APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1985, p. 46-53 Vol. 49, No. 10099-2240/85/010046-08$02.00/0Copyright © 1985, American Society for Microbiology

Kinetics of Microbial Growth on PentachlorophenolG. M. KLECKAL* AND W. J. MAIER2

Environmental Chemistry Research Laboratory, Dow Chemical U.S.A., Midland, Michigan 48640,1 and Department ofCivil and Mineral Engineering, University of Minnesota, Minneapolis, Minnesota 554552

Received 24 July 1984/Accepted 10 October 1984

Batch and fed-batch experiments were conducted to examine the kinetics of pentachlorophenol utilization byan enrichment culture of pentachlorophenol-degrading bacteria. The Haldane modification of the Monodequation was found to describe the relationship between the specific growth rate and substrate concentration.Analysis of the kinetic parameters indicated that the maximum specific growth rate and yield coefficients arelow, with values of 0.074 h-1 and 0.136 g/g, respectively. The Monod constant (Ks) was estimated to be 60,ug/liter, indicating a high affinity of the microorganisms for the substrate. However, high concentrations (K1= 1,375 jig/liter) were shown to be inhibitory for metabolism and growth. These kinetic parameters can beused to define the optimal conditions for the removal of pentachlorophenol in biological treatment systems.

Pentachlorophenol has been extensively used as a woodpreservative, insecticide, and herbicide. In view of its wide-spread application, the feasibility of biological treatment ofpentachlorophenol-containing wastewaters has been the sub-ject of numerous investigations (5, 7, 10, 13, 25). Althoughthe compound has been shown to be extensively degraded inboth laboratory and full-scale systems, relatively few studieshave been conducted to evaluate the basic parameters whichdescribe the kinetics of pentachlorophenol utilization. Sincethe efficient operation of biological treatment systems islargely dependent on the kinetic properties of the microbialpopulation, determination of these parameters is essentialfor the development of operational strategies for the opti-mum removal of pentachlorophenol during wastewater treat-ment.

Stanlake and Finn (21) have recently described the isola-tion and kinetic properties of an Arthrobacter sp. capable ofdegrading pentachlorophenol. Based on cell yield (0.15 g ofcells per g of substrate), pentachlorophenol was a poorsubstrate for growth of the organism, presumably due to thehigh chlorine content of the molecule. The effect of penta-chlorophenol on the growth of the organism was examined inbatch cultures, using a defined medium at substrate concen-trations ranging from 50 to 300 mg/liter. The specific growthrate of the cultures increased with increasing pentachloro-phenol concentrations to a maximum of 0.1 h-1 at 130mg/liter. However, substrate concentrations greater than 130mg/liter significantly decreased the specific growth rate to alevel of approximately 0.05 h-1 at 300 mg/liter.Moos et al. (16) have also examined the kinetics of

pentachlorophenol degradation by a mixed population ofsewage microorganisms. Continuous-flow reactors were op-erated at fixed hydraulic residence times ranging from 3.2 to18.3 days and were supplied a complex medium containing20 mg of pentachlorophenol per liter and an additional 600mg of soluble organic substrates per liter. During operationof the reactors, a high degree of variability was noted ineffluent pentachlorophenol concentrations. These resultswere presumably due to substrate inhibition, since greaterstability was noted in reactors operated at longer residencetimes. Analysis of the reactor performance data by using amathematical model based on the Monod equation indicatedthat the biodegradation of pentachlorophenol was first order

* Corresponding author.

with respect to substrate, with a rate constant (I.LmaxIKs) of0.0017 liters/,ug per day.

Recent concern has been expressed over the use ofmathematical models based on Monod kinetics for describ-ing the biodegradation of inhibitory substrates in wastewatertreatment systems (20). Although such models may be validfor describing the kinetics of substrate removal at lowspecific growth rates (and hence low effluent concentra-tions), they do not permit evaluation of system performanceover a wider possible range of operating conditions. Sincethe kinetics of pentachlorophenol degradation appear toinvolve substrate inhibition, the present study was con-ducted to define the relationship between substrate concen-tration and the specific growth rate of an enrichment cultureof pentachlorophenol-utilizing bacteria.

