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Temperature Effects and Substrate Interactions During the Aerobic Biotransformation of BTEX Mixtures by Toluene-Enriched Consortia and Rhodococcus rhodochrous Rula A. Deeb, Lisa Alvarez-Cohen
Department of Civil and Environmental Engineering, 631 Davis Hall, MC 1710 University of California at Berkeley, Berkeley, California 94720-1710; telephone: (510)-643-5969; fax: 510-642-7483; e-mail: [email protected] Received 18 March 1998, accepted 18 August 1998
Abstract: A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20°C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35°C. A consortium enriched at 35°C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35°Cenriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35°Cenriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and non-
Correspondence to: L. Alvarez-Cohen Contract grant sponsors: NIEHS; NSF; EPA
© 1999 John Wiley & Sons, Inc.
competitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 526–536, 1999.
Keywords: BTEX; gasoline aromatics; Rhodoccus rhodochrous; inhibition; degradation kinetics; substrate interactions
INTRODUCTION The United States has thousands of soil and groundwater sites that are contaminated with petroleum hydrocarbons resulting from industrial activities related to refining, transportation, use, and disposal of petroleum products (Cozzarelli et al., 1990). Of considerable concern are gasoline aromatics or BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene), which are classified as environmental priority pollutants by the EPA (1977), and are either confirmed or suspected carcinogens (Dean, 1985). Because of their solubility in water relative to other petroleum hydrocarbons, BTEX compounds are transported with the groundwater tens to hundreds of meters downgradient of contaminant sources (Cozzarelli et al., 1990). Because it has long been acknowledged that petroleum hydrocarbons including BTEX compounds can be degraded by naturally occurring soil microorganisms, the application of biological remediation for these compounds is appealing (Allen-King et al., 1994). Although the biochemistry of the aerobic degradation of individual BTEX compounds is fairly well understood (Assinder and Williams, 1990; Bayly and Barbour, 1984; Bestetti and Galli, 1984; Davey and Gibson, 1974; Davis et al., 1968; Gibson et al., 1968, 1974; Gibson and Subramanian, 1984; Smith, 1990; Worsey and Williams, 1975), both negative and positive substrate interactions such as inhibition, competition, and cometabolism make biological treat-
CCC 0006-3592/99/050526-11
ment outcomes for BTEX mixtures much more complex (Oh et al., 1994). Bearing in mind that it is unlikely for microorganisms to encounter single environmental pollutants in the subsurface, detailed information regarding the biodegradation kinetics of common industrial chemical mixtures, such as BTEX, is needed (Bitzi et al., 1991; Burback and Perry, 1993). Few studies have been conducted in an effort to understand substrate interactions of monoaromatics in mixtures. Most of these studies have focused on the aerobic degradation of only three BTEX components, benzene, toluene, and p-xylene (Alvarez and Vogel, 1991; Burback and Perry, 1993; Chang et al., 1992; Oh et al., 1994). Oh et al. (1994) investigated the degradation of benzene, toluene, and pxylene, individually and in mixtures, by a mixed consortium and a Pseudomonas species. They found that benzene and toluene can be degraded individually, but not p-xylene. In mixtures, benzene and toluene were removed according to competitive inhibition kinetics, whereas p-xylene was partially removed by a cometabolic process in the presence of either benzene and toluene. Burback and Perry (1993) reported that Mycobacterium vaccae can individually catabolize benzene or toluene; in a mixture of these two compounds, benzene degradation was delayed but toluene degradation proceeded in a manner similar to that when toluene was present alone. Alvarez and Vogel (1991) used pure and mixed cultures to evaluate possible beneficial BT(p-)X interactions and observed that the presence of toluene in mixtures with benzene and p-xylene enhanced the degradation of both aromatics. These researchers observed benzenedependent degradation of toluene and p-xylene, and retardation of both benzene and toluene degradation by pxylene. They also suggested that p-xylene was cometabolically degraded. Chang et al. (1992) used two pure cultures to quantify competitive inhibition kinetics of benzene and toluene, benzene and p-xylene, and toluene and p-xylene mixtures. They observed that the presence of toluene resulted in the competitive inhibition of benzene and the cometabolic degradation of p-xylene. One of their isolates was unable to degrade benzene in a mixture and the other transformed p-xylene to recalcitrant byproducts. Arvin et al. (1989) used two consortia to investigate benzene degradation in the presence of toluene, o-xylene, and a number of polynuclear aromatic hydrocarbons. Their study revealed that, in the presence of either toluene or o-xylene, benzene degradation was stimulated; however, when benzene was present in a mixture with both toluene and o-xylene, the combined effect was smaller than the sum of the stimulatory effects from each of these compounds. For the most part, the variations in the reported observations regarding microbial activity toward BTEX compounds in mixtures have led to some conflicting conclusions. Because the vast majority of contaminated sites involves complex mixtures of petroleum hydrocarbons, and because cosubstrate interactions are crucial for understanding and predicting the behavior of BTEX in biological treatment applications, this study focuses on identifying significant
substrate interactions during the aerobic biodegradation of BTEX mixtures by two consortia and one pure culture derived from a gasoline-contaminated aquifer. Because the role of ethylbenzene has rarely been investigated in previously reported mixture studies, our primary research goal was to characterize the effects of ethylbenzene on the biodegradation of benzene, toluene, and the three isomers of xylene in all possible mixture combinations. Because an initial proposed application of this study involved the use of bioremediation as a polishing step following a thermal gasoline remediation process (Deeb and Alvarez-Cohen, 1994a, 1994b; Udell and Stewart, 1989), a section of this article addresses the effect of temperature on the microbial growth and BTEX degradation rates of a consortium derived from a gasoline-contaminated site. MATERIALS AND METHODS Chemicals Benzene (>99% ACS reagent) was purchased from Mallinckrodt (Paris, KY). Toluene (99.8% ACS reagent), ethylbenzene (99.9% certified grade), and p-xylene (99.8% certified grade) were purchased from Fisher Scientific (Fair Lawn, NJ). o-Xylene (spectro grade) was purchased from J. T. Baker, Inc. (Phillipsburg, NJ). m-Xylene (no grade listed) was purchased from Eastman Kodak (Rochester, NY). Development of the Cultures Two toluene-enriched consortia were used in this study. The first consortium was derived using soil from a gasolinecontaminated aquifer located at Lawrence Livermore National Laboratory (CA). An aseptically collected soil sample was placed in a batch reactor containing toluene as the sole carbon and energy source, and a mineral salts medium with the following composition: CuSO4 ⭈ 5H2O, 0.5 mg/L; NaNO3: 1000 mg/L; K2SO4, 170 mg/L; MgSO4 ⭈ 7H2O, 37 mg/L; CaSO 4 ⭈ H 2 O, 8.6 mg/L; KH 2 PO 4, 530 mg/L; K2HPO4, 1060 mg/L; KI, 0.17 mg/L; ZnSO4 ⭈ 7H2O, 0.57 mg/L; MnSO4 ⭈ H2O, 0.34 mg/L; H3BO3, 0.124 mg/L; CoMoO4 ⭈ H2O, 0.094 mg/L; FeSO4 ⭈ 7H2O, 22.2 mg/L; H2SO4, 9.8 mg/L. Once bacterial growth was observed in the aqueous phase, a liquid sample was used to inoculate a second batch system. This procedure was repeated several times and a sample from the last series of subcultures was transferred to a continuous growth reactor. This consortium was developed at an ambient air temperature of 20°C, which resulted in a liquid temperature of 26° to 28°C. The culture was grown in a 14-L bench-top fermentor (Model No. MF-114, New Brunswick Scientific, New Brunswick, NJ), with a 5-L liter liquid volume, a 5-day detention time, and a mixing rate of 500 rpm. Toluene was added with the influent air which was bubbled through the chemostat liquid at a rate of 400 mL/min. Toluene concentration in the gas-phase influ-
