Kinetics of Multiple Phenolic Compounds

Kinetics of Multiple Phenolic Compounds Degradation with a Mixed Culture in a Continuous-Flow Reactor Author(s): BumHan Bae, Robin L. Autenrieth and James S. Bonner Source: Water Environment Research, Vol. 67, No. 2 (Mar. - Apr., 1995), pp. 215-223 Published by: Water Environment Federation Stable URL: http://www.jstor.org/stable/25044540 Accessed: 17-06-2016 19:22 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at http://about.jstor.org/terms

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Kinetics of multiple phenolic compounds degradation with a mixed culture in a continuous-flow reactor BumHan Bae, Robin L. Autenrieth, James S. Bonner

ABSTRACT: A multiple substrate kinetic model was tested to predict the growth of a mixed culture receiving multiple phenolic compounds. Kinetic constants of the model were estimated from the results of initial

rate experiments with the mixed culture obtained from a continuous

flow reactor (CFR) at steady state. The suggested model was useful to predict the interactions of substrates on the growth of a mixed culture.

In the long-term study using three CFRs with internal cell recycle, 748 to 810 mg/L of influent phenol were completely degraded (hydraulic retention time = 36 hours). The addition of dichlorophenol (DCP) and/ or pentachlorophenol (PCP) into the phenol feed caused system fluc tuation. In a reactor fed phenol and DCP, both compounds were con

sumed completely, whereas the addition of PCP resulted in incomplete

biod?gradation of all phenolic compounds. However, the presence of DCP in a feed increased the removal of PCP up to 43%. The growth of the mixed culture on dual- and triple-phenolic compounds was predicted using the model. The long-term exposure of the mixed culture to DCP

and/or PCP resulted in decreased maximum growth rates and increased substrate inhibition, perhaps caused by a loss in bacterial species diversity

in the mixed culture. Water Environ. Res., 67, 215 (1995).

KEYWORDS: biod?gradation, dichlorophenol, kinetics, pentachlo rophenol, phenol, substrate.

Many microorganisms can use xenobiotic compounds for growth or degrade those compounds by several mechanisms such

as cometabolism. Degradation can occur under aerobic and/or anaerobic conditions, depending on target compound, micro organisms, and environmental conditions. A number of these anthropogenic toxic chemicals are biodegradable if they induce or other substrates induce the synthesis of appropriate enzymes, or if there exists microbial enzymes that accept structurally sim

ilar compounds (H?tzinger and Veerkamp, 1981). Biological treatment of toxic, recalcitrant compounds has become a viable treatment option for these pollutants because of the advance in

biodegradability studies as well as the development of biotech nology (Grady, 1989). Another advantage of biod?gradation is the potential for complete mineralization or, at least, the re duction of toxicity if the treatment system is carefully designed and properly operated to prevent production of secondary pol lutants. The activated sludge process is easy to handle, well understood, and a relatively simple method. It is the most widely used method in wastewater treatment and the best documented in the liter

ature. This process is an engineered system that uses a mixed culture of microorganisms to remove a mixture of pollutants from wastewater. By using a mixed culture, advantages such as enhanced biod?gradation capacity, an increased resistance to toxic compounds, and interactions of multiple species on de grading xenobiotics can be expected (Grady, 1989). Also, the

plasmid transfer between species contributes to the evolution of

degradation pathways for xenobiotic compounds. However, washout or instability may occur by the influent containing toxic

compounds, which results in discharge of pollutants into re ceiving water. Phenolic compounds including chlorinated phenols are fre quently encountered components of industrial wastewater and a major concern for environmental engineers. They comprise 10 out of 114 EPA-designated organic priority pollutants (Eck enfelder, 1989), and chlorinated phenols are frequently suspected carcinogens or teratogens. Chlorophenols are major components of paper pulp bleach plant effluent. 2,4-dichlorophenol (DCP) is used as a precursor for the synthesis of the 2,4-dichlorophen oxyacetic acids (2,4-D), and DCP was successfully treated by a pure Pseudomonad culture in a batch reactor (Tyler and Finn, 1974) and by a mixed culture in a continuous reactor (Chudoba et al, 1989). Pentachlorophenol (PCP) is widely used as an her bicide, fungicide, and wood preservative (Bryant et al, 1991). Also, it is reported to be the second highest used pesticide in the United States (Hickman and Novak, 1984). Because of its highly chlorinated structure, it is a recalcitrant compound and a poor growth-supporting substrate (Stanlake and Finn, 1982). It can even inhibit photosynthesis and growth of aquatic algae (Gotham and Rhee, 1982). However, several studies showed that PCP can be degraded, possibly by cometabolism, by either acclimated microbial consortia or PCP-degrading bacteria (Kirsch and Etzel, 1973; Stanlake and Finn, 1982). Moos et al, (1983) reported the fate of PCP in continuously stirred tank reactors that showed

