Assessment of bacterial growth and total organic carbon removal on granular activated carbon contactors.

K Bancroft, S W Maloney, J McElhaney, I H Suffet and W O Pipes Appl. Environ. Microbiol. 1983, 46(3):683.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1983, p. 683-688 0099-2240/83/090683-06$02.00/0 Copyright 1983, American Society for Microbiology

Vol. 46, No. 3

Assessment of Bacterial Growth and Total Organic Carbon Removal on Granular Activated Carbon Contactors
K. BANCROFT,1 S. W. MALONEY,1 J. McELHANEY,2 I. H. SUFFET,1* AND W. 0. PIPES1 Environmental Studies Institute, Drexel University,1 and City of Philadelphia Water Department,2 Philadelphia, Pennsylvania 19104

Received 25 October 1982/Accepted 23 May 1983

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The overall growth rate of bacteria on granular activated carbon (GAC) contactors at the Philadelphia Torresdale Water Treatment Pilot Plant facility was found to decrease until steady state was reached. The growth rate was found to fluctuate between 6.94 x 10-3 and 8.68 x 10-4 doublings per h. The microbiological removal of total organic carbon (TOC) was calculated by considering the GAC contactors as semiclosed continuous culture systems and using growth yield factors determined in laboratory experiments. After ozonation, the average TOC entering the contactors was 1,488 IAg/liter, and the average effluent TOC was 497 ,ug/liter. Microbiological TOC removal was found to average 240 ,ug/liter on GAC contactors, which was not significantly different from microbiological TOC (220 ,ug/liter) removal across a parallel sand contactor where no adsorption took place. Thus, GAC did not appear to enhance biological TOC removal. Bacterial growth and maintenance was responsible for approximately 24% of the TOC removal on GAC under the conditions of this study.

The identification of potentially harmful or- mented (1, 6, 19, 26), and the maximum numbers ganic chemicals in drinking water has led to an attained have been described as a function of interest in the use of granular activated carbon substrate availability and influent bacterial num(GAC) as a process for their removal (22). The bers (1). Although much effort has been expendpurpose of GAC in water treatment is removal of ed over the last few years to assess the density, toxic organic substances; however, removal of composition, and activity of bacterial populaother background organic compounds as mea- tions associated with GAC contactors (5, 12, sured by total organic carbon (TOC) is also an 13), little consideration has been given to the important consideration because (i) some of actual determination of the growth kinetics. Acthem (e.g., humic acids) may act as precursors cumulation of bacteria that are not active in in the formation of chlorinated compounds removing organics is possible and could be a which may be toxic (2, 17), (ii) there may be confusing factor in attempts to determine microcompetition between compounds of health con- biological removal of TOC in a GAC column. cern and background organics for adsorption Comparing rates of TOC removal on sand and sites (9, 21), and (iii) toxic organics may asso- GAC, Eberhardt (8) concluded that GAC enciate with more poorly adsorbed humic acids (7, hanced the rate of biodegradation. Enhanced 15) and be carried through the carbon column. biodegradation was proposed to occur as a result One mechanism of background organic re- of storage by adsorption of recalcitrant organic moval is biological degradation. The occurrence material on the surface of the carbon particles. of large numbers of bacteria on GAC filters is Storage was postulated to provide a longer conwell documented (1, 8); consequently, bacterial tact time, which could promote acclimation (27). TOC removal has been studied as a mechanism However, it is not clear that organic compounds by which more adsorption sites on GAC could stored by adsorption would be available to be made available for the removal of non-biode- microorganisms growing in the column. gradable compounds (4), thus extending the bed Improved biodegradability of naturally occurlife of GAC columns and reducing operating ring TOC has been observed after ozonation (3, costs. 20). The so-called biological activated carbon The accumulation of large numbers of bacteria process has been based in part on this observain GAC contactors follows a sigmoidal curve tion and in part on Eberhardt's (8) data. It has that has been interpreted as a growth curve (6, been proposed that in the biological activated 13). The bacterial accumulation is well docu- carbon process, carbon bed life can be pro683

