APPuED MICROBIOLOGY, Sept. 1967, p. 1128-1137Copyright 1967 American Society for Microbiology

Aerobic Heterotrophic Bacterial Populations ofSewage and Activated Sludge

III. Adaptation in a Synthetic Waste'T. B. S. PRAKASAM' AND NORMAN C. DONDERO2

Department of Environmental Sciences, Rutgers, The State University, New Brunswick, New Jersey 08903

Received for publication 12 May 1967

The nature and behavior of the microbial population developed on a syntheticwaste containing salts and sorbitol are compared to that of a treatment-plant acti-vated sludge. The population of the adapted sludge developed on the synthetic wasteconsisted approximately of only six kinds of bacteria. Experiments with radioactivesorbitol indicate that the microbial population developed on the synthetic wasteshowed the effects of repression by glucose on the metabolism of sorbitol; in con-trast, the activated sludge from a plant treating primarily domestic waste was able toattack both substrates immediately and simultaneously.

In a series of studies on the applicability of theconcepts of enzyme adaptation to industrialwaste treatment, Gaudy and his associates (3,5-8) reported that specially adapted sludgesconsisting of heterogeneous populations of mi-croorganisms typical of those found in activatedsludge manifested the phenomenon of diauxie,the basis of which was the repression of sorbitolmetabolism by glucose. The experimental systemstudied by Gaudy et al. was similar to that ofMonod (10), except that, instead of pure cultures,an unpurified enrichment culture was tested forthe ability to utilize glucose and sorbitol con-currently. Regardless of whether the mixed pop-ulation derived from sewage was adapted onglucose or on sorbitol, the glucose was consumedbefore the sorbitol was attacked.Although the bacteria of the adapted sludges

were judged by Gaudy to be heterogeneous bymicroscopic inspection, it may be difficult toreconcile the known biochemical versatility of atruly diverse assemblage of microorganisms, inthe aggregate, with the type of limited responsereported. Hence, the implications of sequentialsubstrate removal by mixed cultures are signi-ficant in two ways: (i) biological treatment ofwastes (as Gaudy stated), if the experimentalsystem is truly analogous to practical (treatment-plant) systems; and (ii) in regard to the inference

'Paper of the Journal Series, New Jersey Agri-cultural Experiment Station, Rutgers, The State Uni-versity, New Brunswick.

2Present address: Department of Food ScienceCornell University, Ithaca, N.Y. 14850.

that repression due to glucose is more generallyprevalent among the bacteria than has beenpreviously demonstrated experimentally.The composition of the adaptation media (or

"synthetic wastes"), the rapid transfers of suc-cessive inocula, and the observation that therepression was sometimes absent (7), particularlywith the older adapted sludges, raised somequestions as to the heterogeneity of the adaptedflora. The process of adapting, or acclimating,the experimental sludge seemed to be a par-ticularly critical step. The concentrations in themedium, specifically of sorbitol, ammoniumsalt, and phosphate, in connection with rapidtransfers to fresh media appeared to be conduciveto the development of enrichment culturesdominated by a few opportunistic types of bac-teria capable of rapid growth in the artificialwaste and tolerant of severe environmentalchanges.

This paper reports further experiments on theadaptation of the microbial population by theprocess described by Gaudy et al. An attemptwas made to compare, in a limited way, theaerobic heterotrophic populations of settledsewage, activated sludge, and the adapted sludgein terms of numbers, physiological types, andbiochemical activity on glucose and sorbitol.Some of the initial phases of this research werereported previously (12).

MATERIALS AND METHODSSince Gaudy (5) described the details of the pro-

cedure for developing the acclimated microbial pop-ulations, this paper, except for points of immediate

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interest, describes the adaptation process only ingeneral terms.

Sewage samples, 1, 2, 3, and 5 were taken from theactivated sludge plant of Marlboro State Hospital,Marlboro, N.J., and sample 4, from DonaldsonPark, N.J.The inoculum of settled sewage (20 ml) was incu-

bated in basal medium 1 (BM 1), containing 1,000mg of sorbitol per liter, on a reciprocating ghakerfor 12 hr at 30 C. The basal medium with sorbitol,which has been referred to as synthetic waste or adap-tation medium, resembled the "medium" of Monod(10) with sorbitol added. At each successive 12-hrinterval, 20 ml of inoculum was transferred from thepreviously inoculated flask in the series to a liter offresh medium. At the end of the 3-day acclimationperiod, which entailed six serial transfers, the final cul-tures were subjected to some of the procedures de-scribed by Gaudy and to additional tests.The liquid acclimation medium (BM 1) contained:

NH4Cl, 500 mg; MgSO4c7H20, 500 mg; FeSO47H20, 10 mg; MnSO4V1H20, 10 mg; CaCI2.2H20,10 mg; 1.0 M phosphate buffer (pH 7.0), 10 ml; tapwater, 100 ml; and distilled water to make 1 liter.

