.2 28.0 28.3 6.2 28.0 28.3 6.2 28.0 28.3 6.2 28.0 28.3 6.2 28.0 2.7 3.7 0 2.7 3.7 0 2.7 3.7 0 2.7 3.7 0 2.7 3.7 0 6.0 7.4 6.3 6.0 7.4 6.3 6.0 7.4 6.3 6.0 7.4 6.3 6.0 7.4 6.3 20.6 19.6 18.4 20.6 19.6 18.4 20.6 19.6 18.4 20.6 19.6 18.4 20.6 19.6 18.4 - 1.24Ai -0.238 -0.90A -0.92A -0.108 -0.83A - 1.13* -0.23° -0.618 -0.28* -0.29* -0.34A -u.34A -0.078 -0.098 8C 23B 51 A 12B 50* 47 A 4B 25* 30* 3s 34* 47* 7B 21* 33** * Condition I = aerobic; Condition II = aerobic; and Condition III - anaerobic. ^Figures in a single column with identical letter superscripts are not significantly different. course of several days, indicating no per- sistent fraction of bacteria. The rate of in- activation was substantially lower in these experiments than when inocula were add- ed daily (compare Tables 1 and 2). The greater reduction in supernatant bacteria at the higher temperature (Table 2) is con- sistent with the inoculum experiments. Field Studies Summarized data from field studies are presented in Table 3. Plants 1 and 2 used aerobic digesters, and Plant 3 used an an- aerobic digester. For the aerobic digesters, greater bacterial reductions were achieved in Trial 2 than in Trial 1. The probable ex- planation for this difference was digestion temperature: Trial 1 was conducted during colder months. All of the digesters were completely mixed. With complete mixing, some sludge inevitably short-circuits to the exit with relatively short-term treat- ment. Bacterial reduction was calculated assuming complete mixing and destruc- tion of bacteria except for bacteria in that portion of the feed that leaks through to the exit. Log reductions calculated on this basis (labeled "maximum possible") are presented in Table 3 where they can be compared with experimentally determined reductions. For Trial 2, experimental and calculated values are similar; this result supports the assumptions of the calcula- tion and indicates that use of complete mix reactors may contribute to failure to achieve high bacteria or virus reductions in digesters. Virus Investigations Laboratory Studies Laboratory studies determined the influ- ence of several variables on the survival of viruses during aerobic and anaerobic digestion of sludge under laboratory con- ditions. The variables studied included temperature, dissolved oxygen level, de- tention time, virus type, and the source of the sludge used for digestion studies. The sludge source did not significantly affect the rate of inactivation of poliovirus when sludge was aerobically digested (15-day detention time). At 28 °C and dis- solved oxygen levels of 5 mg/L, the mean daily change (Iog10) in inactivation of seeded poliovirus (Type I, Lsc) for sludges from three sources ranged from -0.71 to -0.97, which was not a significant difference. Table 2 Bacterial Survival and Association with Sludge Floes During Aerobic Digestion of Sludge without Addition of Fresh Sludge Daily Change in Total Bacteria in Supernatant Bacteria Log10 (%) Bacteria 6°C 28°C Salmonella typhimurium -0.15 Streptococcus faecalis —0.05 Total aerobic bacteria -0.07 -0.48 -0.23 -0.19 * Figures with the same letter superscript are not significantly different. 6°C S38* 80A 41C 28°C 3° 2D 3° The temperature of sludge digestion in- fluenced the rate of inactivation of po- liovirus (Table 4), which was highest at 28 °C, intermediate at 17.6°C, and lowest at 5.5 °C. Varying the dissolved oxygen between 1 and 6 mg/L at a relatively constant temperature of 28 °C did not change the inactivation rate. The mean daily change (Iog10) ranged from -0.77 to -1.03. Anaer- obic digestion of the same sludge at 32 °C produced a mean daily change (Iog10) of -0.33, significantly lower than for aerobic digestion. Varying the detention time of aerobic digestion between 16 and 40 days did not change the inactivation rate (log daily change) of poliovirus. Poliovirus 1, echovirus 1, coxsackievirus B3, and the simian rotavirus SA-11 were all inactivated at similar rates during aero- bic digestion of sludge at 28 °C. The range of log daily change was -0.46 to -0.77. Aerobically digested liquid sludge when allowed to dry in a