and American Water Works Service Co., Inc., Voorhees, New Jersey 080432. Received 22 October ... conducted at the New Jersey American Water Co.-Swimming ...... Works Association Water Quality Technology Conference. American Water ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991,
p.
Vol. 57, No. 3
857-862
0099-2240/91/030857-06$02.00/0 Copyright C) 1991, American Society for Microbiology
Bacterial Nutrients in Drinking Water MARK W. LECHEVALLIER,l* WILLIAM SCHULZ,' AND RAMON G. LEE2 American Water Works Service Co., Inc., 1115 South Illinois Street, Belleville, Illinois 62220,' Laboratory, Belleville and American Water Works Service Co., Inc., Voorhees, New Jersey 080432 Received 22 October 1990/Accepted 2 January 1991
Regrowth of coliform bacteria in distribution systems has been a problem for a number of water utilities. Efforts to solve the regrowth problem have not been totally successful. The current project, which was conducted at the New Jersey American Water Co.-Swimming River Treatment Plant, showed that the occurrence of coliform bacteria in the distribution system could be associated with rainfall, water temperatures greater than 15°C, total organic carbon levels greater than 2.4 mg/liter, and assimilable organic carbon levels greater than 50 ,ug of acetate carbon equivalents per liter. A multiple linear regression model based on free chlorine residuals present in dead-end sections of the distribution system and temperature predicted 83.8% of the heterotrophic plate count bacterial variation. To limit the growth of coliform bacteria in drinking water, the study concludes that assimilable organic carbon levels should be reduced to
Temperature (C)
Total Organic Carbon (mg/L)
FIG. 3. Relationship between temperature and average coliform
FIG. 5. Relationship between TOC and 4-day average coliform counts. Coliform data have been offset by 7 days.
occurrences.
RESULTS
Coliform occurrences. Between June 1987 and June 1988 overall coliform densities averaged 0.44 bacteria per 100 ml (geometric mean) in the Monmouth distribution system. During this time treatment plant effluents, which were monitored daily, were uniformly negative for coliform organisms on both m-Endo and m-T7 media (Table 1). Figure 2 shows the coliform occurrences (a rolling 4-day average) in the Monmouth distribution system. A 4-day coliform average was calculated because nutrient data were collected on Mondays and Thursdays. The 4-day average, therefore, includes the coliform data collected on all days and not only those coincident with project sampling days. Coliform occurrences were most frequent during the summer and fall of 1987. Coliform bacteria were not recovered during the winter months and occurred only at low levels during most of the spring. Growth parameters. (i) Temperature. The relationship between water temperature and coliform occurrence is shown in Fig. 3. The monitoring of distribution system coliform levels assumes that the cells are the result of bacterial growth within the pipeline network. However, this assumption may not always be true. Analysis of the data in Fig. 3 shows that most of the coliform occurrences were associated with water temperatures greater than 15°C. The two exceptions to this pattern were the November and April peaks which occurred at water temperatures between 11 and 13°C. It is likely that these peaks are not related to growth in the distribution system but may have been caused by sloughing of the biofilm or other disturbances. Statistical analysis
of these two events showed that none of the statistical models could explain these two coliform peaks. (ii) TOC. TOC (Fig. 4) showed a cyclic pattern, with highest levels occurring in the summer and fall of 1987. This pattern was repeated in 1988, when TOC levels were also high (above 3 mg/liter) during the summer and fall (data not shown). A temporal component of the data is demonstrated by the relationship between TOC levels and coliform occurrences. Coliform data in Fig. 5 are related to TOC levels which occurred 7 days earlier. The time offset may account for the flow of water through the distribution system or may be related to a growth lag. The results show that most coliform occurrences were associated with TOC levels greater than 2.4 mg/liter. Coliform bacteria always occurred in the distribution system when TOC levels were greater than 2.9 mg/liter. TOC levels below 2.2 mg/liter were associated with very low coliform levels (Fig. 5). (iii) AOC. AOC levels in the Swimming River Treatment Plant effluent are shown in Fig. 6. Overall, AOC levels averaged 214 Rig/liter in the treatment plant effluents. However, AOC levels were frequently greater than 500 ,ug/liter during the summer of 1987. The monthly averages for AOC results from the plant effluent and site 4 (dead end) are shown in Fig. 7. AOC levels in the plant effluent were higher than at the dead-end site for 10 of the 13 months. Overall, AOC levels in the plant effluent averaged 73 ,ug/liter higher than levels at the dead-end site. This amount of carbon could provide sufficient nutrients to support 3.5 x 105 bacteria per ml (based on 7 x 10-5 g of carbon per liter divided by 10-'3 g of carbon per cell and
4.4
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Mar-88 Jun-88
FIG. 4. TOC levels in treatment plant effluents.
n1
Jun-87 Aug-87 Oct-87 Jan-88 Mar-88 Jun-88 FIG. 6. AOC in treatment plant effluents.