MATERIALS AND METHODS

Isolation and maintenance of enrichment cultures. Twomixed bacterial cultures, capable of utilizing pentachloro-phenol as a sole carbon source, were isolated from samplesof industrial sewage, using a continuous-culture enrichmenttechnique. Culture vessels were constructed from 2-literPyrex resin kettles, with an overflow tube placed at the1-liter level (Fig. 1). Culture medium was supplied to thereactors from 8-liter reservoirs via glass and silicone rubbertubing. The medium flow rate was controlled by a Manostatmodel 72-500-000 peristaltic pump. Cultures were aeratedwith filtered and humidified air at a rate of 250 ml/min andagitated with a star-head magnetic stirrer. The reactors weremaintained in a constant-temperature incubator at 20°C.The two continuous cultures were maintained on a mineral

salts medium containing either 10 or 100 mg of pentachlo-rophenol per liter as the sole carbon source. The mediumwas composed of, per liter: K2HPO4, 8.7 g; MgSO4, 0.1 g;NH4NO3, 0.05 g; tap water, 50 ml; and deionized water, 950ml. Pentachlorophenol was added to the medium from astock solution (10 g/liter) prepared in 0.01 N NaOH. Themedium was adjusted to pH 7.5 with H3PO4. The reactorswere initially operated on a discontinuous basis until con-sistent substrate removal was noted. At that point a contin-uous flow of medium was supplied to the reactors. Sampleswere removed daily from the reactors for pentachlorophenolanalysis and periodically for cell mass determinations. Oncestable operation of the reactors was noted, the continuous

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KINETICS OF MICROBIAL GROWTH ON PENTACHLOROPHENOL 47

MediaSampling i1FfAssembly

Media Reservoir8 Liter Glass Carboy

BacterialAir Filter

AirSupply

FlowController

Stirrer

ReactorEffluent

Continuous CultureReactor

2 Liter Resin Ketle

FIG. 1. Continuous-flow enrichment culture reactor.

cultures provided the source of inoculum for all subsequentkinetic experiments.

Kinetic experiments. Batch culture experiments were con-ducted in 2-liter flasks containing 450 ml of the mineral saltsmedium previously described. Flasks were amended withvarious concentrations of pentachlorophenol (200 to 2,000p.g/liter) and inoculated with 50 ml of the mixed cultureobtained from the appropriate continuous-culture reactor.Flasks were incubated at 20°C on a rotary shaker at 200 rpm.

Samples of the cultures were removed periodically andanalyzed for pentachlorophenol concentration as describedbelow.A fed-batch experiment was also conducted to examine

the kinetics of pentachlorophenol degradation. To performthe test, the volume of the continuous-culture reactor wasinitially decreased by removing approximately 500 ml of theculture. The reactor was then allowed to fill to the originalvolume by the slow continuous addition of mineral salts

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48 KLECKA AND MAIER

medium containing pentachlorophenol. Samples were re-moved periodically from the fed-batch culture and analyzedfor pentachlorophenol concentration as described below.

Analytical methods. Pentachlorophenol was analyzed byhigh-pressure liquid chromatography. Culture samples wereinitially filtered through 0.2-p.m polycarbonate membranefilters (Nuclepore Corp., Pleasanton, Calif.). During samplepreparation, the first 3 ml of culture filtrate was discarded.The second portion of the filtrate was collected in a samplevial and analyzed as described below. Preliminary experi-ments indicated that >90% of the pentachlorophenol presentin standard solutions was recovered in the second portion ofthe filtrate.