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ent was maintained at 40 mg/L with an effluent of 20 to 30 mg/L. The consortium was sustained at a pH range of 6.8 to 7.0 in the aforementioned mineral salts medium. A second consortium was derived from the 20°C-enriched chemostat culture, but was grown in another continuous flow bioreactor at 35°C, a temperature, which was shown to enhance microbial activity and to optimize BTEX degradation rates. The cell detention time, as well as the influent and effluent toluene concentrations, were analogous to those of the 20°C-enriched consortium. The 35°C-enriched consortium was monitored on a regular basis for microbial stability using FAME (fatty acid methylester) analysis. A pure culture was isolated from the 35°C-enriched consortium on agar plates using toluene as the sole carbon and energy source. This isolate was identified by Microbial ID, Inc. (Newark, DE) as Rhodococcus rhodochrous. R. rhodochrous was grown aseptically in batch reactors consisting of 250-mL clear glass bottles (Alltech Co., Deerfield, IL) sealed with Teflon-lined Mininert valves and containing 100 mL of the mineral salts medium. The batch reactors were maintained at 35°C in a controlled environment incubator/shaker (Model No. G25, New Brunswick Scientific) with a mixing rate of 175 rpm; this mixing rate was experimentally shown to overcome potential oxygen mass-transfer limitations (Chang and Alvarez-Cohen, 1995). The growth reactors were amended with toluene (80 mg/L) and oxygen gas as needed to maintain approximately 15% to 21% oxygen by volume in the headspace. The cells were harvested during exponential growth phase. Stock solutions of R. rhodochrous were prepared by centrifuging the cell suspensions from the growth reactors at maximum speed for 20 min using an MSE GT-2 centrifuge (VWR Scientific, West Chester, PA) and by resuspending the pellets in a mineral salts medium. Experimental Procedures
Evaluating Temperature Effects on Microbial Activity Temperature studies were performed in batch reactors consisting of either 250-mL clear glass bottles (Alltech Co.) or 385-mL side-armed Nephelo culture flasks (Bellco Glass, Inc., Vineland, NJ). The reactors were sealed with Teflonlined Mininert valves (Alltech Co.). Culture growth on toluene was monitored over a broad range of temperatures (7° to 65°C) using a light spectrophotometer (Milton-Roy Spectronic 20D, Spectronic Instruments, Inc., Rochester, NY). A sample containing mineral salts medium (but no cells) was used as a reference for the light spectrophotometer prior to each absorbance measurement. Absorbance readings of liquid culture samples were taken at 600 nm and correlated to gravitametrically measured dry cell mass. Dry cell mass was measured in duplicate by taking the difference of sample weights after drying at 105°C for 8 h and after combustion at 550°C for 30 min. A temperature growth curve was ob-
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tained by plotting initial exponential growth rates on toluene (g cells/L per day) vs. growth temperature (°C), and a temperature optimum for culture growth and activity was determined. Arrhenius parameters, E and A, were obtained by linear regression of the exponential growth curve data as follows: lnrg = −
E 1 ⭈ + lnA R T
where rg is the cell growth rate (g/L per day), E is the activation energy (J/mol), R is the gas constant (atm.m3/ K.mol), T is the temperature (degrees K), and A is the Van’t Hoff–Arrhenius coefficient (frequency factor).
BTEX Biodegradation Rate Experiments (a) Experiments testing the biodegradation of each of the BTEX compounds were conducted in batch reactors consisting of 250-mL clear glass bottles sealed with Mininert valves. Each of the batch reactors contained 125 mL of liquid (mineral salts medium and cells), 123 mL of headspace, and 6 glass beads (6 mm, volume approx. 2 mL) to promote mixing and to prevent cell aggregation. The mass of aromatics added (M) was calculated using dimensionless Henry’s constants (Hc), reactor liquid and gas volumes (VL, VG), and aromatic substrate concentrations in the liquid and gas phases (SL, SG) using the following equation: M ⳱ SL ⭈ VL + SG ⭈ VG ⳱ SG(VL/HC + VG). HC values for BTEX compounds at standard temperature and pressure were adapted from Mackay and Shiu (1981). At 35°C, Hc values were calculated as described by Ashworth et al. (1987) (Table I). Pure phase BTEX compounds were added to the batch reactors using a high-precision 20-L syringe (Hamilton Co., Reno, NV) to result in an initial BTEX aqueous concentration of approximately 80 mg/L. All biodegradation rate experiments were conducted at 35°C. Prior to cell inoculation, the reactors were placed for 3 h in a 35°C controlled-environment incubator/shaker with a mixing rate of 175 rpm to ensure complete equilibrium BTEX partitioning between the liquid and gas phases. In the case of the consortia, the cell inoculum consisted of a 5- to 15-mL sample directly removed from the continuous growth bioreactor and purged with N2 for a short period of time to remove any residual toluene. In the case of the pure culture, the cell inoculum consisted of a 1- to 3-mL sample from the harvested cell stock solution. The volume of liquid culture added to the reactors was precalculated to result in uniform initial cell densities in all the batch reactors within one experiment. Immediately following the introduction of the cells to the batch reactors, the bottles were shaken vigorously for 1 min after which initial measurements of toluene were taken. The batch reactors were placed back in the 35°C incubator/shaker for the duration of the experiment and were only removed briefly for sampling. BTEX concentrations in the batch reactors were determined from headspace analyses. Samples of the head-
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Table I.
Henry’s constants for BTEX compounds at 35°C as calculated using a temperature regression equation described by Ashworth et al. (1987).
BTEX
A [-]
B [-]
Hb ⳱ exp(A − B/T), T ⳱ 308 K (35°C) [atm ⭈ m3/mol]
Benzene Toluene Ethylbenzene o-Xylene m-Xylene p-Xylene
5.534 5.133 11.92 5.541 6.280 6.931
3194 3024 4994 3220 3337 3520
0.00793 0.00923 0.0136 0.00734 0.0105 0.0111
a
a
Hcc ⳱ H/RT [-]
Densityd [g/L]
MW [g/mol]
0.314 0.365 0.540 0.291 0.416 0.440
0.879 0.867 0.866 0.880 0.868 0.861
78.1 92.1 106.1 106.1 106.1 106.1
a
A and B are constant parameters for the temperature regression equation. bHenry’s constant. cDimensionless Henry’s constant. dFrom the Merck Index (1989).
space gas (50 to 200 L) were withdrawn from the bottles using a Hamilton CR-700 constant rate gas-tight syringe (Hamilton Co.), and analyzed for BTEX using a HewlettPackard 5880 gas chromatograph equipped with a flameionization detector and a 0.75 mm × 30 m glass capillary column (Supelco Co., Bellefonte, PA). The oven, injector and detector temperatures of this gas chromatograph were fixed at 85°C, 250°C, and 300°C, respectively. Headspace sampling proceeded at frequent intervals until BTEX concentrations in the batch reactors dropped below the detection limits of the gas chromatograph (1 to 10 g/L). The gas chromatograph was calibrated using standards prepared by adding known amounts of pure phase aromatics or aromatic-saturated aqueous solutions to Teflon-sealed 250-mL glass bottles. CO2 production and O2 consumption were measured using a Hewlett-Packard 5890 gas chromatograph equipped with a thermal conductivity detector. This gas chromatograph was calibrated using Scotty certified multi-component gas mixtures (Alltech Co.). A set of cell-free controls was used to monitor abiotic losses of aromatics; abiotic BTEX losses never exceeded 5% throughout the duration of any experiment. Another set of controls, containing cells, but no aromatic substrates, was used to measure CO2 production from cell decay or other processes. Each experiment was repeated at least twice to ensure reproducibility of results. (b) Experiments testing the biodegradation of BTEX mixtures were conducted following a similar experimental procedure to the one just described. Each of the aromatics was present in a BTEX mixture at equal initial aqueous phase concentrations.
