ApPuED MICROBIOLOGY, Nov. 1973, p. 796-803Copyright @ 1973 American Society for Microbiology

Vol. 26, No. 5Printed in U.SA.

Transient Response of Continuously CulturedHeterogeneous Populations to Changes in

TemperatureT. K. GEORGE AND A. F. GAUDY, JR.

Bioengineering Laboratories, School of Civil Engineering, Oklahoma State University, Stillwater, Oklahoma74074

Received for publication 19 June 1973

Completely mixed, once-through continuous culture systems of heterogeneousmicrobial populations of sewage origin were systematically examined for re-

sponse to changes in reactor temperature. Systems were operated at two dilu-tion rates of 0.125 and 0.25 per h. "Steady state" conditions of the systemswere assessed with the reactors operating at 25 C. From this base line, tempera-ture was decreased to as low as 8 C and increased to as high as 57.5 C. Responsewas assessed in the ensuing transient phase as the system approached a new"steady state." The response was measured by changes in amount and typeof carbon source in the reactor effluent as determined by the chemical oxygendemand test, the anthrone test, and gas chromatography. Biological solids con-centration and cell composition (protein, carbohydrate, ribonucleic acid anddeoxyribonucleic acid) were also determined. These systems responded morefavorably to increases than to decreases in temperature. Regardless of the direc-tion of change, the system with the lowest dilution rate (D = 0.125 per h) re-sponded more successfully; i.e., there was less leakage of carbon source in theeffluent and less dilute-out of cells during the transient phase.

Although there has been much investiga-tional interest regarding the effects of tempera-ture on the growth and composition of chemo-statically grown cells under "steady state" con-ditions, there is scant experimental data in theliterature regarding the transitional response tostep increases or decreases in temperature, orboth. Such aspects are of general interest in thearea of continuous culture of microorganismsand of considerable applied interest regardingthe understanding and control of microbialprocesses such as biological treatment, e.g., byactivated sludge, of organic-laden wastewaterswherein heterogeneous populations rather thanpure or specific mixtures of species are em-ployed.Much of the work on effects oftemperature on

microbial growth and physiology through 1966has been reviewed by Farrel and Rose (5). Therehas been recent interest in the caloric values ofcells grown at various temperatures, and itwould appear that the calories per gram of cellsremain essentially unchanged regardless ofgrowth temperature (12, 14). There is continu-ing controversy regarding the effect of growth

temperature on chemical composition (deoxyri-bonucleic acid [DNA], ribonucleic acid [RNA],protein, etc.) (2, 6, 10). The differences in dataappear to arise from variations in experimentalor environmental conditions of growth.

Little or no quantitative data describing thetransient response to changes in temperatureare available. With regard to heterogeneouspopulations, Dougherty and McNary (4) per-formed some pilot plant studies on activatedsludge by using orange juice as substrate andnoted changes in predominant species and somedeterioration in effluent quality after gradualincreases in temperature over the range of 21 to36 C. Brezonick and Patterson (1) noted anincrease in adenosine 5'-triphosphate content ofactivated sludge over the temperature range of 9to 37 C, with a marked decrease at 45 C.The results presented herein form a part of a

long-term and continuing investigation of theeffect of various environmental perturbances tochemostatically growing mixed microbial popu-lations, i.e., shock loadings to activated sludgeprocesses. Of particular interest in these studieswas response to both increases and decreases in

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VOL. 26, 1973 RESPONSE OF MIXED SYSTEMS TO TEMPERATURE CHANGE

temperature from a common base-line tempera-ture, and we were interested in assessing theeffect, if any, which initial growth rate ordilution rate might have on the nature andextent of the transient phase between initialand final steady states. These results are pre-sented to provide possible guidelines for limitsof tolerance of activated sludge processes totemperature shock and because the experimen-tal picturization of changes in system parame-ters in the transient phase may have someusefulness in the general area of microbialkinetics and physiology, even though the re-sponse involves transient ecological as well asphysiological reaction to the environmentalstress.