high instability due to its toxicity. Kinetic studies using an en richment culture in a batch reactor showed development of mixed culture with high affinity to PCP (Ks = 60 /ug/L) (Klecka

and Maier, 1985). In another study, PCP concentrations up to 12 mg/L was degraded to less than 10 ug/L in activated sludge reactors with 10 to 20 days of solids retention time (Melcer and Bedford, 1988). For these inhibitory compounds, the Haldane equation is re garded as the most acceptable kinetic expression for the growth

of microorganisms (Pawlowsky and Howell, 1973; Chi and Howell, 1976;Beltrame^a/., 1980; D'Adam?la/., 1984; Roz ich and Gaudy, 1985; Rebhun and Galil, 1988; Hobson and Mills, 1990; Buswell, 1975; Sokol and Howell, 1981). However, many studies have focused on a single substrate, although actual wastewater always contains multiple carbon sources (Grady, 1989). Considering the coexistence of noninhibitory and inhib itory substrates in wastewater, the effect of multiple substrates on microbial growth is not adequately characterized under single substrate conditions. Previous studies showed that the presence

March/April 1995 215


Bae et al.

Sp *$DCP Ksp Ksdcp

of growth-supporting noninhibitory substrate stimulated the synthesis of active cells, which increased the stability of reactors and consequently increased the removal rate of the target com

pound (Papanastasiou, 1982; Topp and Hanson, 1990). Other studies showed preferential use of a less inhibitory substrate (phenol) over a more inhibitory substrate (2,4-DCP) that resulted

in reduced removal rate of 2,4-DCP (Namkoong et al, 1989). Kinetic studies regarding multiple substrates are very limited.

After the observation of diauxie effects by Monod (Gaudy and Gaudy, 1980), substrate interactions on the microbial growth were of concern. Substrates in the culture medium can be used sequentially, simultaneously, or competitively. A kinetic model for the growth of an organism on multiple substrates assumed a competitive inhibition by one substrate on the other substrate

uptake (Yoon et al, 1977). According to this model, the coex istence of several species in a continuous mixed culture is possible

during steady state if more than one substrate is available. Bader (1978) suggested the presence of two substrates could affect the

? = Mmax

Sp, Sdcp, Spcp = phenol, DCP, and PCP concentrations, respectively, mg/L; KsP, Ksdcp = half-saturation constants for phenol and DCP, respectively, mg/L; and Kip, KiDCp, Kipcp = inhibition constants for phenol, DCP, and PCP, respectively, mg/L.

Assuming that removal of each growth-supporting substrate (phenol and DCP) contributes to the growth of total cell mass with respective cell yield (YP and Ydcp):

~-df-?P?X (3)

less than the saturating concentrations. If the interaction is small,

The main objective of this study was to characterize the deg radation of inhibitory multiple phenolic compounds in a mixed culture CFR and adapt the interactive multiple-substrate model

(McCreary, 1990) appropriately. A second objective was to in terpret the estimated kinetic coefficients to predict the effects of

multiple inhibitory substrates on the reactor operation. This study showed that the suggested inhibitory multiple-substrates kinetic model was a useful tool to predict the growth of a mixed

culture on the multiple phenolic compounds. Long-term op eration of CFRs showed that the mixed culture arrived at the first steady state when phenol was the sole carbon source. How

ever, transition into another steady state was observed in all reactors after the introduction of DCP and/or PCP in the phenol

feed.

Model Development For an inhibitory substrate, the Haldane equation can be ap

plied:

(1)

S2

Ks + S + ? Ki

p = cell growth rate coefficient, hr-1;

Where

?dT-rn1* (4)

?p? Ydcp = cell yield for phenol and DCP, respectively; and X = total biomass concentration, mg/L. The respective cell yields for individual substrate to the total cell mass (X) can be measured with the initial rate experiment coupled with the method of excess that minimizes the effects of other substrates (Grady and Lim, 1980). This kinetic model re duces to the Monod equation if only one noninhibitory substrate exists. If only one inhibitory substrate exists, this model reduces

to the Haldane equation.