Colony counts were obtained by spreading ap-propriate dilutions of the thoroughly dispersed sam-ples of sewage, activated sludge, or adapted sludgewith bent, sterile glass rods on the surfaces of severaldifferent agars. Six sets of plating agars correspondingto the two basal media, each supplemented in threeways were made, i.e., BM 1 plus (i) sorbitol (300 mgper liter), (ii) glucose (300 mg per liter), and (iii)glucose plus sorbitol (300 mg of each per liter), andbasal medium 2 (BM 2; 6) in three lots with the samesupplements at the 400-mg level. The two basal mediawere essentially similar, differing only in that BM 2contained (NH4)2S04 instead of NH4Cl, and FeCl3instead of FeSO4. Colony counts were made also onstandard Nutrient Agar (Difco) and MacConkeyAgar (Difco).

Replica plating of spread plates was performed bythe technique of Lederberg and Lederberg (9) by useof, in addition to the culture media previously men-tioned, Eosin Methylene Blue Agar (BBL), Tergitol7 Agar (BBL) and sodium azide agar [Azide BloodAgar Base (BBL) plus 1.5% agar].

Respirometric oxygen uptake experiments wereperformed by the usual Warburg techniques, by use of125-ml flasks with alkali in the center wells (14).

Chemical oxygen demand (COD) was determinedaccording to standard methods (2). Glucose wasestimated with anthrone (11).

For the radioisotope experiments, a suspension ofwashed adapted sludge in basal medium along withlabeled sorbitol (Volk Radiochemical Co., Burbank,Calif.), unlabeled sorbitol, and glucose was placed inreaction vessels connected to a train of two tubes con-taining saturated Ba(OH)2 solution. Filtered air waspassed through the reaction vessel, and the CO2evolved was collected as BaCO3. The tubes were re-placed every 0.5 hr with fresh tubes of Ba(OH)2solution. The BaCO3 precipitate was filtered, dried,and impregnated in Cab-O-Sil gel, and the countswere recorded by a liquid scintillation counter.

Cell counts were obtained with the Coulter elec-tronic particle counter (model B).

Calculation of results in plating experiments. Sincebacterial viable plate counts follow the Poisson dis-tribution, statistical procedures designed for normallydistributed data can not be used in analyzing platecounts. To normalize the distribution of plate counts,the square root transformation was made. Thistransformation involves taking the positive squareroot of each plate count. The means of plate countsin the results of plating experiments were obtained notby averaging the plate counts as such, but by squaringthe arithmetic mean of all the positive square roots.The t test and analysis of variance were used to analyzethe transformed plate count data (4).

RESULTSPopulation studies. Very little morphological

heterogeneity was seen in the, cells of the adaptedsludge. The predominant bacteria were rods ofvarying length and slenderness, many showingslight curvature. There appeared to be three orfour classes of cell sizes. Many cells were activelymotile.

Flocculation or aggregation of cells, beyondoccasional microscopic clumps and incompletelyseparated cells undergoing division, was notevident. The adapted culture which developed inour experiments, referred to as adapted sludge,was a dense, well-dispersed suspension of rod-shaped bacteria which would, however, settleafter standing quietly for a time.At the time of inoculating the flask of sorbitol

adaptation medium with settled sewage, samplesof the sewage and of the activated sludge takenfrom the same treatment plant (Marlboro) atthe same collection were also dispersed and platedon several types of agar to obtain some approxi-mation of the numbers and types of aerobicheterotrophs of the seed and of the plant-activatedsludge. After the period of adaptation, samplesof the adapted sludge were plated on the samearray of media. Plates were inoculated in quad-ruplicate and counted (Table 1) after 2 days at30 C.For individual sewage samples or adapted

sludge samples, the differences between countson the three supplemented forms of BM 1 werenot statistically significant, although there weredifferences between sewages sampled at thedifferent times. This was generally also true forthe counts of BM 2 media. The differences be-tween counts on the two basal media were eithersmall or not statistically significant.The counts for activated sludge were 5 to 40

times more than those for the settled sewage,but the colony counts for the adapted populationwere as much as 13 to 600 times greater thanthose for activated aludge. The equal counts for

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TABLE 1. Colony counts of settled sewage, activatedsludge, and adapted sludgea

Plating medium Settled Activated Adaptedsewage sludge sludge

Sample IBasal agar 1

Glucose.Glucose + sorbitol..Sorbitol.. .