MATERIALS AND METHODSThe continuous-flow growth reactors employed

were once-through chemostats (2.5-liter Pyrex glass)and have been previously described in detail (8). Priorto receiving a temperature shock, the units weremaintained at 25 C 0.5 C. Temperature was con-trolled by a Precision Lo/Temptrol (Precision Scien-tific) which was connected to the water bath in whichthe reactors were placed. Aeration through carborun-dum diffusers was maintained at 5,000 ml/min. Thegrowth medium contained: glucose, 1,000 mg/liter;(NH4),SO,, 500 mg/liter; MgSO4 7HO, 100 mg/liter;MnSO4-HO, 10 mg/liter; CaCl, 2HO, 7.5 mg/liter;FeCl, 6H20, 0.5 mg/liter; tap water, 100 ml/liter; 1 Mphosphate buffer solution (pH, 7.0), 10 ml/liter, anddistilled water to volume.Each experiment was initiated by inoculating the

synthetic waste with a sample of sewage obtainedfrom the effluent of the primary clarifier of themunicipal sewage treatment plant at Stillwater,Okla. The reactors were run 2 to 4 days undercontinuous-flow conditions at the "steady state" toestablish the base-line condition prior to the change intemperature. Each step change in temperature wasapplied by adjusting the temperature of the waterflowing into the water bath. Thus, the "shock" wasnot immediate. In all cases, the temperature wasclosely monitored so that the rate of temperaturechange was known. Experiments were conducted attwo dilution rates: D = 0.125 per h (8-h meanhydraulic retention, t) and 0.25 per h (t = 4 h).

Frequent samples were taken to assess the responseof the biomass. The concentration of biological solidswas determined by the membrane filter technique(15) by using 0.45-jum pore size filters. The filtrate wasanalyzed for chemical oxygen demand (COD) (15)and carbohydrates by the anthrone test (7). Thefiltrate was also analyzed by gas-liquid chromatogra-phy for volatile (acetic) acids by using a Polypac-2column (model 810 Hewlett-Packard Co., Avondale,Pa.). Protein and carbohydrate contents of the biolog-ical solids were determined, respectively, by thebiuret and anthrone analyses (7). RNA and DNAcontents of the biological solids were determined,

respectively, by the orcinol (13) and the diphenyla-mine (3) reactions, by using a trichloroacetic acidextract of the cells. Frequent checks on the pH of thereaction liquor were made, and the reactors were alsochecked frequently for complete mixing (11).

RESULTSFive long-term continuous-flow experiments

were conducted in which like changes in tem-perature, increases and decreases from the basetemperature at 25 C, were administered to che-mostat systems operating at dilution rates of0.125 and 0.25 per h. The temperature shockrange was from 8 to 57.5 C.

In Fig. 1, as in all succeeding figures, data tothe left of the dashed vertical line (negativetime scale) depict the preshock "steady state"condition. The temperature change was initi-ated at time zero, and the responses are shownto the right (positive values on the time scale).In all cases, the graph on the left depictsresponse at D = 0.125 per h; response at D =0.25 per h is shown on the right. The curveidentified as "substrate dilute-in curve" in Fig.1 represents the calculated value of the reactor(or effluent) COD in the absence of metabolism,i.e., if metabolism had stopped at the time ofchanging the temperature. The curve labeled"T-COD" depicts the total chemical oxygendemand of the filtrate. The curve labeled "A-COD" is the carbohydrate concentration in thefiltrate calculated to its COD value as hexosesugar (e.g., COD glucose = mg of glucose perliter x 192/180). The difference between T-COD and A-COD may be taken as a measure ofnoncarbohydrate metabolic intermediates orend products produced by the organisms, orboth. The curve labeled "acetic acid COD"results from gas-liquid chromatographic analy-sis. In the experiment shown in Fig. 1, the onlychromatographic peak detected corresponded toacetic acid, and the amount was very small (±30 mg/liter) in the system operating at D = 0.25per h; none was detected at the lower dilutionrate. Prior to the change in temperature, bothsystems provided excellent substrate removalefficiency. The cell yield was somewhat higherfor the higher dilution rate. The change from 25to 8 C was effected in 12 h. It is apparent thatneither system responded successfully as thetemperature was decreased to the psychrophilicrange; there was no indication of impendingrecovery after 200 h of operation at the post-shock temperature. It appears that the lowerdilution rate permitted a greater degree ofdissimilation of substrate (compare A-CODcurves) and slightly greater utilization of the

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798 GEORGE AND GAUDY

organic carbon (compare T-COD curves). Therewas some indication of "overshoot" with regardto A-COD in the system with lower D, whereasthere was a smoother transition at the higherdilution rate.When a less severe decrease in temperature,

from 25 to 17.5 C, was applied (see Fig. 2), theresponse of the system growing more slowly inthe preshock state was much more successful

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than that of the more rapidly growing one. Thechange in temperature was effected in 6 h, i.e.,0.75 and 1.5 mean hydraulic retention times,respectively, for the two systems. There wasessentially no leakage of anthrone-reactive ma-terial in the system of lower D, and there wasonly a short-lived transient rise in T-CODwhich corresponded to a transient decrease inbiological solids concentration. During the tran-

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FIG. 2. Response of a heterogeneous microbial population growing in a chemostat at 25 C to a decrease intemperature to 17.5 C. Left, dilution rate = 0.125 per h; right, dilution rate = 0.25 per h.