centrifuge tube from about 1 to 50 g/L over 28 days showed essentially no surviving viruses (polio- virus), whereas 5% survived in a tightly capped control. Field Studies Viruses were detected in samples of mixed liquor solids and aerobically di- gested sludge. The lowest levels were found in sludge from the second aerobic digester at the plants studied. However, the numbers of viruses were so variable that statistical analyses did not show that the differences were significant except in a few cases. Results ranged from 0 to 20 ------- Table 3. Treatment Plant* Summary of Field Data on Bacterial Reduction During Aerobic and Anaerobic Digestion of Sludge Bacteria Bacterial Reduction^ (Log10) Trial 1 Trial 2 Plant 1 (aerobic digester) Plant 2 (aerobic digester) Plant 3 (anaerobic digester) Total coliforms Fecal coliforms Fecal streptococci Aerobic bacteria Maximum possible1 Total coliforms Fecal coliforms Fecal streptococci Aerobic bacteria Maximum possible' Total coliforms Fecal streptococci Maximum possible' Plant 1: Influent sludge and second of two 8.5-day residence time aerobic digesters in series Plant 2: Sludges in first and second of two aerobic digesters, each with 50 days of residence time Plant 3: Influent sludge and sludge from an anaerobic digester with 10 days of residence time. Log bacterial density in digested sludge - log density in undigested sludge. January through December 1981. August through October 1983. - 7.53 - 7.52 - 7.03 -0.35 - 1.86 - 1.02 - 1.09 -0.77 -0.80 - 1.70 — — - 1.99 — - 1.63 — - 1.86 - 1.92 — - 7.77 _ - 7.70 -0.91 - 1.10 - 1.00 Calculated assuming total destruction of bacteria except for leakage caused by complete mixing: 1:72.2 for Plant 1, 1:50 for Plant 2, and 1:10 for Plant 3. Table 4. Effect of Temperature on Poliovirus (Type 1, Lsc) Survival in Laboratory-Scale Aerobic Digesters Temperature 28 17.6 5.5 Dissolved Oxygen (mg/L) 5.8 5.2 5.8 Total Solids (g/U 10.4 7.9 7.9 Volatile Solids (g/U 4.8 5.7 5.2 pH 5.3 6 5.3 Mean Daily Change in Virus Survival* (Log10) - 0. 77A -0.5B -0.21C Means within the same column with the same letter are not significantly different at the p = 0.05 level. plaque-forming units (PFU/g) of sludge solids. Poliovirus and coxsackievirus serotypes were isolated from the different sludge samples. Compared with wasted sludge before digestion, the aerobically digested sludge contained relatively few types of viruses. Parasite Investigations Laboratory Studies Three methods of mixing (magnetic stir- ring, shaking, and aeration with air pumps) were used to test the effects of aerobic sludge digestion on the embryonation of Ascaris ova. When sludge samples were mixed mechanically by magnetic stirrers, 85% to 90% of the ova were physically destroyed. Those ova that were recovered showed gross abnormalities such as vac- uolation, cracked egg coats, and granulation. Shaking the sludge sample on recipro- cating shakers during aerobic digestion resulted in 32% embryonation after 40 days of shaking and 19% embryonation after 35 days. In only one experiment were 52% of the recovered ova embryonated. No ova were embryonated in one experi- ment that shook the sludge sample for 49 days. Most of the nonembryonated ova in this trial were vacuolated, showed in- creased granulation, and were physically distorted. The percentages of unfertilized ova present in shaken sludge samples de- creased within 10 to 14 days. After 10 days of shaking in one trial, 40% to 60% of the initially seeded ova were arrested in the 2-, 4-, and 8-cell stages. This result indicated that embryonation had been initi- ated but stopped early in the cleavage stage of development. Aeration of aerobic sludge using an air pump produced the highest percentages of ova embryonation. After 30 to 35 days of aeration, 62% to 68% of the ova were embryonated in sludges. The percentages of nonembryonated ova consistently de- creasesd over a 30- to 42-day period of aeration. Increasing the detention time of digestion up to 42 days produced little change in the percentage of the embry- onation of the Ascaris ova. Embryonated ova recovered from aerated sludge di- gesters