Liquid chromatography was performed with a systemcomposed of a Waters model 710B automatic sample proc-essor and model M6000A pump and an LDC SpectromonitorIII variable-wavelength detector adjusted to 214 nm. Sepa-rations were achieved on a ZORBAX tetramethylsilanereverse-phase analytical column (DuPont Co., Wilmington,Del.), using a solvent system composed of acetonitrile-water(45:55) and 0.02 M H3PO4. The flow rate was 0.9 ml/min atapproximately 1,000 lb/in2. Output of the detector wasconnected to a Waters model 730 recording integrator.Pentachlorophenol concentrations were calculated on thebasis of peak height measurements by comparison with anexternal standard. The detector response was linear over theconcentration range of interest (10 to 2,000 ,ug/liter), with adetection limit of approximately 10 ,ug/liter.

Cell mass concentrations were determined by a totalorganic carbon analysis. Two samples (10 ml) of the culturewere removed for each determination; one sample wassonicated for 1 min at the full output of a Labline Ultrasonicsystem, whereas the cell mass was removed from the othersample by centrifugation at 20,000 x g for 20 min. The totalorganic carbon contents of both the culture supernatentsolution and the sonicated sample were determined with aBeckman model 915B total carbon analyzer. The instrumentwas calibrated with potassium biphthalate as the standard.Total carbon concentration of the cell mass was calculatedfrom the difference of the organic carbon present in thesonicated sample and the supernatent solution. By assumingthat the molecular formula for a microbial cell was C5H7NO2(8), the organic carbon concentration was multiplied by afactor of 1.88 (C5H7NO2/C5) to obtain the dry weight of thecell mass.Data analysis. Results of the kinetic experiments were

analyzed with an IBM model 370 computer, using the Dowadvanced computer simulation language (DACSL [1]). Batchtest data were analyzed by using modified Monod equationsfor growth and substrate utilization (2, 6),

dt = Lmax X K, + S + S2/Kj - kdX (1)

dS max X S (2)dt Y Kv + S + S2/K) (

where ILmax is the maximum specific growth rate coefficient,X is the cell mass concentration, Y is the cell yield coeffi-cient, S is the substrate concentration, Ks is the Monodconstant, K, is an inhibition constant, and kd is the endoge-nous decay coefficient. To simplify the model calculations,the value for the endogenous decay coefficient (kd) wasassumed to be 0.002 h-1, which is within the range of valuesfrequently observed for heterogeneous microbial popula-

tions (8). The remaining kinetic parameters (Pmax, Y, Ks, K1)were estimated by computerized numerical integrations byfitting the equations for growth and substrate utilization tothe shape of the substrate concentration-versus-time curves.The Adams-Moulton algorithm was used for the integrations(1). Note that the differential forms of the equations wereused during the computer analysis, since this method hasbeen found to provide more reliable estimates of pmax K,and Y than are obtained by linear analysis with integratedforms of the Monod equations (19).

Results of fed-batch experiments were analyzed as de-scribed above, using mass balance equations for the reactorvolume and cell mass and substrate concentrations

dVdt= Qi - Qedt

d = Amax X KS + 7+S2IK) - kdX

QeX--

(ddV

V V VdtJ

dS VQi Si _ LmaxX xX S

dt V Y K + S +JS2IKi

-Qe S-(dV)V V \ dt

(3)

(4)

(5)

where Qi and Qe are the influent and effluent flow rates,respectively, Si and S are the substrate concentrations in theinfluent medium and reactor, respectively, and V is thereactor volume. Note that the term dV/dt occurs in the massbalance equations for both cell mass and substrate concen-tration to account for the dilution effect associated withincreasing the volume of the fed-batch reactor. This termbecomes zero when the reactor fills to the original level andresumes normal continuous-culture operation (Qi = Qe).Thus, the mass balance equations describing the operationof fed-batch cultures are analogous to those describing theperformance of continuous cultures, except that the volumeis time dependent during fed-batch operation.