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VOL. 26, 1973 RESPONSE OF MIXED SYSTEMS TO TEMPERATURE CHANGE

sient stage, there was an increase in protein andRNA content of the biomass and a slightdecrease in carbohydrate content. Although thissystem successfully accommodated the de-crease in temperature, the one at D = 0.25 per hcould not accommodate the change. During thefirst 20 h, the effluent COD rose sharply butthere was essentially no leakage of carbohy-drate. During this period, acetic acid concentra-tion rose to 114 mg/liter. The concentration ofbiological solids decreased from 510 to 240mg/liter. The system appeared to be on theverge of recovery approximately 40 h afterchanging the temperature, but both T-CODand A-COD rose to nearly 600 mg/liter 100 hafter initiating the shock. There was an unex-pected recovery of the concentration of biologi-cal solids to nearly 390 mg/liter concomitantwith the rise in effluent COD, leading to a cellyield of approximately 90% as compared to oneof approximately 50% in the preshock condition.The high cell concentration was not due toincomplete mixing, i.e., retention of cells in thereactor, since the concentrations of cells in thereactor and the effluent were the same. As in thecase of the system with lower D there was,during the transient stage, an increase in RNAand protein content and a decrease in carbohy-drate content of the biomass.The mildest increasing temperature shock

studied was one from 25 to 36 C. The changewas effected over a period of approximately 40 h(5 x t at D = 0.125 perh and 10 x t atD = 0.25per h). Successful responses occurred at bothdilution rates (Fig. 3). At the lower dilutionrate, there was only slight fluctuation in efflu-ent quality and a slow, but completely reversi-ble, decrease in the concentration of biologicalsolids. There was a decrease in protein contentand a concomitant increase in carbohydratecontent, but these parameters, along with theconcentrations of effluent COD (S) and biologi-cal solids (X), returned to the preshock level. Atthe higher dilution rate, there was initially arapid loss of biological solids but the biomassconcentration recovered rapidly, followed by aslow decrease to the new "steady state" level.The fluctuations and decreasing trend in X didnot result in any deterioration in purificationefficiency. The most noticeable effects were thedecrease in cell yield and increase in proteincontent of the biomass.When the systems were subjected to a more

severe increase in temperature, from 25 to 47 Cover a period of 26 h, a severe transient leakageof substrate ensued at both dilution rates. Theeffect of lower dilution rate in attenuating the

severity of dilute-out in X and leakage in S inthe transient phase is amply demonstrated inthese experiments. It is also seen that, for thesystem with lower D, dissimilation of the sub-strate proceeded without interruption, whereasin the system of higher D, the leakage ofanthrone-reactive material paralleled the T-COD concentration. Both systems recovered thepreshock level of treatment efficiency, and thehigher operating temperature led to a lower cellyield (see Fig. 4).An increase in temperature to the thermo-

philic range, i.e., from 25 to 57.5 C, led not onlyto severe transient disruption of the system butto inability to recover treatment efficiencywithin 200 h after changing the temperature.Again the system with lower D evidenced themore successful response. Analyses for sludgecomposition were not performed, since the cellconcentration was extremely low in the tran-sient phase. It is important to note that thedeleterious response could not be attributableto deficiency of dissolved oxygen. The lowestdissolved oxygen concentration recorded was 3mg/liter, a value much in excess of oxygenconcentration usually found to limit metabo-lism of microorganisms (see Fig. 5).

DISCUSSIONIn these studies, the temperature shocks,

either increases or decreases, were applied atequal rates of change to each of two comparablesystems which were growing at specific growthrates of 0.25 and 0.125 per h in the initial steadystate, and it is amply apparent that, regardlessof the direction of temperature change, thesystem with lower D exhibited a greater degreeof accommodation to the shock. The same effecthas been observed in other shock-loaded sys-tems for which the carbon source was thegrowth-limiting nutrient. For example, systemswith lower D have been observed to leak lesssubstrate during transient response to qualita-tive shock, i.e., changes in the type of com-pounds comprising the carbon source in mul-ticomponent substrate systems, e.g., carbohy-drate-amino acid systems (9) and carbohydrate-alcohol systems (unpublished data, Komolritand Gaudy). We have also observed in otherstudies (Krishnan and Gaudy, unpublisheddata) that systems with lower D respond morefavorably to quantitative shock, i.e., changes inthe concentration of the inflowing carbonsource.