Materials and Methods The experiments were divided into two phases: long-term ex periments for a steady-state determination and short-term batch experiments to measure kinetic constants using the initial rate method. Short-term experiments were conducted on steady-state

acclimated cultures from CFRs. All experiments and analyses were performed at room temperature (25 ?C ? 1?C). Long-term experiments. The degradation of phenolic com pounds and the change in biomass were examined through long term experiments with three identical CFRs. In each CFR, a stable mixed culture at steady state was obtained. A flow sche matic of the CFR is presented in Figure 1. An internal settling tube was used to recycle effluent biomass. Sterile, humidified air ensured aerobic conditions at airflow rates that varied slightly

from 2.32 to 3.25 L/min with dissolved oxygen concentrations

Where /?max = maximum cell growth rate coefficient, hr"

(2)

Where

growth of the organisms if the concentrations of substrates are the noninteractive type model may predict the behavior of mi croorganisms (Bader, 1978). McCreary (1990) developed a mul tiple-substrate interactive model based on the Monod and Hal dane equations, and the dual-substrate enzyme equations (Bailey and Ollis, 1986), assuming the interactions of substrates on the growth of microorganisms.

1 4. ^p i ^DCP , Sp , ^DCP , iVSp JvSdcp Klp tVtjDCP Kip

i.

S = substrate concentration, mg/L; Ks = half-saturation constant mg/L; Ki = inhibition constant, mg/L. The interactive multiple-substrates model (McCreary, 1990) was expanded for two growth-supporting inhibitory substrates (phe

nol and DCP) and one nongrowth-supporting inhibitory sub strate (PCP).

of 4.3 ? 0.5 mg/L in all reactors. Design hydraulic retention time (HRT) was 36 hours with a constant influent flow rate of 3.3 mL/min. At start-up, three reactors were inoculated with activated sludge obtained from the Texas A&M University wastewater treatment plant (College Station, Tex.). The mixed culture was acclimated to synthetic phenol wastes by slowly increasing the feed rate up to design flow rate. Initially, phenol was the only available carbon source to ensure selection of phenol-degrading organisms. After the steady state under phenol-only condition (first steady state) in all CFRs, chlorinated phenols (DCP and/ or PCP) were introduced to the influent under the same reactor 216 Water Environment Research, Volume 67, Number 2


Bae et al.

INFLUENT

l.Carbon Adsorption Column 2.Humidifier 3.Air Flow Regulator

4.Reactor

5.1nfluent Sampling Port 6.1nfluent Jar

7.EffluentJar

Figure 1 ?Continuous-flow reactor with an internal recycling tube. operational conditions at day 186. The chlorinated phenols were added for the phenol-acclimated cultures to degrade chlorinated

to the method described by Autenrieth (1986). Using a phosphate buffer, pH was maintained at 7.2 ? 0.2. Synthetic phenolic feeds

phenols and to obtain multiple substrates-acclimated cultures so that the mixed culture in the steady state under multiple phenolic compounds (second steady state) could be used for the measurement of kinetic constants. The substrate combinations

were prepared by adding nutrient stock solution, 15.0 g of crystal

of phenolic compounds to each reactor are as follows: 750 mg/ L phenol with 1.0 mg/L PCP (reactor PP); 750 mg/L phenol with 1.0 mg/L DCP (reactor PD); 750 mg/L phenol with 1.0 mg/L DCP and 1.0 mg/L PCP (reactor PDP). Short-term experiments. A series of batch reactors were op erated to measure the growth rate and substrate consumption

was monitored by measuring total suspended solids (TSS), total organic carbon (TOC), and individual substrate concentrations. During each sampling time, 10 mL of reactor sample was taken and filtered through a tared 0.45-um glass fiber filter (Gelman Inc.). The filter was used to measure TSS concentration, and the filtrate was used to measure TOC and individual phenolic compound concentration. An influent sample was taken from a sampling port located in the feed line. The TSS was analyzed using a microwave suspended solids analyzer, Model No. AVC 80 (CEM Industries). The TOC was analyzed (Dohrmann DC 800) in duplicate with an injection volume of 40 pL. High per formance liquid chromatography was used to quantify phenol, DCP, and PCP with a Supelcosil? LC-8 reversed-phase column at 280 nm. Throughout the experiments, 1.0% acidified methanol and 1.0% acidified water were used as eluents. The flow rate was

of the mixed culture in steady-state CFRs. From the steady-state

reactor, five aliquots of 45 mL of culture suspension were col lected and centrifuged at 33 000 g (0?C) for 5 minutes, and the supernatant was decanted. The biomass pellet was resuspended with equal amount of nutrient solution (without a carbon source) at the same strength of phosphate buffer. This washing and re suspension step was iterated twice. The biomass was dispersed thoroughly with manual and mechanical mixing before pouring into a batch reactor. Besides the preparation of biomass, five 1 L batch reactors containing 300 mL of nutrient solution at the same strength of phosphate buffer were preaerated with 1.0 L/ min airflow rate. To start the initial rate experiments, either single substrate or a combination of substrates solution, plus 45 mL of biomass suspension, and nutrient solution was combined to give a final volume of 500 mL. Initial conditions in the batch experiments were quantified for biomass and substrates concen trations. After 1.5 to 3.0 hours, samples were taken to measure the final condition. All samples in the short-term experiments were taken in duplicate. The changes in biomass and individual substrate concentration were measured to calculate the growth rate.