Basal agar 2Glucose ............

Glucose + sorbitol..Sorbitol ......Nutrient Agar..MacConkey Agar. ..

Sample 2Basal agar 1GlucoseGlucose + sorbitol..Sorbitol

Basal agar 2GlucoseGlucose + sorbitol..SorbitolNutrient Agar.MacConkey Agar...

Sample 3Basal agar 1GlucoseGlucose + sorbitol..Sorbitol

Basal agar 2GlucoseGlucose + sorbitol..SorbitolNutrient Agar..MacConkey Agar...

0.63b0.750.51

0.250.370.271.100.13

0.060.050.03

0.060.0060.0280.140.225

0.3940.330.45

0.260.410.390.490.04

9.19.78.6

1.73.11.85

11.02.9

1.360.941.28

0.460.640.543.880.98

2.241.742.5

0.940.981.832.000.46

a The entries of this table and subsequent tables(2 and 3) represent the averages derived from thetransformed plate counts. These averages are ofthe same order of magnitude as the arithmeticmean of the original plate counts.

b Colony counts are expressed in millions permilliliter.

glucose agar, sorbitol agar, and glucose plussorbitol agar showed equivalent numbers ofbacteria in adapted sludge capable of usingeither or both carbohydrates. The. counts foradapted sludge samples 1 and 2 were not signi-ficantly different from each other, despite thefact that the counts of the settled sewage fromwhich they originated were different. The countsforadapted sludge 3 were statistically much higher,in absolute values, than for samples 1 and 2.

It was notable that the colonial morphologyon the plates inoculated with adapted sludge was

quite uniform, whereas the Nutrient Agar platesof sewage and activated sludge contained coloniesof many forms, sizes, and different pigmentation.The enumeration data do not adequately illus-trate these differences. The counts of the adaptedsludge with Nutrient Agar showed few, if any,statistically significant differences from countson the carbohydrate-supplemented basal agars.MacConkey Agar was included among the

media for enumeration of the adapted sludge, inspite of its known inhibitive effect on coliforms,because the carbohydrate content of the artificialwaste seemed likely to favor coliforms. Theadaptation procedure resulted in a trend towardhigher numbers of bacteria and, based on theMacConkey counts, toward a marked enhance-ment of low-temperature coliforms to the extentof 100- to 1,000-fold.

Notwithstanding the indications that coliformswere probably numerous in the adapted sludge,the colony counts made from nonidenticalinocula were not adequate to estimate the pro-portion of coliforms on the different culturemedia. To help resolve the problems of theproportion of coliforms (and possibly of othertypes of bacteria) among the various media, weresorted to the replica plating method. An accountof the validation and limitations of the replicaplating technique for mixed bacterial inocula,which are too lengthy and involved to be in-cluded here, is the subject of a separate report(13). It will suffice to say that the method isreproducible to about 86% at 0.05 significancelevel when the population is heterogeneous. Inthe experiments reported here, all theS colonieson the master plates were of a physical textureto be replicated, and it was ascertained experi-mentally that neither nutrients nor inhibitingagents were transferred by imprinting from plate-to-plate in sufficient amounts to affect the colonycounts.The replica plating scheme used in analyzing

the population of the adapted sludge entailedspreading an inoculum from the adapted sludgeon each kind of agar. After 48 hr of incubationat 30 C, each plate was used as a master platefrom which each other kind of medium was im-printed. The scheme is diagrammed in Fig. 1.The replication was carried out with two seriesof plates, one at 108 dilution and one at 107dilution. After 48 hr of incubation at 30 C ofthe imprinted plates, there was an exact colony-to-colony correspondence on each master plateand its replicas, except on the azide plates wherethere were no colonies. This pattern of dis-tribution, with colonies giving typical coliformreactions on selective media, indicated that allthe colonies on the master plates were coliforms