Substrate Interactions in Mixtures Experiments aimed at characterizing the inhibition effects of BTEX compounds in mixtures were performed in 250mL glass batch reactors similar to the ones just described. Lineweaver–Burk-type plots were produced using initial biodegradation rates of one BTEX compound in a binary mixture with an inhibitor. Initial biodegradation rates were determined from the linear regression of the first six to eight data points from BTEX disappearance plots within the first hour of the experiment. The concentrations of the substrate
and the inhibitor were varied to get a range of kinetic rates while maintaining a fixed cell density. A set of reactors containing the aromatic substrate, but no inhibitor, served as inhibitor-free controls. Cell-free controls as well as BTEXfree controls were also used. Inhibition characterization experiments were repeated three to six times to ensure reproducibility of results. Cometabolism was evaluated by measuring culture growth on each BTEX compound, alone and in the presence of a growth substrate, while monitoring BTEX disappearance.
Culture Growth on BTEX Compounds In experiments in which cell growth on each of the BTEX components was evaluated, the batch reactors consisted of 385-mL side-armed Nephelo culture flasks (Bellco), which allow absorbance measurements without liquid culture removal. The Nephelo flasks were equipped with 38-mm Teflon-lined screw caps (Bellco) and gas-tight Mininert valves on the sidearm (Alltech). They were also equipped with baffles to promote mixing and to prevent cell aggregation. The liquid volume in these reactors was set at 150 mL and consisted of the mineral salts medium and the liquid culture sample. The growth reactors were incubated at 35°C throughout the duration of the experiment. Absorbance readings of liquid culture samples were taken at 600 nm and correlated to gravitametrically measured dry cell mass as described earlier. A sample containing mineral salts medium (but no cells or substrate) was used as a reference for the light spectrophotometer. Cell-free controls were maintained throughout the duration of the experiment; negligible abiotic losses of BTEX were noted. RESULTS Evaluating Temperature Effects on Microbial Activity A microbial consortium, derived from a gasoline-contaminated aquifer, was enriched on toluene and grown in a chemostat at 20°C. Growth rate studies conducted over a
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range of temperatures to optimize microbial growth and degradation activity of this culture revealed that cell growth on toluene increased with temperature from 7° to 35°C, decreased sharply at incubations of 36° to 40°C, and was inhibited entirely at temperatures above 45°C (Deeb and Alvarez-Cohen, 1994a). A summary of the temperature effect studies is presented here in the form of a temperature growth curve (Fig. 1). The exponential growth phase data in this graph can be represented by the Arrhenius equation (rg ⳱ A ⭈ e−E/RT); linear regression of this data on a semilogarithmic scale (logarithm of the growth rate plotted against the reciprocal of the temperature in degrees K) yielded a straight line with a slope of −E/R and a y-intercept of lnA. The Arrhenius constant parameter, A, was calculated to be 2.1 × 1016 g/L per day, while the activation energy E was found to be 97 kJ/mol. The optimum temperature for culture growth and toluene degradation activity was determined to be 35°C. Because toluene oxidation was optimized at 35°C, cells from the 20°C-enriched consortium were transferred to a continuous flow bioreactor maintained at 35°C, and were grown under conditions analogous to those of the parent culture. A comparison was then made of the degradation kinetics of BTEX compounds by both of these consortia to determine whether the higher temperature caused fundamental changes in the overall degradation activity of the microbial culture. Results indicated that for the degradation of BTEX compounds, both individually and in representative mixtures, cells that were enriched and maintained at 35°C had increased degradation rates in most cases, compared with cells that were enriched and maintained at 20°C (Table II). In addition, the 35°C-enriched culture exhibited shorter BTEX degradation lag times than the 20°C-enriched culture. The only exception to these two observations was in the case of ethylbenzene; in a BTEX mixture, the rate of
TABLE IIa. Degradation rates of individual BTEX compounds by the 20°C- and 35°C-enriched cultures (initial B, T, E, and o-X aqueous phase concentrations ⳱ 80 mg/L). Individual BTEX compound
20°C-Enriched culture degradation rate (mg/mg per day)
35° C-Enriched culture degradation rate (mg/mg per day)
Benzene Toluene Ethylbenzene o-Xylene
0.49 0.79 0.41 NAa
1.2 1.4 1.0 0.64
a
Not available. The degradation of o-xylene by the 20°C-enriched culture was inhibited at concentrations >60 mg/L.
ethylbenzene degradation was faster for the 20°C-enriched culture (Table IIb). All kinetic rate experiments were repeated several times and the results were reproducible over time. Figure 2 shows representative comparisons of benzene and o-xylene degradation rates by the 35°C- and 20°Cenriched consortia. Substrate Utilization Patterns During BTEX Degradation by the Toluene-Enriched Consortia Both toluene-enriched consortia were capable of degrading all BTEX compounds, individually and in mixtures, over a broad range of hydrocarbon concentrations (up to 80 mg/L for the majority of BTEX compounds). Substrate degradation patterns observed for individual aromatics were compared with patterns for aromatics in mixtures and found to differ significantly. Toluene was repeatedly degraded faster by the toluene-enriched cultures than benzene, ethylbenzene, or the xylenes when each was present individually; this is shown in Figure 3a, and illustrates degradation patterns by the 35°C-enriched culture for each of the BTEX compounds in single-substrate experiments. This pattern was manifested in an even more distinct manner at higher BTEX concentrations, especially in the case of o-xylene, which had a degradation rate that decreased considerably at concentrations >60 mg/L due to substrate toxicity (Deeb and Alvarez-Cohen, 1994b). When cells were exposed to mixtures of aromatics, substrate degradation patterns were altered; ethylbenzene was degraded fastest, followed by toluene, benzene, and the xylenes. Fig-
Table IIb. Degradation rates of BTEX compounds in a mixture (total initial BTEX aqueous phase concentration ⳱ 80 mg/L; B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 20°C- and 35°C-enriched cultures
Figure 1. Growth curve depicting the effect of temperature on the growth rates of a toluene-oxidizing consortium enriched at 20°C. Linear regression of the exponential growth phase data on a semilogarithmic scale (logarithm of the growth rate plotted against the temperature) yielded a straight line.