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longed by converting a portion of the recalcitrant organics to biodegradable organics by preozonation. The microorganisms attached to the GAC then convert the biodegradable portion to biomass, carbon dioxide, and waste products before that material can occupy adsorption sites on the GAC. It has also been hypothesized that increased biodegradation in GAC columns occurs due to the larger surface area provided by the carbon particles for attachment and growth of bacteria (1). Existing data do not show conclusively that extended service time of GAC beds is a result of enhanced biodegradation. Slow adsorption kinetics (10, 14) could also explain long-term TOC removal. Whether GAC provides a more favorable environment for bacterial growth and attachment than other media is open to question. The approach taken in this study was to consider the GAC contactor as a "black box" in which heterotrophic microorganisms function for individual maintenance and to produce new cells at the expense of energy and carbon sources entering the contactor. We need small GAC contactors to examine bacterial growth rates during the initial increase in the bacterial populations. Larger contactors containing GAC or sand were used to measure growth rates and bacterial TOC removal during long-term pseudosteady state TOC removal.
MATERIALS AND METHODS columns. Two sizes of columns were used in GAC these studies. Columns used to assess initial bacterial growth rates in short-term experiments were con-

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FIG. 1. Diagram of a column used as a contactor for the studies. Columns used for the growth rate studies were 25 in. long. Columns used for pseudosteady state experiments (including the sand column) were 40 in. long. addition (FeCl3 and NaOH). After the clarifier, the water passes through a rapid sand filter and an ozone contactor (ozone dose, 1.2 mg/liter) to a retention tank (30 min) for dissipation of residual ozone. From the retention tank the water is pumped to the GAC contactors. The contact times in the GAC contactors were 7.5 and 15 min for the short-term and long-term experiments, respectively. Bacterial enumeration. Influent and effluent water samples were collected in sterile 500-ml Nalgene bottles. To determine bacterial numbers in the contactors, 1-g carbon samples were taken from the ports located in the columns with a sterile spatula. The samples were suspended in 10 ml of sterile water in test tubes and placed in a sonicator for 30 min by the method of McElhaney and McKeon (12). All samples were then diluted in sterile buffered water for plate count analysis. Triplicate plates of soil extract agar were prepared for each dilution. The plates were incubated for 7 days at 28C before counting. Organic carbon. Samples for TOC analysis were collected in 250-ml amber bottles sealed with caps with Teflon liners. Bottles had been previously washed with detergent, and rinsed with tap water, dilute HCI, distilled water, acetone, and hexane (three times each). After the hexane rinse the bottles were air dried. Approximately 250 ml of sample was acidified with 1 ml of concentrated phosphoric acid and stored at 4C until analysis (11). A Dohrman 54-D low-level TOC analyzer was employed to measure TOC.

structed of segmented Teflon with side ports for removing carbon samples without taking the columns out of service. The Teflon segments (Savillex Corp., Minnetonka, Minn.) were 4.75 in. (ca. 11.9 cm) long with an inside diameter of 1.6 in. (ca. 4 cm). Five segments were coupled together for an overall length of approximately 25 in. (ca. 63.5 cm). Water to one column (designated as column A) was passed through a 0.22-jLjm membrane filter (Millipore Corp., Bedford, Mass.) which reduced, but did not entirely eliminate, bacterial loading before the GAC column. The column that received water that was not prefiltered was designated as column B. Figure 1 is a diagram of the columns showing sample ports. Columns of similar construction, but 40 in. (ca. 1 m) in length and containing ca. 1,000 g of GAC were used for the longer-term experiment on pseudo-steady state growth. For these experiments a similar column containing sand was run in parallel to assess bacterial TOC removal in the absence of adsorption. The sand was sieved to give the same particle size range as the carbon. Water source. The source of influent water for these studies was the Pilot Plant at the Philadelphia Torresdale water treatment facility located on the Delaware River. The Pilot Plant has been described elsewhere (16). The raw water is pumped from the river into a basin and then to an upflow clarifier with chemical

