Pentachlorophenol biodegradation—I

Water Rer. Vo[. 17. No 1I. pp 1575-15S4. 1983 Printed in Great Britain. All rights reser,.ed

0043-1354 ~3 53 00 - 0.00 Cop?right q 1983 Pergamon Pre,,s Lid

PENTACHLOROPHENOL BIODEGRADATION--I AEROBIC L. P. Moos*. E. J. KIRSCH, R. F. WUKASCH a n d C. P. L. GRADY JR+++ Environmental Engineering Laboratory. School of Civil Engineering. Purdue University, West Lafayette. IN 47907. U.S.A. (Receiced January 1983)

Abstract--The biodegradation of pentachlorophenol (PCP) was tested in a three phase protocol. Phase I involved acclimation; phase II allowed determintion of biodegradation kinetics through use of continuous stirred tank reactors (CSTR) operated at solids retention times of 3.2, 7.8. 12.8 and 18.3 days: phase Ill assessed the importance of volatilization and sorption in PCP removal and evaluated the extent of biodegradation. Over the range of SRT's studied, PCP was found to be biodegradable with first order kinetics: the rate constant O~,/K~) had a value of 0.0017 l it g - td- t. The minimum concentration of PCP attainable in a CSTR was found to be 27 #g I-~. Additional studies suggested that the full relationship between the PCP degradation rate and the PCP concentration followed an inhibition-type function, with the maximum rate occurring at a PCP concentration of around 350 ,u g I - t. Radioisotopic studies revealed that PCP was mineralized by the culture, with the liberation of CO, and the incorporation of carbon into cell material. Neither volatilization nor sorption removed signific;mt amounts of PCP from the reactors.

NOMENCI,ATURE

P e n t a c h l o r o p h e n o l (PCP) is the second most heavily used pesticide in the United States, with approx.

8 0 m i l l i o n p o u n d s being m a n u f a c t u r e d in 1977 (Cirelli, 1978). The most significant source of PCPc o n t a i n i n g wastewaters is the wood preserving industry a l t h o u g h P C P has also been used as a fungicide/bactericide in cooling tower water, adhesives, c o n s t r u c t i o n materials, textiles, leather, paint, paper, oil well drilling m u d and m a n y other products. Because of its toxic properties it is also an environmentally significant chemical and is on the U.S. E n v i r o n m e n t a l Protection Agency's list of priority pollutants. T h e literature contains a fairly large n u m b e r of reports c o n c e r n i n g the aerobic biodegradation of PCP, a l t h o u g h most deal with its fate in soil (Moos, 1980). F u r t h e r m o r e , even t h o u g h n u m e r o u s a u t h o r s have found P C P to be degraded to a high degree in c o n t i n u o u s flow biological reactors (Dust et al., 1971: C h u a n d Kirsch, 1972, 1973; Dust and T h o m p s o n , 1973; Kirsch and Etzel, 1973; Etzel and Kirsch, 1975; W h i t e et al., 1976; Reiner et al., 1978), relatively few have a t t e m p t e d to delineate the kinetics of its biod e g r a d a t i o n in a way which would be useful for predicting its fate in wastewater treatment systems (Moos, 1980). Consequently, as part of a larger project (Kirsch et al., 1981), work was undertaken to determine those kinetics as well as to assess the i m p o r t a n c e of volatilization and sorption to its removal.

*Present address: Technical Services Branch, Bureau of Pollution Control, City of Atlanta, P.O. Box 93761, Atlanta, GA 30377, U.S.A. ?Present address: Environmental Systems Engineering, 501 Rhodes Engineering Research Center, Ciemson University. Clemson, SC 29631. U.S.A. {To v, hom all correspondence should be addressed.

Ot'erciew o f testing protocol The testing protocol employed in this stud? was developed to determine the fate of priority pollutants in biological wastewater treatment systems (Moos, 1980: Kirsh et al.. 1981). It contained three phases: acclimation: kinetic analysis: and evaluation of ultimate fate. Because of the potential

,4 hhreciations

CSTR = HRT = MLSS = PCP = RCF = SCOD = SRT = SS =

continuous stirred tank reactor hydraulic retention time mixed liquor suspended solids pentachlorophenol relative centrifugal force soluble chemical oxygen demand solids retention time suspended solids.

Symhols

b = specific decay rate of microorganisms (T- ') b, = specific decay rate of microorganisms responsible for degradation of a particular substrate (T-~) K = saturation constant (M L-3) K, = first-order rate constant for volatilization (T-~) - r , V = mass removal rate of PCP by volatilization ( M T -L) S = PCP concentration (M L -j) V = reactor volume (L 3) Y¢ = true growth yield ~l = specific growth rate of microorganisms ( T - t) gm= maximum specific growth rate of microorganisms (T-n).

INTRODUCTION

MATERIALS AND METHODS

1575

5"¢,

L . P . M{x)s et al.