Similar trends for various types of systemperturbances by no means imply similar mech-anisms of metabolic or ecological response, but

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the general trend does tend to explain the rathergood ability of activated sludge systems tosuccessfully withstand, without serious meta-bolic malfunction, the various environmentalstresses which are imposed upon them. Thespecific growth rate, At, for such systems isnaturally rather low because of cell feedback,i.e., = D(1 + a - aXR/X), wherein a is thehydraulic feedback ratio, XR is the cell concen-

tration in the feedback, and X is the concentra-tion of cells in the aeration tank. In addition,cell feedback provides a much greater concen-tration of biomass in the reactor than couldexist without feedback, and we have observed insome experiments that a higher cell concentra-tion also attenuates the transient leakage ofsubstrate. Both results of operation with cellfeedback (lower and higher X) can combine toprovide apparent greater protection against var-ious types of environmental stress. Indeed,there may be some doubt as to which factormost affects the resistance to environmentalshock. For this reason, studies in once-throughsystems are apropos to activated sludge processresearch because they provide the investigatorwith a tool with which to separate the effects.The present study on temperature shock, as

well as other shock load study results mentioned

above, would seem to leave little doubt that thespecific growth rate at which the cells weregrowing in the preshock steady state has aseparate and a rather significant influence on

the response. It is also quite possible that theresponse is more greatly influenced by thehydraulics of the system than by the preshockphysiological condition of the cells as influencedby ;t or X at the time of applying the stress. Alonger mean hydraulic retention time (i.e., 1/D= t) may, simply by retaining cells in thereactor longer, provide more time for adjustingto the new conditions.

In studies cited above on changes in type andconcentration of substrate, the imposed changewas usually administered at a rate governed bythe hydraulic feed rate, D. Thus, the change inconcentration or type of substrate, or both,would be administered more slowly for lowervalues of D in accordance with the calculatabledilute-in curves. However, for the presentstudy, the rate of temperature change in eachsystem was the same, since the temperatureshock was not administered via a change in theinflowing medium, and the difference in re-

sponse can be attributed solely to the differentdilution rates (with allowance for possible dif-ferences in the populations in the reactors prior

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VOL. 26, 1973 RESPONSE OF MIXED SYSTEMS TO TEMPERATURE CHANGE 801

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to the shock). It would appear that the lowerdilution rate provides a greater intra- or inter-cellular response time which permits the morefavorable metabolic response.

With regard to the composition of the bi-omass, the lowering of temperature (see Fig. 2)caused an early decrease in carbohydrate andan increase in RNA and protein contents during

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GEORGE AND GAUDY

the transient stage. The cell composition in thefinal "steady state" was approximately thesame as in the preshock state, and there was aslight rise in cell yield. The increase in proteinand RNA accompanied by a decrease in car-bohydrate is indicative of rapid growth. Sincethe lower temperature would be expected todecrease the growth rate of the populationpredominating prior to the shock, the change inbiomass composition may be indicative of anadaptive response in which cells which couldgrow rapidly at the cooler temperature werebeing selected, while cells incapacitated at thelower temperature were diluting out of thesystem. The higher carbon source concentrationin the reaction liquor during the transient stagewould also tend to increase the specific growthrate of the cells which were able to grow. It isnoted, however, that if such a change in pre-dominance took place it was not reflected in anoticeable change in the morphological appear-ance of the biomass as adjudged by frequentmicroscope examination during the experiment.

Dilution rate apparently affected the changein protein content in response to an increase intemperature (Fig. 3, 4). In both cases, thepopulation which was growing more slowly inthe preshock condition responded with a de-crease in protein content, whereas in the morerapidly growing system protein content in-creased during the transient phase. There was,in general, a decreased cell yield at the elevatedtemperature. During the period of cell dilute-out and recovery, there was some evidence forchanges in species predominance for all threetemperature increases as indicated by changesin morphology; however, an ecological (or, inany event, a morphological) shift was particu-larly evident at the 47 and 57.5 C temperatures.Prior to the shock, short, thick rods predomi-nated in the biomass. These began to dilute outas the concentration of biological solids de-creased, and they were replaced in the recoveryphase by thin, elongated cells.Although there was, in these experiments, a