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BTEX compounds in a BTE(o-)X mixture
20°C-Enriched culture degradation rate (mg/mg per day)
35°C-Enriched culture degradation rate (mg/mg per day)
Benzene Toluene Ethylbenzene o-Xylene
0.90 1.2 2.7 0.71
1.5 1.4 1.2 0.93
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Figure 2. Representative data comparing the degradation rates of (a) benzene and (b) o-xylene (initial concentration of B ⳱ o-X ⳱ 80 mg/L) by two toluene-enriched consortia grown at 20°C and 35°C, respectively.
ure 3b shows representative results of substrate degradation patterns within a BTEX mixture with o-xylene exemplifying the behavior of xylenes. Similar gas chromatograph retention times for ethylbenzene and two of the xylenes made it difficult to differentiate individual compound degradation rates in comprehensive mixtures. Studies to evaluate substrate degradation patterns in all possible BTEX combinations revealed that, in each of these mixtures, the three isomers of xylene were always the last to be degraded, whereas ethylbenzene was unfailingly the first to be degraded (Table III). These substrate degradation patterns were analogous for both the 20°C- and 35°C-enriched consortia, and were reproducible over time. These patterns were also manifested in the presence of other common gasoline monoaromatics such as n-propylbenzene and 1,2,4trimethylbenzene.
Substrate Interactions During the Degradation of BTEX Mixtures by the 35°C-Enriched Consortium Substrate interactions were investigated in all possible BTEX combinations. Both positive and negative substrate interactions were observed during the biodegradation of BTEX mixtures by the 35°C-enriched consortium. These interactions were found to be different from those previously reported in the literature. In general, the presence of xylenes in most mixtures had little to no effect on the degradation of benzene, toluene, and ethylbenzene. However, in some cases, the presence of o-xylene may have enhanced the degradation of benzene to some extent and that of toluene to a lesser extent. For example, when benzene was present alone at a concentration of 20 mg/L, it took 1.6 h to degrade 50% of the initial aqueous phase concentration (10 mg/L) compared with 1.3 h when benzene was in a mixture with o-xylene. Similarly, when toluene was present alone, it took 0.8 h to degrade 10 mg/L compared with 0.68 h when present with o-xylene (Fig. 4). The presence of either toluene, benzene or ethylbenzene with one of the xylene isomers in binary mixtures consistently had a negative effect on xylene degradation rates; although the degradation of the xylene isomers was not entirely inhibited, a retardation in xylene degradation rates was observed. Figure 5 portrays these observed effects using the ortho isomer as a representative xylene. As can be seen, when o-xylene was present alone at a concentration of 40 mg/L, it was degraded the fastest. When o-xylene (40 mg/L) was present in a mixture with either benzene or toluene (40 mg/L), degradation was delayed by 2 h. The effect of ethylbenzene in a mixture with o-xylene was the strongest; the degradation of o-xylene was retarded by over 4 h in the presence of ethylbenzene. Further studies revealed that ethylbenzene was the most potent inhibitor of BTX in all mixtures tested. Figure 6a illustrates this inhibition by showing that the negative effect of ethylbenzene on toluene degradation was much more significant than that of either benzene or o-xylene. On the other hand, the presence of benzene, toluene, or xylenes in mixtures with ethylbenzene seemed to exert little or no effect on the degradation rate of this compound (Fig. 6b). Other inhibition effects observed in BTEX mixtures included the retardation of toluene degradation in the presence of benzene and the retardation of benzene degradation in the presence of toluene (Fig. 4). Because ethylbenzene was found to be the most potent inhibitor of BTX biodegradation in mixtures, kinetic rate studies were performed using the 35°C-enriched consortium in an effort to identify and quantify the observed inhibition effects. Representative data shown in Figure 7 illustrate the retardation of toluene degradation with increasing concentrations of the inhibitor, ethylbenzene. Double reciprocal (Lineweaver–Burk) plots prepared using observed initial biodegradation rates of toluene in binary mixtures with varied concentrations of ethylbenzene indicated no specific inhibition pattern, but rather suggested a combination of in-
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Figure 3. (a) Degradation of individual BTEX compounds (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ m-X ⳱ p-X ⳱ 20 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.27 g/L). (b) Degradation of a representative BTEX mixture (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.37 g/L).
hibition patterns, including both competitive and noncompetitive inhibition (data not shown). Substrate Interactions During the Degradation of BTEX Mixtures by R. rhodochrous R. rhodochrous was able to degrade all six BTEX compounds, individually and in mixtures, over a broad range of
hydrocarbon concentrations (0 to 40 mg/L). In BTEX mixtures, both positive and negative substrate interactions were observed. In studies similar to the ones performed using the consortium, ethylbenzene was shown to be the most potent inhibitor of BTX degradation in binary mixtures; representative data from such studies is shown in Figure 8. Figure 9
Table III. Order of biodegradation of mixtures of benzene (B), toluene (T), ethylbenzene (E), o-xylene (o-X), m-xylene (m-X), and p-xylene (p-X) by the 35°C-enriched toluene-grown culture. BTEX mixture
B, T B, E B, o-X T, E T, o-X T, m-X T, p-X E, o-X E, m-X B, T, E B, T, o-X B, E, o-X T, E, o-X B, T, E, o-X
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T
B
E
1
2 2 1
1
2 1 1 1
2 1 2 2
o-X
m-X
p-X
2 1 2 2 2
3 2 2 3
1 1 1 1 1 1
2 2 3 3 3 4
Figure 4. Time in hours to degrade 50% of each of the BTEX compounds (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium alone or in binary mixtures with one other BTEX component.
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tion results are consistent with the limited data that has been reported previously for enhanced degradation rates of BTEX with increased temperatures. For example, in one study, toluene mineralization rates were shown to triple due to an increase in temperature from 11° to 25°C (Armstrong et al., 1991). When a consortium was enriched in a continuous growth reactor at 35°C, BTEX degradation rates increased and lag times decreased relative to the 20°C-enriched consortium. Comparisons of the degradation kinetics of BTEX compounds by the consortia enriched at 20° and 35°C were made in order to determine whether the increased degradation rates observed at the elevated temperature were due solely to temperature-induced increases in enzymatic reaction rates, or whether enrichment at the elevated temperature caused fundamental changes in the microbial consorFigure 5. o-Xylene degradation alone and in binary mixtures with B, T, and E (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 40 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.27 g/L).
illustrates the retardation of toluene degradation in the presence of increasing concentrations of ethylbenzene. The presence of o-xylene had no significant effect on the degradation of toluene in binary mixtures, whereas benzene had a slight inhibitory effect on toluene degradation (Fig. 8). Culture Growth on BTEX Compounds Studies were performed to evaluate the growth of the mixed and pure cultures on each of the BTEX aromatics. The 20°C- and the 35°C-enriched consortia, as well as R. rhodochrous, were able to effectively utilize toluene, benzene, and ethylbenzene as primary carbon and energy sources. Toluene, benzene, and ethylbenzene biodegradation corresponded to increases in cell mass, CO2 production and O2 consumption. Cell growth was evidenced by increases in optical density at 600 nm; for example, in the case of the growth of the 20°C-enriched consortium using benzene, a change in absorbance from 0.27 to 0.60 corresponded to an increase in cell dry weight from 0.14 to 0.35 g/L. Although initial additions of each of the xylene isomers to the batch reactors were degraded in the absence a growth substrate, the cultures were not able to grow using the xylenes. In fact, significant decreases in cell density were observed after repeated additions of o-xylene and m-xylene. DISCUSSION In batch studies conducted to quantify the effect of temperature on the microbial activity of the 20°C-enriched consortium, cell growth on toluene increased approximately fourfold due to an increase in the incubation temperature from 20° to 35°C. A temperature optimum of 35°C was determined from a range of temperatures tested (7° to 65°C). Although this is the first study to report the effect of temperature on microbial growth on toluene, our degrada-
Figure 6. (a) Toluene degradation alone and in binary mixtures with B, E, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium. (b) Ethylbenzene degradation alone and in binary mixtures with B, T, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium.
