Regrowth of coliforms and fecal coliforms in chlorinated wastewater effluent

Water Research Pergamon Press 1973. Vol. 7, pp. 537-546. Printed in Great Britain

REGROWTH OF COLIFORMS AND FECAL COLIFORMS IN CHLORINATED WASTEWATER EFFLUENT HILLEL I. SHUVAL, JUDITH COHEN a n d ROBERT KOLODNEY Environmental Health Laboratory, Department of Medical Ecology, Hebrew University--Hadassah Medical School, Jerusalem, Israel

(Received 19 May 1972) Abstract--Observations made both in the field in chlorinated effluent, and in laboratory experiments show that coliforms and fecal coliforms are capable of regrowth in chlorinated wastewater. Under field conditions reg~owth of coliforms in chlorinated effluent held in a storage reservoir for about 3 days appeared inversely correlated to: (1) The residual chlorine in the storage reservoir and (2) The number of coliforms surviving chlorination. In the laboratory experiments regrowth occurred after initial doses as high as 11 ppm total chlorine even when there was no chemical inactivation of the chlorine. Fecal coliforms did not generally show regrowth to the same extent as coliforms. Regrowth occurred even when coliforms were not detectible in 10-ml of samples after chlorination. Since coliforms and fecal coliforms are capable of regrowth in chlorinated sewage effluent and admixtures of it, the sanitary Significance of the number of coliforms after storage or in receiving bodies of water is difficult to interpret. Thus standards might be based on the number of coliforms, or fecal coliforms detected in effluents immediately after chlorination. However, this would not be justified if in addition to coliforms, pathogenic bacteria can regrow in chlorinated effluents. INTRODUCTION

IN LIGHT of the trend in many countries to establish bacteriological standards for rivers and lakes used as sources of water supply or for recreation, the disinfection of wastewater effluents is becoming more common. Thus it is appropriate to reconsider the efficacy of sucfi classical methods of wastewater disinfection as chlorination, in relation to the bacteriological tests used in their evaluation. The primary objective of wastewater disinfection by chlorine is the inactivation of pathogenic microorganisms including bacteria and viruses. The usual criteria used to determine the effectiveness of such disinfection measures is the concentration of coliform bacteria or more recently fecal coliforms in the treated wastewater or receiving stream (GELDR~XCH, 1967). However, the question arises as to whether effective coliform or fecal coliform reduction in a wastewater effluent at the treatment plant site will, in fact, lead to a subsequent reduction of these organisms in streams or lakes receiving the treated wastewater. RLrDOLrSand G ~ (I 936) reported on coliform regrowth after chlorination of sewage and others have considered this problem more recently (ELIASSON, 1968). However D ~ and ~ (1969) were unable to detect regrowth of fecal coliforms below a chlorinated sewage outfaU in the American River. The study of chlorination of wastewater and coliform regrowth reported on here grew out of a study aimed at establishing the feasibility of setting criteria for the unrestricted agricultural use of wastewater effluent in Israel where undiluted effluent was to be used for irrigation. The proposed bacteriological criteria for such use was 1000 coliforms (I00 m1-1) in 80 per cent of the samples. Although this standard could be achieved under controlled laboratory conditions with reasonable levels of applied chlorine, a field study, undertaken in Jerusalem at the Hadassah Medical Centre Wastewater Treatment Plant 537

535

HILLEL L SHUVAL,JUDITH COHEN and ROBERT KOLODNEY

which handles 1000 m 3 day-~, revealed that e~en when such a standard could be obtained in the plant effluent after 15 rain detention in the chlorine contact chamber the coliform counts in the reservoir, having an average retention time of about 3 days, holding the efftuent for ultimate auicultural irrigation, were usually higher than the recommended limit. The results of these field and laboratory studies are reported upon here. METHODS AND MATERIALS