RESULTS The total numbers of bacteria on the GAC and the total bacteria per day in the influents and effluents for the prefiltered (column A) and nonfiltered (column B) contactors are presented in Fig. 2 and 3, respectively. These numbers were calculated from bacterial densities on the carbon times the amount of carbon in the contactors and the average influent and effluent

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FIG. 2. Bacterial numbers for column A (prefiltered). The number of bacteria in the influent (O) increased from 106 per day to more than 108 per day. The number of bacteria in the effluent (A) increased from 101 to 109 per day. The total number of bacteria in the contactor (O) shows an exponential increase followed by a leveling off.

densities times the flow rate. Average influent densities to column A (Fig. 2) increased from 1.47 x 102 cells per ml to 3.32 x 103 cells per ml over the first 25 days. Average influent bacterial
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densities to column B (Fig. 3) were found to be fairly constant (xi= 7.43 x 103 cells per ml) for the duration of the experiment. Effluent bacterial densities for both contactors were found to be relatively constant. However, the average effluent bacterial density for column A (xe = 2.94 x 104 cells per ml) was 56% of the average for column B (M = 5.18 x 104 cells per ml) throughout the study period. Figures 2 and 3 show that growth and accumulation of bacteria on both filtered and nonfiltered carbon contactors exhibited the typical increase followed by a constant density, which has been observed in batch culture and other GAC experiments (6). The time to reach pseudo-steady state bacterial concentrations for prefiltered column A (7.21 x 107 cells per g of carbon) was approximately 50 days compared with 20 days for nonfiltered column B (7.39 x 107 cells per g of carbon). Occasional fluctuations in bacterial densities on the carbon and loss of bacteria were noted. The decreases in GAC bacterial densities were not accompanied by obvious increases in effluent bacterial densities. The net change in the number of organisms in the contactor with time can be determined by the relative rates of input, growth, and washout as follows: Rate of change = input + growth - washout M(dX/dt) = QXi + pXM - QXe (1) where X is the bacterial density per gram of carbon, Xi is the influent density per ml, Xe is the effluent density, M is the mass of carbon in the contactor, Q is the flow rate, and ,u is the specific growth rate. At pseudo-steady state:

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FIG 3. Bacterial numbers for column B (nonfiltered). The number of bacteria per day in the influent (0) and effluent (A) remained relatively constant, but the total number of bacteria in the contactor (O) increased from 108 to more than 1010.

Prior to pseudo-steady state, dX/dt > 0 and the specific growth rate can be calculated by iterative solution of: IAXM = (Xe - Xi)Qe' - (Xe - Xi)Q (4) Equation 4 was derived from equation 1 by integration. For making the calculations, XM is the total number of bacteria in a contactor calculated from the number per gram times the grams of carbon in the contactor, and the term (Xe - Xj)Q is obtained by multiplying the flow rate times the difference between the effluent and influent densities. These calculations were performed for the filtered and nonfiltered 25-in. GAC contactors. No statistical difference was found by a t-test (a = 0.05) in calculated growth rates; therefore, the averages of the two are presented in Fig. 4. The results indicate a decrease in the growth rate of

686

BANCROFT ET AL.
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APPL. ENVIRON. MICROBIOL.