economic and ecological significance of finding a test compound to be nonbiodegradabte, phase I (acclimation) was conducted in a manner which maximized the potential for de`,elopment of an acclimated biomass. The key principle employed ~'.as long-term exposure of the biomass to the test compound `,~hile maintaining continuous growth on a complex carbon source at ~er,, low specific rates, thereby alIo~'.ing m a x i m u m opportunity for enzyme induction and outgro`,'.th of PCP-degrading organisms. Furthermore. con* tinua[ inoculation was practiced throughout the acclimation period to maximize the chance input to the system of genetically capable organisms. Phase II testing for determining the kinetics of biodegradation was conducted primaril? using continuous culture techniques because of the advantages associated with them for such studies (Calcott. 1981). Although continuous culture experiments are time consuming, they ensure that the organisms are maintained in a constant average physiological state for each specific gro`,'.th rate and, when natural microbial populations are emplo?ed, they result in kinetic data which incorporate the changes in population diversity associated with changes in specific growth rate. Furthermore, as long as the specific gro~'.th rates employed are consistent with those utilized in actual waste~'.ater treatment systems the resulting rate data are applicable to predicting the fate of the test c o m p o u n d in those systems, even if the data do not fit any of the classical models. On the other hand. because wastewater treatment systems generally employ very low specific growth rates, continuous culture techniques are not likely to delineate the inhibition kinetics associated with c o m p o u n d s like PCP. Additional information can be gained by conducting batch experiments with organisms removed from the continuous culture reactors. Consequently, experiments of that type were also performed. The purpose of phase Ill testing `,'.as to determine the ultimate fate of PCP by estimating the import:race ofabiotic removal mechanisms. Two potentially important ones are volatilization (stripping) and sorption (e.g. physical adsorption, chemisorption, partitioning, etc.) (Matter-Muller ct al.. I981), 1981). In addition, evidence concerning the extent of biodegradation was obtained from batch experiments employing radiolabeled PCP and from continuous culture experiments in which PCP served as the soic carbon and energy source.

Phase l--accli.lation A iibre wall reactor, similar to the one described by Etzel and Kitsch (1975), was operated at a hydraulic residence time (HRT) of 12 h to produce an acclimated biomass. Prior to start-up the fiber wall was sealed by slowly adding 201. of activated sludge, obtained from a local treatment plant, while allowing the strained effluent to pass out through the overllow device. This formed a filter coat inside the enclosure thereby ensuring the production of a clear effluent. The use of the fiber wall vessel was equivalent to employing cell recycle from ti perfect settler. Furthermore. since no biomass was wasted during acclimation the solids retention time (SRT) was very long and the biomass concentration increased throughout the acclimation phase. This provided the reactor with a large inoculum of diverse organisms. The reactor was also seeded with the contents of a flask which had been incubated for two weeks after having 50 g of garden soil. 250 ml of raw sewage. 25 mg of Difco nutrient broth and 5 mg of PCP added to it. Acclimation was accomplished by continually pumping to the reactor a feed consisting of raw sewage, dog food extract and PCP. The sewage, originating from several dormitories and housing units on the Purdue University campus, plus the University Hospital and Pharmacy Building. was settled for 30 rain and filtered through a plug of glass wool. The soluble chemical oxygen demand (SCOD) was then determined and sufficient dog food extract was added to bring the SCOD to 200 mg I t. The dog food extract was prepared by placing approx. 900 g of dry-moist dog food (Gaines Top

Choice, General Foods Corp.) into 5 1. of deionized v.ater and autoclaving the mixture for 30 rain. Upon cooling, the supernatant was filtered through glass wool foLlo~'.ed by diatomaceous earth and the clear extract was frozen in small aliquots for future use. This extract contained approx. 53 g 1-* SCOD, 4 0 m g l - * N H ~ - N , 800 mg I- t organic nitrogen and 1800 mg 1-* orthophosphate. Reagent grade PCP (Aldrich Chem. Co., Milwaukee. WI) was added to the feed as the sodium salt. Initially the concentration v, as 1,0 mg 1 as PCP but this was gradually increased to 20 mg 1- * over a 90 day period. Performance analyses (e.g. SCOD, suspended solids, etc.) were not made routinely during acclimation although they were performed occasionally. Due to equipment problems. routine determinations of the effluent PCP concentration were not performed during the first month and a half of acclimation. After that they `,~ere made with the 4-aminoantipyrine technique.

Phase ll--kinetic analysis Continuous stirred tank reactors (CSTR) without cell recycle (i.e. H R T = SRT) were utilized during phase II testing. Eight reactors were employed, two at each of four HRT's. One reactor of each pair received feed which did not contain PCP in order to determine whether the presence of PCP influenced the removal of SCOD. The nominal H R T ' s employed were 3, 7, II and 15days. Because each reactor received a flow of 500 ml d a y - ~, the liquid volumes in the reactors were 1.5, 3.5, 5.5 and 7.5 t. The feed for the kinetic analysis was similar to that used during acclimation except that the SCOD was increased to 6 0 0 m g 1-~ with dog food extract and the PCP concentration was held constant at 20 mg I- ~. Four liters of feed were prepared every other day and it was kept refrigerated at 6 C to minimize growth of the organisms present in the sewage. Because of those organisms it ,,,.'as impossible to eliminate growth and SCOD loss in the feed lines which were tit room temperature (-~21 :C). as were the reactors. Consequently, several precautions were taken which reduced the loss of SCOD to less than 20",,,. Initial reactor operation took place for a period of approx. 4 months during which several problems with the original design were corrected (Moos, 1980). Operation continued for 3 m o n t h s after that and samples were taken 3 days per week for determinations of the concentrations of" SCOD and PCP. Those samples were removed directly from the reactors and were immediately centrifuged at 12,000 R C F (Sorvall RC2-B centrifuge) for 10rain at 4 C. The centrate required for SCOD analysis was added directly to a prepared C O D flask whereas the volume required for PCP analysis was placed into a 20 ml glass snap-top vial and refrigerated for 24 h or less prior to analysis. Twice a week the solids removed from the samples were used to measure the suspended solids concentration.

Phase lll--ecaluation o f ultimate ./~,te Three types of analyses were conducted during Phase lie sorption isotherms onto biosolids: determination of the rate constant for volatilization: and degree o1" biodegradation. The procedures employed are described in the Results section.