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Figure 7. Toluene degradation alone (initial concentration ⳱ 15 mg/L) and in the presence of ethylbenzene at a range of concentrations by the 35°C-enriched consortium (cell density ⳱ 0.21 g/L).
tium. Although degradation rates for individual BTEX compounds were higher for the 35°C-grown cells relative to 20°C-grown cells, the rate of ethylbenzene degradation in a BTEX mixture by the 35°C-enriched consortium was consistently lower than that by the 20°C-enriched consortium. These results suggest that fundamental changes in the composition of the consortium were promoted by enrichment at a higher temperature. Both the 20°C- and the 35°C-enriched consortia were able to degrade all BTEX compounds at higher concentrations than those commonly present in gasoline-contaminated subsurface environments. Additionally, the 35°Cenriched consortium was shown to mineralize all the BTEX compounds (14C-labeled) to 14CO2 (Deeb and Alvarez-
Figure 8. Toluene degradation alone and in binary mixtures with B, E, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by R. rhodochrous (cell density ⳱ 28 mg/L). The plotted data is the average of duplicates samples and the error bars represent the range.
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Figure 9. Toluene degradation alone (initial concentration ⳱ 15 mg/L) and in the presence of ethylbenzene at a range of concentrations by R. rhodochrous (cell density ⳱ 35 mg/L). The plotted data is the average of duplicates samples and the error bars represent the range.
Cohen, 1998). As expected, both consortia degraded the enrichment substrate toluene the fastest when BTEX compounds were supplied individually. The fact that the xylenes were degraded the slowest among the BTEX compounds is consistent with what has been reported in the literature previously (Alvarez and Vogel, 1991). When cells were exposed to mixtures of aromatics present at equal concentrations, degradation patterns were modified substantially from those of individual BTEX compounds with disappearance in the order of ethylbenzene, toluene, benzene, and the xylenes. Surprisingly, ethylbenzene underwent preferential degradation over the enrichment substrate toluene. This pattern was also maintained in the presence of other monoaromatics such as n-propylbenzene and 1,2,4-trimethylbenzene, and even in a complex mixture such as gasoline (Deeb and Alvarez-Cohen, 1994b). In view of the fact that the bioremediation of gasoline contaminated sites requires the microbial degradation of complex waste mixtures, it is important to understand the potential enhancement or inhibition effects caused by the concurrent presence of multiple BTEX compounds in the subsurface. In this study, our major objective was to identify both negative (i.e., inhibition) and positive (i.e., stimulation, cometabolism) substrate interactions in comprehensive BTEX mixtures; to our knowledge, no previously reported work focusing on substrate interactions during the bacterial degradation of BTEX has included mixtures that incorporated ethylbenzene and all three xylene isomers. In the case of the 35°C-enriched consortium, the enhancement of BTEX degradation in mixtures was observed to a small extent for benzene and toluene when each was present in a binary mixture with o-xylene. This was an interesting observation, but is difficult to explain from the available information, especially because the cells were incapable of sustained o-xylene degradation in the absence of
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toluene, benzene, or ethylbenzene. On the other hand, the presence of toluene, benzene, or ethylbenzene with any of the xylene isomers in binary mixtures had a negative effect on xylene disappearance rates, although the degradation of the xylene isomers was not entirely inhibited. The most potent inhibitor of BTX degradation was ethylbenzene, whereas the effect of benzene, toluene, and xylenes on the degradation of ethylbenzene was found to be inconsequential. This is a significant finding, because, to our knowledge, this is the first report of inhibition in monoaromatic mixtures involving ethylbenzene. The use of Lineweaver–Burk plots for toluene degradation in the presence of ethylbenzene as the inhibitor did not yield conclusive results, but did suggest a mixed inhibition pattern for the 35°C-enriched consortium that may encompass both competitive and noncompetitive inhibition. These mixed inhibition effects are not surprising, because the culture tested is a mixed culture that can be expected to exhibit mixed inhibition patterns due to the presence of multiple species with multiple BTEX degradation pathways. Other inhibition effects that were noted, but not characterized due to lesser degrees of magnitude, included toluene inhibition in the presence of benzene and benzene inhibition in the presence of toluene. Because R. rhodochrous was isolated from the 35°Cenriched consortium, we expected to observe similar substrate interactions during the biodegradation of BTEX mixtures by this isolate. As anticipated, ethylbenzene was found to be the most potent inhibitor of benzene, toluene, and xylene degradation in binary mixtures. Moreover, this inhibition was more pronounced in the case of R. rhodochrous than in the case of the parent consortium; in fact, the degradation of BTX in binary mixtures did not proceed until all the ethylbenzene was degraded, although the interim lag period characteristic of diauxy was not observed. As a result, we were unable to obtain initial biodegradation rates of BTX in the presence of ethylbenzene over a range of ethylbenzene concentrations, which prevented us from generating Lineweaver–Burk plots and from identifying or quantifying the observed inhibition effects. In comparing the results of this work with previously published studies, we found some similarities and some differences. For example, the presence of toluene in previous studies has been shown to inhibit the degradation of benzene in mixtures (Burback and Perry, 1993; Chang et al., 1992; Oh et al., 1994) as was shown here. Although the presence of p-xylene in previous work has been shown to inhibit benzene and toluene degradation (Alvarez and Vogel, 1991; Oh et al., 1994), this was not observed in our work; in fact, we found some of the xylene isomers (i.e., o-xylene) to exhibit a slight stimulatory effect on BTE degradation. In this study, benzene and ethylbenzene were effectively utilized by the three toluene-grown cultures as primary carbon and energy sources, whereas the xylene isomers were not. Although initial additions of the xylenes were degraded by the three toluene-degrading cultures, repeated additions of o- and m-xylene caused marked decreases in cell densi-
ties. The inability of the three cultures to grow on the xylenes, combined with the failure of the cultures to degrade repeated additions of these compounds in the absence of a growth substrate, suggest that the xylenes were cometabolically degraded. This assumption is reasonable, because the observed losses in biomass upon the biotransformation of the xylenes can be explained by the dependency of the toluene oxygenase enzyme systems on cell-reducing power. The cometabolic degradation of the xylenes in BTEX mixtures in this work is consistent with what has been reported in previous mixture studies (Alvarez and Vogel, 1991; Chang et al., 1992; Oh et al., 1994). In conclusion, we have observed several patterns of substrate interactions in comprehensive mixtures of benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene by two mixed consortia and one pure culture, R. rhodochrous. These patterns included no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. Our results suggest that substrate interactions among monoaromatics in mixtures are complicated despite the similarities in the chemical properties and structures of these compounds. Our understanding of these interactions can contribute to a better approach for modeling BTEX biodegradation within a complex mixture such as gasoline. Nevertheless, it would be equally important to understand whether these interactions affect the mineralization (degradation to CO2) of BTEX mixtures. Most of the studies that have been referenced in this work have focused on the effect of mixtures on the biotransformation rates of BTEX. Studies investigating the effect of mixtures on the mineralization of BTEX using 14C-labeled aromatics have been performed in our laboratory. Manuscripts describing the results of these studies are currently in preparation. We thank Dr. Hsiao-Lung Chang for his assistance in enriching the 20°C-enriched consortium, Ms. Ruth Richardson for isolating R. rhodochrous, Prof. David Jenkins and Ms. Linda Sawyer for their suggestions on the analysis of our temperature study results, and Susan Hou and Harold Tully for their assistance in the laboratory.