860
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APPL. ENVIRON. MICROBIOL.
LECHEVALLIER ET AL.
100-
6 1
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the daily coliform levels in the distribution system 7 days later (i.e., 9 to 19 August). The peaks in coliform occurrences corresponded to the peaks in AOC levels 7 days earlier. During this time TOC levels ranged from 3.5 to 4.4 mg/liter, and water temperatures were 25 to 27°C. (iv) Rainfall. Figure 10 shows the relationship between rainfall and coliform occurrences. Because most of the coliform occurrences were during the summer and fall of 1987, the data for June through November are shown. Once again, the coliform occurrences were offset 7 days after the rainfall. Figure 10 shows that nearly every coliform peak was associated with rainfall; however, the magnitude of the coliform occurrence was not necessarily related to the magnitude of the rainfall. (v) Discriminate analysis. Data shown in Table 3 have been partitioned according to when distribution system coliform occurrences were positive or negative. Parameters which showed a positive relationship to coliform occurrences included site 4 HPC bacterial levels,- rainfall 7 days earlier, AOC levels 7 days earlier, rainfall 4 days earlier, AOC levels 4 days earlier, the change in AOC 4 days earlier, water temperature, plant effluent AOC, and plant effluent HPC bacterial levels. Statistical models. It was of interest to combine all the measured factors into a model which could be used to predict HPC bacterial occurrences. Because of new coliform regulations, control of HPC densities in potable water supplies will also be a concern to water utilities. A multiple regression model was developed for HPC bacteria in the distribution dead-end mains. The model (Table 4 and Fig. 11), which included free chlorine and temperature as param-
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FIG. 9. Relationship between daily fluctuations in AOC levels (-----) and distribution system coliform levels ( ), 1 to 12 August 1988. Coliform data have been offset by 7 days (8 to 19 August).
50% assimilation). The difference between AOC levels in the plant effluent and at the dead-end site (site 4) was greatest during the summer, fall, and spring. During August 1987, the difference in AOC levels between the plant effluent and the dead-end site averaged almost 300 ,ug/liter. Table 2 shows the average values (partitioned by site) for various nutritional parameters. Of the nutritional parameters examined, only AOC showed a reduction as the water flowed through the distribution system. The results show that once the AOC entered the distribution system it was consumed rapidly, within a very short distance of the treatment plant. Analysis of the data with the paired t test showed that the difference in AOC levels between the plant effluent and site 2 was statistically significant (P = 0.01; n = 96). A scatter diagram showing the relationship between coliform occurrences and treatment plant AOC levels is shown in Fig. 8. For these data, the AOC value was calculated as the level which occurred in the treatment plant effluent 7 days earlier. Once again, the time lag appeared to be related to growth of coliform bacteria in the distribution system biofilms. AOC levels greater than 50 ,ug/liter (1.7 logs) were associated with higher levels of coliform bacteria. An experiment conducted during the first 12 days of August 1988 was performed to examine the daily fluctuation of AOC levels in the treatment plant effluent. Results presented in Fig. 9 show two peaks in AOC levels (171 pg of acetate carbon equivalents per liter on 3 August and 910 p.g/liter on 9 August). AOC values progressively increased and then decreased from the peaks. Also shown in Fig. 9 are
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FIG. 10. Relationship between rainfall (----) and daily coliform levels ( ). Coliform data have been offset by 7 days.
VOL. 57, 1991
BACTERIAL NUTRIENTS IN WATER
861
TABLE 2. Changes in bacterial nutrients at various points in the distribution system
Sitea
Nitrate-N
Nitrite-N
Ammonia-N
Orthophosphate
Total P
(mg/liter)
(mg/liter)
(mg/liter)
(mg/liter)
(mg/liter)
1
0.64 0.64 0.64 0.66
0.05 0.05 0.04 0.04
0.01 0.01 0.02 0.03
0.13 0.12 0.14 0.14
0.15 0.15 0.18 0.14
Sitea
2 3 4 a
TOC
(mg/liter)
AOC
2.31 2.31 2.32
(pg/liter) 214 145 134 141
2.31
For a description of the study sites, see Materials and Methods.
eters, predicted 83.8% of the variation of HPC bacteria. By using the model, it was calculated that at 20°C, a 1-mg/liter free chlorine residual would be necessary in the dead-end pipe to maintain HPC bacterial levels (R2A counts) below 500 CFU/ml. Typical summertime free chlorine levels at site 4 (dead end) ranged from 0 to 0.3 mg/liter.
