Reductions of enteric microorganisms during aerobic sludge digestion

Wat. Res. Vol. 24, No. II, pp. 1377-1385, 1990 Printed in Great Britain.All rights t~erved

OM3-1354/90$3.00+ 0.00 Copyright ~ 1990PergamonPress plc

REDUCTIONS OF ENTERIC MICROORGANISMS DURING AEROBIC SLUDGE DIGESTION JOHN H. M~d~nN JR t'*O, HAR~Y E. BOSTI^S2 and GERALDSTERN2"~" tCenter for Environmental Research, Cot'nell University, Ithaca, NY 14853, U.S.A. and 2Risk Reduction Engineering Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, U.S.A.

(First received January 1990; accepted in revisedform May 1990) Abstract--Seasonal variations in the reductions of total coliform, fecal coliform, fecal streptococci and enterovirus densities that occur during conventional aerobic sludge digestion in cold climates were characterized. Also, the potential to improve reductions in the densities of these four groups of enteric microorganisms in cold climates by simple modifications that increase process temperature by reducing beat losseswas demonstrated. To obtain this data, two 32 m~aerobic digesters located at a small municipal wastewater treatment plant were operated continuously over a period of 20 months. One digester was a conventional digester while the other was designed to minimize heat losses and thus, facilitate autoheating. When the results obtained during 11 separate periods of steady-state operation at mean mixed liquor temperatures ranging from 8 to 40°C and at residence times of 10, 15 and 20 days were combined for analysis, it was evident that significant reductions in the densities of the four groups of enteric microorganisms were dependent on both residence time and temperature. Using the Arrhenius equation, it was possible to describe mathematically the temperature dependence of the rate of logto reduction in density of each of these four groups of enteric microorganisms. The four mathematical relationships developed provide a rational basis to determine residence times necessary to achieve desired levels of indicator organism and enterovirus reductions during aerobic sludge digestion at mixed liquor temperatures ranging from 8 to 40°C.

Key words--sewage sludge, aerobic digestion, total coliforms, fecal coliforms, fecal streptococci, enteroviruses, temperature

INTRODUCTION With the exception of anaerobic digestion, aerobic digestion is probably the most widely used process for the stabilization of wastewater treatment sludges in the U.S. Both ease of operation and relatively low capital costs have made aerobic digestion particularly attractive for small municipal wastewater treatment plants such as those common in rural areas. A number of problems, however, are also associated with this sludge stabilization process. One of these problems, variation of process performance, is due to the influence of climate on process temperatures. Due to relatively long residence times, usually a minimum of 10--15 days, and the use of open tanks, mixed liquor temperatures can vary by as much as 25°C between summer and winter operation in northern climates. As with all biological waste treatment processes, the performance of the aerobic sludge digestion process is temperature dependent. As temperature decreases, the rate of microbial activity and thus the rate of oxidation of biodegradable organics, which translates into the rate of stabilization, is reduced. *Author to whom all correspondence should be addressed. ?Present address: P.O. Box 385, Dayton, NV 89403, U.S.A.

For example, an empirical relationship that was developed by the Municipal environmental Research Laboratory (1979) suggests that it is necessary to increase the solids residence time (SRT) from 22.5

days at 25°C to 45 days at 10°C to realize a 4 0 % reduction in the concentration of volatile solids. At 5°C, a 90-day S R T appears to be necessary. Available evidence suggests that temperature not only affects the rate of sludge stabilization but also the rates of inactivation of pathogens and indicator organisms. For example, Kuchenrither and Benefield 0983) reported that with aerobic digestion, the densities of both fecal coliform and fecal streptococci groups of indicator organisms decreased more rapidly with time as temperature increased. At a residence time of 15 days, an increase in temperature from 20 to 30°C increased the reduction, Iogto basis, of fecal coliforms from 0.91 to 1.56.At 40°C, no fecal coliforms were detectable. For fecal streptococci, a temperature increase from 20 to 30°C increased Io&0 reductions from 0.64 to 1.24. At 40°C, the observed fecal streptococci Iogj0 reduction was 1.71. Similar temperature effects for aerobic digestion also have been reported by Farrah et al. (1986). At a residence time of 15 days, an increase in temperature from 6 to 28°C increased Iogmo reductions in the density of Escherichia coli from 0.26 to 1.26. For Streptococcus

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JOHN H. MPa~TZNJR et al.

faecalis, log~o reductions increased from 0.05 to 0.82, and an increase from 0.21 to 1.29 was observed for the pathogen, Salmonella typhimurium. When the disposal o f sewage sludge via land application is practiced, pathogen reduction is an important prerequisite due to the potential for direct or indirect public contact. With the exception of the previously cited studies, little information as noted by Pedersen (1981) has been available, however, concerning the effectiveness of aerobic sludge digestion in reducing the densities o f either indicator organisms or pathogens, particularly at psychrophilic and mesophilic temperatures. Thus, an investigation was proposed to: (I) characterize the seasonal variations in sludge stabilization and reductions in the densities of indicator organisms, Salmonella spp and enteroviruses that occur with conventional aerobic digestion in cold climates, and (2) demonstrate the improvement in both sludge stabilization and reduction in densities o f the indicator organisms, Salmonella spp and enteroviruses that can be achieved by simple modifications o f the aerobic digestion process that increase process temperatures by reducing heat losses. The results of this study related to reductions in the densities of enteric indicator organisms are presented and discussed in this paper. The complete project report (Martin, 1988) is available from the National Technical Information Service, Springfield, VA 22161, U.S.A. (Order No. PB 89-138 846/AS). MATERIALS AND METHODS