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Add pted SludgeI I I I I I

MIM PlI I

RepliccteI r I I I I2 3 4 5 6 7 8

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MP3

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MP4

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1 2 3 5 6 7 8

MIP 5 NP6I I

MP7 MIP8Repl Icc3te

1 2 3 4 56 7

Repl cc3te

1 2 3 4 5 6 8

Repl iccte

I I I71 2 3 4 5 7 8

Replicdte

I 12 3 8l1 2 3 4 G 7 8FIG. 1. Outline of replica plating scheme. MP = master plate. Numerals indicate type of agar: 1, basal agar

I + glucose; 2, basal agar I + glucose + sorbitol; 3, basal agar I + sorbitol; 4, MacConkey Agar; 5, EosinMethylene Blue Agar; 6, Nutrient Agar; 7, Tergitol 7 Agar; and 8, sodium azide agar.

and that the streptococci were excluded. Suchuniformity seemed improbable, and the resultsof further experimentation indicated that theuniformity in this case was either erroneous orfortuitous.

Several repetitions of the adaptation processdeveloped populations which produced non-coliform colonies on MacConkey Agar. Thediscrepancy may arise from two possible sources;the great variability of institutional waste, orthe elimination of the smaller numbers of non-coliforms through choice of dilutions for plating.The experimental results thus far led to the

conclusion that the colony-forming populationfrom the initial sewage seed had been narroweddown through adaptation to bacteria of whichall were capable of utilizing both glucose andsorbitol. The possibility remained, however,that there were viable bacteria in the adaptedpopulation incapable of forming colonies onthe carbohydrate plates because of the lack ofan adequate supply of necessary nutrient factorsor because of otherwise unsuitable environmentalconditions. The heavy growth in the liquidadaptation medium seemed likely to provide ahaven for microaerophilic bacteria which mighthave difficulty in growing exposed to air.

TABLE 2. Pour plate counts and surface plate countsofadapted sludge on different substrates

Pour plate Surface plateSubstrate counts counts

(106/ml) (106/ml)

Glucose ................. 198 230Sorbitol ................. 179 250Glucose and sorbitol 171 257

To determine whether obligate microaero-philic organisms formed a proportion of theadapted sludge population sufficient to elevatethe count, pour plate counts were compared withsurface-spread plate counts. Table 2 representsthe counts obtained on the various substrates.

Statistical analysis of these results indicatedthat the surface plate counts were higher thanthe pour plate counts; thus, there was probablyno significant amount of obligate microaero-philes. The cell counts obtained with the elec-tronic particle counter (Coulter) were discardedas unreliable because of the interference byinconstant numbers of the noncellular par-ticles in the basal medium during the growthperiods. The Petroff-Hausser microscopic cell

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counts were higher, however, than the viablecounts by about twofold. The lower viable countwas not due to possible harmful effects of thediluent, because there was no significant differencebetween the counts obtained with the usualdistilled water as the diluent and those obtainedwith basal medium. Although the visual im-pressions of different-size classes in the adaptedpopulation did not necessarily mean that thebasal agars with carbohydrates were incapableof supporting their growth, there was, however,coupled with the higher microscopic count, someindication of at least a low degree of hetero-geneity not yet resolved and a possible bias ofthe plate counts toward low values.To investigate whether there were types of

bacteria incapable of multiplying on the sorbitoladaptation medium unless supplied with sub-stances (autogenous nutrients) formed by thegrowth of the less fastidious population, the72-hr population of an acclimated sludge wasplated on the basal medium plus sorbitol andalso on plates of the staled sorbitol medium.The staled agar was prepared from a sterilized

portion of the 72-hr flask of sorbitol adaptationmedium, with its content of cells and growthproducts.Table 3 represents the results obtained with

acclimated sludges developed by using severalsewages as initial seed.No significant differences were found between

the counts obtained on basal agar and those onstaled medium plates, indicating the absence ofa significantly large cryptic population dependenton materials in addition to those of the definedsorbitol medium.

Population characterization. To estimate thenumber of types of bacteria in the adapted sludge,all colonies grown from the plates seeded at adilution of 107 for counting on basal agar and

the staled medium plates were isolated by platingand subjected to several differential tests. Coli-forms were characterized by the confirmed test,by use of Brilliant Green Bile Broth (BBL). Table4 indicates the types of organisms found in theadapted sludge characterized on the basis of thedifferential tests used in this investigation. Onlya few types of organisms dominated in theadapted sludge. The data suggest further thatan enrichment of type 1 bacteria, coliforms, andtype 4 bacteria had occurred during the ac-climation, since these accounted for about 80%of the population.