Chemicals. High-purity pentachlorophenol (99+%) wasobtained from the Aldrich Chemical Co., Milwaukee, Wis.,and was recrystallized from tetrachloroethylene before use.Acetonitrile (Distilled in Glass grade) was purchased fromBurdick and Jackson Laboratories, Inc., Muskegon, Mich.All other chemicals were reagent grade and were from theJ. T. Baker Chemical Co., Phillipsburg, N.J.

RESULTS

Operation of enrichment cultures. Pentachlorophenol-de-grading bacterial populations were obtained by continuousenrichment from a sample of industrial sewage. Two contin-uous cultures were maintained during the investigation onmineral salts media containing pentachlorophenol as the solecarbon source. Reactor 1 was operated with a mediumcontaining 10 mg (nominal) of pentachlorophenol per liter;the medium for reactor 2 contained 100 mg (nominal) of thecompound per liter. During the initial enrichment, the reac-tors were operated for a few days on a discontinuous basisuntil consistent substrate removal was noted. At that point,

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KINETICS OF MICROBIAL GROWTH ON PENTACHLOROPHENOL 49

TABLE 1. Summary of performance of the continuous-culture reactors during the period of stable operation

Hydraulic/ Effluent (pentachlorophe- Cell mass in reactorFlow solids Influent nol) (p.g/liter)a (mg/liter) Cellyoeld

Reactor Reactor rate residence (pentachlorophenol) (g of cells/Vol (ml) (ml/h) time (mg/liter) Mean Range Mean Range g of

(days) ~~~~~~~~~~~~~~~~~~~~~~~~substrate)1 1,050 3.4 12.9 8.0 <10 <10 0.13 0.094-0.17 0.01-0.022 1,110 3.2 14.5 80.7 37.8 <10-142.0 2.6 1.7-3.0 0.02-0.04

Detection limit was 10 Fg/liter; a value of 5 ,ug/liter was used for calculations.

a continuous flow of medium was supplied to the cultures.Initial operation of both reactors at a flow rate of 10 ml/hproved unstable, and thus the flow rates were decreased toapproximately 3 ml/h. After 55 days of continuous opera-tion, stable performance was noted in both reactors (Table1). Pentachlorophenol removal efficiencies of >99.8% weremaintained in the cultures during the following 3 months ofincubation.During the period of stable operation, the mixed cultures

in the two reactors consisted primarily of small suspendedflocculant particles. Microscopic examination of the parti-cles indicated the presence of bacteria and protozoa in thecultures; both suspended and stalked protozoa were noted.Qualitative examination of samples of the two culturesindicated that a greater number of protozoa were present inthe culture maintained on the higher concentration of penta-chlorophenol. As a result, the low yields of cell mass (0.01 to0.04 g/g) in the continuous cultures may be attributed topredation.

Kinetics of pentachlorophenol degradation. Batch experi-ments were conducted to examine the effects of both cellmass and substrate concentration on the rate of pentachlo-rophenol degradation. Studies were conducted at two dif-ferent initial cell mass concentrations by using a 10% (vol/vol)inoculum of mixed culture obtained from either reactor 1 orreactor 2. Biodegradation rates were examined at nominalpentachlorophenol concentrations ranging from 200 to 2,000jig/liter.