This research was conducted at the Trumansburg, N.Y., wastewater treatment plant. The climate of the area, humid continental, made the Trumansburg wastewater treatment plant an appropriate site for the study. Average monthly air temperatures range from a low of -5.6~C in January and February to a high of 20°C in July. During the period from mid-December through mid-March, daily minimum temperatures below - 12°C are not uncommon, and temperatures as low as - 2 3 to -29°C can occur. Wastewater treatment plant details The Trumansburg wastewater treatment plant which was designed for an average flow of 946m~/day employs the conventional activated sludge process without primary clarification to provide secondary treatment for the Village's wastewater. Waste activated sludge is thickened without chemical conditioning using a gravity thickener and then is stabilized using conventional aerobic digestion. Following stabilization, Trumansburg sludge is iagnoned and ultimately disposed of by spreading on agricultural land. lnvestigatit'e facilities To provide facilities necessary to satisfy the objectives of the investigation, two above-ground 32 m 3 aerobic digesters were added to the Trumansburg wastewater treatment plant. One digester, a closed-tank with a man-hole located in the cover to permit access for aerator installation and *Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

removal, was designed to minimize heat losses and facilitate autoheating. The second digester was an open tank. Both digesters were insulated with a 7.6 cm coating of 32 kg/m 3 density urethane foam. After 17 months of operation, an insulated cover was added to the conventional digester. In the closed-tank autoheated digester, a Framco* submersible, self-aspirating aeration unit was used. This aeration unit has a tap water oxygen transfer efficiency of ca 22% as compared to 4-8% for coarse bubble diffusers commonly used for conventional aerobic sludge digestion. Effluent gas heat losses thus were minimized. Originally, Chicago Pump Discfuser* coarse bubble diffusers were installed in the open-tank conventional digester. After 17 months of operation, and in conjunction with the addition of an insulated cover to this digester, the coarse bubble diffusers were replaced with Wyss Flex-A-Tube* fine bubble diffusers in an attempt to increase oxygen transfer efficiency and reduce diffuser fouling problems. Data collection To develop the data base necessary to satisfy the objectives of this investigation, the two previously described 32.2 m 3 aerobic digesters were operated continuously from 29 July 1985 through 30 March 1987. During this period, the autoheated digester was operated at residence times of 10, 15 and 20 days while the residence time of the conventional digester was held constant at 20 days. Operation of the autoheated digester at a residence time of 5 days also was attempted but was terminated before steady-state conditions were established. The oxygen transfer capacity of the Framco aerator was found to be inadequate for the exerted oxygen demand at this residence time. Digested sludge was withdrawn and raw sludge was added daily. The draw and fill mode of operation was selected to eliminate the possibility of effluent characteristics being influenced by short-circuiting of raw sludge additions. Thus, a minimum of 24 h of treatment always was insured. Typically, aerobic as well as anaerobic sludge digesters are operated using a fill and draw mode of operation which produces an effluent containing some raw sludge. This reduces the magnitude of enteric microorganism reductions. During periods of steady-state operation, raw sludge samples were collected on Mondays and Wednesdays, and digested sludge samples were collected on Tuesdays and Thursdays for analysis to determine the densities of the total coliform, fecal coliform and fecal streptococci groups of indicator organisms and the enterovirus group of viruses. These samples also were analyzed to determine densities of Salmonella spp during the first three periods of steady-state data collection, through 13 May 1986. At this time, Salmonella enumerations were discontinued since the group of pathogens were only infrequently detected in the raw sludge. Thus, no meaningful data were being obtained. During periods of steady-state data collections, a minimum of seven sets of raw and digested sludge samples were collected and analyzed for the microbiological parameters noted above. The raw sludge samples were collected during the daily transfer of raw sludge from a mechanically mixed feed tank to the two digesters. The digested sludge samples were mixed liquor samples taken prior to effluent withdrawal and subsequent raw sludge addition. Raw sludge and mixed liquor temperatures in both digesters were routinely measured. Raw sludge temperatures were measured and recorded daily during raw sludge additions using a digital thermometer, while a continuous record of mixed liquor temperatures was provided by a dual recorded thermometer. In addition, a continuous record of ambient temperatures at the field site was obtained using a recording thermograph located in a standard weather instrument shelter. Mean daily mixed liquor and ambient temperatures were calculated by taking the average of the minimum and maximum temperatures recorded in a 24 h period beginning at 0800 h.