Isolates obtained in the above experimentsfrom both staled medium and sorbitol mediumwere transferred to two sets of tubes, one setcontaining basal medium with sorbitol and theother sorbitol plus 0.1% yeast extract, to de-termine whether yeast extract stimulated thegrowth of the isolates. All the tubes containingthe yeast extract yielded luxuriant growth after24 hr, whereas 32 and 40% of the isolates fromthe staled medium and sorbitol agar, respectively,did not exhibit visible growth in the tubes devoidof yeast extract. Microscopic examination ofthe yeastless tubes after 4 days of incubation,however, revealed the presence of motile bac-teria.

TABLE 3. Viable counts ofadapted sludge on sorbitolmedium and staled medium

Viable counts

Adapted sludge developedfrom sewage of Sorbitol Staled

medium medium(107/ml) (107/ml)

Marlboro .................. 53.3 42.9Donaldson Park ........... 17.9 11.9Marlboro.................. 46 37.3

TABLE 4. Differential reactions of the bacteria present in sorbitol adapted sludgea

Per cent ofGram ~~~~~~~~~~~~~~~~~~~~~~~~totalisolates

Type Gtran Gelatin Glucose Lactose Maltose Sorbitol Starch SucroseSorbitol Staledagarb mediumc

1 - - - - - - - - 40 43.92 - - A - - - - - 16.7 7.43 - - A - A A - A 3.4 4.94 - - A A A A A A 6.6 24.45 - + A - A - A -2.46d 33.3 17.0

a Abbreviations: A, acid production, and -, no reaction or gram-negative.Total isolates = 30.

c Total isolates = 41.d Coliforms, based on confirmation in Brilliant Green Bile Broth.

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The substantial growth of the yeast-stimulatedcolonies on both kinds of plates, viz, those con-taining the basal medium plus sorbitol and thosecontaining staled medium, may have been due tothe presence of some stimulatory nutrients inthe agar used in the preparation of the platesor to syntrophism between neighboring colonies.These two factors could not operate when in-dividual colonies were isolated in broth cultureslacking agar, so that the stimulation may beregarded as some indication for a tendency tosyntrophism in those cultures requiring yeastextract.Comparison of the adapted sludge colonies

growing on sorbitol agar and on staled mediumshowed a different distribution of types. Thecounts on the staled medium probably representa truer picture of the population. Althoughstatistical analysis of the counts indicated thatthe total viable counts were not significantlydifferent (i.e., a difference may have existed,but was not proven by counts), the fact that asubstantial proportion of isolates from the staledmedium were benefited by yeast extract can beregarded as evidence that a population of bac-teria builds up nutrients autogenously, and thatbacteria in the population become dependentupon these additional nutrients for growth eitherwith or without the exogenous nutrients suppliedin the medium.

Chemical studies. The adapted populationswere grown on the synthetic waste (BM 1 plussorbitol) as previously described.

After the 3-day acclimation period, the cellsfrom about 320 ml of mixed liquor were washedthrice in about 30 ml of 0.05 M phosphate buffer,then resuspended in a small volume of basalmedium, and dispersed with a blendor. Thedispersed cells were suspended in an eventualvolume of 3,000 ml of basal medium, of which500-ml portions were dispensed into three 1-literflasks. Glucose and sorbitol were added toseparate flasks in amounts of 300 mg per liter;glucose plus sorbitol were added to give 300 mgof each per liter to a third flask. The flasks werethen shaken at 30 C. The conditions were thoseof growing cultures in a complete medium. Athourly intervals, approximately 40 ml of liquorwas withdrawn from the flasks, was freed fromcells by centrifuging, and was analyzed forresidual sorbitol and glucose by the methodsused by Gaudy at that time; i.e., the anthronemethod for glucose and the subtraction of theCOD equivalent of the anthrone value from thetotal COD when both carbohydrates were to-gether for sorbitol.