Results of batch kinetic experiments inoculated with cellsfrom reactor 1 are shown in Fig. 2. The initial cell massconcentration (X0) in the tests was 16 ,ug/liter. Note that therate of pentachlorophenol degradation increased with time,due to growth of the microorganisms. However, high con-centrations (800 to 1,600 jig/liter) of pentachlorophenol wereinhibitory for growth, as the rates of substrate utilizationwere greater at low initial concentrations (160 to 400 jig/liter).The results were analyzed with a computer simulation modelto obtain values for the kinetic parameters (jmax, Y, Ks, K,).During computer analysis, the values for the kinetic param-eters and the initial cell mass concentration were variedindividually to obtain the best fit of the experimental data.Final values for each of the coefficients were obtained byaveraging the results of numerous simulations of each dataset. Average values estimated for the parameters were asfollows: Rmax = 0.074 h-'; Xo = 17.3 ,ug/liter; Y = 0.136 g/g;Ks = 60 jig/liter; and K, = 1,375 jig/liter. The validity of theaverage values was tested by simultaneously applying thecoefficients to describe the kinetics of pentachlorophenoldegradation over the entire range of initial substrate concen-trations. Excellent agreement was noted between the meas-ured and predicted pentachlorophenol concentrations forthree of the four data sets (Fig. 3). Although the agreementbetween the measured and predicted values was not as goodfor the remaining data set, this may be due to differences inthe quantity of cell mass actually present in the batch tests.

However, the reasonable agreement observed over the en-tire range of initial substrate concentrations suggests that theaverage values adequately describe the kinetics of pentachlo-rophenol degradation.

Batch experiments were conducted in an identical mannerto investigate the biodegradation of pentachlorophenol bycells from reactor 2. The initial cell mass concentration inthe tests was 170 jig/liter. Results of the study are shown inFig. 4. Note that the rate of pentachlorophenol utilizationwas greater than observed in the previous batch tests due tothe higher level of cell mass used. Analysis of the resultswith the computer simulation model indicated that thekinetic coefficients previously obtained from the analysis ofreactor 1 batch cultures also described the rate of pentachlo-rophenol degradation by cells from reactor 2.

Kinetics of pentachlorophenol degradation in fed-batchculture. Conventional continuous-culture kinetic experi-ments were not feasible since washout of the populationwould be expected to occur as a result of substrate inhibitionas steady-state concentrations of pentachlorophenol in-creased in the reactor with increases in the dilution rate.Consequently, a fed-batch technique was used to examinethe kinetics of pentachlorophenol utilization under condi-tions resembling continuous-culture operation. Theoreticalcalculations for the operation of a fed-batch culture withpentachlorophenol indicated that decreasing the volume ofthe culture in the reactor (and hence the level of active cellmass) should result in a transient increase in substrateconcentration in the reactor, followed by a decline to theoriginal level. These calculations were tested in a fed-batchexperiment conducted with reactor 2. The culture wasinitially adjusted to a volume of 500 ml and then allowed tofill to the original level by the constant addition of mineralsalts medium containing 86.2 mg of pentachlorophenol perliter at a flow rate of 2.9 ml/h. During the experiment, theconcentration of cell mass in the reactor was nondetectable.This was presumably due to technical difficulties in measur-ing low concentrations of cell mass or the inability to obtaina representative sample because of the flocculant nature ofthe culture. However, the transient increase in pentachlo-rophenol concentration observed during operation of thefed-batch culture was shown to be predicted by the com-puter simulation model, using values of gumax = 0.074 h-', Ks= 60 jig/liter, K, = 1,375 jig/liter, and Y = 0.136 g/g for thekinetic coefficients (Fig. 5). The best fit of the data wasobtained when the initial cell mass concentration was as-sumed to be 920 jig/liter, which is consistent with the lowlevels of cell mass previously measured during continuousoperation of the reactor.

DISCUSSIONPentachlorophenol has often been considered to be rela-

tively resistant to biodegradation due to the high chlorinecontent of the molecule. However, numerous reports haveappeared in the literature describing the microbial degrada-

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50 KLECKA AND MAIER

2000 _lmax = 0.083 h-' 1600

X. = 20.8 pig/L1600~~~~~~~y = 0.136 9/'gK, = 60ug/L

1200 - K, = 1375 pg/L \

800~~~~~~~~~~~~~~~~~00 ~~~~~0o-

400 L a wi400

0~~~~~~~~~000 15 30 45 60 75 90 105 0 10 20 30 40 50 60 70

1000 Time (Hours)

Plmax = 0.086 h-1 FIG. 3. Comparison of measured (0) and predicted (-) penta-800 o xO = 11.0 pg/L chlorophenol concentrations for batch cultures of cells from reactor