showed no distortion, vacuolation, or granulation. The infectivity of ova recovered from sludge aerated either by shaking or with air pumps was examined in two trials. In each trial, rats were dosed with ova from sludge, embryonated ova from hog feces (positive control), and physiological saline containing no ova (negative control). In the first trial, an average of 9% (11% and 8%) of the ova recovered from shaken sludge were infective to rats, as demonstrated by the recovery of third-stage larvae from rat lungs. An average of 93% (95% and 91%) of the embryonated positive controls and no larvae from the negative controls were recovered. In the second trial, an average of 12% (10% and 14%) of the ova recover- ed from sludge aerated by air pumps were infective. Postivie controls showed an average infectivity of 79% (83% and 75%), and no larvae were demonstrated in the lungs of negatively controlled rats. Recovered larvae in all experiments ap- peared to be normal and active. Ascaris ova are highly resistant to var- ious acids, alkalies, and corrosive chem- icals. The inner, lipoid, vitelline membrane is primarily responsible for this effect. Ova surrounded only by these membranes are resistant to these chemicals. The primary lipid component of the vitelline membrane is a waxy alcohol known as ascaryl alco- hol. Ascaryl alcohol appears to be a mix- ture of three closely related compounds — ascosides A, B, and C. The lipid nature of this membrane makes it susceptible to organic solvents, surface active agents, and noxious gases. Possibly, the nonem- bryonation and physical deformities of ova may be caused by various chemicals or gases that are generated during aerobic ------- digestion and that destroy or alter the physiological integrity of this vitelline membrane. Once the membrane is altered, the primary protective barrier is breached, and the developing embryo is unprotected. Alterations occur at this point. Field Studies A number of human parasites were de- monstrated in locally collected, undigested sludge and sludge obtained from aerobic and anaerobic digesters. Four human para- sites were recovered from sludge: Ascaris lumbricoides, Trichuris trichiura, Enter- obius vermicularis, and hookworm. These are the most common of the intestinal hel- minth parasites of man. The most com- mon parasite present in sludges from the four collection sites was the common pin worm or seat worm, E. vermicularis. The second most common parasite was the large intestinal roundworm, A. lumbri- coides. Hookworm and the whip worm, T. trichiura, were also present, but to a small extent. Sludge samples from treatment plants in Tallahassee, Florida, showed the highest incidence of indigenous parasite ova, followed (in order) by the Main Street, Kanapagha, and University of Florida treat- ment plants in Gainesville, Florida. Parasite ova were recovered from all sludges, re- gardless of their treatment. An unexpected observation during ex- amination of the sludge samples was the high incidence of the intestinal helminth E. vermicularis in Tallahassee, Florida. This parasite has a wide geographic range in the United States. Cheng reported an in- fection rate of 32.9% in the American population. Enterobius is the most com- mon helminth infection in the United States, sometimes reaching infection rates of 60% in children and adults in various institutions in Florida. An infection rate of 26.8% was reported among students of five elementary schools in and around Tallahassee, Florida, These various studies suggest that Tallahassee, Florida, is an endemic loci of enterobiasis in the state of Florida. Ascaris and Trichuris are the most com- mon and cosmopolitan helminth infections in the world. The prevalence of these in- fections in the population of North Amer- ica has been estimated at 4 million for Ascaris and 2.2 million for Trichuris. These two infections are still extremely common in the rural areas of the southern United States. With the prevalence of these infec- tions in the population and with the fecun- dity of these parasites (Ascaris produces 200,000 ova/day per female, and Trichuris produces 