In each of the three experimental runs (Fig. 2),the results corresponded rather closely to those

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2 4 G 2 4 6 2 4 6

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FIG. 2. Utilization of sorbitol by adapted sludge.Symbols: 0, total COD (glucose + sorbitol); *,sorbitol COD by difference; 0, corrected sorbitolCOD; and *, COD of sorbitoll aone.

of Gaudy (5). The pattern of utilization of glucosealone, as traced by the dichromate COD andanthrone-equivalent COD, is shown in Fig. 3.The anthrone-equivalent COD of glucose wasconsistently lower than the dichromate COD,but not by a constant value; hence, determinationof the sorbitol COD in the sorbitol-glucose cul-ture by subtraction from the total COD of theanthrone-equivalent COD of glucose showed afalse trend in utilization. Nevertheless, the evi-dence confirmed that we were working with thesame sort of system as previously reported forthe batch cultures and that the utilization ofsorbitol seemed blocked at least partially, ifnot completely by glucose.The requirement for a sensitive and specific

method for detecting the utilization of sorbitolin the presence of glucose was fulfilled by feedingradioactive sorbitol and trapping the evolved14CO2 Because of the possibility of accumulationof incompletely oxidized products in the cells orin the medium, an experiment was performed withsorbitol labeled in either the 1 or the 6 carbonand with a mixture of both. A 20-,uc amount of14C-sorbitol was added to three reaction vesselscontaining 50 ml of washed cells of adaptedsludge suspended in 0.05 M phosphate buffer.Then, unlabeled s6rbitol and glucose were added

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HOURSFIG. 3. Utilization ofglucose by adapted sludge. Symbols: 0, COD of glucose alone (Cr2O72); *, COD of

glucose alone (anthrone); and *, COD of glucose in glucose-sorbitol mixture (anthrone).

to yield a resultant concentration of 300 mg ofeach per liter. The concentration of adaptedsludge solids was 110 mg per liter. The utilizationexperiment was performed at room temperature(27 C). The evolved C02 was swept from theculture vessels, precipitated as BaCO3 in thecollecting tubes, processed as described, andcounted in a liquid scintillation counter. Col-lecting tubes were replaced by fresh tubes at30-min intervals.Labeled CO2 was evolved at equal rates, re-

gardless of the position of the "C. The resultsobtained with the sorbitol labeled C-1 are pre-sented in Fig. 4. It is clear that a constant butrelatively small fraction of the sorbitol wasattacked during the first 3.5 hr (approximately4 X 104 counts per hr), following which theutilization of sorbitol was much more rapid(about 13 X 104 counts between the 5th and6th hr).Numerous reproducible experiments measuring

the utilization of glucose both chemically andmanometrically in this system have shown thatthe glucose is largely consumed in about 4 hr.These results were interpreted as proof that theutilization of sorbitol by the adapted sludge wasvery largely, although not completely, suppressedin the presence of glucose. The results agreedvery closely with those Monod (10; p. 180-181)obtained from corresponding experiments withEscherichia coli growing in a mixture of mannitoland xylose. The xylose was utilized at a low rateuntil mannitol was consumed, whereupon therate of xylose utilization sharply increased.

Experiments with activated sludge from the

70

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FIG. 4. Utilization of sorbitol-1-'4C by adaptedsludge. Symbols: 0 = cumulative counts, and* =rate of counts (counts per hr).

treatment plant were performed manometricallyand with radioactive sorbitol for comparison withthe sorbitol-adapted system. The details of 14Ctracer experiments were similar to the adaptedsludge experiments, except for the followingmodifications: 5 ,uc of sorbitol-1-'4C instead of20 ,uc; a 150 mg per liter concentration each ofglucose and sorbitol; a concentration of 300 mg

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POPULATION ADAPTATION

per liter when sorbitol was used alone; and anactivated sludge mixed liquor suspended solidsconcentration of 245 mg per liter.

In the Warburg respirometer studies, bothadapted and treatment-plant activated sludgewere used. For oxygen consumption measure-ments, 50 ml of mixed liquor, freshly collectedfrom the aeration tank (2,450 mg of mixedliquor suspended solids per liter), was placed in125-ml Warburg flasks with alkali in the centerwell. The adapted sludge was washed in 0.05 Mphosphate buffer and resuspended in BM 1 togive 110 mg of cell solids per liter. In one run withadapted sludge, 300 mg of each carbohydrateper liter was used. In a second run with activatedsludge only, 300 mg of each, carbohydrate perliter was used when taken individually, and 150mg of each per liter, when used together. Res-pirometer experiments were performed at 30 C.The evolution of '4CO2 is shown in Fig. 5.

In contrast to the suppression of sorbitol metabo-lism by adapted sludge, the treatment-plantactivated sludge utilized the sorbitol in thepresence of glucose as readily as in its absence.In both curves, there was an initial period of 4to 5 hr during which utilization was somewhatslower, apd this lag therefore cannot be at-

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FIG. 5. Utilization of sorbitol-1-14C by activatedsludge. Symbols: * = utilization ofsorbitol in presenceofglucose, and a = utilization of sorbitol alone.