phenol, and 20.0 mL of 1 000 mg/L DCP and/or PCP stock solution into a Nalgene plastic jar, subsequently bringing the volume to 20 L with deionized water. The behavior of CFRs

1.6 mL/min, and the gradient condition was 70% H20 to 30% MeOH to 10% H20 to 90% MeOH over 30 minutes. According to preliminary experiments, TSS was insensitive to detect the small changes of biomass during the initial rate experiments. Because the change of biomass was too small to be detected by TSS measurement, a calibration curve of TSS versus cumulative volume of biomass measured by a Coulter particle counter was used instead. Duplicate samples were col lected from each batch reactor and sonicated for 5 seconds in

Chemicals and analysis. For the preparation of synthetic phe nolic wastes, 99.9% purity phenol (Sigma Chemical Company),

pulse mode to reduce floe size. Sonicated samples were diluted to 1/100 with ISOTON? II solution and a drop of ionic dis persant to minimize the coincidence level to less than 5% during particle counting. Isolation and identification of bacterial culture. The microbial

99.8% purity of DCP (Sigma Chemical Company), and 93%

populations in each reactor were isolated and identified revealing

purity pentachlorophenol sodium acetate (Aldrich Chemical Company) were used. All chemicals used for the preparation of

count agar (Standard Methods, 1991) during steady state. After

nutrients solution were of reagent grade. A thermodynamically balanced nutrient stock solution (10X) was prepared according

were transferred to a TSA medium for identification by a whole

several species. Each reactor sample was streaked on a plate incubation at 28?C ? 0.5?C, all the colonies in the petri dish



Bae et al._ cell, fatty acids analysis (Miller and Berger, 1985). The isolated species identification was subcontracted to a commercial mi crobial laboratory for identification (Silliker Laboratory, College Station, Tex.). The whole cell, fatty acids analysis and compar ison with database were performed in the Department of Plant Pathology and Microbiology, Texas A&M University. The iso lation and enumeration was performed in our laboratory. For the preparation of culture medium, DIFCO? technical grade agar and tryptone glucose yeast extract agar were used. Analysis of experimental data. Cell growth rate from the results

of initial rate experiments was determined using the following

equation:

*???- (6) AS/at

Where

AS = the change of substrate concentration during the time interval, mg/L; and q = specific substrate removal rate (mg/biomass h). The calculated growth rate was then fitted either to the Haldane

model or to the interactive model by using a parameter esti mation routine (Ernest et al, 1991 ) to estimate kinetic constants of mixed culture.

Results and Discussion

Where AX = the change of biomass concentration during the incu bation time interval, mg/L; X = average of concentration of biomass at time zero and time U mg/L; and At = time elapsed for the reaction, hr. For the growth-supporting substrates, the specific substrate use

rate is

Continuous-flow reactor. The inoculated mixed culture quickly acclimated to synthetic phenol-only (750 mg/L) wastes. After 1 month, TSS concentrations in all reactors were greater than 1 500

mg/L with a color change from brown to bright yellow. The acclimation process of three CFRs is presented in Figure 2. The PD reactor reached a steady state 32 days after inoculation. The steady state of PP reactor and PDP reactor was achieved by day 38 and day 66, respectively. During this period, phenol was completely removed in all three reactors. The performance of three CFRs during the first steady state is summarized in Table

a. TOC and TSS in reactor PD

+ TSS o Influent TOC Effluent TOC

OX)

S 3000

fi O

2000

c

1000

-T?r~

fi O

100

lJS?_ _

200 ?. .. . 300

Time (days)

b. TOC and TSS in reactor PP

400

500

+ TSS o Influent TOC Effluent TOC

CL

B 3000 2000 1000 ?

500

Time (days) c. TOC and TSS in reactor PDP

0 100

+ TSS O Influent TOC Effluent TOC

200 ^ ,. v 300 Time (days)

400

500

Figure 2?Monitored TSS and TOC concentration in continuous-flow reactors. 218

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Bae et al.

Table 1?The performance of three CFRs during the first

steady state that was provided phenol only as the substrate.