References Allen-King RM, O’Leary KE, Gillham RW, Barker JF. 1994. Limitations on the biodegradation rate of dissolved BTEX in a natural, unsaturated, sandy soil: Evidence from the field and laboratory experiments, In: Hinchee RE, Alleman BC, Hoeppel RE, Miller RN, editors. Hydrocarbon bioremediation. Boca Raton, FL: Lewis. p 175–191. Alvarez PJ, Vogel TM. 1991. Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries. Appl Environ Microbiol 57:2981–2985. Armstrong AQ, Hudsen RE, Hwang HM, Lewis DL. 1991. Environmental factors affecting toluene degradation in ground water at a hazardous waste site. Environ Toxicol Chem 10:147–58. Arvin E, Jensen BK, Gundersen AT. 1989. Substrate interactions during aerobic biodegradation of benzene. Appl Environ Microbiol 55: 3221–3225. Ashworth RA, Howe GB, Mullins ME, Rogers TN. 1987. Air–water partitioning coefficients of organics in dilute aqueous solutions. J Hazard Mater 18:25–36.
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Assinder SJ, Williams PA. 1990. The TOL plasmids: Determinants of the catabolism of toluene and the xylenes (vol. 31), In: Rose AH and Tempest DW, editors. Advances in microbial physiology. London: Academic Press. p 1–69. Bayly RC, Barbour MG. 1984. The degradation of aromatic compounds by the meta and gentisate pathways, In: Gibson DT, editor. Microbial degradation of organic compounds. New York: Marcel Dekker. p 253–294. Bestetti G, Galli E. 1984. Plasmid-coded degradation of ethylbenzene and 1-phenylethanol on Pseudomonas fluorescens. FEMS Microbiol Lett 21:165–168. Bitzi U, Egli T, Hamer G. 1991. The biodegradation of mixtures of organic solvents by mixed and monocultures of bacteria. Biotechnol Bioeng 37:1037–1042. Burback BL, Perry JJ. 1993. Biodegradation and biotransformation of groundwater pollutant mixtures by Mycobacterium vaccae. Appl Environ Microbiol 59:1025–1029. Chang H-L, Alvarez-Cohen L. 1995. Transformation capacities of chlorinated organics by mixed cultures enriched on methane, propane, toluene, or phenol. Biotechnol Bioengin 45:440–449. Chang M-K, Voice TC, Criddle C. 1992. Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and pxylene by two Pseudomonas isolates. Biotechnol Bioeng 41: 1057–1065. Cozzarelli I, Eganhous RP, Baedecker MJ. 1990. Transformation of monoaromatic hydrocarbons to organic acids in anoxic groundwater environment. Environ Geol Water Sci 16:135–141. Davey JF, Gibson DT. 1974. Bacterial metabolism of para- and metaxylene: Oxidation of a methyl substituent. J Bacteriol 119:923–929. Davis RS, Hossler FE, Stone RW. 1968. Metabolism of p- and m-xylene by species of Pseudomonas. Can J Microbiol 14:1005–1009. Dean BJ. 1985. Recent findings on the genetic toxicology of benzene, toluene, xylenes and phenols. Mutat Res 145:153–181. Deeb RA, Alvarez-Cohen L. 1994a. Thermally enhanced bioremediation of a gasoline-contaminated aquifer using toluene oxidizing bacteria. In: Ryan JN and Edwards M, editors. Critical issues in water and waste-
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water treatment. Proceedings of the 1994 National Conference on Environmental Engineering. New York: American Society of Civil Engineers. p 400–407. Deeb RA, Alvarez-Cohen L. 1994b. Degradation of BTEX compounds: Temperature and mixture effects. In: Proceedings of the 67th Annual Conference and Exposition of the Water Environment Federation. p 185–193. Deeb RA, Alvarez-Cohen L. 1998. Biotransformation and mineralization of BTEX mixtures by toluene-oxidizing mixed and pure cultures (manuscript in preparation). Gibson DT, Koch JR, Kallio RE. 1968. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry 7:2653–2662. Gibson DT, Mahadevan V, Davey JF. 1974. Bacterial metabolism of paraand meta-xylene: Oxidation of the aromatic ring. J Bacteriol 119: 930–936. Gibson DT, Subramanian V. 1984. Microbial degradation of aromatic hydrocarbons, In: Gibson DT, editor. Microbial degradation of organic compounds. New York: Marcel Dekker. p 361–369. Mackay D, Shiu EY. 1981. Critical review of Henry’s constants for chemicals of environmental interest. J Phys Chem Ref Data 10:1175–1199. Merck index. 1989. 11th Edition. Rahway, NJ: Merck and Co. Oh Y, Shareefdeen Z, Baltzis BC, Bartha R. 1994. Interactions between benzene, toluene, and p-xylene during their biodegradation. Biotechnol Bioeng 44:533–538. Smith MR. 1990. The biodegradation of aromatic hydrocarbons by bacteria. Biodegradation 1:191–206. Udell KS, Stewart LD. 1989. Field study of in situ steam injection and vacuum extraction for recovery of volatile organic solvents. UCBSEEHRL report no. 89-2. U.S. Environmental Protection Agency. 1977. Serial no. 95-12. U. S. Government Printing Office, Washington, DC. Worsey MJ, Williams PA. 1975. Metabolism of toluene and xylenes by Pseudomonas puptida (arvilla) mt-2: Evidence for a new function of the TOL plasmid. J Bacteriol 124:7–13.
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Department of Civil and Environmental Engineering, 631 Davis Hall, MC 1710 University of California at Berkeley, Berkeley, California 94720-1710; telephone: (510)-643-5969; fax: 510-642-7483; e-mail: [email protected] Received 18 March 1998, accepted 18 August 1998
Abstract: A microbial consortium derived from a gasoline-contaminated aquifer was enriched on toluene (T) in a chemostat at 20°C and was found to degrade benzene (B), ethylbenzene (E), and xylenes (X). Studies conducted to determine the optimal temperature for microbial activity revealed that cell growth and toluene degradation were maximized at 35°C. A consortium enriched at 35°C exhibited increased degradation rates of benzene, toluene, ethylbenzene, and xylenes in single-substrate experiments; in BTEX mixtures, enhanced benzene, toluene, and xylene degradation rates were observed, but ethylbenzene degradation rates decreased. Substrate degradation patterns over a range of BTEX concentrations (0 to 80 mg/L) for individual aromatics were found to differ significantly from patterns for aromatics in mixtures. Individually, toluene was degraded fastest, followed by benzene, ethylbenzene, and the xylenes. In BTEX mixtures, degradation followed the order of ethylbenzene, toluene, and benzene, with the xylenes degraded last. A pure culture isolated from the 35°Cenriched consortium was identified as Rhodococcus rhodochrous. This culture was shown to degrade each of the BTEX compounds, individually and in mixtures, following the same degradation patterns as the mixed cultures. Additionally, R. rhodochrous was shown to utilize benzene, toluene, and ethylbenzene as primary carbon and energy sources. Studies conducted with the 35°Cenriched consortium and R. rhodochrous to evaluate potential substrate interactions caused by the concurrent presence of multiple BTEX compounds revealed a range of substrate interaction patterns including no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. In the case of the consortium, benzene and toluene degradation rates were slightly enhanced by the presence of o-xylene, whereas the presence of toluene, benzene, or ethylbenzene had a negative effect on xylene degradation rates. Ethylbenzene was shown to be the most potent inhibitor of BTEX degradation by both the mixed and pure cultures. Attempted quantification of these inhibition effects in the case of the consortium suggested a mixture of competitive and non-
Correspondence to: L. Alvarez-Cohen Contract grant sponsors: NIEHS; NSF; EPA
© 1999 John Wiley & Sons, Inc.
competitive inhibition kinetics. Benzene, toluene, and the xylenes had a negligible effect on the biodegradation of ethylbenzene by both cultures. Cometabolism of o-, m-, and p-xylene was shown to be a positive substrate interaction. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 526–536, 1999.