Y = 0.136 g/g 1. Values used for the computer simulation were as follows: P-max =0 K, = 60 ,g/L 0.074 h-'; XO = 17.3 p.g/liter; Y = 0.136 g/g; K = 60 pSg/liter; and K,

600 K1 = 1375 jug/L=1,7pgltrtion of pentachlorophenol in soil (14, 17, 24) and water (3,

400 0 15, 18) and in systems simulating aerobic wastewater treat-cm \ment (5, 7, 10, 13, 25). In addition, bacteria isolated from a_1 \variety of sources have been shown to utilize pentachloro-

.0O 200 phenol as a sole carbon source (4, 21-23). These resultssuggest that pentachlorophenol-degrading bacteria are widely

0 I| \ss l distributed in the environment.o 0 15 30 45 60 75 90 105 This study was conducted to examine the kinetics of

pentachlorophenol degradation by enrichment cultures of0.066

50 pentachlorophenol-utilizing bacteria. During the initial en-

o P1max = 0.066 h-' richment, considerable instability was noted in the continu-°0 400 W = 18.9 pg/L ous cultures at dilution rates of 0.24 day-', but was not\ Y = 0.136 g/g observed at a dilution rate of 0.07 day-'. Similar difficulties

Ks = 60 /g/L have been previously reported by Moos et al. (16). These300 ~ Qv K1 = 1375 Mg/L results are consistent with the effects of substrate inhibition

0\ and suggest that adaptation of bacterial populations to200 pentachlorophenol can best be achieved by continuous ex-

posure to low substrate concentrations. After 55 days ofcontinuous operation, stable performance was noted in both

100 reactors. Pentachlorophenol removal efficiencies of >99.8%were maintained in the cultures during the following 3

0 months of incubation.015 30 45 60 75 90 1 t During the period of stable operation, the mixed cultures

consisted primarily of small suspended flocculant particles.200 The flocculant nature may have been due to the growth

Pmax = 0.058 h-1 conditions, since Gaudy and Gaudy (8) have noted that160 K = 19.3 pg/L flocculation is frequently observed in heterogeneous cul-

Y = 0.136 g/g tures at low growth rates. Low cell yields were noted duringKs = 60 pg/L operation of the reactors, with yield coefficients estimated in

120 K1 = 1375 pug/L the range of 0.01 to 0.04 g (dry weight) of cell mass per g ofsubstrate consumed. These low yields were presumably due

80 to predation by protozoa present in the cultures. However,Yang and Humphrey (26) have noted that cell yield de-creases as the specific growth rate decreases. Thus, low

40 yields would be expected for continuous cultures operatingat dilution rates of approximately 0.07 day-'. Computer

0 analysis of the batch test data indicated that the actual cell08 16 24 32 40 48 51 ; yield is much higher, with a value estimated to be 0.136 g/g.

Time (Hours)

FIG. 2. Kinetics of pentachlorophenol degradation in batch cul-tures of cells from reactor 1. Batch tests were conducted in 2-liter tures were incubated on a rotary shaker at 200 rpm and 20°C. Kineticflasks containing 500 ml of mineral salts medium, 8 ,g of cells from parameters were estimated by using the computer simulation modelreactor 1, and various concentrations of pentachlorophenol. Cul- as described in the text.

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KINETICS OF MICROBIAL GROWTH ON PENTACHLOROPHENOL 51

1600_0

0

8 1200

c 80000

c 400

00 5

FIG. 4. Comparison of rrin pentachlorophenol concereactor 2. Values estimatedfollows: ILmax = 0.074 h-1; A,ug/liter; and K, = 1,375 jig/

This value is consistenreported by Stanlake an(pentachlorophenol as the

Batch culture experimckinetics of pentachloroptdation was found to be p

2000

c

C

._

0'U

c

0

L.400U

0

0.