Analytical techniques The SCOD in the samples was determined by the dilute dichromate reflux method described in Standard Metho&" (APHA. 1975). An error analysis of this technique gave a coefficient of variation of 3% at 22.5 mg 1 Two methods of PCP analysis were used in the study. A modification of the 4-aminoantipyrine method, as described by Etzel and Kirsch (1975). was used during Phase 1. A more sensitive G C analysis was developed for the work in Phases II and I[1 (Moos, 1980). It involved benzene extraction of the PCP from a clarified, acidified sample: derivatization

Pentachlorophenol biodegradation--I v,ith 2". chloroacetic anh.,,dride: and clean-up v,ith NazCO> The instrument v,as a '*arian Model 3700 equipped with microprocessor control and automatic peak area integration. The column was a 3.2mm* 50cm stainless steel column packed ~ith Supelcoport G-1620. 1% SP-1240DA. 100 [20 packing. The minimum sensitiqty of the instrument ~as approx. 51~g I ~ PCP and the response was linear to 250 u g I t. An error analysis ga~e a coefficient of variation o r S ' , at ll)()ltg I ' The concentration of suspended solids (SS) was determined b~ using 0.45,~m pore size membrane filters (Millipore Corp.). The coefficient of variation was 6.6"i, at 133 mg 1- ~. RESLLTS

AND

DISCUSSION

Phase [--acclimation Although no analyses tbr PCP were performed during the first month and a half of acclimation the PCP concentration was gradually increased to 4 mg I E. When routine PCP analyses were begun the concentration in solution was found to be below the detectable limit of 1001tgl-~ and continued to be so during the remainder of the acclimation period as the influent PCP concentration was increased to 20 mg I - ' . Furthermore, spot checks of the S C O D concentration in the reactor showed it to be less than 40 mg I -~. Thus, it was concluded that acclimation bad been achieved and the decision was made to move to Phase II testing. The relative ease of acclimation indicates that organisms existed in the seed stock which possessed the genetic capability for degrading PCP and that the acclimation procedure was satisfactory for allowing their enrichment within the biomass. Although acclimation of microorganisms to PCP has been achieved by others as cited earlier, other published reports seem contradictory to this. For example, Heidman et al. (1967) were unable to acclimate a biomass when PCP was at a concentration of l0 mg I-~. Watanabe (1977) showed that PCP at a concentration of 10 mg I ~ significantly inhibited the growth of PCPdegrading organisms. Consequently, it appears critical to start acclimation at low concentrations and to

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Phase ll--kinetic analysis Because the reactors were mixed by aeration, relatively high air flow rates were required. This. coupled with the high H R T ' s employed, caused significant evaporative losses of water in spite of the fact that the air supply was humidified. By* collecting and measuring the etfluent volumes, the magnitude of this loss was determined, thereby allowing correction of the H R T (and SRT) in the reactors. The corrected values were 3.2, 7.8, 12.8 and 18.3days. The evaporation also tended to concentrate the insoluble and nondegradable soluble matter in the reactors. Ahhough this has little significance to determination of the normal kinetic parameters, it affected calculations involving suspended solids concentrations. Figures I and 2 present the routine operational data for the reactors during the three month stable operating period. Reactors R I - R 4 were the test reactors which received PCP whereas reactors C l - C 4 were the controls. Except for some minor perturbations, the concentrations of S C O D and SS stabilized quite nicely throughout the operating period, with the S C O D concentration falling below 30 mg I - ' . Since the concentration of S C O D in the influent was 600 mg I- z during this phase, the S C O D removal efficiency generally exceeded 95";. With the exception of reactor R4 ( S R T = 1 8 . 3 d a y s ) . the S C O D in the reactors decreased as the SRT was increased, in conformance with most kinetic concepts. It was interesting to note that reactor R4 contained dense granular floc not seen in the other reactors. When examined microscopically, each aggregate was found to consist of a central core of bacteria entirely surrounded by' rotifers typical of the genus, Philodena. It is likely that mass transfer requirements into the dense central core were the cause of the higher S C O D in that reactor. Finally', several spikes in effluent S C O D can be observed for both the PCP and the control reactors. These coincided with operational episodes which stressed the reactors for short periods of time. Figure 3 shows the changes in PCP concentration with time. A high degree of variability in effluent ~-

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Fig. 3. PCP concentration in test reactors during phase II testing. concentration was noted, although considerably greater stability was achieved at longer SRT's. Jank and Fowlie (1981) also observed considerable variability during pilot scale treatment of a wood preserving wastewater containing PCP. The periods of highest PCP concentration seemed to correlate with periods of highest SCOD concentration, although the magnitude of the increase was far greater for PCP than for SCOD. It is important to note, however, that given time, the systems invariably recovered with restoration of both SCOD and PCP degradation. Apparently the biological system was able to adapt to increasing PCP concentration. The irregularities in response in reactor RI (and perhaps R2) make it difficult to argue that it achieved steady state, and from a practical point of view, suggest that the SRT must exceed 7.8 days for consistent PCP degradation. In spite of these variations, the mean level of PCP in the reactor effluents decreased with increasing SRT, even though during some periods of operation the PCP concentration in reactor R4 was higher than in reactor R3. This was particularly true when the floc in reactor R4 was dense and heavily burden with rotifers. When the SCOD and SS data were plotted on log-normal probability paper they were found to follow a straight line and thus log-normal distributions were used to compute the means and standard deviations. PCP data below 350pg I -~ also fit a log-normal distribution although data above that value did not, as shown in Fig. 4. The reason for this will become apparent later, but the result was that

only concentrations below 3501tg I-~ were used to calculate means and standard deviations for operation in the noninhibitory region. Extremely low data points which did not fall on the line were also excluded. Table 1 presents the mean values and 95'~.~, confidence limits on those means as calculated using the Student's t distribution. The objective of this study was to obtain biodegradation data in such a way that it could be used to predict the fate of PCP in an aerobic wastewater treatment system. Cursory examination of the data collected clearly showed that the effluent PCP concentration was related to the SRT. If kinetic constants were obtainable from these data. one should be able to predict the level of PCP in the effluent at any chosen SRT. The question of the prediction of the concentration of a single component in a multicomponent substrate is one that has received little attention. Believing that a simple and straight-forward approach is the most practical when it works, it was decided to try a noninteractive model (Bader, 1978) to allow prediction of the PCP level. Basically, such a model assumes that only a portion of the biomass is contributing to the removal of the specific pollutant in question and that there is a functional relationship between the specific growth rate of that portion and the concentration of the pollutant in the reactor. A widely used functional relationship in the noninhibitory concentration range is that of Monod (Grady and Lira, 1980). That expression may be simplified to a first-order relationship when the substrate concentration, S, is small with respect to the saturation constant, K<. The PCP data in Table l indicate that

T a b l e 1. Steady-state performance o f C S T R ' s C O D (mg I -~) Reactor No.