Concentration,

mg/L Reactor PD Reactor PP Reactor PDP

TSS 2 848 ? 202 2148 ? 186 2 745 ? 176 TOC influent 747.8 ?160.1 780.6 ? 37.7 809.9 ? 7.6 TOC effluent 42.0 ? 15.6 60.9 ? 43.5 40.8 ? 12.5

Phenol influent 759.8 ? 71.2 728.8 ? 23.2 731.2 ? 8.1 Phenol effluent N.D.a N.D. N.D. aN.D., nondetectable.

I. Reactor PDP showed large variation of TSS and TOC con

Table 2?The performance of three CFRs during the

second steady state with addition of phenol and

chlorinated phenols fed condition.

Concentration, mg/L Reactor PD Reactor PP Reactor PDP

TSS TOC influent TOC effluent Phenol influent Phenol effluent

DCP influent DCP effluent PCP influent PCP effluent

1 633 ? 292 1 405 ? 506 1 616 ?318 757.1 ? 1.2 855.6 ? 17.7 814.3 ? 10.6 42.1 ?1.6 81.0 ? 10.8 46.0 ? 10.6 747.8 ? 10.3 766.3 ? 23.2 744.5 ?6.1 0.8 ?0.1 0.5 ? 0.7 0.1 ?0.2 1.1 ?0.2 0.84 ?0.1 N.D. 0.14 ?0.1 0.91 ? 0.01 0.86 ? 0.07 0.88 ? 0.07 0.53 ?0.1

N.D., nondetectable.

centrations during the acclimation period (Figure 2c), sometimes

resulting in incomplete removal of phenol. Despite the same reactor setup, the PP reactor showed a lower concentration of TSS (2 148 mg/L) during the phenol-only condition. This might be caused by the lower cell recycle rate (95%) compared with the other two reactors (98%). The solids retention time (SRT) for each reactor was calculated based on the observed TSS con centrations in the reactor and in the reactor effluent. Calculated

SRT values in reactors PD, PP, and PDP were 75, 30, and 75 days. The large variation of SRT results from the measured dif ferences in the effluent TSS concentration for the three reactors.

Because the CFRs had an internal settler with no cell wastage, the SRT became solely dependent on the ratio of TSS in the reactor and in the effluent at a fixed HRT. Moreover, the SRT became extremely sensitive to the ratio of TSS because of the higher HRT (36 hours) and cell recycle (>95%). Therefore small changes in effluent TSS concentration (20 to 60 mg/L) resulted in large variations of SRT. Consequently, differences in SRT are overmagnified because of the lower sensitivity of the TSS measurement technique. The addition of DCP and PCP caused a gradual decrease in TSS and increase in effluent TOC concentrations in all reactors. This effect was most severe in reactor PDP which was fed both DCP and PCP. The TSS concentration decreased as low as 210

mg/L in reactor PDP. To prevent washout, the influent flow rate was reduced to 2.2 mL/min from 3.3 mL/min from day

Because TSS measurement could not detect the small change of microbial concentration during the short incubation time, the change of microbial volume was used to detect the change of biomass. The linear relationship between cumulative micro bial cell volume and TSS is presented in Figure 3. Evaluation of steady-state mixed cultures receiving individual substrates was performed using initial rate experiments. Results of initial rate experiments on single substrate for the mixed cul

tures at both steady states showed that phenol, DCP, and PCP are all inhibitory substrates. Table 3 summarizes the kinetic coef ficients for each reactor under two conditions: the first at a steady

state with cultures acclimated only to phenol and the second, at a new steady state with cultures acclimated to multiple sub strates. Experimental results for PCP showed that the total cell volume in a batch reactor increased with the decrease in PCP concentration. However, PCP was not considered a growth-sup porting substrate for the parameter estimation of multiple sub strates. This was assumed because the estimated growth for PCP was an order of magnitude smaller than the growth rate of the

other two compounds.

3000

223 to day 327. During this time, large clumps of microbial floe accumulated in the recycling tube. Proper recycling of biomass and removal of effluent stream was hindered by the presence of

the huge mass of bacterial floes (diameter of 1 to 2 cm) inside of the settling tube. The SRT in reactor PD, PDP, and PP was as low as 24.2, 3.9, and 2.4 days, respectively. However, SRT slowly recovered to the first steady-state condition as the reactors

became stable. The second steady state in reactor PP was achieved at day 400. The steady state in reactor PDP was achieved at day 424. The PD reactor had steady state at day 454. The reactor performance during the second steady state is

summarized in Table 2.

Initial rate experiments. The kinetic constants for the mixed

culture in all CFRs at both steady states were measured using initial rate techniques (Grady and Lim, 1980). Initial rate ex periments on the phenol-only reactors were performed only on single substrate. Initial rate experiments on the multiple-sub strates reactors were performed by first evaluating microbial growth on the individual compounds and then in combinations.