Field Experiments (a) Sampling. Grab samples of wastewater were taken twice weekly from a number of points at the Hadassah Medical Center Wastewater Treatment Plant which consists of high rate biDfiltration followed by slow sand filtration and disinfection with about I0 ppm chlorine added. For the purposes of this study only the samples taken before and after the chlorination and in the storage reservoirs (3000 m 3) will be reported. Samples were taken in sterile bottles containing sodium thiosulphate, refrigerated and brought to the laboratory in insulated containers within 2-3 h. (b) Coliform coemts. The multiple fermentation tube procedure using lactose broth (Difco) incubated at 35°C was used for the presumptive test. All positives were confirmed in Brilliant Green bile broth (Difco) tubes at 35°C (STANDARDMETHODS, 1965). Five tubes were used for each decimal dilution. The results were reported as the most probable number (MPN). In the second year of the study, coliforms were also estimated by membrane filter (MF) counts on m-Endo broth (Difco) (STANDARD METHODS, 1965). (C) Fecal coliform counts. Positive presumptive coliform tubes were subcultured into tubes containing E.C. medium (Difco) or Eijkman medium (Difco) and incubated at 44.5 ° 4- 0'5°C in a water bath (STANDARD METHODS, 1965). The results for fecal coliforms were also reported as MPN. (d) Residual chlorine. In the case of field samples residual chlorine was determined by the O-T test with a visual colour comparator. Laboratory experiments (a) Chlorination and sampling. Two to three litre grab samples of treatment plant effluent were used for laboratory chlorination and regrowth experiments. The samples were thoroughly mixed before being divided into aliquots for parallel experiments and controls. The 10-min chlorine demand and pH were determined on each sample. Sodium hypochlorite solution was added to give the calculated chlorine dose. The samples were held in reactor vessels with cotton closures at room temperature (20 4- 2°C) during the experiment for periods up to 1 week. Thoroughly mixed afiquots were withdrawn at intervals for residual chlorine determinations and bacterial assay. All samples withdrawn for bacterial assay were treated with appropriate amounts of sodium thiosulphate to neutralize the remaining chlorine. In some experiments the chlorine in the reactor vessels was neutralized after a specified period by the addition of appropriate amounts of sodium thiosulphate. (b) Bacterial counts. Coliform, and fecal coliform counts were estimated by the methods described above (field experiments). (c) Residual chlorine. In all laboratory experiments residual chlorine was determined

Regrowth of Coliformsin ChlorinatedWastewaterEffluent

539

by the iodiometric back titration method with a visual end point (STANDARDME'mOPS, 1965). Residuals were assumed as total available combined chlorine. RESULTS (1) Field experiments Except for a few isolated cases there were usually higher monthly average coliform counts in the holding tank samples than in those taken just after chlorination (FIG. 1). Out of I01 days of testing 67 showed higher coliforms in the holding tank with a log

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FIG. 1. Monthlymean number of coliformsin effluentbeforeand after chlorinationand in the storage reservoir. mean of 120 (100 ml -x) after chlorination and 800 (I00 ml -I) in the holding tank (FIG. 2). Increases as great as 4 orders of magnitude were detected on individual days. However, the number of coliforms in the holding tank never approached those in the unchlorinated sewage, with a log mean of 5 × 106 (100 ml-l). Chlorine residual estimates showed that there was usually some chlorine in the holding reservoir. (0.1-0.75 ppm). F[otr]~ 3 shows that when the number of observations in which the coliform counts were higher in the holding tank than shortly after chlorination are expressed as a percentage of the total number of observations at any given level of residual chlorine, there was regrowth on 75 per cent of the days on which 0 ppm residual chlorine was recorded, regrowth on 60 per cent of the days at 0.35 ppm and only 25 per cent at 0.75 ppm. FIGURE 4 shows a similar relationship with the number of coliforms surviving chlorination. Thus when less than 103 coliforms (I00 m1-1) survived chlorination, 80--85 per cent of the samples showed regrowth. With survivals of between 104-105 coil (100 ml-i), the regrowth feU to 20-30 per cent and where more than 106 coil (100 ml-1) survived chlorination no regrowth was observed. In each case (FIGs. 3 and 4) the calculated regression fine is shown.

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Regrowth o f coliforms occurred in all samples o f effluent chlorinated in laboratory experiments with applied chlorine doses up to 11 ppm except that receiving 20 ppm added chlorine. Fecal coliforms were generally more sensitive to chlorination and active regrowth was most apparent at the lower concentrations o f applied chlorine.