SRT days

Lower limit"

RI R2 R3 R4 CI C2 C3 C4

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Pentachlorophenol biodegradation--[ this condition was probably met in these experiments and thus the Monod equation was simplified to

u = (It,,, K,)S

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(2)

where h, represents the loss of PCP degrading organisms by decay'. The similarity of b, for the PCPdegrading organisms to the specific decay rate of the biomass as a whole, b. is unknown, and thus for the time being it must be assumed to have a value characteristic of the PCP-degrading biomass alone. Other work conducted in our laboratory, but not presented here, suggested, in fact, that the two decay rates were quite similar. A simple equation relating the reactor PCP concentration to the SRT can be obtained by combining equations (I) and (2) and rearranging terms: S = (K~/~L.,) (1/SRT) + (l~/~.,)b,.

(3)

Thus a plot of the PCP concentration in the noninhibitory range as a function of 1/SRT should yield a straight line with a slope equal to KJ,u., and an ordinate intercept of (K,/~l,.)b,.. Figure 5 shows such a plot made from the data in Table I. Examination of it shows that an excellent fit was obtained (correlation coefficient = 0.998), which suggests that on pragmatic grounds the use or the noninteractive model and the first-order approximation were

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1579

justified. The slope of the line was 593~(g day 1 ' making the first order rate constant (lz,, K.J equal to 0.0017 I ,ug-' day-*. The intercept was 27.ug I giving a value of b,. of 0.046. A physical meaning may be attached to the intercept value of 27,ug 1-' in a truly noninteractive system. It is the minimum concentration possible in a single CSTR, provided that PCP is being used to drive the growth reactions needed to balance the cells lost by decay. McCarty et ul. (198l), on the other hand, have proposed the concept of secondary utilization whereby the concentrations of individual substrates may be driven to lower values through use of other primary substrates for maintenance energy needs. This represents one type of interactive system. Another type of interactive system has been demonstrated by Law and Button (1977) who observed that the concentration of one constituent in the teed to a CSTR was driven to lower and lower concentrations in the reactor as the number of compounds in the feed was increased. Both of these suggest that it may indeed be possible to drive individual substrate concentrations to values lower than the valve given bv the ordinate intercept and thus care must be taken in application of these results. Nevertheless, the restihs presented here suggest that it may be difficult to obtain PCP concentrations lower than about 30 .ug I -~ in completely' mixed activated sludge reactors, regardless of how long the SRT is made. One question of significance when PCP is being degraded in the presence of other carbon sources is whether the PCP had any' influence on the kinetics of degradation of that other organic matter. This question can be addressed by performing a kinetic analysis using the SCOD data. When standard techniques were used to evaluate the true growth yield (Ye) and the specific decay rate (by as well as the first order rate constant (p,,/K,) no significant differences (down to the 751~,; level) were found in the slopes of the lines drawn through control reactor and test reactor data (Moos, 1980; Kirsch et al., 1981). Thus it appears that PCP had little impact on the removal of other organic matter in the reactors. An important benefit of the kinetic approach used here is that no knowledge is required of the abiotic removal mechanisms or of the amount of biomass bringing about the removal of a particular pollutant when determining its steady-state concentration in a single CSTR, whether with or without cell recycle. This follows from the fact that the concentration of that pollutant is functionally related to the specific growth rate of the microorganisms involved, which is in turn controlled by the characteristics of the reactor and its operation. It should be recognized, however, that this is only true for a single CSTR at steady state. If the reactor cannot be approximated as completely mixed, then the contribution of the other removal mechanisms must be considered as well. Furthermore, the importance of those mechanisms is important from an ecological viewpoint. Thus the', `,,.ere

580

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studied during Phase Ill testing and ~ill be discussed later. First, ho~vexer, it is important to consider the kinetics of PCP removal at concentrations above the noninhibitory range. It has hypothesized that the ~ariabilit.,, of the PCP data, particularly from reactors v, ith short SRT's. was related to the inhibitory properties of PCP to bacteria responsible for its degradation (Chu, 1972: Watanabe, 1973. 1977: Suzuki, 1977). PCP inhibition was suggested bv the data in Fig. 4. particularly for reactor RI (SRT = 3.2 days} where two log-normally distributed data populations appeared, the second being apparent abo~e PCP concentrations of 350 ug I-E. Furthermore. the data from the other reactors also deviated from the log-normal distribution at concentrations above that value. All of these data support the hypothesis that above this level of PCP the degradation rate is redvced as the PCP concentration increases. This phenomenon has been described as substrate inhibition (Edwards, 1970) and leads to a situation wherein two steady' states can exist at a single SRT: a stable one at a low, noninhibiting concentration of substrate and an unstable one at a higher, inhibiting concentration. To determine if substrate inhibition was affecting the degradation of PCP, the following experiment was performed. Four reaction flasks '*ere prepared by diluting a concentrated slurry' of test reactor cells with filtered control reactor eMuent to give suspended solids concentrations of 50, 50, 300 and 3000 mg I r. The two flasks containing 50 mg I ~ of cells were spiked with PCP at concentrations of 100 and 500 !lg 1-~: the flask containing 300mg 1 j of cells was spiked to 2000/.tg 1 -~ while the one with 3000 mg 1 -E of cells was ,.riven 12,0001~g l -~ of PCP. The solids concentrations were chosen to give an approximately constant percent removal of PCP per hour for each of the flasks based upon the specific removal rates in the test reactors and inhibitory effects its reported in the literature. Before the cells used in the flasks containing 2000 and 12,000,ug I * PCP were used in the test they were exposed to their respective concentrations of PCP for 1 h. This was done to minimize any possible sorption of PCP onto the cells which might be interpreted as biodegradation during the actual experiment. Pre-exposure was not employed for the cells spiked with 100and 5 0 0 1 1 g l - t of PCP because the cell and PCP concentrations employed were so low as to make any potential sorption negligible. After the cells were placed into the flasks and spiked with the appropriate quantities of PCP, the concentration of PCP present in the liquid phase was determined at various times over a 12 h period. Smooth curves were dra~vn through the points and the resulting curves were graphically differentiated to obtain the specific degradation rate of PCP as a function of its concentration. Figure 6 shows the result of that analysis for flasks containing 50 and 300mg I t of cells. The flask containing 3000rag I ~ of cells and t2,000/~g I ' of PCP was