Water temperature was found to be an important ratecontrolling factor in bacterial regrowth. Analysis of the data showed that there were essentially two seasonal episodes, one when water temperatures were warm (greater than 15'C) and the other when water temperatures were cold (less than 15'C). Coliform organisms occurred almost exclusively during the warm water periods (summer and early fall). Rainfall occurrences or nutrient peaks during periods of low temperature would not result in discernible bacterial growth. Temperature can have indirect effects on bacterial growth such as increasing chlorine demand reactions, which would result in loss of chlorine residuals in dead-end sections. Operational factors such as increased plant production during the summer months could increase the nutrient flux into the system. Plant production, however, did not correlate with coliform bacterial levels. AOC was the only nutrient observed to decline as the water moved through the distribution system (Table 2). During the summer months, coliform regrowth could be directly related to changes in AOC levels (Fig. 9). The removal of AOC in the distribution system occurred rapidly, within 1 mile (1-h flow time) of the treatment plant. These results are consistent with previous observations (5). The change in AOC could be related to reasonable HPC bacterial levels in the distribution system. HPC bacterial levels at the dead-end site for the summer of 1988 were approximately 10 times lower than plate counts for the summers of 1986 and 1987. This change in bacterial levels could be accounted for by a lowering of AOC levels in the plant effluent (Fig. 7). One objective of this study was to determine treatment goals for removal of bacterial nutrients. In this study, AOC levels greater than 50 Fig/fiter were associated with an increased incidence of coliform bacteria (Fig. 9). This level is consistent with the findings of Van der Kooij et al. (8, 9), who indicated that HPC bacterial growth may be limited in distribution water containing AOC levels less than 50 p.g/ liter. Previous research (5) showed that growth of an E. coli isolate from a distribution system biofilm was inhibited by AOC levels less than 54 ,ug/liter. On the basis of these results, a treatment objective of AOC levels less than 50 ,ug/liter should be established for systems experiencing regrowth problems.
DISCUSSION The current study shows that environmental (rainfall and temperature) and nutritional (AOC and TOC) parameters could be related to the occurrence of coliform bacteria in distribution water. This study did not address the issue of whether bacterial growth occurred in the water column or in distribution system biofilms, although previous studies of this system showed that coliform bacteria were found in pipeline biofilms (5) and that these organisms had the same biochemical profile as isolates found on the pipe surface. Because the treatment plant effluent was consistently free of indicator bacteria, the occurrence of coliform organisms in the water column is consistent with the hypothesis of bacterial regrowth within the distribution system. Additional research needs to relate AOC levels to bacterial growth within the biofilm itself. Rainfall probably washed detritus into the watershed, where dissolved organic material eventually enters the distribution system. These soluble compounds were not removed by conventional treatment practices, nor did they correlate with traditional operating parameters (i.e., turbidity, pH, alkalinity, hardness, raw water coliform levels, or HPC bacterial counts). The 7-day lag was found to be an important factor in relating rainfall to coliform occurrences. Because nutrient observations were made only twice a week, these relationships were limited to day 3, 4, or 7. A more detailed study might be able to more closely define the relationship between rainfall and coliform occurrences. In addition, this study did not show a correlation between the magnitude of the rainfall and the coliform density. However, the hydrology of the rainfall was not evaluated. For example, a small amount of rainfall following a long dry spell may have a greater impact on nutrient loading than a large amount of rainfall in the wet season. The observation that AOC levels fluctuated in the plant effluent suggests that a number of hydrologic factors influence nutrient conditions.
TABLE 3. Data partitioned by positive and negative coliform occurrence in distribution system' 7-day
Coliform occurrence (4-day avg)
Site 4 HPC bacteria/ml
rainfall (in.)
Negative
18,000 69,000
0.10 0.16
Positive
7-day (/t
156 247
4-day
4-day
4-day change.
rainfall (in.)
(pAgliter)
AOC (,ug/liter)
0.06 0.19
176 236
6.3 78.6
Temp 11.0 17.7
AOC
(g/liter)
HPC bacteriaml
165 246
26 44
a Site 4 HPC bacteria, HPC bacteria at dead-end site; 7-day rainfall, rainfall 7 days earlier (1 in. = 2.54 cm); 4-day rainfall, rainfall 4 days earlier; 4-day AOC, AOC 4 days earlier; 4-day change, AOC, difference between plant effluent and dead-end AOC values 4 days earlier.
862
APPL. ENVIRON. MICROBIOL.
LECHEVALLIER ET AL. TABLE 4. Multiple linear regression model for log HPC bacteriaa
Parameter
RCb
SE
t statistic
SCC
Contribution, R-square
Intercept Free4d Temp
3.238 -2.578 0.087
0.438 0.380 0.017
7.394 -6.78 4.879
-0.552 0.397
0.076 0.040
a Corrected R-square, 0.838; F test, 253. b RC, Regression coefficient. c SC, Standard coefficient. d Free4, Free chlorine residual at dead-end distribution site.