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542

HILLEL I. SHUVAL,JUDITH COHEN and ROBERT KOLODNEY

Some variation in the detailed behaviour of different batches of sewage were observed. F[GUm~ 5 shows that regrowth of coliforms and fecal coliforms occurred at the 3 and 7 ppm levels with fecal coliform re~owth reaching lower levels than coliforms. However, the numbers were never as high as those in the unchlorinated control at any given time within the duration of the experiment. In this case, practically all the

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Regrowth of Coliformsin ChlorinatedWastewater Effluent

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chlorine was consumed before the addition of the sodium thiosulphate. The residual chlorine just prior to inactivation was 0-06 ppm in the 3 ppm treatment (pH 7.5) and 0.2 ppm in the 7 ppm treatment (pH 7"68). In contrast, the results illustrated by FIG. 6 (pH 7.9) the residual chlorine level just prior to dechlorination was 2.25 ppm in the 3 ppm treatment and 4.5 ppm in the 7 ppm and no coliforms were detected in 10-ml samples. In this case considerable regrowth of both coliforms and fecal coliforms occurred in the 3 ppm treatment, the numbers eventually approaching those in the unchlorinated control. However, in the 7 ppm treatment the fecal coliforms showed no regrowth and the coliforms only showed slight regrowth after some 40 h. FIGURE 7 shows that regrowth occurred even when the sample was not chemically dechlorinated and there was a chlorine residual at the end of 50 h. Here too fecal coliform regrowth was less than that of the coliforms, which reached the predisinfection level in 4 days. DISCUSSION Regrowth of coliforms after chlorination was common in the holding tank of the Hadassah Medical Centre Wastewater Treatment Plant. There appears to be a direct, inverse relationship between the occurrence of regrowth in the holding tank and: (1) the residual chlorine in the tank (FIG. 3), (2) the number of bacteria surviving chlorination (FIG. 4). Thus it appears that when high chlorine residuals are maintained in the effluent while being retained in the storage reservoir regrowth is minimized. Also when the number of coliforms remaining shortly after chlorination are drastically reduced, this appears to allow for the regrowth of coliforms. This may be due to the general absence of competitive microflora and/or to the fact that the wastewater effluent provides sufficient nutrients to allow for the regrowth and maintenance of a certain level of coliforms. When the coliform counts are reduced to below that level they may be able to regrow, but when the coliform count after chlorination remains higher than that level there are insufficient nutrients to allow for further regrowth. The variation in the results of the regrowth studies, both in the field (FIG. 1) and the laboratory (FIGs. 5, 6 and 7) indicate the chemical and physical as well as the mircobiological variability of sewage. Thus in one laboratory experiment the residual chlorine was almost completely consumed before dechlorination after 3 h and regrowth of coliforms and fecal coliforms occurred at both levels of applied chlorine (3 and 7 ppm) and yet in a similar experiment (FIG. 6) no coliforms or fecal coliforms were detected in 10-ml samples by multiple tube method 3 h after chlorination and apparently hardly any chlorine was consumed prior to the thiosulphate treatment. Coliform regrowth was suppressed and fecal coliform regrowth completely eliminated at the 7 ppm applied chlorine level. Fmum~ 7 shows the regrowth of coliforms and fecal coliforms in one of the typical experiments in which there was no active chemical dechlorination. In one such experiment regrowth occurred with 0-82 ppm of residual chlorine detectable after 48 h. Fecal coliforms were generally more sensitive to chlorination than the coliforms and regrew to lower levels. Although several of the bacteriological tests showed no organisms in 10-ml samples after chlorination, regrowth nevertheless occurred, indicating that some bacteria were