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I I 400 600 PCP

I I [ 1 I I [ 800 I000 1200 1400 1600 1800 2000 conc. (k~g l - ' )

Fig. 6. Effects of PCP concentration on its own removal rate as determined in batch experinaents. severely inhibited, although some PCP removal did occur. Examination of Fig. 6 reveals several important points. First and [bremost is that the curves have the classical shape of substrate inhibition (Edwards, 1970), i.e. a maximum removal rate is attained at some finite substrate concentration. When the cell concentration was 50 mg 1- t the maximum removal rate occurred at a PCP concentration of 3 5 0 - 4 0 0 # g I ', a value close to the threshold concentration for the second family of log-normally distributed data observed in the continuous flow experiments (Fig. 4). The use of a higher cell concentration shifted the PCP concentration associated with the maximum removal rate to a higher value, in the range of 8 0 0 - 1 2 0 0 # g I t. Furthermore. the maximum specific degradation rate observed with the higher cell concentratin was also higher. Chu (1972) reported a similar effect in studies related to the biodegradation of PCP by Arthrohacter KC3. In spite of this shift in the maximum point, the case For substrate inhibition by PCP is strong and its implication to reactor performance is evident. The previous experinaents were done using PCP as the sole substrate wheras in the continuous flow reactor studies PCP was supplied along with other biodegradable organic matter. Consequently. the experiments were repeated with the addition of dog food extract to make the S C O D 100 mg 1-~. Another modification was to increase the cell concentration from 50 to 100 mg I - ' in the flasks receiving 100 and 500 fig 1-~ PCP. When the data were analyzed it was found that the addition of extraneous organic matter increased the maximum specific PCP removal rate by' an order of magnitude, strongly suggesting that cometabolism or secondary utilization may be involved in PCP biodegradation, It did not alter the substrate

Pcntachlorophenol biodegradation--[ inhibition character of PCP remo,,al however, nor did it significantly change the PCP concentration at which the maximum PCP removal rate occurred. This striking influence of extraneous organic substrates clearly demonstrates the necessity t\~r conducting biodegradation studies of this type with multicomponent media. If the continuous culture studies had been done with PCP alone the,, ~,,ould have greatly underestimated the kinetics of PCP removal likely to occur in wastewater treatment plants. It is apparent from these studies that the bioremoval of inhibitory substances may be associated with very complex, concentration dependent interactions. Such interactions may make the use of simple st, bstrate removal rate relationships difficult because such rates have been shown to depend upon the substrate concentration, the nature of the substrate and the cell concentration. It is ot, r contention, however, that kinetic analyses based upon the results from continuous flow reactors receiving a complex multicomponent media provide the most reliable approach to the prediction of the fate of inhibitory substrates in wastewater treatment systems especially when coupled with ancillary experiments to delineate the inhibitory characteristics of the compound. In any event, one can begin to understand the re~tson for the greater variability of PCP concentration encountered in the reactors with short SRT's. A CSTR operated at a short SRT will operate at a higher average PCP concentration than one operated at a long SRT. The higher the average PCP concentration, the closer to the maximum point on the removal curve the reactor is operating and the greater the probability that natural variations in the influent or in system performance will push the PCP concentration across the peak into the region ,,',,here the PCP removal n, te decreases with increasing concentration. When such a shift occurs the PCP concentration in the reactor will continue to rise until some factor, at this point unknown, allows the biomas to adapt to the higher concentration of PCP and remove it rapidly enough to return the removal rate to the left side of the peak. A reactor with a long SRT, on the other hand, will be operating farther to the left of the peak so the likelihood of the peak concentration being exceeded is small. Under that condition perturbations in the character of the influent or the performance of the system will be selILcorrecting and there ,,`.ill be less variation in the PCP concentration. An important point to arise from this work is that decisions concerning the design characteristics of an activated sludge plant removing inhibitory material must consider the perturbations likely to occur in the system. Not only will a longer SRT reduce the average pollutant concentration, it will also reduce the variability associated with that concentration regardless of its source. In addition, the observation that the degree of inhibition exhibited by PCP is influenced by the biomass concentration introduces another consideration into activated sludge process

158.1

design. According to generall~ accepted design concepts (Grad> and Lira. I980) once the SRT has been chosen, an'. reasonable ,,alue ma,, be selected for either the MLSS concentration or the aeration basin ,.olume so long as their product is held constant. The results of this study suggest, hov`.ever, that the choice of a high MLSS concentration for a given SRT `.`.ill result m a more stable process because it ,.`.ill increase the biomass concentration in relation to the concentration of inhibitor,, substrate. No data are yet available which address this contention directly, so this constitutes an area ~vhich requires further research.