Keywords: BTEX; gasoline aromatics; Rhodoccus rhodochrous; inhibition; degradation kinetics; substrate interactions
INTRODUCTION The United States has thousands of soil and groundwater sites that are contaminated with petroleum hydrocarbons resulting from industrial activities related to refining, transportation, use, and disposal of petroleum products (Cozzarelli et al., 1990). Of considerable concern are gasoline aromatics or BTEX compounds (benzene, toluene, ethylbenzene, o-xylene, m-xylene, p-xylene), which are classified as environmental priority pollutants by the EPA (1977), and are either confirmed or suspected carcinogens (Dean, 1985). Because of their solubility in water relative to other petroleum hydrocarbons, BTEX compounds are transported with the groundwater tens to hundreds of meters downgradient of contaminant sources (Cozzarelli et al., 1990). Because it has long been acknowledged that petroleum hydrocarbons including BTEX compounds can be degraded by naturally occurring soil microorganisms, the application of biological remediation for these compounds is appealing (Allen-King et al., 1994). Although the biochemistry of the aerobic degradation of individual BTEX compounds is fairly well understood (Assinder and Williams, 1990; Bayly and Barbour, 1984; Bestetti and Galli, 1984; Davey and Gibson, 1974; Davis et al., 1968; Gibson et al., 1968, 1974; Gibson and Subramanian, 1984; Smith, 1990; Worsey and Williams, 1975), both negative and positive substrate interactions such as inhibition, competition, and cometabolism make biological treat-
CCC 0006-3592/99/050526-11
ment outcomes for BTEX mixtures much more complex (Oh et al., 1994). Bearing in mind that it is unlikely for microorganisms to encounter single environmental pollutants in the subsurface, detailed information regarding the biodegradation kinetics of common industrial chemical mixtures, such as BTEX, is needed (Bitzi et al., 1991; Burback and Perry, 1993). Few studies have been conducted in an effort to understand substrate interactions of monoaromatics in mixtures. Most of these studies have focused on the aerobic degradation of only three BTEX components, benzene, toluene, and p-xylene (Alvarez and Vogel, 1991; Burback and Perry, 1993; Chang et al., 1992; Oh et al., 1994). Oh et al. (1994) investigated the degradation of benzene, toluene, and pxylene, individually and in mixtures, by a mixed consortium and a Pseudomonas species. They found that benzene and toluene can be degraded individually, but not p-xylene. In mixtures, benzene and toluene were removed according to competitive inhibition kinetics, whereas p-xylene was partially removed by a cometabolic process in the presence of either benzene and toluene. Burback and Perry (1993) reported that Mycobacterium vaccae can individually catabolize benzene or toluene; in a mixture of these two compounds, benzene degradation was delayed but toluene degradation proceeded in a manner similar to that when toluene was present alone. Alvarez and Vogel (1991) used pure and mixed cultures to evaluate possible beneficial BT(p-)X interactions and observed that the presence of toluene in mixtures with benzene and p-xylene enhanced the degradation of both aromatics. These researchers observed benzenedependent degradation of toluene and p-xylene, and retardation of both benzene and toluene degradation by pxylene. They also suggested that p-xylene was cometabolically degraded. Chang et al. (1992) used two pure cultures to quantify competitive inhibition kinetics of benzene and toluene, benzene and p-xylene, and toluene and p-xylene mixtures. They observed that the presence of toluene resulted in the competitive inhibition of benzene and the cometabolic degradation of p-xylene. One of their isolates was unable to degrade benzene in a mixture and the other transformed p-xylene to recalcitrant byproducts. Arvin et al. (1989) used two consortia to investigate benzene degradation in the presence of toluene, o-xylene, and a number of polynuclear aromatic hydrocarbons. Their study revealed that, in the presence of either toluene or o-xylene, benzene degradation was stimulated; however, when benzene was present in a mixture with both toluene and o-xylene, the combined effect was smaller than the sum of the stimulatory effects from each of these compounds. For the most part, the variations in the reported observations regarding microbial activity toward BTEX compounds in mixtures have led to some conflicting conclusions. Because the vast majority of contaminated sites involves complex mixtures of petroleum hydrocarbons, and because cosubstrate interactions are crucial for understanding and predicting the behavior of BTEX in biological treatment applications, this study focuses on identifying significant
substrate interactions during the aerobic biodegradation of BTEX mixtures by two consortia and one pure culture derived from a gasoline-contaminated aquifer. Because the role of ethylbenzene has rarely been investigated in previously reported mixture studies, our primary research goal was to characterize the effects of ethylbenzene on the biodegradation of benzene, toluene, and the three isomers of xylene in all possible mixture combinations. Because an initial proposed application of this study involved the use of bioremediation as a polishing step following a thermal gasoline remediation process (Deeb and Alvarez-Cohen, 1994a, 1994b; Udell and Stewart, 1989), a section of this article addresses the effect of temperature on the microbial growth and BTEX degradation rates of a consortium derived from a gasoline-contaminated site. MATERIALS AND METHODS Chemicals Benzene (>99% ACS reagent) was purchased from Mallinckrodt (Paris, KY). Toluene (99.8% ACS reagent), ethylbenzene (99.9% certified grade), and p-xylene (99.8% certified grade) were purchased from Fisher Scientific (Fair Lawn, NJ). o-Xylene (spectro grade) was purchased from J. T. Baker, Inc. (Phillipsburg, NJ). m-Xylene (no grade listed) was purchased from Eastman Kodak (Rochester, NY). Development of the Cultures Two toluene-enriched consortia were used in this study. The first consortium was derived using soil from a gasolinecontaminated aquifer located at Lawrence Livermore National Laboratory (CA). An aseptically collected soil sample was placed in a batch reactor containing toluene as the sole carbon and energy source, and a mineral salts medium with the following composition: CuSO4 ⭈ 5H2O, 0.5 mg/L; NaNO3: 1000 mg/L; K2SO4, 170 mg/L; MgSO4 ⭈ 7H2O, 37 mg/L; CaSO 4 ⭈ H 2 O, 8.6 mg/L; KH 2 PO 4, 530 mg/L; K2HPO4, 1060 mg/L; KI, 0.17 mg/L; ZnSO4 ⭈ 7H2O, 0.57 mg/L; MnSO4 ⭈ H2O, 0.34 mg/L; H3BO3, 0.124 mg/L; CoMoO4 ⭈ H2O, 0.094 mg/L; FeSO4 ⭈ 7H2O, 22.2 mg/L; H2SO4, 9.8 mg/L. Once bacterial growth was observed in the aqueous phase, a liquid sample was used to inoculate a second batch system. This procedure was repeated several times and a sample from the last series of subcultures was transferred to a continuous growth reactor. This consortium was developed at an ambient air temperature of 20°C, which resulted in a liquid temperature of 26° to 28°C. The culture was grown in a 14-L bench-top fermentor (Model No. MF-114, New Brunswick Scientific, New Brunswick, NJ), with a 5-L liter liquid volume, a 5-day detention time, and a mixing rate of 500 rpm. Toluene was added with the influent air which was bubbled through the chemostat liquid at a rate of 400 mL/min. Toluene concentration in the gas-phase influ-