5-14

HILLF.L 1. SHUV.X.L,JUDITH COHEN and ROBERT KOLODNEY

present in the chlorinated effluent and that art eiguent with apparently no cotiforms as shown in small volumes with conventional tests (both MPN and MF) immediately after chlorination may still be a source of coliform regrowth. Testing larger volumes of effluent of 200--500 ml by the M F method might overcome this apparent error. The main obstacle, however, is the clogging of filters in the case of wastewater laden with suspended solids. Another method of obtaining a more reliable coliform count after chlorination might be to hold the dechlorinated samples for a few hours before testing. MCKEE et al. (1958) showed that holding dechlorinated settled sewage 4 h, increased the M F coliform count from 90 to 2.2 × 103 (100 ml- i). Experiments in our laboratory using a pure strain ofE. coli which had been held in contact with 0'08-0-05 ppm residual chlorine in buffer showed that holding the dechlorinated suspension in an excess of sodium thiosulphate for 1 h at room temperature increased both the M F and MPN counts by one order of magnitude. Unchlorinated controls showed that this would not be accounted for by multiplication of the coliforms. It may be hypothesized that some chlorine, not detectable by conventional "residual chlorine" tests is loosely bound to the bacterial cell preventing growth and multiplication. Prolonged contact with thiosulphate or organic substrate appears to neutralize the effect of this " b o u n d " chlorine. HEINMET et al. (1954) claimed that incubation for 24 h in a mixture of the tricarboxylic acid cycle metabolites, though not in nutrient broth, revitalized "killed" E. coll. Attempts to repeat these findings with a pure strain of E. coli in our laboratory failed. Coliforms were recovered from chlorinated bacterial suspension after 24 h incubation in nutrient broth as well as from the metabolites medium. Since the unchlorinated E. coli were capable of growth in the metabolites medium it may be assumed that regrowth in addition to any recovery was responsible for the higher numbers isolated after incubation. The regrowth of bacteria in the presence of chlorine has been observed before in sewage by RCrDOLrS and GEHM (1936), ALLEN and BROOKS (1952), and in swimming pool water by McLEaN et al. (1961). It has been suggested that chlorine resistant strains may in some cases br responsible for this. Such strains have been reported in samples taken from swimming pools by FARKAS-HIMSLEY (1953) who also reports inducing such resistant strains in the laboratory. However, KOTT and BEN ARI (1967) did not observe any resistant strains in sewage even after prolonged chlorination. Although in general, there appeared to be a significant inverse correlation between coliform regrowth and both residual chlorine levels and post-chlorination coliform counts, in the field studies, the absence of regrowth was not always associated with high residual chlorine in the storage reservoir or a large number of bacteria surviving chlorination. Thus, other factors, such as the availability of nutrients, the presence of natural predators (RUDOLFSand GEHM, 1936), toxic substances, etc. cannot be ruled out. The necessity for a rich medium in the reactivation of chlorine-inactivated E. coli was noted by MILBAUER and GROSSOWICZ (1959) who also observed that bacteria grown on a rich nutrient medium were less sensitive to subsequent chlorination than those grown on a minimal medium. In order to determine the factors responsible for the regrowth of bacteria after chlorination, further work into the physical, chemical and biological conditions of raw and chlorinated sewage is needed. The field observations under prevailing local conditions showed that even when coliform regrowth did occur in the storage reservoir, the number of coliforms [usually

Regrowth of Coliforms in Chlorinated Wastewater Effluent

545

103-104 (I00 ml-1)] never approached that of the unchlorinated effluent [106-107 coli (I00 ml-1)]. Even though the coliform levels in the efffluent shortly after chlorination usually met the proposed coliform standards set [I000 (100 re.l-1) in 80 per cent of the samples], the effluent taken for irrigation from the storage reservoir usually did not meet the standard due to regrowth ofcoliforms. The sanitary significance of such regrowth has not been fully assessed. It is known that viruses cannot multiply outside the living host cell and to the extent that their numbers are reduced by chlorination no regrowth can occur. Other studies have indicated, however, that enteroviruses are generally more resistent to chlorine than are coliforms (S~UVAL et al., 1967). Pathogenic bacteria, e.g. Salmonellae and Vibrio cholera can survive in sewage for extended periods, and theoretically can even multiply in such an environment. HENDgaCKS and Mog~soN (1967) found that when Salmonella seftenberg was suspended in a dialysis sac in the Poudre River at the site near the point of discharge of a sewage treatment plant the number of bacteria feU at first but then reached a peak of double the original number after 6 days. After 8 days only 1 per cent of the original inoculum survived. A similar trend was found for Shigella flexneri. Since these organisms were protected from many of their natural predators it is possible that such an increase may not have occurred had the bacteria been free in the river water. The 1970 cholera outbreak in Jerusalem was associated with vegetables irrigated with raw sewage. Subsequent heavy chlorination (15 ppm) of this sewage appears to have assisted in containing the spread of the disease. In view of the possibility of regrowth of both coliforms and fecal coliforms in chlorinated sewage and in the absence of clear evidence that pathogens behave similarly, it may be necessary to reconsider the coliform standards used in assessing the sanitary condition for rivers receiving chlorinated effluent or of chlorinated effluent used for irrigation. One possible approach would be to evaluate the effectiveness of the effluent disinfection procedure shortly after its application rather than apply the standards to the receiving body of water. However, if it can be shown that pathogenic bacteria regrow in effluent similarly to coliforms, this approach would be unsafe. SUMMARY AND CONCLUSIONS Observations made both in the field, at the Hadassah Medical Centre Wastewater Treatment Plant, and in the laboratory showed that coliforms and fecal coliforms were capable of regrowth in chlorinated sewage. In the field, regrowth generally appeared to be inversely proportional to: (1) the residual chlorine in the storage reservoir and, (2) the number of coliforms surviving chlorination. In the laboratory experiments regrowth occurred after initial doses as high as 11 ppm total chlorine even when there was no chemical inactivation of the chlorine. Only an initial dose of 20 ppm total chlorine prevented regrowth of coliforms during the course of the experiment. Regrowth occurred even when collforms were not detectible in 10 ml of samples after chlorination. Fecal cotiforms generally showed less regrowth than coliforms. Since coliforms and fecal coliforms are capable of regrowth in chlorinated sewage