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250

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FIG. 6. Oxygen uptake ofactivated sludge. Symbols:0, sorbitol (300 mg per liter); A, glucose (300 mg perliter); and O, glucose + sorbitol (150 mg of each perliter).

tributed to repression by exogenous glucose. Itshould be noted here that the initial period inthese experiments did not correspond to a sup-pression of sorbitol utilization by glucose, since'4CO2 was evolved at an accelerating rate fromthe beginning of the experiment. There was nocorresponding lag in the oxygen uptake curves;the high rate of endogenous respiration of theactivated sludge may, however, have oblit-erated the lag in oxygen uptake due to exogenoussubstrates.

In spite of the rapid initial rates of sorbitolutilization by the activated sludge in both cases,the evolution of 14C02 from the reaction vesselcontaining sorbitol almost ceased at atout 60%of that evolved from the vessel containing bothglucose and sorbitol. We had insufficient infor-mation to hazard speculation on this phenom-enon.The conclusions that can be drawn from the

Warburg respirometer experiments (Fig. 6 and 7)are limited, but the principal information wesought was available: the activated sludge wascapable of using either or both carbohydrateswithout prior adaptation. Comparison of ratesof oxygen uptake between activated and adaptedsludges could not be fairly made because of the

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HOURSFIG. 7. Oxygen uptake of adapted sludge. Symbols:

0, sorbitol (300 mg per liter); A, glucose (300 mg perliter); and O, glucose + sorbitol (300 mg of each perliter).

differences in cell solids concentration, those ofthe adapted sludge being much lower. Further-more, there was no way of comparing the numbersof active organisms. Nevertheless, taking mano-metric and isotopic experiments together, itmay be said that, although the adapted sludgeshowed the effects of repression by glucose, theactivated sludge was able to attack both substratesimmediately and simultaneously.

DIscussIoN

The substitution of artificial wastes for sewageor industrial wastes in laboratory investigationsis an established practice. It is not our purposeto analyze the merits and drawbacks of thepractice beyond pointing out that rather unusualmixtures may be used with justification to simulateindustrial wastes or to isolate and study partic-ular phenomena under controlled conditions.One of the problems is to evaluate model systemsin the light of our meager knowledge of the

microbial flora and of the influences that directthe manner of their adaptive responses.Although the synthetic waste chosen by Gaudy

(5) qualitatively resembled the formula for themedium used by Monod (10) to show diauxicgrowth with E. coli and Bacillus species, therewas little reason to expect a priori that a microbialpopulation, truly heterogeneous in its nutritionalrequirements and physiological activities, wouldrespond in the same fashion as a pure culture,unless the population had been drastically nar-rowed down to a few types of bacteria.Concerning the variety of types of bacteria

in the sewages and the activated sludges used inthese experiments, the enumeration data do notgive adequate evidence of the variety of colonytypes seen on the plates in the absence of re-pression in the activated sludge; however, thefundamental physiological difference betweenthe adapted sludge and the activated sludge isexpressed. The divergence of the two populationswas the result of different sets of environmentalinfluences on the same type of seed.The experiments discussed in this report dealt

only with the population developed in the initialphases of the acclimation of a freshly adaptedseed. The simplification of the population duringadaptation provided an explanation for therepression of sorbitol metabolism by glucose.From the evidence presented as to the re-

stricted composition of the adapted population,it may be concluded that the adaptive responsesof such a population are largely confined to thoseassociated with physiologically uniform cultures.The most immediate effective reaction consistsof revising the enzyme economy of the cells inresponse to the nutrient substrate. When in apopulation all the cells are subject to enzymerepression, the result is to delay the utilizationof some available substrate. In a truly mixedpopulation, whatever enzyme repression takesplace appears to affect only a part of the popula-tion, leaving the remainder free to attack theavailable substrates immediately.The appearance of auxotrophic bacteria to

the extent of 30 to 40% during the first few daysof adaptation provided a hint of the beginningsof diversity in the developing population. What-ever the nature of the nutrients, prolongation ofthe adaptation procedure would have introducedmore competitors for the by-product materials,since the culture medium was not protected fromexterior contamination. Provided that multipli-cation of the invaders was rapid enough to preventattrition on transfer of the enrichment culture,they would then become established, at leasttemporarily, as a segment of the population.Lengthening the period between transfers, re-