Aerobic digestion of enteric microorganisms Table I. Periods of steady-state operation used to characterize autoheated and conventional digester performance

digester foaming making the collection of representative samples impossible. The factor or factors responsible for this atypical period of excessive autoheated digester foaming remain unclear. Thus, there was a total of I I rather than 12 periods of steadystate operation as plCnned. In the following, the results obtained during the steady-stateoperation of the two digesters are summarized and discussed.

time (daD) Dates 6 Nov-5 Dec 1985 6 Jan-30 Jan 1986 9 Apt-13 May 1986 28 July-3 Sept 1986 15 Sept-9 Oct 1986 2 Feb-26 Mar 1987

Autoheated

Conventional

20 10 15 -10 15

20 20 20 20 20 20

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Temperature

Analytical procedures The densities of the total coliform, fecal coliform and fecal streptococci groups of indicator organisms in both raw and digested sludge samples were determined employing the

methods used by Farrah eta/. (1986). Sample preparation, dispersion of sludge flocs to release bacteria, was as described by Gaylord and Richards (1970). The modification by Venosa et aL (1979) of the Kenner and Clark procedure (Kenner and Clark, 1974) was used for the detection and enumeration of Salmonella spp. For primary enrichment, the following procedure was followed: (1) three tubes with 10rul of double-strength nutrient broth, dulcitol seleniteenrichment (DSE) broth, were inoculated with 10ml of sludge; (2) three tubes containing 10 ml of single-strength broth were inoculated with I ml of sludge; and (3) three tubes with 10ml of single-strength broth received 0.1 ml of sludge. These enumerations were performed by the New York State Diagnostic Laboratory, Cornell University College of Veterinary Medicine. Enterovirus enumerations were performed by the Environmental Monitoring and Support Laboratory (EMSL), USEPA. The method used for recovering viruses from the sludges and the BGM cell technique used was as described by Berg et al. (1984). Influent sludge samples also were analyzed in accordance with Standard Methods (APHA, 1985) to determine total solids concentrations. RESULTS AND DSICUSSION

Mixed liquor temperatures and reductions in the densities of the three groups of indicator organisms and enteroviruses were characterized during five separate periods of steady-state operation (Table 1). Also noted in Table 1 are the dates of concurrent data collection to characterize the performance of the conventional digester using the same parameters that were used for the autoheated digester. The additional period of data collection for the conventional digester, 28 July through 3 September, 1986, without concurrent data collection for the autoheated digester was due to an atypical period of excessive autoheated

With respect to mixed fiquor temperatures, both digesters performed as anticipated. In the conventional digester, daily mean mixed liquor temperatures ranged from 5°C with some surface ice formation during extended periods of cold weather to 28°C during summer months. Monthly means of mixed liquor temperatures ranged from 8 to 26°C and varied seasonally and linearly with ambient air and influent sludge temperatures. Mixed liquor temperatures for each of the five periods of autoheated digester and each of the six periods of conventional digester steady-state operation (Table 1) are summarized and compared with each other and with ambient air and influent sludge temperatures in Table 2. As shown in this table, the design of the autoheated digester provided substantially higher mixed liquor temperatures as compared to both ambient air and influent sludge temperatures and also to mixed liquor temperatures in the open conventional digester. Sludge characteristics

During the 18-month period beginning in November 1985, the densities of the total coliform, fecal coliform and fecal streptococci groups of indicator organisms and the enterovirus group of viruses in the Truraansburg wastewater treatment plant raw thickened sludge were determined during six separate sampling periods (Table 1). Duration of these sampling periods ranged from 24 to 50 days with between seven and nine samples per sampling period. Mean thickened sludge total solids concentrations ranged over the six separate sampling periods from 10.5 to 15.4 g per I. The results of these enumerations, expressed on a colony-forming or plaque-forming unit as appropriate per gram of total solids, are summarized in Table 3. As shown in Table 3, substantial variations in the densities of each of the three groups of indicator

Table 2. Summary of ambient air, influent sludge, and autoheated and conventional digester mixed liquor temperatures during periods of steady-state operation Dates 6 Nov-5 Dec 85 6 Jan-30 Jan 86 9 Apr-13 May 86 28 July-3 Sept 86 15 Sept-9 Oct 86 2 Feb-26 Mar 87

Ambient air (°C)

lnfluent sludge (°C)

3.9*+4.6 -4.3 4- 7.0 9.3+5.8 18.9 + 3.1 13.94-4.4 -3.8 4- 7.2

12.8+ 1.4 6.8 + 0.7 12.2+ 1.4 18.5 + 1.7 17.4± 1.0 7.7 _+ 1.0

*Mean + SD. tConventional digester with insulated cover.