1,000 to 46,000 ova/day per female), it is not surprising that these parasitic ova were found in locally col- lected sewage sludge. Conclusions Indicator and pathogenic bacteria were inactivated during aerobic digestion of sludge under laboratory conditions. Proto- zoans and other predators were the pri- mary agents responsible for inactivating bacteria. Bacterial densities in undigested sludge are reduced during aerobic treat- ment of sludge under field conditions. The addition of fresh, undigested sludge to aerobic digesters results in contamina- tion of the digested sludge with undi- gested sludge and thus diminishes the ef- fectiveness of the process for reducing bacterial densities. Enteric viruses are also inactivated dur- ing the aerobic digestion of sludge under laboratory conditions. In contrast with bacteria, the presence or absence of pre- dators had little influence on the inactiva- tion of viruses. Aerobic treatment of sludge under field conditions reduces the density of enteric viruses. As with bac- teria, contamination of digested sludge with fresh, undigested sludge leads to the presence of viruses in the digested sludge. Aeration of Ascaris suum ova in the presence of 0.1 N H2SO4 contamination with fungi led to embryonation of more than 90% of the ova. Approximately 80% of these ova were infective for rats. In con- trast, aeration in aerobically digested sludge resulted in embryonation of 50% or fewer of the ova. Only some 10% of these ova were infective for rats. Thus aerobic treatment of sludge reduces the ability of ova in the sludge to embryonate and reduces the infectivity of those ova that are embryonated. The relatively low numbers of parasitic ova in undigested and aerobically digested sludge from treatment plants made it difficult to determine the effects of aerobic sludge treatment on these ova under field conditions. Recommendations The presence of bacterial pathogens, enteric viruses, and parasitic ova in aero- bically digested sludge suggests two ma- jor areas of future research: (1) the fate of these microbes during subsequent treat- ment or after disposal (including during sludge drying and application to land), and (2) modif^ition of existing sludge treat- ment processes to reduce pathogens fur- ther. One modification of sludge treatment that should be considered is the aeration of sludge in batches without addition of fresh, undigested sludge. All sludge re- moved from digesters operated in this manner would be treated for the same length of time and would not be contam- inated with the bacterial pathogens, enter- ic viruses, and parasitic ova that are often found in undigested sludge. The possibility of raising the temperature of sludge diges- tion should also be considered. Additional studies are needed on the fate of parasitic ova during aerobic sludge di- gestion. Additional laboratory studies are required to confirm our findings on the ef- fects of aerobic sludge digestion on the embryonation and infectivity of Ascaris suum ova. Larger volumes of sludge from treatment plants should be processed to provide the large numbers of ova needed for embryonation and infectivity studies. The full report was submitted in fulfill- ment of Grant No. R806290 by the Univer- sity of Florida under the sponsorship of the U.S. Environmental Protection Agency. .S. GOVERNMENT PRINTING OFFICE: 1986/646-116/20852 ------- ------- ------- Samuel R. Far rah, Gabriel Bitton, and Stephen G Zam are with the University of Florida, Gainesville, FL 32611. B. V. Salotto was the EPA Project Officer (see below for present contact). The complete report, entitled "Inactivation of Enteric Pathogens During Aerobic Digestion of Waste water Sludge, "(Order No, PB 86-183 084/A S; Cost: $11.95, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 For further information, contact Joseph B. Farrell at: Water Engineering Research Laboratory U.S. Environmental Protection Agency Cincinnati, OH 45268 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 BULK RATE POSTAGE & FEES PAID EPA PERMIT No. G-35 Official Business Penalty for Private Use $300 EPA/600/S2-86/047 ------- |