Overall, the AOC averaged 8.9% of the TOC. This value was within the 0.1 to 9.0% range for AOC/DOC values reported by Van der Kooij et al. (8-10). Although TOC appeared to be associated with coliform regrowth (Fig. 5), the levels did not decrease as water moved through the distribution system (Table 2). These conflicting results suggest that TOC may be a good predictor of growth episodes but may not act as a bacterial nutrient or that most of the total organic material is not readily digestable by microorganisms. Statistical analysis showed that there was only a small correlation (r = 0.21; P = 0.02) between TOC and AOC. These results support the conclusion of Van der Kooij et al. (8-10) that most of the TOC is not available for bacterial regrowth. The Surface Water Treatment Rule (7) includes provisions for control of HPC bacterial densities in potable water supplies. The rationale of the regulation is that HPC bacteria may interfere with the coliform analysis. It would be useful, therefore, if utilities could predict HPC levels in dead-end distribution lines. Results shown in Table 4 indicate that temperature and free chlorine residuals could be used to predict 84% of the variation in HPC bacterial densities. Application of the multiple linear regression model indicates that 1.0 mg of free chlorine residuals per liter would be necessary to limit HPC
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Sample No. FIG. 11. Comparison of actual HPC data and multiple linear actual HPC data; regression HPC model. Symbols: predicted value; ...., 95% confidence intervals.
bacterial levels (R2A agar counts) to less than 500 CFU/ml. To achieve a 1.0-mg/liter residual, much higher chlorine doses would be required. These levels would probably result in unacceptable trihalomethane levels. The fact that nutrients were not predictive of HPC bacteria suggests that these organisms were not nutrient limited. Van der Kooij et al. (8-10) have shown that AOC levels less than 10 ,ug/liter are necessary to inhibit growth of HPC bacteria. Application of treatment schemes to reduce AOC levels could control HPC bacterial densities without excessive disinfectant doses. ACKNOWLEDGMENTS We thank Martin J. Allen, project officer, and Ed Geldreich, Ed Means, and Joyce Kippen for their helpful suggestions and comments. The comments of Richard Moser were also appreciated. This research was funded by the American Water Works Research Foundation (grant 309-87) and by the American Water System. REFERENCES 1. American Public Health Association. 1985. Standard methods for the examination of water and wastewater, 16th ed. American Public Health Association, Washington, D.C. 2. Bordner, R., and J. Winter (ed.). 1978. Microbiological methods for monitoring the environment. EPA-600/8-78-017. U.S. Environmental Protection Agency, Cincinnati. 3. Characklis, W. G., D. Goodman, W. A. Hunt, and G. A. McFeters. 1988. Bacterial regrowth in distribution systems. American Water Works Association Research Foundation, Denver. 4. Gaidish, T. J., R. L. Calderon, and J. G. Grochowski. 1987. Abstr. Annu. Meet. Am. Soc. Microbiol. 1987, N-31, p. 249. 5. LeChevallier, M. W., T. M. Babcock, and R. G. Lee. 1987. Examination and characterization of distribution system biofilms. Appl. Environ. Microbiol. 53:2714-2724. 6. Symons, J. M., T. A. Bellar, J. K. Carswell, J. MeMarco, K. L. Kropp, G. G. Robeck, D. R. Seeger, C. J. Slocum, B. L. Smith, and A. A. Stevens. 1975. Natural organics reconnaissance survey for halogenated organics. J. Am. Water Works Assoc. 67:634-647. 7. U.S. Environmental Protection Agency. 1989. National primary drinking water regulations; filtration and disinfection; turbidity, Giardia lamblia, viruses, Legionella and heterotrophic bacteria; final rule. Fed. Regist. 54:27486-27541. 8. Van der Kooij, D. 1987. The effect of treatment on assimilable organic carbon in drinking water. p. 317-328. In P. M. Huck and P. Toft (ed.), Proceedings of the Second National Conference on Drinking Water, Edmonton, Alberta, Canada, 7 and 8 April 1986. Pergamon Press, London. 9. Van der Kooij, D., and W. A. M. Hijnen. 1985. Measuring the concentration of easily assimilable organic carbon in water treatment as a tool for limiting regrowth of bacteria in distribution systems, p. 729-744. In Proceedings of the American Water Works Association Water Quality Technology Conference. American Water Works Association, Denver. 10. Van der Kooij, D., A. Visser, and W. A. M. Hinen. 1982. Determining the concentration of easily assimilable organic carbon in drinking water. J. Am. Water Works Assoc. 74:540545.