general pattern of cell dilute-out and substrateleakage followed by recovery, and although theseverity and duration of dilute-out were greaterwith greater changes in temperature from thebase of 25 C, it is somewhat difficult to deter-mine if these responses can be modeled mathe-matically. The ultimate utility of a model forthe transient stage depends upon the adequacyof its physiological (mechanistic) basis. Al-though there have been attempts, by using asystems approach, to devise such predictivemodels for single species systems without dem-

onstration of a mechanistic basis for them, theutility of such approaches for heterogeneouspopulation systems would seem rather minimal.For natural populations, the problem is muchmore complicated, and successful modelingwill, in any event, depend upon the availabilityof experimental results obtained in controlledexperiments which provide a record of theresponse as measured by a number of signifi-cant parameters. The results herein presentedare intended to help satisfy this need. Also, theyprovide some guidelines regarding the magni-tude of change which a natural system canaccommodate, and it may be tentatively con-cluded that systems operating at reasonablymoderate temperatures, e.g., ± 25 C, can morereadily accommodate increases than decreasesin temperature. This may be due in part to thefact that the most general effect of a nonlethalrise in temperature is an increase in growth rate,either of the existing predominants or of cellsselected by the higher temperature.

ACKNOWLEDGMENTSThe experimental phases of this work were conducted

under research grant WP-00075 from the Federal Environ-mental Protection Agency. Portions of the analysis of the dataand manuscript preparation were supported by a researchgrant A-043 from the Oklahoma Water Resources ResearchInstitute, U.S. Department of the Interior.

LITERATURE CITED1. Brezonick, P. L., and J. W. Patterson. 1971. Activated

sludge ATP; effects of environmental stress. J. Sanit.Eng. Div. Amer. Soc. Civil Eng. 97:813-824.

2. Brown, C. L., and A. H. Rose. 1969. Effects of tempera-ture on composition and cell volume of Candida utilis.J. Bacteriol. 97:261-272.

3. Burton, K. 1956. A study of the conditions and mecha-nism of the diphenylamine reaction for the calorimetricdetermination of deoxyribonucleic acid. Biochem. J.62:315-322.

4. Dougherty, M. H., and R. R. McNary. 1958. Elevatedtemperature effect on citrus waste activated sludge. J.Water Pollut. Contr. Fed. 30:1263-1265.

5. Farrel, J., and A. R. H. Rose. 1967. Temperature effects ofmicroorganisms. Annu. Rev. Microbiol. 21:101-120.

6. Frank, H. A., A. Reed, L. M. Santo, N. A. Lum, and S.T. Sandler. 1972. Similarity in several properties ofpsychrophilic bacteria grown at low and moderatetemperatures. Appl. Microbiol. 24:571-574.

7. Gaudy, A. F., Jr. 1962. Colorimetric determination ofprotein and carbohydrate. Ind. Water Wastes 7:17-22.

8. Gaudy, A. F., Jr., M. Ramanathan, and B. S. Rao. 1967.Kinetic behavior of heterogeneous populations in com-pletely mixed reactors. Biotechnol. Bioeng. 9:387-411.

9. Grady, C. P. L., Jr., and A. F. Gaudy, Jr. 1969. Controlmechanisms operative in a natural microbial popula-tion selected for its ability to degrade L-lysine. III.Effects of carbohydrates in continuous-flow systemsunder shock load conditions. Appl. Microbiol.18:790-797.

10. Harder, W., and H. Veldkamp. 1967. A continuousculture study of an obligately psychrophilicPseudomonas species. Arch. Mikrobiol. 18:790-797.

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11. Komolrit, K., and A. F. Gaudy, Jr. 1966. Biochemicalresponse of continuous-flow activated sludge processes

to qualitative shock loadings. J. Water Pollut. Contr.Fed. 38:85-101.

12. Mennett, R. H., and T. 0. M. Nakayama. 1971. Influenceof temperature on substrate and energy conversion inPseudomonas fluorescens. Appl. Microbiol.22:772-776.

13. Morse, M. L., and C. E. Carter. 1949. The synthesis of

nucleic acid in cultures of Escherichia coli strains Band B/R. J. Bacteriol. 58:317-322.

14. Powers, J. J., A. J. Howell, and S. J. Vacinek. 1973. Heatof combustion of cells of Pseudomonas fluorescens.Appl. Microbiol. 25:689-690.

15. American Public Health Association. 1965. Standardmethods for the examination of water and wastewater,12th ed. American Public Health Association, Inc.,New York.

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