5Z6

HILLEL l, SHUVAL,JUDITH COHEN and ROBERT KOLODNEY

effluent and admixtures of it, the sanitary significance of the number of co!iforms in such waters after storage or in receiving bodies of water is difficult to interpret. Thus standards might be based on the number of coliforms, or fecal col.iforms detected in effluents immediately after chlorination. However, this would not be justified ff further research indicates that in addition to coliforms pathogenic bacteria can regrow in chlorinated effluents. Pathogenic viruses cannot multiply in sewage or water but such information on bacterial pathogens is generally lacking and requires careful evaluation. REFERENCES ALLE:,,' L A. and B~ooKs E. (1952) Some factors affecting the bactericidal action of chlorine. Proc. Sac. appl. Bact. 15, 155-165. DEANERD. G. and KERR! K. D. (1969) Regrowth of fecal coliforms. J. Am. Wat. Wks Ab, 61,465-..468. ELtASSO.~ R. (1968) Coliform aftergrowths in chlorinated stream overflows. J. san#. Engng Div. Am. Sac. cir. Engrs 94, 371-380. FARK~-HIMsLEV H. (1964) Killing of chlorine-resistant bacteria by chlorine-bromine solutions. Appl. Microbial. 12, 1-16. GEt.OREICtt E. E. (1967) Fecal coliform concepts in stream pollution. Water Sew. Wks R, 98-110. HEI.~ME'rs F., TAVCORW. W. and LEHMANJ. J. (1954) The use ofmetabolites in the restoration of the viability of heat and chemically inactivated E. Coll. J. Bact. 67, 5-12. HEt~DRICKS C. W. and MogRtsos S. M. (1967) Multiplication and growth of selected enteric bacteria in clear mountain stream water. Water Research 1,567-576. K o r r Y. and BEN-Am (1967) Chlorine dosage versus time in sewage purification. Water Research, I, 451-459. McKEE J. E., McLACrGHCL~R. T. and LESGROUGESP. (1958) Application of molecular filter techniques to bacterial assay of sewage. Sewage ind. Wastes 30, 245-252. McLE,~,s D. M., BROWN J. R. and NxxoN M. C. (1961) Microbiological and chemical investigations of outdoor public swimming pools. Can. J. Public Health 52, 61-67. MIt.aAUER R. and GRossowIcz N. (1959) Reactivation of chlorine-inactivated Escherichia coll. App. Microbial. 7, 70-71. RUDOLFS W. and G~HM H. W. (1936) Sewage chlorination studies, Bulletin 601, New Jersey Agric. Expt. St., New Brunswick, N.J. SmJVAL H. I., CVMBA.CtS'rAS., WACHS A., ZOHAR Y. and GOLDat-tSM N. (1967) The inactivation of enteroviruses in sewage by chlorination. Ia Advances in Water Polh~tion Research Vol. 2. W.P.C.F. Washington D.C. Standard Methods for the Examination of Water and Wastewater, 12th Edn. (1965). American Public Health Association, New York.