R e s i d e n c e Autoheated time (days) digester (=C) 20 10 15 -10 15

38.2__.0.9 31.0 4- 1.2 39.8 : 2.4 -37.5__. 1.3 29.0 ~- 1.5

Residence time (days) 20 20 20 20 20 20

Conventional digester (°C) 14.6+2.1 8.0 4- 1.6 17.54-2.3 25.6 4- 1.8 21.7+2.0 23.7t + I.I

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JOHN H . M A g T I N

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Table 3. Statistical comparisons of geometric means of Trumansburg, N.Y., raw thickened sludge indicator organism and enterovirm densities* 11/6-12/4/85

I/6-1/29/86

4/9-5/12/86

7/28,-9/2/86

9/15-10/8/86

2/2-3/23/87

6.65'I" 5.60* 4.70" 3. I I*~

6.04*~ 5,02 b 4.32 b 3.01 *c

6.14" 4.58 b 4.07 ~ 2.17 b

5.49k 3.81 e 3.72~ 3.28 ~

5.28 c 4.61 b 3.91 ~ 2.94'

6.52* 4.65b 4.34*b 2.27 ~

Total coliforms Fecal coliforms Fecal streptococci Entesoviruses

*Colony-forming or plaque,forming units per gram of total solids, log,0 basis, as appropriate. ?Geometric means in the same row with a common superscript are not significantly different (P < 0.05).

organisms and also the enterovirus group of viruses occurred within each sampling period. Results of one-way analyses of variance (Snedecor and Cochran, 1980) indicate that statisticallysignificant differences (P <0.01) also occurred among the geometric mean densitiesof all four groups of microorganisms between sampling periods. The resultsof comparisons of each mean value with every other mean value within each group of microorganisms using Fisher's protected least significantdifference test (Steel and Torrie, 1980) are summarized in Table 3. It appears that these differences among sampling periods were random, since no seasonal pattern or statistically significant correlation (P <0.05) with influent sludge temperature are apparent. The data base of the densitiesof indicator organisms and enteroviruses in the raw thickened sludge from the Trumansburg wastewater treatment plant (Table 3) provided the opportunity to examine the nature of the relationshipsamong the four groups of microorganisms in municipal wasewater treatment sludges. Of specific interest was the question of dependence. Because the geometric means of the densities of each group of microorganisms for the six sampling periods, n = 6, were found to be normally distributed ( P < 0 . 0 1 ) using the KolmogorovSmirnov Test (Rohatgi, 1976), it was possible to use correlation analysis (Devote, 1982) to test for linear dependence among these four groups of microorganisms using the sample correlation coefficient, R, as a point estimate for the population correlation coefficient, p. Where the sample correlation coefficients were not equal to zero suggesting linear independence, the Student's t distribution (Devore, 1982) with n-2 degrees of freedom was used to test the null hypothesis p = 0. The results of the correlation analyses summarized in Table 4 show a strong and highly significant Table 4. Results of correlation analyses to test for linear dependence among the three groups of indicator organisms and enteroviruses in Trumansburg thickened raw sludge Correlation

Correlation coefficient, R

Total coliforms and fecal coliforms Total coliforms and fecal streptococci

0.668"

Total Fecal Fecal Fecal

0.396* 0.925~ 0.003* 0.103"

coliforms and enteroviruses coliforms and fecal streptococci coliforms and enteroviruses streptococci and enteroviruses

*Not significantly different from p = 0 (P > 0.01). tSignificantly different from p = 0 (P < 0.05). ++Significantly different from p = 0 (P < 0.01).

0.880t

(P < 0.01) linear correlation between the densities of fecal coliforms and fecal streptococci. The null hypothesis that p = 0 also was rejected at the P < 0.05 level of significance for the relationship between total coliforms and fecal streptococci. The correlation coefficients for the relationships between total coliforms and fecal coliforms and between the enterovirus group of viruses and each of the three groups of indicator organisms were not significantly different from zero (P < 0.05), however, suggesting statistical indel~ndence. The absence of strong linear correlations (P > 0.01) between the densities of total coliforms and fecal coliforms and between total coliforms and fecal streptococci in the raw sludge was unexpected. It was assumed that the ratio of the densities of fecal coliforms to total coliforms would be relatively constant. As noted in Standard Methods (APHA, 1989), the total coliform group of bacteria includes organisms that are present in the gut and feces of warm-blooded animals as well as coliform organisms from other sources. One example of a non-fecal coliform is Enterobacter aerogenes (Brock, 1979). Given the absence of the expected strong correlations, it appears that there was substantial variation in the ratio of fecal to non-fecal coliforms over time. The strong correlation (P < 0.01) between the densities of fecal coliforms and fecal streptococci seems to support this hypothesis. In contrast, the absence of even moderately strong correlations (P < 0.05) between enterovirus densities and the densities of each of the three groups of indicator organisms was not unexpected. It would be illogical to expect such correlations because enteroviruses, as well as other pathogens, are not normal inhabitants of the human gastro-intestinal tract, but rather present only in infected individuals. The absence of even moderately strong correlations between enterovirus densities and the densities of each of the three groups of indicator organisms in raw sludges also has been reported by Berg and Berman (1980). Conversely, the strong correlation between raw sludge densities of fecal coliforms and fecal streptococci was not surprising since both of these groups of indicator organisms include only organisms of enteric origin. Microbial reductions

The observed reductions in the densities of the three groups of indicator organisms and the enterovirus group of viruses for both the conventional and

Aerobic digestion of enteric microorganisms

1381

Table 5. Summaryof observed reductionsin the densitiesof indicatororganismsand enterovirusesduringautoheated and conventionalaerobicdigestion