0 500 1000 1500 2000 2500 3000 3500 4000 Mixed culture cumulative volume (106pm3/mL)

Figure 3?Relationship between cumulative cell volumes determined by a particle size analyzer and TSS concen trations.



Bae et al.

Table 3?Estimated model coefficients from the Haldane equation for single substrate conditions in continuous-flow reactors.

Reactor PDP

PD

PP

Substrate

Steady state

Mmax? hr

Ks, mg/L

Ki, mg/L

266.2 125.7 26.4 1.8 0.6 2.8 74.6

82.9 12.3 9.4 3.2 3.5 28.9

Phenol

First3

DCP

First

PCP

First

Phenol

First

0.91 0.90 0.76 0.31 0.02 0.07 0.96

DCP

First

0.48

19.9

11.7

Phenol

First

PCP

207.9 121.2 3.6

37.5 35.6

First

0.90 0.85 0.08

Second6

Second Second

Second

Second

Second Second

1.1

1.1

3 Under the condition of single substrate. b Under the condition of multiple substrates.

Small values for Ki indicates large inhibition because Ki is in the denominator in the Haldane equation. As the value of in hibition coefficient (Ki) shows, higher substrate inhibition was observed in the order of PCP, DCP, and phenol. The growth rates of mixed cultures in phenol-only fed condition varied from 0.90 to 0.96 hr"1. Compared with other studies on a pure culture

the absence of phenol, which was used as a sole carbon source. The presence of these species may explain why the mixed culture had lower Ki, higher Ks, and higher growth rate than those of

pure cultures. Comparison of kinetic constants for the first steady state (phe

(Table 4), the mixed cultures used in our experiments showed

nol-only condition) and the second steady state (multiple-sub strates condition) shows that the mixed cultures became more

significantly higher growth rates. However, there are two major differences between the mixed cultures in this experiments and

addition. In reactor PDP, the growth rates decreased from 0.91

the pure cultures in Table 4. First, the mixed cultures used in our experiments exhibited greater inhibition than the pure Pseudomonas cultures. The inhibition constant (Ki) for phenol in our experiments, under phenol-only conditions, varied from 28.9 to 82.9 mg/L compared with 106 to 470 mg/L of the pure cultures (Table 4). Second, the pure cultures had higher substrate affinities than those of our mixed cultures, that is, the range of Ks values in the pure cultures is significantly lower than the range of Ks value in the mixed cultures. The pure cultures, ca pable of phenol utilization as a sole energy source, can be char acterized as a system with less inhibition (higher Ki), high affinity

for substrate (lower Ks), and slow growth, whereas the reverse applies to the mixed cultures in this study. In a mixed culture, the interactions between microbial species may be an important

factor for the degradation of xenobiotic compounds (Grady, 1985). A portion of mixed culture with phenol degradation ca pability removes phenol and secretes metabolites or cell lysis materials into the culture medium. Then other species may use these as carbon sources. The average effluent TOC concentrations in Table 1 shows that small amounts of carbon were present in

inhibited to phenolic compounds as a result of chlorinated phenol

to 0.90 hr1 (phenol) and from 0.76 to 0.31 hr1 (DCP). Also, the inhibition constant (Ki) decreased in all cases reported, in dicating greater inhibition. The inhibition constant for phenol decreased from 82.9 to 12.3 (PDP) and from 37.5 to 35.6 (PP). However, the cultures had more affinity for the substrates, one of the characteristics of the pure culture. These results are con sistent for PCP and DCP as shown in Table 3. Rozich and Gaudy (1985) reported the response of phenol-acclimated activated sludge system to quantitative shock load. In this study, they suggested the longer transient behavior of the activated sludge system may occur because of the ecological changes of biomass, which is more serious in the system treating toxic compounds. Therefore, by the addition of PCP and/or DCP in the feed, the characteristics of mixed cultures shifted, resulting in more in hibitions when only phenol was fed. The estimated kinetic constants using the results of initial rate experiments on dual and triple substrates are presented in Table 5. The initial rate experiments on dual or triple substrates were conducted during the second steady state by varying the con centration of one substrate while maintaining constant concen

Table 4?The reported kinetic constants of a phenol-degrading bacterial culture and respective culturing conditions. Authors Hill and Robinson (1975) Yang and Humphery (1975) Chi and Howell (1976) Kotturieia/. (1991)