1600

1200

800

400

cell mass used in the test. Degradation rates were alsorelated to the pentachlorophenol concentration; however,high substrate concentrations (800 to 1,600 ,ug/liter) wereinhibitory, as the removal rates were greater at low concen-trations (160 to 400 ,uglliter). These observations indicatedthat the relationship between the specific growth rate andpentachlorophenol concentration deviates from the classicalhyperbolic function described by the Monod equation. Var-ious kinetic relationships have been devised to depict the

°\ joint dependence of the specific growth rate on substrateconcentration when the compound furnished for growthserves as both a substrate and an inhibitor. One that is oftenused to describe experimental data is based on the equationdeveloped by Haldane for the kinetics of enzyme catalyzedreactions (2, 6). The Haldane modification of the Monodequation has been previously used to describe the kinetics ofmicrobial growth on phenol (9, 12, 20, 26) and was found inthe present study to be suitable for describing the kinetics of

10 15 20 25 30 pentachlorophenol utilization. The relationship between spe-Time (Hours) cific growth rate and pentachlorophenol concentration is

ieasured (0) and predicted (-) changes shown in Fig. 6. The specific growth rate increases withntration in batch cultures of cells from increases in pentachlorophenol concentration and reaches afrom the computer simulation were as maximum at approximately 300 ,ug/liter. Concentrations

Y0 = 140 ,ug/liter; Y = 0.136 g/g; K, = 60 above this level decrease the specific growth rate as theliter. effects of substrate inhibition become more pronounced.

These effects would not be predicted by the hyperbolicMonod function and indicate that the Haldane modification

t with the cell yield of 0.15 g/g should be used to describe the growth rate-substrate rela-d Finn (21) for bacterial growth on tionship for inhibitory compounds. Rozich et al. (20) havesole carbon source. also stressed the importance of including substrate inhibition

ents were conducted to evaluate the effects into predictive models for describing the removal ofhenol utilization. The rate of degra- inhibitory compounds in biological systems.roportional to the concentration of Due to the inhibitory nature of pentachlorophenol, con-

ventional continuous-culture kinetic studies were not feasi-ble since washout of the microbial population was likely athigh dilution rates. Moos et al. (16) have previously ob-served considerable instability during operation of continu-ous cultures on pentachlorophenol at high flow rates, com-plicating a kinetic analysis of the results. Thus, a fed-batchapproach was used in the present study to examine thekinetics of pentachlorophenol utilization under conditionsresembling continuous-culture operation. Computer analysis

0.05

zUc

0 30 60 90

Time (Hours)FIG. 5. Comparison of measured (0) and predicted (-) changes

in pentachlorophenol concentration during fed-batch operation ofreactor 2. Values estimated from the computer simulation were as

follows: P-rax = 0.074 h'; X0 = 920 ,g/liter; Y = 0.136 g/g; K, = 60,g/liter; and K, = 1,375 ,ug/liter.

0.04

0.03

0.02

0.01

0 400 800 1200 1600 2000

PmtachkrophenI Concentation (WUL)FIG. 6. Relationship between pentachlorophenol concentration

and microbial growth rate calculated by using the Haldane modifi-cation of the Monod equation. Values of the parameters used forcalculation were as follows: PLmax = 0.074 h-'; K, = 60 ,ug/liter; andK, = 1,375 ,ug/liter.

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70

2aa.