Logt0 reduction Residence Mixedliquor time temperature Total Fecal Digester (days) (°C*) coliformst coliformst 10 31.1 ± !.2 0.84 1.04 Autohcated 10 37.5 + !.3 0.90 1.10 15 29.0 ± i.5 1.44 1.32 15 39.8 + 2.4 2.20 1.58 20 38.2 + 0.9 2.55 2.42 20 8.0 + 1.6 0.68 0.64 Conventional 20 14.6± 2.1 1.27 1.01 20 17.5+ 2.3 1.70 !.38 20 21.7 ± 2.0 0.69 !.i8 20 23.7 + !.1 2.11 1.74 20 25.6 + 1.8 1.43 0.56 *Mean+ S D . ?Colony-formingunits per 100ml basis. :~Plaque-formingunits per I00 ml basis. §No plaque-formingunits recovered from digestedsludge samples. autoheated digesters during the eleven periods of steady-state operation are summarized in Table 5. In this table, reductions are expressed on a volumetric, per 100 ml, basis and not on a per gram of total solids basis as in Table 3 to eliminate the influence of total solids reductions as an added variable. Table 5 shows that a number of general patterns are readily discernible when the observed mean reductions in the three groups of indicator organisms and the enterovirus group of viruses are summarized. One pattern is the difference in resistance to destruction among these four groups of microorganisms. Generally, total coliforms were the most easily destroyed while fecal streptococci were the most resistant. Reduction of enteroviruses was comparable to total coliforms in some situations but comparable to fecal streptococci in other instances. Similar observations in studies involving both aerobic (Farrah et al., 1986; Appleton et al., 1986) and anaerobic (Berg and Berman, 1980) sludge digestion have been reported. These observations led to the examination of the nature of the relationships among these four groups of microorganisms with respect to reductions during aerobic digestion of wastewater treatment sludges. Again the question of statistical dependence was of specific interest. As the observed geometric mean reductions for each group of microorganisms were found to be normally distributed (P < 0.01) using the

Table 6. Resultsof correlationanalysesto test for linearindependenceamonglogmreductionsof total coliforms,fecalcoliforms,fecal streptococci and enterovirusesduring aerobicdigestion Correlation Correlation coe/~cient, R Total coliforrasand fecal coliforms 0.793* Total coliformsand fecal streptococci 0.838* Total coliformsand enteroviruses 0.569? Fecal coliformsand fecal streptococci 0.868* Fecal coliformsand enteroviruses 0.650~ Fecal streptococciand cnteroviruses 0.512§ • Significantlydifferentfrom p =0 (P <0.01). tNot significantlydifferentfrom p = 0 (P > 0.05). ,~:Significantlydifferentfrom p 1 0 (P < 0.05). §Not significantlydifferentfrom 0 = 0 (P > 0.10).

Fecal streptococci? 0.60 0.82 0.80 1.23 1.60 0.33 1.07 1.17 1.00 1.42 0.72

Enteroviruses~ 1.08 2.43 1.03 > 2.33~ > 3.16~ 0.72 0.95 0.98 0.85 1.06 1.28

Kolmogorov-Smirnov test (Rohatgi, 1976), again it was possible to use correlation analyses (Devore, 1982) to test for linear independence. When the results of the correlation analyses (Table 6) are compared with the results of the raw sludge correlation analyses (Table 4), a number of similaritiesare apparent. One is the strength of the correlations between fecal coliforms and fecal streptococci. Another is the lack of strong correlations between the enterovirus group of viruses and each of three groups of indicator organisms, although the correlations for reductions were stronger. A notable difference is the stronger correlation between reductions in total coliforms and fecal coliforms. From the data summarized in Table 5, it also can been seen that both residence time and temperature appear to be important factors in reducing the density of each of the three groups of indicator organisms, whereas residence time appears to be of lesser importance as compared to temperature with respect to reductions in enterovirus densities. This apparent dependence of reductions in the densities of the three groups of indicator organisms on both residence time and temperature becomes even more obvious when the reductions summarized in Table 5 are first grouped by residence time and then ordered with respect to temperature (Table 7). Interestingly, the same pattern of increasing reductions with increases in temperature for each residence time also applies to the enteroviruses. The apparent dependence of microbial reductions on both residence time and temperature suggested that it might be possible to use the Arrhenius equation [equation (1)] to describe the temperature dependence of the reductions mathematically, if the nature of the reactions could be characterized.

k = A exp(-/~/RT)

(I)

where: k = the temperature dependent reaction-rate coefficient; ,4 -- constant; ~ = the temperature characteristic; R--- the universal gas constant; T = the absolute temperature.

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JOHN H. M ~ N

J g et al.