220

Temperatures, ?C ??max? hr 1 Ks, mg/L 30 30 30 10

0.534 0.567 0.369 0.119

0.015 2.39 5.94 5.27

Ki, mg/L

Species

470 106 227 377

P. putida P. putida Pseudomonas P. putida

Fermentation mode Batch and continuous

Continuous Transient Batch

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Table 5?Estimated coefficients of the proposed multiple substrate interactive model for dual- and triple- substrate conditions in continuous-flow culture reactors. Reactor ??max, hr 1 KsP, mg/L KsDCP, mg/L KiP, mg/L KiDCP, mg/L KiPCP, mg/L PDP

PCP + phenol (92.7 mg/L) 0.62 8.38 DCP + phenol (108 mg/L) 0.79 4.98

? 1 874 ? 0.37 0.85 110.4 1.60 ? 2.82 167.7 1.17 0.28

PCP + DCP (11.3 mg/L) + phenol (111 mg/L) 0.78 9.37

PP

PCP + phenol (84 mg/L) 0.96 11.0

? 154.4 ? 0.25

trations of the other substrate. The experimental concentration is also presented along with the kinetic constants. The results

culture in the presence of PCP and phenol was 0.62 hr-1, whereas

ment of cell volume. The aggregation of cells was most severe in triple-substrate experiments. Mixed-culture characterization. The primary bacterial species were tentatively identified as Bacillus firmus, B. subtilis, B. pumilis, B. sphaerieus, Pasteurella multucida, Pseudomonas

the individual ^max on phenol and PCP during the second steady

aeruginosa, and P. testosteroni during the first steady state. After

showed that the interaction of substrates significantly affects the behavior of the mixed cultures. In reactor PDP, ixmax of the mixed

state were 0.90 and 0.02 hr-1, respectively. The trend of substrate

the addition of DCP and/or PCP to feed, only three dominant

interactions was consistent on all the results presented in Table

species from each reactor were isolated and identified by a whole

5. In general, the presence of phenol seems to enhance the growth

cell, fatty acids analysis. All three dominant species were ten tatively identified as a Pseudomonas species; P. cepacia gas Chromatograph (GC) Subgroup B, P. cepacia intermediates of Subgroup A and Subgroup B, and P. testosteroni. P. cepacia is known as a d?grader of phenol and other chlorinated hydrocar bons (Folsom, 1990). Karns et al (1983) reported that P. cepacia AC1100 can dechlorinate several chlorophenols including PCP. From our studies, it is not clear whether these species are re sponsible for the degradation of PCP and why this species was not isolated during the first steady state because no pure culture study was conducted. The two P. cepacia species were different in shape and color of the colony on the agar medium. This intermediate species is thought to be a mutant of P. cepacia GC Subgroup B because 71.6% of the fatty acid chromatogram matched to the Subgroup B. This evolution is strengthened by the lengthy acclimation period in excess of a year. Heipieper et al (1991) observed the modification of cell membrane in bac

of mixed cultures on chlorinated phenols. The results of exper iments were successfully fitted to our suggested model (Figures 4, 5, and 6). The substrates do not act independently; there are interactions on microbial growth that were not characterized by other models that did not consider the interaction effects. As shown in Figures 4 and 5, the results of initial rate experiments on dual substrates, phenol, and different concentrations of PCP

or DCP were successfully fitted to Equation 2. As the concen tration of DCP or PCP increases, the growth rate of a mixed culture decreased because of the inhibition of DCP or PCP. However, DCP slightly contributed to the growth of a mixed culture (Figure 5). In the case of triple substrates, the results of initial rate experiments fluctuated over a wide range as shown in Figure 6, but the model still fits the trend of the results. The variation in experimental value may be caused by the aggregation

of cells during the experiments that caused inaccurate measure

0.4

0.16 ^? Model fitted

0.3

A Observed

Q 0.14 u

Model fitted

? 0.12

A Observed

0.10

0.2 1

0.08

0.06

0.1

0.04 0.0

0.0 2.0 4.0 6.0 8.

10.0

PCP Concentration (mg/L)

0.02-1- -1-?-1-?-r 0.0 10.0 20.0 30.0 40.0

50.0

DCP Concentration (mg/L)

Figure 4?Comparison of the observed growth rates of a mixed culture (reactor PDP) on dual substrates in the

Figure 5?Comparison of the observed growth rates of a mixed culture (reactor PCP) on dual substrates in the

presence of 92.7 mg/L phenol and the model-fitted results

presence of 108 mg/L phenol and the model-fitted results

as a function of PCP concentration.

as a function of DCP concentration.