0

0)

0

(U)

CL0

0-

601-

501-

40

301

20

10

0

0 10 20 30Residence Time (Days)

40 50

FIG. 7. Relationship between mnean cell residence time (0c) andeffluent pentachlorophenol concentration for operation of a contin-uous-flow biological reactor. Values of the parameters used forcalculation were as follows: P-max = 1.78 day-1; Y = 0.136 g/g; Ks =

60 pg/liter; K, = 1,375 ptg/liter; and kd = 0.05 day-'.

indicated that the kinetic coefficients determined in batchexperiments also described the behavior of fed-batch cul-tures. The transient increase in pentachlorophenol concen-

tration during fed-batch operation can be attributed to theeffects of substrate inhibition and to decreasing the amountof active cell mass in the system. The good agreementbetween the experimental data and model predictions indi-cate that the kinetics of pentachlorophenol utilization are

adequately described by the Haldane modification of theMonod equation. Furthermore, the studies illustrate theutility of fed-batch experiments for examining the kinetics ofmicrobial growth on inhibitory substrates.The enrichment conditions used to isolate the mixed

culture preferentially selected for organisms capable ofgrowth at low pentachlorophenol concentrations. Analysisof the kinetic properties of the culture indicated that both themaximum specific growth rate (1max) and the Monod con-

stant (K5) are low, with values of 0.074 h-1 and 60 pg/liter,respectively. Jannasch (11) has previously noted that a

correlation may exist between microbial growth rates andsubstrate affinities. Organisms capable of high growth ratesat high substrate concentrations typically grow less effi-ciently at lower concentrations due to low substrate affinities(high K5). Alternatively, organisms that grow efficiently atlow substrate concentrations generally exhibit low growthrates at high substrate affinities (low K5). Note that thekinetic properties of the mixed culture differ considerablyfrom those reported by Stanlake and Finn (21) for an

Arthrobacter sp. capable of growth on pentachlorophenol.The latter organism was isolated from soils highly contami-nated with pentachlorophenol, using batch culture enrich-ment techniques at high substrate concentrations. Analysisof the kinetic properties of the Arthrobacter sp. suggest that

the Monod constant is high, with a value estimated to beapproximately 60 mg/liter. These observations illustrate thatthe conditions used for enrichment play an important role inselecting for a particular type of microbial population.An understanding of the kinetics of pentachlorophenol

degradation provides a foundation for process analysis anddesign for the optimum removal of the compound in waste-water treatment systerns. The low growth rate, cell yield,and effects of substrate inhibition indicate that relativelylong cell residence times will be required for the efficientremoval of pentachlorophenol. Mean cell residence time,that is, the average time a cell remains in the reactor beforeit is wasted or lost, has been widely used as a controlparameter for describing the steady-state performance ofbiological treatment systems. Mean cell residence time isinversely proportional to the net specific growth rate, andthus the effluent substrate concentration for a given resi-dence time can be calculated from knowledge of the kineticparameters. The relationship between effluent pentachlo-rophenol concentration and mean cell residence time for acontinuous-flow reactor is shown in Fig. 7. Operation of thesystem at short residence times leads to high effluent con-centrations; washout of the population is likely to occur at aresidence time of <2 days. Alternatively, high removalefficiencies are predicted at cell residence times of >10 days,which is consistent with the results obtained during opera-tion of the laboratory enrichment culture reactors. Thus,long mean cell residence times are an important controlparameter for the efficient removal of pentachlorophenol inbiological systems. Further applications of the kinetic pa-rameters in conjunction with predictive models, such as theone recently described by Rozich et al. (20), may revealadditional process control strategies for the optimum per-formance of wastewater treatment facilities.

ACKNOWLEDGMENTS

We thank F. T. R. McElroy III for many helpful discussionsduring the development of the project. We also thank G. L. Agin andL. S. Lickly for assistance with the computer model, C. G. Mendozaand S. J. Gonsior for analytical advice, and L. K. Roy for assistancein preparing the manuscript.

LITERATURE CITED1. Agin, G. L., and G. E. Blau. 1982. Application of DACSL (Dow

advanced continuous simulation language) to the design andanalysis of chemical reactor systems, p. 108-118. In R. S. H.Mah and G. V. Reklaitis (ed.), Selected topics on computer-aided process design and analysis. AIChE Symposium Series214, vol. 78. American Institute for Chemical Engineers, NewYork.

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