Table 7. Reductions in the geometric mean densities of indicator organisms and enteroviruses grouped by residence time and then ordered with r n l ~ t to temperature Log~0 reductions Residence time (days)

Mixed liquor tempsratute (°C)

Total coliforms*

Fecal coliforms*

Fecal streptococci*

Enteroviruses+

10

3 I. I 37.5 29.0 39.8 8.0 14.6 17.5 21.7 23.7 25.6 38.2

0.84 0.90 1.44 2.20 0.68 1.27 1.70 0.69 2.11 t.43 2.55

1.04 1.10 1.32 1.58 0.64 1.01 1.38 I. 18 1.74 0.56 2.42

0.60 0.82 0.80 1.23 0.33 1.07 1.17 1.00 1.42 0.72 1.60

1.08 2.43 1.03 > 2.33~ 0.72 0.95 0.98 0.85 1.06 1.28 > 3.16~

15 20

*Colony-forming units per 100 ml basis, tPlaque-forming units per 100 ml basis. ~No plaque-forming units recovered from digested sludge samples.

If one assumes that the Arrhenius equation does describe the temperature dependence of a reaction, the linearized form of the Arrhenius equation [equation (2)] can be used to determine the nature of that reaction. /~ 1

Ln(k ) = -~(-~) + Ln(A ).

(2)

A plot of the natural logarithms of the temperature dependent reaction rate coefficients (Ln k) vs the reciprocals of absolute temperature (l/T) should yield a straight line if the assumed nature of the reaction is correct. Initially, it was assumed that the observed reductions in the densities of each of the three groups of indicator organisms and the enterovirus group of viruses could be characterized as either zero-order [equation (3)] or first-order [equation (4)] relationships.

So Sl -

k0 =

0

(3)

where: ko = the zero-order reaction rate coefficient, CFU or PFU per 100ml per day; So--geometric mean influent microorganism density, CFU or PFU per !00m l; S~=geometric mean effluent microorganism density, CFU or PFU per 100ml; 0 = residence time, days.

So - S l kl = ~ S1 (0)

where: k~-~ the first-order reaction rate coefficient, days-*. No evidence of strong or even moderately strong linear relationships existed, however, when the natural logarithms of the calculated zero-order and first-order reaction rate coefficients for each of the four groups of microorganisms were plotted vs the reciprocals of absolute temperature. The failure of both the zero-order and first-order Arrbenius type models to describe the temperature dependence of the microbial reductions observed in this study led to the formulation of a simple empirical rate model [equation (5)] using logl0 rather than absolute reductions as an alternative.

Se-S, k =--T-

(5)

where: k = the empirical reaction rate coefficient, Iogm0 reduction per day; Si = influent microorganism density, Io&0 CFU or PFU per 100 ml; Se = effluent microorganism density, log~0 CFU or PFU per 100 ml; 0 -- residence time, days. When the natural logarithms of the empirical reaction rate coefficients for the total coliform, fecal coliform and fecal streptococci groups of indicator

Table 8. Summary of empirical model re,action rate coefficients for reductions in the densities of the three groups of indicator organisms and the enteroviruses Empirical model reaction rate coefficients* Residence time (days)

Mixed liquor tempsrature (°C)

Total coliforms

Fecal coliforms

Fecal streptococci

Entroviruses

20 20 20 20 20 20 15 l0 10 20 15

8.0 14.6 17.5 21.7 23.7 25.6 29.0 31 .I 37.5 38.2 39.8

0.03 0.06 0.08 0.03 0. I I 0.07 0.10 0.08 0.09 0.13 0.15

0.03 0.05 0.07 0.06 0.09 0.03 0.09 0.10 0.11 0.12 0.11

0.03 0.05 0.06 0.05 0.07 0.04 0.05 0.06 0.08 0.08 0.08

0.04 0.05 0.05 0.04 0.05 0.06 0.07 0.1 I 0.24 0.16 0.16

*Log,0 reduction per day.

(4)

Aerobic digestion of enteric microorganisms

1383

-2..0 °

-I.0]

y = 7.0662 - 2854.3056x R ffi0.85

-I.5]

(WithoutOutlier)

' ~i l -2.5 -2.0

3

•

•

•

•

-3.0

-3.0 -3.5

3244.2881x R •0.94 (WithoutOudier)

y ffi8 . 2 9 2 4 -

-3.$

•' 4 .

ol

i

i

i

I

"

I

0.0031 0.0032 0.0033 0.0034 0.0035 0.0036

-4.0 0.0031 0.0032 0.0033 0.0034 0.0035 0.0036 •

t

lfr~nl~amm, OK

"

|

"

I

•

!

s

Ifrempemtm~ "K

Fig. 1. Empirical model Arrhenius plot without outlier for rate of Iogl0 reduction of total coliforms.