Pha,w III

czaluu:ioH q/ ultimate /i~:~'

So far. the tacit assumption has been made that PCP disappearance was due to biodegradation and indeed the fact that there was a strong functional relationship between organism specific growth rate and PCP concentration suggests that this was the case. As discussed earlier, however, it is necessary to strengthen this contention by evaluating the importance of abiotic removal mechanisms to its loss. Furthermore, even if abiotic mechanisms are unimportant, the possibility exists that only primary biodegradation was occurring, i,e. PCP removal represented a minor alteration in structure which made PCP undetectable by the analytical technique used, but, did not lead to significant loss of PCP carbon. Thus it was necessary to estimate the extent of biodegradation and describe the ultimate fate of the PCP molecule. SorFlion. Ideally. the sorptive characteristics of the biomass for PCP should be assessed by inactivating acclimated biomass from a test reactor to stop metabolism and then using it as the sorbent in a classical isotherm determination. The problem with this approach is that any measure severe enough to stop biological activity is also likely to alter the sorptive properties of the biomass. Alternatively. unacclimated biomass might be used provided it is reasonably representative of the acclimated biomass. Under the experimental conditions employed, in which the organic carbon contributed by the PCP constituted less than 5"{; of the organic carbon in the feed to the CSTR's. the bulk of the biomass ,.,,ill have arisen from the background organic matter rather than from the PCP. This suggests that the use of unacclimated biomass is reasonable and alter a preliminary experiment in which it was found that the concentration of PCP in solution was stable for at least 16 h after sorption had occurred (i.e. there was no metabolism occurring) the sorptive characteristics of the biomass for PCP were determined using unacclimated cells. This w~s done by performing a classical adsorption isotherm experiment: biomass with concentrations from I000 to 3000mg I-~ was brought into contact with PCP at concentrations from 100 to 10.000/lg I -t in shake flasks, mixing was continued for 20 h, the biomass was removed by

[5S2

L.P. M(x)s e[ al.

2000

7

1800

o_ o O.

1600

~. 1400

G

12o0

X o o

I000

~,

eoo

~

6oo 400

~. zoo 03

I I000

I 2000 PCP

I 3000

conc. (,u.g

l 4000

t-')

Fig. 7. Isotherm for sorption of PCP onto unacclimated biomass. centrifugation and the residual equilibrium PCP concentration was measured by gas chromotography. The amount of PCP sorbed by the biomass was determined as the difference between the initial and final concentrations and the specific capacity at the various equilibrium PCP concentrations was calculated by dividing the loss of PCP by the cell concentration employed. The resulting isotherm curve is shown in Fig. 7. Attempts to fit the data to the Freundlich and Langmuir models were unsuccessful. The contribution of sorption to the removal of PCP can be determined from the isotherm curve because the long HRT's employed in the test reactors make it likely that equilibrium was approached closely. The amount of PCP sorbed per unit of biomass was determined for each reactor by entering Fig. 7 with the mean PCP concentration in the reactor and reading the sorptive capacity from the graph. Multiplication of the sorptive capacity by the mass of solids grown and removed daily gave the mass of PCP removed daily by sorption. Performance of these computations revealed that the sorptive losses were only 0.31, 0.15, 0.10 and 0.08~o of the mass applied for reactors with SRT's of 3.2, 7.8, 12.8 and 18.3 days, respectively. It should be recognized that for a given SRT and influent characteristic the amount of PCP removed from a biological reactor by sorption will be independent of the MLSS concentration employed. This is because the mass of MLSS associated with a given SRT is constant and independent of the MLSS concentration. Furthermore, so is the mass of MLSS wasted per day. Since the mass of PCP removed by sorption is the product of the mass of MLSS wasted per day and the sorptive capacity associated with those solids, the concentration of MLSS is irrelevant.

On the other hand, the importance of sorption as ~ mechanism of PCP removal is very much dependent upon the relative concentrations of biodegradable SCOD and PCP in the influent. The higher the concentration of organic matter in the influent the greater the mass of MLSS associated with a given SRT and the greater the mass which must be wasted daily. Since the sorptive capacity depends on the average PCP concentration (which depends on the SRT) more PCP will be removed as more cells are wasted. Thus sorption could ,,,,'ell be a significant mechanism if the ratio of SCOD:PCP in the influent is very high. In this case, however, sorption was insignificant. Volatilization. A long term, unsteady-state air stripping test was performed to determine the extent of volatilization of PCP from the reactor contents even though its physical/chemical characteristics suggested that it was not susceptible to such removal. This was done by spiking filtered and autoclaved control reactor effluent with 100,ug I-~ of PCP in a sterile reaction vessel. The vessel was aerated by passing a stream of sterile, humidified air through fritted glass diffusors. Any water lost by evaporation was replaced daily with sterile deionized water. Over a 2week period samples For PCP analysis were periodically removed from the vessel through a rubber septum using a syringe. Assuming that the equilibrium concentration of PCP with the incoming air was zero, the first-order rate constant for the volatilization process, K,., was determined by plotting the natural log of the PCP concentration versus time and measuring the slope. The value was 0.0076 day An estimate of the contribution of volatilization to the removal of PCP from each reactor can be obtained from:

- r , . V = K,.SV

(4)

where --r,.V is the mass removal rate of PCP by volatilization (M T - ' ) ; K, is the first-order rate constant ( T - '); S is the mean PCP concentration in the reactor (M L-3); and V is the reactor volume (LJ). Calculations of the stripping losses were made for each reactor, based upon the asumption that the value of K,. is the same in all reactors and equal to the value measured experimentally in the stripping test, i.e. 0.0076 d a y - ' . These showed that the maximum volatilization loss was only 0.037"i;. Actually, K,. will be different for each reactor because the reactor geometries and air flow rates were different. However, the estimated loss of PCP by volatilization was so small that more accurate estimates based upon corrected K,. values were considered to be unnecessary. The contribution of volatilization to the removal of PCP in actual activated sludge reactors would be even lower because the hydraulic retention times in those systems are much smaller than the values employed in this research. The shorter the hydraulic retention time the less the importance of volatilization. Thus,