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POPULATION ADAPTATION

taining high proportions of cellular solids, orreintroducing foreign organisms repeatedly wouldencourage diversity in the populations. Since thepreceding factors are to some extent operative inpractical and continuous culture systems, theirinfluence would help to explain the absenceof repression in older acclimated sludges.In trying to account for all the bacteria in the

adapted sludge, it was necessary to reconcile thetotal microscopic cell count with the colonycounts on the basal media supplemented withcarbohydrates. If it cannot be accepted that theexcess of the cell count over the colony does, infact, represent largely experimental error, al-ternative possibilities must be considered. Thesurplus of cells by the Petroff-Hausser methodmay be conceived as falling into two categories:viable cells incapable of forming colonies on thesorbitol agar and nonviable cells. The proportionin each category is uncertain, but, if the viablecells happened to be in the majority in the rapidlymultiplying population, the results of the 14Csorbitol experiment point to either of two alter-natives for this hypothetical, noncolony-formingfraction: (i) a high degree of repression byglucose, a property it would share with the restof the population, or (ii) a small capacity fordissimilation of sorbitol. The bacteria unable todissimilate sorbitol would be unlikely to survivethe acclimation process in any appreciable quan-tities.

In the model glucose-sorbitol system reportedupon here, the process of cellular adaptation wasconfirmed. The concomitant loss of ability toutilize the double substrate was, however, duenot to the repression of enzymes in a hetero-geneous population but to repression in a physio-logically homogeneous one.

ACKNOWLEDGMENTSWe wish to extend our sincere thanks to D. R. K.

Murty, E. R. Squibb & Sons, New Brunswick, N.J.,for his help in conducting the radioactivity experi-ments.

This investigation was supportecdy research grantWP-00645 from the Federal WaterlPollution ControlAdministration.

LITERATURE CIrED1. ALLEN, L. A. 1944. The bacteriology of activated

sludge. J. Hyg. 43:424-431.2. AMERICAN PUBLIC HEALTH ASSoCIATION. 1960.

Standard methods for the examination of waterand waste water, 11th ed. American PublicHealth Association, Inc., New York.

3. BHATLA, M. N., AND A. F. GAUDY, JR. 1964.Studies on the causation of phasic oxygen up-take in high energy systems. Proc. Ind. Waste.Conf. 19th Purdue Univ. p. 871-886.

4. DIXON, W. J., AND F. J. MASSEY, JR. 1957. Intro-duction to statistical analysis. McGraw-HillBook Co., Inc. New York.

5. GAUDY, A. F., JR. 1962. Studies on induction andrepression in activated sludge systems. Appl.Microbiol. 10:264-271.

6. GAUDY, A. F., JR., E. T. GAUDY, AND K. KoMOL-RIT. 1963. Multicomponent substrate ultiizationby natural populations and a pure culture ofEscherichia coli. Appl. Microbiol. 11:157-162.

7. GAUDY, A. F., JR., K. KoMoLRrr, AND M. N.BHATLA. 1963. Sequential substrate removal inheterogeneous populations. J. Water PollutionControl Federation 35:903-922.

8. GAUDY, A. F., JR., K. KOMOLRIT, AND E. T.GAUDY. 1964. Sequential substrate removal inresponse to qualitative shock loading of acti-vated sludge systems. Appl. Microbiol. 12:280-286.

9. LEDERBERG, J., AND E. M. LEDERBERG. 1952.Replica plating and indirect selection of bac-terial mutants. J. Bacteriol. 63: 399-406.

10. MONOD, J. 1942. Recherches sur la croissance descultures bacteriennes. Hermann & Cie, Paris.

11. MORRIS, D. L. 1948. Quantitative determinationof carbohydrates by Dreywood's anthrone rea-gent. Science 107:254-255.

12. PRAKASAM, T. B. S., AND N. C. DONDERO. 1964.Observations on the behavior of a microbialpopulation adapted to a synthetic waste. Proc.Ind. Waste. Conf., 19th, Purdue Univ., p. 835-845.

13. PRAKASAM, T. B. S., AND N. C. DONDERO. 1967.Aerobic heterotrophic bacterial populations ofsewage and activated sludge. II. Method ofcharacterization of activated sludge bacteria.Appl. Microbiol. 15.1122-1127.

14. UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF-FER. 1957. Manometric techniques. Burgess Pub-lishing Co., Minneapolis.

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