Fig. 2. Empirical model Arrhenius plot without outlier for rate of logic reduction of coliforms

organisms and the enterovirus group of viruses, Table 8, were plotted versus the reciprocals of absolute temperature, the anticipated linear relationships were realized. Each indicator organism data set appeared, however, to contain a discrepant or outlying observation. Specifically, the reaction rate coefficients for total coliforms at 21.7°C and fecal coliforms and fecal streptococci at 25.6°C (Table 8) appeared to be suspect. All three of these reaction rate coefficients have standardized residuals laying outside the interval of - 2 to + 2. No assignable causes such as errors in analytical methodology, recording of data, or subsequent calculations could be identified to explain the presence of these apparently discrepant observations. Thus, statistical methodology outlined by Snedecor and Cochran (1980) for testing suspiciously large deviations in regression analysis was used to test the validity of these observations. This methodology uses the Student's t distribution to calculate the probability that a random value will have a deviation exceeding the deviation of the extreme observation. The results of these calculations indicated that all of the observations considered to be suspect were unusual. For the empirical model, the calculated probabilities that random values for total coliforms, fecal coliforms and fecal streptococci would have deviations exceeding the extreme observations were 0.23, <0.01 and 0.18 respectively. In contrast, all of the calculated probabilities for the reaction-rate coefficients with the second highest standardized residuals, absolute values, exceeded 0.90. All three of the suspect reaction rate coefficients, therefore, were judged to be invalid. Results of the empirical model linear regression and associated correlation analyses for total coilforms, fecal coliforms and fecal streptococci, without

those previously identified reaction-rate coefficients judged to be invalid, are presented in Figs 1 through 3. The results of these same statistical analyses for the enterovirus group of viruses are presented in Fig. 4. As indicated by the noted correlation coefficients, the strength of the linear relationships between reaction rate coefficients and the reciprocals of absolute temperature is universally strong for the empirical model. In order to locate the data necessary to test the validity of the empirical Arrhenius type relationships, a review of the pertinent literature was conducted. The review yielded only two sets of suitable data. Both studies (Kuchenrither, 1981; Farrah et al., 1986) -2.4 " -2.6 -2.8

"~ -3.0

-3.2

(WithoutOutlier)

-3.4 '

-3.6

0.0031

i

0.0032

|

I

i

•

i

0.0033 0.0034 0.0035 0.0036

1/Temperanu~, °K Fig. 3. Empirical model Arrhenius plot without outlier for rate of logt0 reduction of fecal streptococci.

1384

JouN H. Mxgl'tr~ JR et

al.

Table 10. Comparisonof predictedempiricalmodelreaction rate coefficientsfor fecalstreptococciwith valuesreportedby Kuchenrither (1981) and Farrah et al. (1986) Empiricalreaction rate coefficients Temperature Observed (°C) Predicted* by others

-1 y = 13.9923 - 4950.6759x R ffi0.90

6 20 27 28 30 40

-2 ~e

,.d

0.04 0.05 0.06 0,06 0.07 0.08

>0.01"[" 0.08~, 0.02I", 0.05t" 0.05 + 0.11 +

0.03 + 0,01 ** 0.02"1" 0.02** 0.01~

*From regressionequation. tFarrah et al. (1986). SKuchenrither (1981) (n = 6).

-3'

=4

i

0.0031

i

i

i

i

0.13032 0.0033 0.0034 0.0035 0.0036 1/Temperaa~, ~ :

Fig. 4. Empirical model Arrhenius plot without outlier for rate of logl0 reduction of enteroviruses. were comparable to the study in that both mixed liquor residence time and temperature were variables with similar ranges of values. Data were available, however, only for the fecal coliform and fecal streptococci groups of indicator organisms. In spite of these limitations, both of these data sets were of value in testing the validity of this empirical modeling approach. As shown in Tables 9 and 10, there was a reasonable degree of agreement between the empirical reaction rate coefficients predicted by the regression equations (Figs 1-4) for both fecal coliforms and fecal streptococci and the values calculated from the data obtained from the two other studies. From the results of this model validation process, it can be concluded, at least tentatively, that the relationships between residence time and temperature and reductions in the densities of fecal eoliforms and fecal streptococci can be best described by the empirical equations developed as part of this study. This tentative conclusion also can be extended by inference for total coliforms, since fecal coliforms are a component of the total coliform group. The only evidence available to test the validity of the empirical Table 9. Comparisonof predictedempiricalmodelreaction rate coefficientsfor fecalcoliformswith values reportedby Kuchenrither and Ikmefleld(1983) and Farrah et al. (1986) Empirical reaction rate coefficients Temperature Observed (°C) Predicted* by others 6 0.04 0.02t" 20 0.06 0.081", 0.05=t:0.025 27 0.08 0.03t', 0.08, 28

0.08

0.08"~

30 0.09 0.11 ± 0.01~. *From regressionequation. tFarrah et al. (1986). **Kuchenritherand Bcnefield(1983) (n = 6).