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FIG. 4. The specific growth rate of the populations on the 25-in. contactors decreased exponentially for the first 40 days and remained constant thereafter.

gradual decrease. The average doubling time during this second period (day 180 to 250) was found to be approximately 50 days. Total TOC removal (the sum of biological removal and adsorption) for this 40-in. GAC contactor and TOC removal on a parallel sand contactor are presented in Fig. 6. TOC removal for the GAC contactor exhibited an initial rapid decrease as adsorption sites were occupied, followed by pseudo-steady state removal apparently due to slow adsorption and bacterial removal. The sand column exhibited a more gradual change initially and an average TOC removal of 220 ,ug of carbon per liter during the pseudosteady state period. We interpret the two brief periods of no TOC removal on the sand columns as indicating inhibitory substances in the influent. TOC removal on the GAC columns continued during these periods due to adsorption. DISCUSSION The use of the 0.22-,um Millipore filter cartridge to prevent introduction of bacteria to the GAC contactors was found to be unsatisfactory. This apparently was the result of grow-through. The rate of growth of bacteria on the contactors was therefore determined by mathematically eliminating the bacteria applied to the contactors in the influent water as outlined above (equation 4). Accumulation and growth of bacteria on GAC shows the typical sigmoidal curve commonly observed (6, 13). However, the proposed increase in growth rate (6) was not observed during this study. Using the non-steady state equation to calculate growth rates, we found a decrease in growth rate which levels off as GAC and effluent bacterial densities become stable,
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bacteria until pseudo-steady state is attained. During pseudo-steady state, fluctuations in growth rate occur with the mean population doubling time being approximtely 6 days. The average bacterial densities on the GAC and in the effluent for the 40-in. GAC carbon columns were 5.27 x 107 cells per g of carbon and 1.55 x 104 cells per ml, respectively, during the pseudo-steady state experiment. The growth rates of bacterial populations in the 40-in. GAC contactors during pseudo-steady state were also calculated (Fig. 5). The results, while showing considerable fluctuation, indicate a mean doubling time of approximately 20 days for the period up to 180 days. Thereafter, the population doubling time increased, followed by a
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FIG. 5. The specific growth rate of the populations the 40-in. contactors fluctuated between 10-4 and 5 x 10-4 per h from day 60 to day 260.

FIG. 6. The total TOC removal on the 40-in. GAC contactor (O) averaged 992 ,ug/liter from day 60 to day 260, whereas the bacterial TOC removal on the sand contactor (A) averaged 220 ,ug/liter for the same

period.

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similar to a continuous culture apparatus without attachment surfaces. In this case however the "reactor" (40-in. column) cell density (x = 5.27 x 107 cells per g of carbon or approximately 10.54 x 107 cells per ml) is much greater than the effluent cell density (x = 1.55 x 104 cells per ml) due to attachment of bacteria on the carbon. This type of continuous culture has been described by Rose (18) as a semiclosed continuous culture system where the deviation from a normal open system can be described by the closure index (C): (5) C = 100 [(1 - Xe)lXcI where Xe is the effluent cell density and Xc is the reactor cell density. From this equation it is therefore possible to determine cell production if one knows the average effluent and GAC cell densities. Since biomass production is a function of substrate (TOC) availability, it is possible to determine the biological TOC removal on the GAC contactors if the average growth yield (cells per g of TOC) is known. Van der Kooij et al. (23-25) have reported yield factors for several species of bacteria which are the organisms commonly isolated from GAC contactor. For concentration ranges of 25 ,ug to 1.0 mg of glucose carbon per liter, average yield factors for a Flavobacterium sp., two Pseudomonas spp., and Aeromonas hydrophila were 4.7 x 10, 5.0 x 103, and 3.8 x 103 CFU/ml per ,ug of carbon per liter, respectively. In our laboratory we reproduced these results with an isolate of Pseudomonas aeruginosa from the Torresdale water treatment plant to determine a growth yield factor (K. Bancroft, W. 0. Pipes, S. W. Maloney, G. Burlingame, I. H. Suffet, J. McElhaney, and J. Santo, Abstr. Annu. Meeting Am. Soc. Microbiol. 1982, Q31, p. 215) by the technique of Van der Kooij and Hijnen (24). An average value determined was 4.05 x 103 cells per ml per ,ug of glucose carbon per liter. Using this value and the closure index, we determined biological TOC removal on the GAC contactor with the following equation: Bacterial TOC removal = (1/closure index) (6) x (effluent cell/ml)/yield factor It should be noted that the bacterial TOC removal can be calculated from equation 6 without knowing the number of bacteria in the contactor. Over the duration of the experiment no statistical difference (t test, a = 0.05) was found between the GAC microbial TOC removal and the average removal across sand, which is in agreement with the results determined by Maloney et al. (11) at the Torresdale Water Treatment Plant. Moreover, by using the mean sand TOC removal and the average GAC bacterial densities, the growth yield factor determined by