Bae et al._ 0.10

The results of initial rate experiments and the estimation of kinetic constants showed that strong interactions of substrates

0.091 0.08

? Model fitted

0.07

Observed

exist for the growth of a microbial culture with multiple substrates

and mixed-culture conditions. In the presence of phenol, the growth rate of the culture on PCP or DCP significantly increased,

0.06 0.05

whereas the growth rate on phenol decreased in the presence of chlorinated phenols. The estimated kinetic parameters suggest

0.04

a change in the mixed-culture characteristics between the cultures

0.03

0.021 0.01 0.00 0.0

?I?

?I?

2.0

4.0

?i?

6.0

?i?

8.0

10.0

PCP Concentration (mg/L)

Figure 6?Comparison of the observed growth rates of a mixed culture (reactor PDP) on triple substrates in the presence of 111 mg/L phenol and 11.3 mg/L DCP and the model-fitted results as a function of PCP concentration.

of the first steady state and the cultures of the second steady state. Identification of dominant species in the mixed cultures revealed a significant reduction in species diversity after the ad dition of chlorinated phenols. Because of the introduction of DCP and/or PCP into the reactors, less tolerant and adaptive species to chlorinated phenols had gradually disappeared, in spite of the internal cell recycle resulting in the dominance of three highly adaptive Pseudomonas species in the reactors. The re maining species had more affinity for the phenolic compounds; however, the estimated value of /?max and Ki decreased. For treatment systems receiving toxic wastewater, long-term exposure to inhibitory substrates causes the change of microbial characteristics that leads to inadequate treatment. As alternatives, periodic enrichments of the reactor to provide species diversity

or batch type reactor may be adequate so that the treatment terial cell that was previously cultured in phenol. They suggested that this modification is the response of cells to phenol to reduce

system still retains microbial diversity.

the permeability to phenol through the membrane. Scanning

Acknowledgments

electron micrographs showed us that the cell membrane of two

P. cepacia is quite different even though the size and shape is almost identical (micrographs not shown). In this context, the mutant observed in our cultures was thought to be a membrane modified species of P. cepacia. Other species, P. testosteroni, was consistently identified throughout the experiments. The shift of microbial species in the CFR system may be due to the adverse environments, especially the toxicity of chlorinated phenols. The presence of chlorinated phenols was apparently too toxic for the survival of other original species present. Species diversity was reduced and the kinetic constants were altered, but specifically how these two phenomena are related was not revealed in our study. However, the Pseudomonas species, known to be phenol degraders, prevailed in the mixed culture, and the affinity for substrates increased as seen in pure cultures (Table 4). The loss in species diversity in the presence of multiple substrates rendered the bacterial culture more vulnerable to the effects of substrate

inhibition as indicated by the Ki values (Table 3). Also, the decrease in growth rate may represent less effective use of carbon

sources in a less diverse population.

Credits. Larry Barns in the Department of Plant Pathology and Microbiology at the Texas A&M University identified the bacterial species. Andy T. Ralph assisted in the experiments and operation of the reactors. Part of this manuscript was presented to 65th Annual Conference of Water Environment Federation, 1992. This research was funded by the Gulf Coast Hazardous Substance Research Center, Contract No. 100TAM0203, which is supported under cooperative agreement R818197 with the U.S. Environmental Protection Agency. The contents do not necessarily reflect the views and policies of the U.S. EPA nor does the mention of trade names or commercial product con stitute endorsement or recommendation for use. Authors. BumHan Bae is a Ph.D. student in the Department of Civil Engineering at the Texas A&M University. Robin L.

Autenrieth and James S. Bonner are Associate Professors in the Department of Civil Engineering at the Texas A&M University.

Correspondence should be addressed to Robin L. Autenrieth, Department of Civil Engineering, Texas A&M University, TX

77843-3136.

Submitted for publication February 16, 1994; revised manu script submitted May 13, 1994; accepted for publication July 15,

Conclusions Phenolic wastewater was successfully treated by a mixed cul ture in three continuous-flow reactors. Using continuous-flow reactors with an internal settling tube, 729 to 760 mg/L of phenol

were completely removed at 36 hours of HRT. The addition of 1.0 mg/L DCP and/or PCP in a phenol feed inhibited the growth of mixed cultures. As a result of chlorinated phenol addition, 0.1 to 0.8 mg/L of phenol was detected in all reactor effluents. The PCP was persistent in all reactors. In the reactor receiving 1.0 mg/L PCP with 750 mg/L phenol, only 3% of PCP was removed. However, in the reactor receiving 1.0 mg/L PCP, 1.0 mg/L DCP, and 750 mg/L of phenol, 38% of PCP was removed. The presence of DCP seems to encourage the removal of PCP. The DCP was completely removed when fed with phenol.

1994. Deadline for discussions of this paper is July 15, 1995. Discussions should be submitted to the Executive Editor. The authors will be invited to prepare a single Closure for all discus sions received before that date.

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