Pentachlorophenol biodegradation--I

for a given SRT,

the choice of reactor volume will have an impact upon the importance of volatilization but not upon the importance of sorption. Extent of biodegradution. Having found that volatilization and sorption were not removing significant amounts of PCP from the test reactors, the last question to be addressed was whether mineralization of PCP was actually occurring or whether the culture was merely altering PCP's structure sufficiently to prevent its detection. This was done by first looking for the release of labeled CO, by microorganisms degrading ['aC]PCP and second, by maintaining a CSTR with PCP as the sole carbon and energy source. For the radioisotope study six Warburg flasks (125ml) were charged with 25ml each of a suspension containing 500 mg I ' of biomass taken from a CSTR actively degrading PCP. Randomly labelled [~aC]PCP with a nominal specific activity of 500 cpm #g-~ was added to give a PCP concentration of 2000 #g 1-'. Two wicks, made of filter paper moistened with a 10% solution of hyamine hydroxide in methanol, were placed in the center wells of the flasks to absorb CO: from the gas space. The flasks were sealed and continuously mixed to allow metabollsm to occur. At selected intervals 0.5 ml of 5"/ H,SO4 was added to one of the flasks to stop metabolism and release any radioactive bicarbonate to the gas phase. The CO, collector strips were placed in 25 ml of scintillation fluid consisting of 0.4'~ 2,5-diphenyloxazole in 50"~, 2-ethoxyethanol and radioactivity was analyzed in an ISOCAP/300 liquid scintillation counter. Appropriate corrections for background were made. Figure 8 shows that '4CO, was rapidly evolved for around 6h, at which time

20,000 o

18,000

/

o

~E 16,000 o... <.)

approx. 67°0 of the PCP carbon had been collected as CO,. This value is typical of the amount of CO, released during cell growth on an organic substrate and is very similar to the work by Chu (1972) who showed release of 68° o of PCP carbon as CO, by Arthrobacter KC3 which was capable of using PCP as a sole source of carbon and energy. Suzuki (1977) showed that 50°o of the PCP carbon fed to a culture ended up as CO: with the remainder being incorporated into cell material. Thus it appears quite likely that PCP underwent ultimate biodegradation and that its disappearance from the CSTR's was due to mineralization. To further demonstrate that PCP was being mineralized in the CSTR's the influent to test reactor R2 (SRT = 7.8 days) was gradually depleted of all organic contituents except PCP in three stages. Initially the reactor contained approx. 185 mg 1-t of cells and received 600 mg 1-' of SCOD in the feed. However, when it was shifted to a feed containing filtered control reactor effluent (SCOD g 22 mg 1- ') to which 1 mg 1- ' nitrogen as urea and 0.2 mg l - ' phosphate were added, the cell concentration began to dilute out. This feed was maintained for 14days (---2 SRT's). For the next 21 days (---_3SRT's) standard BOD dilution water made with well water plus 5 nag I - ' yeast extract was supplied. Finally, for 14days BOD dilution water made from deionized water plus 15,?~ well water and l mg 1-' nitrogen added as NH4HCO3 was supplied. All of these feeds contained 20mg I - ' PCP. When the PCP concentratin in the effluent was monitored, the PCP levels were shown to never exceed 6 0 0 # g I -~, as shown in Fig. 9. Since there was ample time ( ~ 7 SRT's) for cell washout to occur, because the reactor walls were scraped routinely on a daily basis, and since stripping was a neglibible factor, de not'o synthesis of cells must have occurred to keep the concentration of PCP well below the input level of 20rag I - ' . In other words, new PCP-degrading cells must have replaced those which were washed out, demonstrating that mineralization of PCP was occurring.

la,O00

700 StageI

0 £3

E

1583

12,000

I0,000

/

Stage

2

Stage

3

'"600

8,000 CPM added

to slurryinitially

~ 500

v

=~ 400 o u

~~-o ~-~ °6,0008'00024,~000,000 Cell conc.=500mgk-

O_ 3 0 O 2OO I00

o 1

0

2

4

I

I

I

I

I

6

8

IO

12

14

Elapsed

I

time ( hours )

Fig. 8. Release of radiolabeled CO, by a microbial slurry degrading randomly labeled [14C]PCP.

5

K)

15

20

Elapsed

I

1

I

I

1

25

30

35

40

45

time

(days)

Fig. 9. Concentration of PCP in the effluent from a CSTR receiving PCP at a concentration of 20 mg I-~. Each stage represents a period in which PCP constituted a greater percentage of the influent carbon. During stage 3 PCP was the sole carbon and energy source. SRT = 7.8 days.

1584

L.P. Moos etal. SI_TMMAR~ AND CONCLUSIONS

The b i o d e g r a d a t i o n of p e n t a c h l o r o p h e n o l (PCP) was tested in a three phase protocol. Phase I involved acclimation: phase II allowed d e t e r m i n a t i o n of biDd e g r a d a t i o n kinetics t h r o u g h use of c o n t i n u o u s stirred tank reactors operated at solids retention times of 3.2, 7.8, 12.8 and 18.3days: phase III assessed the i m p o r t a n c e of volatilization and sorption in PCP removal and evaluated the extent of biDdegradation. F r o m the results of the experiments the following conclusions can be drawn. (1) PCP is biodegradable and acclimation can be readily achieved using c o n t i n u o u s e n r i c h m e n t techniques provided that the c o n c e n t r a t i o n of P C P is kept low d u r i n g initial exposure and is increased slowly over a long time period. (2) At low c o n c e n t r a t i o n s ( < 2 5 0 , u g 1 t) the relationship between the bacterial specific growth rate and the PCP c o n c e n t r a t i o n can be expressed as a first order relationship with a rare c o n s t a n t (~L,,,/KO of 0.0017 I r a g - ' day -~ when PCP is supplied in a mixture of c a r b o n sources. (3) At higher c o n c e n t r a t i o n s PCP inhibits its own b i o d e g r a d a t i o n but the PCP c o n c e n t r a t i o n at which the m a x i m u m b i o d e g r a d a t i o n rate occurs is a function o f the biomass c o n c e n t r a t i o n . Higher biomass c o n c e n t r a t i o n s can tolerate higher PCP concentrations. (4) S o r p t i o n is a m i n o r mechanism for PCP removal from c o n t i n u o u s flow reactors like those employed herein. (5) Volatilization of P C P is insignificant in continuous flow reactors like those employed herein. (6) P C P undergoes ultimate biodegradation. Acknowledgement--This research was supported by the U.S. Environmental Protection Agency through Cooperative Agreement No. 12-805858 with Purdue University.