reverse calculation, was found to be 4.705 x 103 cells per ml per ,ug of carbon per liter. More importantly, in a semiclosed system such as that described here, the bacterial populations can be heterogeneous or plug flow (18), a condition frequently encountered in GAC contactors (6, 12), which until now has made discussion of growth characteristics difficult. Using the modified continuous culture approach we have been able to explain the operation of these contactors and to determine biological TOC removal. The calculated mean biological TOC removal on the GAC column was found to be 240 ,ug of carbon per liter compared with sand removal of 220 ,g of carbon per liter. The average influent TOC was 1,488 jig of carbon per liter, and the average effluent TOC was 497 ,ug of carbon per liter; thus, 24% of the TOC removal was attributed to biological activity. More importantly since the biological removal was not significantly different from the sand removal it appears that in these columns GAC did not enhance biodegradation. This finding is in agreement with an independent study conducted at the same water treatment facility by Maloney et al. (11). The generality of the results obtained at this location can be checked by applying equation 6 to results from other locations.
ACKNOWLEDGMENTS This research was completed under a cooperative program (CR 80625-02) between the City of Philadelphia Water Department, William Marazzo, Commissioner and Drexel University, Philadelphia, Pa., supported by the U.S. Environmental Protection Agency, Cincinnati, Ohio, Project Officer Keith Carswell. We also thank J. Santo and G. Burlingame for sample collection and analysis.
LITERATURE CITED 1. American Water Works Association. 1981. An assessment of microbial activity on GAC. J. Am. Water Works Assoc.

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73:447-454. 2. Bellar, T. A., J. J. Lkchtenberg, and R. C. Kroner. 1974. The occurence of organohalides in chlorinated drinking water. J. Am. Water Works Assoc. 66:703-706. 3. Benedek, A. 1979. Ozone and activated carbon adsorption: mechanistic analysis of water treatment data. Ozonews 6:1-6 4. Benedek, A. 1980. Simultaneous biodegradation and activated carbon adsorption, p. 273-302. In M. J. McGuire and I. H. Suffet (ed.), Activated carbon adsorption of organics from the aqueous phase, vol. 2. Ann Arbor Science, Ann Arbor, Mich. 5. Brewer, W. S., and W. W. Carmichael. 1979. Microbiological characterization of granular activated carbon filter systems. J. Am. Water Works Assoc. 71:738-740. 6. Cairo, P. R., J. McElhaney, and I. H. Suffet. 1979. Pilot plant testing of activated carbon adsorption systems. J. Am. Water Works Assoc. 71:660-673. 7. Carter, C. W., and I. H. Suffet. 1982. Binding of DDT to dissolved humic materials. Environ. Sci. Technol. 16:735740. 8. Eberhardt, M. 1976. Experience with the use of biologically effective activated carbon, p. 331-347. In H. Sontheimer (ed.), Reports on special problems in water technol-