equation for enteroviruses is the correlation coefficient associated with the regression analysis. As noted in Fig. 4, the correlation coefficient for the empirical equation for enteroviruses was 0.90. Thus, it also appears, at least for this study, that the empirical Arrbenius equation provided a reasonable description of the observed relationship between mixed liquor temperature and residence time and reduction of enterovirus density during aerobic sludge digestion. The objective of this mathematical modeling exercise was to develop a methodology for determining the residence time necessary for a given mixed liquor temperature that will provide a desired reduction in the densities of the three groups of indicator organisms and the enterovirus group of viruses during aerobic sludge digestion. Using the empirical model regression equations (Figs 1--4), it was found that even a modest 10°C increase in mixed liquor temperature generally results in a significant reduction in required residence time and thus, aeration basin volume. It is interesting that the residence times of 60 days at 15°C to 40 days at 20°C specified for aerobic digestion to be a "Process to Significantly Reduce Pathogens" in Section 257.3--6 of 40 C F R 257 (U.S. Code of Federal Regulations, 1979) have predicted reductions of at least two log~0 in the densities of the indicator organisms and enteroviruses. At 30 and 40°C, the minimum predicted residence times to achieve two log~0 reductions are 29 and 25 days, respectively, with fecal streptococci being the most resistant of these four groups of microorganisms. Given the limited data base used to develop the four predictive equations, these relationships should be used with caution. work was supported in part by the USEPA under Cooperative Agreement CR-811776 between Cornell University and the Risk Reduction Engineering Laboratory, USEPA. Partial support also was provided by the Village of Trumansburg, N.Y. The assistance of Dr Sang Shin, Director of Bacteriology, New York State Diagnostic Laboratory, Cornell University College of Veterinary Medicine, and of Dr Robert Stafferman, Chief and Ms Mary-Ellen Morris, Microbiologist, Virology Section, Environmental Monitoring and Support Laboratory, USEPA and of their staffs in general is geatefully acknowledged. Technical assistance provided by Ms Amy Slebert and Mr Leslie Kusmerik also is acknowledged. Acknowledgements--This

Aerobic digestion of enteric microorganisms REFERENCES

APHA (1985) Standard Methoda for the Examination of Water and Wastewater, 16th edition. American Public Health Association, Washington, D.C. APHA (1989) Standard Method~ for the Examination of Water and Wastewater, 17th edition. American Public Health Association, Washington, D.C. Appleton A. R. Jr, Leong C. J. and Venosa A. D. 0986) Pathogen and indicator organism destruction by the dual digestion system. J. War. Pollut. Controlled. 58, 992-999. Berg G. and Berman D. (1980) Destruction by anaerobic mesophilic and thermophilic digestion of viruses and indicator bacteria indigenous to domestic sludges. Appl. envir. Micro. 39, 361-368. Berg G., Safferman R. S., Dahung D. R., Berman D. and Hurst C. J. (1984) USEPA Manual of Methods for Virology. EPA-600/4-84-013, USEPA, Cincinnati, Ohio. Brock T. D. (1979) Biology of Microorganisms, 3rd edition. Prentice Hall, Engiewood Cliffs, N.J. Devote J. L. (1982) Probability and Statistics for Engineering and the Sciences. Brooks/Cole, Monterey, Calif. Farrah S. R., Bitton G. and Zam S. G. (1986) Inactivation of Enteric Pathogens during Aerobic Digestion of Wastewater Sludge. EPA-600/2-86-047, USEPA, Cincinnati, Ohio. Gaylord C. G. and Richards J. P. (1970) Isolation and enumeration of aerobic heterotrophic bacteria in activated sludge. J. appl. Bact. 33, 342-340. Kenner B. A. and Clark H. P. (1974) Detection and enumeration of Salmonella and Psuedomonas aeruginosa. J. War. Pollut. Control Fed. 46, 2163-2171. Kuchenwrither R. D. (1981) The investigation of mortality patterns of indicator organisms during aerobic digestion.

WR 24, I I - - F

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Unpublished M.S. thesis, Department of Civil, Environmental, and Architectural Engineering, University of Colorado, Boulder. Kuchenwrither R. D. and BenefieldL. D. (1983) Mortality patterns of indicator organisms during aerobic digestion. J. Wat. Pollut. Control Fed. 55, 76-80. Martin J. H. Jr (1988) Reduction of Enteric Microorganisms during Aerobic Sludge Digestion: A Comparison of Conventional and Autoheatud Digestion. EPA-600/2-88072, USEPA, Cincinnati, Ohio. Municipal Environmental Research Labortory (1979) Process Design Manual for Sludge Treatment and Disposal. EPA-625/I-79-001, USEPA, Cincinnati, Ohio.

Pedersen D. C. (1981) Density Level~ of Pathogenic Organisms in Manicipai Wastewater Sludge: A Literature Review. EPA-600/2-81-170, USEPA, Cincinnati, Ohio. Rohatgi V. K. (1976) An Introduction to Probability Theory and Mathematical Statistics. Wiley, New York. Snedecor G. W. and Cochran W. G. (1980) Statistical Methods, 7th edition. The Iowa State University Press, Ames, Iowa. Steel R. G. D. and Torrie J. W. (1980) Principles and Procedures of Statistics, 2nd edition. McGraw-Hill, New York. U.S. Code of Federal Regulations. (1979) Tiotle 40, Part 257, "Criteria for the Classification of Solid Waste Disposal Facilities and Practices." Fedl Register 44, 336-344. Venosa A. D., Opatken E. J. and Meckles M. C. (1979) Comparison of Ozone Contactors for Municipal Wastewater E.~uent Disinfection, Packed Column versus Jet Scrubber. EPA-600/2-79-098, USEPA, Cincinnati, Ohio.