REFERENCES

APHA (1975) Standard Methods for the Examination o f Water and Wastewater, [4th Edition. American Public Health Association, Washington, DC. Bader F. G. (1978) Analysis of double-substrate limited growth. Biotechnol. Biogengng 20, 183-202. Calcott P. H. (1981) Continuous Culture o f Cells, Vols. I and II. CRC Press, Boca Raton, FL. Cirelli D. P. ([978) Patterns of pentachlorophenol usage in the United States of America. In Pentachlorophenol; Chemisto', Pharmacology and Encironmental Toxicity (Edited by Rao K. R.), pp 13-18. Plenum Press, New York. Chu J. P-H. (1972) Microbial degradation of pentachlorophenol and related chlorophenols. Ph.D. Thesis, Purdue University, West Lafayette, IN. Chu J. P-H. and Kirsch E. J. (1972) Metabolism of pentachlorophenol by an axenic bacterial culture. Appl. Microbiol. 23, I033~t035.

Chu J. P-H. and Kirsch E. J. I1'173~ Utilization of halophenols b? a pentachlorophenol metaboli,,ing bacterium. Dec. Ind..~licrohiol. 14, 260-273. Dust J. V. and Thompson W. S. (1973) Pollution control in the wood preserving industr?. Part IV Biological methods of treating wastev, ater. For. Prod. J. 23, No. 9.50-66. Dust J. V., Thompson W. S.. Shindala A. and Fancinque~ N. R. {1971) Chemical and biological treatment of ~astewater from the wood-preserving industr}. Proc. 2&h lnd Waste Con/. Purdue Unit. En'4n'4 Bull. Ext. Set No. 140. 227-243. Edwards V. H. (1970) The influence of high substrate concentration on microbial kinetics. Beotechnol. Biocny, n.k, 12, 697-712. Etzel J. E. and Kirsch E. J. (1075) Biological treatment of contrived and industrk, l ~aste~ater containing pentachlorophenol. Dec. Ind..~licrohio[. 16, 287-295. Grady C. P. L. Jr and Lira H. C. 11980) Bioh,t#cal Waswwater Treatmettt: Theory and .4pplication.v. MarceI Dekker, New York. Heidman J. A.. Kincannon D. F. and Gaud.v A. F. ([967) Metabolic response of ,'Lctivated sludge to sodium pentachlorophenol. Proc. 22nd Ind. Waste Cont. Purdue L'nic. Engng Bull. Ext, Ser. No. I2% 661-674. Jank B. E. and Fowlie P. J. A. (1981) Treatment o f a ,aood preserving elfluent containing pentachlorophcnol b~ activated sludge and carbon adsorption. Proc. 35th Ind. Waste ConJ] 198"0 Purdue Unit'.. pp. 63-79. Ann Arbor Science, Ann Arbor, M[. Kirsch E. J. and Etzel J. E. ([973~ Microbial decomposition of pentachlorophenol, d. Wag. Pollut. Cemtrol Fe~L 45, 359-363, Kirsch E. J., Grady C. P. L. Jr and Wukasch R. F. (1981) Protocol development for the prediction of the fate of organic priority pollutants in biological ~astewater treatment systems. Report to the U.S. Environmental Protection Agency on Cooperative Agreement No. 12-805858. Law A. T. and Button D. K. {19771 Multiple-carbon-sourcclimited growth kinetics of a marine cor.~neform bacterium. J. Baet. 129, I15-123. Matter-Muller C., Gujer W. and Giger W. (I9811 Transl'er of volatile substances from water to the atmosphere. I,Valer Res. 15, 1271-1279. Matter-Muller C., Gujer W., Giger W. and Stumm W. (1980) Non-biological elimination mechanisms in a biological sewage treatment plant. Pro',,,. Wag. Technol. 12, 299-314. McCarty P. L.. Reinhard M. and Rittmann B. E. (1981) Trace organics in groundwater. Encir. Sci. Technol. 15, 40-51. Moos L. P. (1980) The development of a testing protocol to determine the biodegradability of pcntachlorophenol in activated sludge systems. MSCE Thesis. Purdue University. West Lafayette, IN. Reiner E. A., Chu J. and Kirsch E. J. (1978) Microbial metabolism of pentachloropheno[. In Pentachlorophenol: Chemistry. Pharmacology and 7}J.vieiO'{Edited by Rao K. R.), pp 67-81. Plenum Press. New York. Suzuki T. (1977) Metabolism of pcntachlorophenol by a soil microbe. J. encir. Sci. tflth BI2, 113-127, Watanabe I. (1973) Isolation of pentachlorophenol decomposing bacteria from soil. Soil Sci. Plant Nutr. 19, 109-116. Watanabe I. (1977) Pentachlorophenol decomposing and PCP-tolerant bacteria in field soil treated ~ith PCP. Soil Biol. Biochem. 9, 99-103. White J. T. et al. (1976) Treating wood preserving plant wastewater by chemical and biological methods. U.S. Environmental Protection Agency Report No. EPA600/2-76-23 I.