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ogy (translation), vol. 9, Adsorption, EPA-600/9-76-030. U.S. Environmental Protection Agency, Cincinnati, Ohio. Frick, B., R. Bartz, H. Sontheimer, and F. A. DiGiano. 1980. Predicting competitive adsorption effects on granular activated carbon filters, p. 229-242. In I. H. Suffet and M. J. McGuire (ed.), Activated carbon adsorption of organics from the aqueous phase, vol. 1. Ann Arbor Science, Ann Arbor, Mich. Mallevialie, J. 1979. Transformations of humic acids by ozone, p. 291-308. In W. Kuhn and H. Sontheimer (ed.), Oxidation techniques in drinking water, EPA-570/9-79020. U.S. Environmental Protection Agency, Cincinnati, Ohio. Maloney, S. W., K. Bancroft, W. 0. Pipes, and I. H. Suffet. 1982. Bacterial TOC removal on sand and GAC, p. 600-608. In Proceedings 1982 ASCE National Conference on Environmental Engineering. American Society of Civil Engineers, New York. McElhaney, J., and W. R. McKeon. 1978. Enumeration and identification of bacteria in granular activated carbon columns, p. 63-68. In Proceedings of the 6th Annual American Water Works Association Water Quality Technology Conference. American Water Works Association, Denver, Co. Parsons, F., P. R. Wood, and J. DeMarco. 1980. Bacteria associated with granular activated carbon columns, p. 271-2%. In Proceedings of the 8th Annual American Water Works Association Water Quality Technology Conference. American Water Works Association, Denver, Co. Peel, R. G., and A. Benedek. 1980. Dual rate kinetic model of fixed bed adsorbers. J. Env. Engin. Div. Am. Soc. Civil Engrs. 106:797-813. Pirbazari, M., and W. J. Weber. 1982. Removal of dieldrin from water by activated carbon, p. 506-513. In Proceedings 1982 American Society of Civil Engineers National Conference on Civil Engineering. American Society of Civil Engineers, New York. Radziul, J. V. 1977. Philadelphia's advanced water treatment plant. Water Sew. Works. 124:76-78.

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17. Rook, J. J. 1974. Formation of haloforms during chlorination of natural waters. J. Water Treatment Exmination 23:254-259. 18. Rose, A. H. 1976. Chemical microbiology: an introduction to microbial physiology. Plenum Publishing Corp., New York. 19. Schalekamp, M. 1979. The use of GAC filtration to ensure quality in drinking water from surface waters. J. Am. Water Works Assoc. 71:638-647. 20. Schalekamp, M. 1979. Experiences in Switzerland with ozone, particularly in connection with hygienically undesirable elements present in water. Ozonews 7:1-8. 21. Snoeyink, V. L., J. J. McCreary, and C. J. Murin. 1977. Activated carbon adsorption of trace organic compounds, EPA-600/2-77-223. U.S. Environmental Protection Agency, Cincinnati, Ohio. 22. Suffet, I. H. 1980. An evaluation of activated carbon for drinking water treatment: a National Academy report. J. Am. Water Works Assoc. 72:41-50. 23. Van der Kooij, D. 1978. Drinking water, (technical report 11/78). The Netherlands Waterworks Testing and Research Institute, KIWA Ltd., Rijswijk, Netherlands. 24. Van der KoolJ, D., and W. A. M. Hlnen. 1981. Utilization of low concentrations of starch by a Flavobacterium species isolated from tap water. Appl. Environ. Microbiol. 41:216-221. 25. Van der KooJJ, D., A. Visser, and W. A. N. Hijnen. 1980. Growth of Aeromonas hydrophila at low concentrations of substrates added to tap water. Appl. Environ. Microbiol. 39:1198-1204. 26. Weber, W. J., M. Pirbazarl, and G. L. Nelson. 1978. Biological growth on activated carbon: An investigation by scanning electron microscopy. Environ. Sci. Technol. 12:817-819. 27. Ying, W., and W. J. Weber. 1979. Biophysical chemical adsorption systems for wastewater treatment: Predictive modelling for design and operation, p. 128-136. In Proceedings of the 33rd Industrial Waste Conference, Purdue University. Ann Arbor Science, Ann Arbor, Mich.

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