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ent was maintained at 40 mg/L with an effluent of 20 to 30 mg/L. The consortium was sustained at a pH range of 6.8 to 7.0 in the aforementioned mineral salts medium. A second consortium was derived from the 20°C-enriched chemostat culture, but was grown in another continuous flow bioreactor at 35°C, a temperature, which was shown to enhance microbial activity and to optimize BTEX degradation rates. The cell detention time, as well as the influent and effluent toluene concentrations, were analogous to those of the 20°C-enriched consortium. The 35°C-enriched consortium was monitored on a regular basis for microbial stability using FAME (fatty acid methylester) analysis. A pure culture was isolated from the 35°C-enriched consortium on agar plates using toluene as the sole carbon and energy source. This isolate was identified by Microbial ID, Inc. (Newark, DE) as Rhodococcus rhodochrous. R. rhodochrous was grown aseptically in batch reactors consisting of 250-mL clear glass bottles (Alltech Co., Deerfield, IL) sealed with Teflon-lined Mininert valves and containing 100 mL of the mineral salts medium. The batch reactors were maintained at 35°C in a controlled environment incubator/shaker (Model No. G25, New Brunswick Scientific) with a mixing rate of 175 rpm; this mixing rate was experimentally shown to overcome potential oxygen mass-transfer limitations (Chang and Alvarez-Cohen, 1995). The growth reactors were amended with toluene (80 mg/L) and oxygen gas as needed to maintain approximately 15% to 21% oxygen by volume in the headspace. The cells were harvested during exponential growth phase. Stock solutions of R. rhodochrous were prepared by centrifuging the cell suspensions from the growth reactors at maximum speed for 20 min using an MSE GT-2 centrifuge (VWR Scientific, West Chester, PA) and by resuspending the pellets in a mineral salts medium. Experimental Procedures
Evaluating Temperature Effects on Microbial Activity Temperature studies were performed in batch reactors consisting of either 250-mL clear glass bottles (Alltech Co.) or 385-mL side-armed Nephelo culture flasks (Bellco Glass, Inc., Vineland, NJ). The reactors were sealed with Teflonlined Mininert valves (Alltech Co.). Culture growth on toluene was monitored over a broad range of temperatures (7° to 65°C) using a light spectrophotometer (Milton-Roy Spectronic 20D, Spectronic Instruments, Inc., Rochester, NY). A sample containing mineral salts medium (but no cells) was used as a reference for the light spectrophotometer prior to each absorbance measurement. Absorbance readings of liquid culture samples were taken at 600 nm and correlated to gravitametrically measured dry cell mass. Dry cell mass was measured in duplicate by taking the difference of sample weights after drying at 105°C for 8 h and after combustion at 550°C for 30 min. A temperature growth curve was ob-
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tained by plotting initial exponential growth rates on toluene (g cells/L per day) vs. growth temperature (°C), and a temperature optimum for culture growth and activity was determined. Arrhenius parameters, E and A, were obtained by linear regression of the exponential growth curve data as follows: lnrg = −
E 1 ⭈ + lnA R T
where rg is the cell growth rate (g/L per day), E is the activation energy (J/mol), R is the gas constant (atm.m3/ K.mol), T is the temperature (degrees K), and A is the Van’t Hoff–Arrhenius coefficient (frequency factor).
BTEX Biodegradation Rate Experiments (a) Experiments testing the biodegradation of each of the BTEX compounds were conducted in batch reactors consisting of 250-mL clear glass bottles sealed with Mininert valves. Each of the batch reactors contained 125 mL of liquid (mineral salts medium and cells), 123 mL of headspace, and 6 glass beads (6 mm, volume approx. 2 mL) to promote mixing and to prevent cell aggregation. The mass of aromatics added (M) was calculated using dimensionless Henry’s constants (Hc), reactor liquid and gas volumes (VL, VG), and aromatic substrate concentrations in the liquid and gas phases (SL, SG) using the following equation: M ⳱ SL ⭈ VL + SG ⭈ VG ⳱ SG(VL/HC + VG). HC values for BTEX compounds at standard temperature and pressure were adapted from Mackay and Shiu (1981). At 35°C, Hc values were calculated as described by Ashworth et al. (1987) (Table I). Pure phase BTEX compounds were added to the batch reactors using a high-precision 20-L syringe (Hamilton Co., Reno, NV) to result in an initial BTEX aqueous concentration of approximately 80 mg/L. All biodegradation rate experiments were conducted at 35°C. Prior to cell inoculation, the reactors were placed for 3 h in a 35°C controlled-environment incubator/shaker with a mixing rate of 175 rpm to ensure complete equilibrium BTEX partitioning between the liquid and gas phases. In the case of the consortia, the cell inoculum consisted of a 5- to 15-mL sample directly removed from the continuous growth bioreactor and purged with N2 for a short period of time to remove any residual toluene. In the case of the pure culture, the cell inoculum consisted of a 1- to 3-mL sample from the harvested cell stock solution. The volume of liquid culture added to the reactors was precalculated to result in uniform initial cell densities in all the batch reactors within one experiment. Immediately following the introduction of the cells to the batch reactors, the bottles were shaken vigorously for 1 min after which initial measurements of toluene were taken. The batch reactors were placed back in the 35°C incubator/shaker for the duration of the experiment and were only removed briefly for sampling. BTEX concentrations in the batch reactors were determined from headspace analyses. Samples of the head-
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Table I.
Henry’s constants for BTEX compounds at 35°C as calculated using a temperature regression equation described by Ashworth et al. (1987).
BTEX
A [-]
B [-]
Hb ⳱ exp(A − B/T), T ⳱ 308 K (35°C) [atm ⭈ m3/mol]
Benzene Toluene Ethylbenzene o-Xylene m-Xylene p-Xylene
5.534 5.133 11.92 5.541 6.280 6.931
3194 3024 4994 3220 3337 3520
0.00793 0.00923 0.0136 0.00734 0.0105 0.0111
a
a
Hcc ⳱ H/RT [-]
Densityd [g/L]
MW [g/mol]
0.314 0.365 0.540 0.291 0.416 0.440
0.879 0.867 0.866 0.880 0.868 0.861
78.1 92.1 106.1 106.1 106.1 106.1
a
A and B are constant parameters for the temperature regression equation. bHenry’s constant. cDimensionless Henry’s constant. dFrom the Merck Index (1989).
space gas (50 to 200 L) were withdrawn from the bottles using a Hamilton CR-700 constant rate gas-tight syringe (Hamilton Co.), and analyzed for BTEX using a HewlettPackard 5880 gas chromatograph equipped with a flameionization detector and a 0.75 mm × 30 m glass capillary column (Supelco Co., Bellefonte, PA). The oven, injector and detector temperatures of this gas chromatograph were fixed at 85°C, 250°C, and 300°C, respectively. Headspace sampling proceeded at frequent intervals until BTEX concentrations in the batch reactors dropped below the detection limits of the gas chromatograph (1 to 10 g/L). The gas chromatograph was calibrated using standards prepared by adding known amounts of pure phase aromatics or aromatic-saturated aqueous solutions to Teflon-sealed 250-mL glass bottles. CO2 production and O2 consumption were measured using a Hewlett-Packard 5890 gas chromatograph equipped with a thermal conductivity detector. This gas chromatograph was calibrated using Scotty certified multi-component gas mixtures (Alltech Co.). A set of cell-free controls was used to monitor abiotic losses of aromatics; abiotic BTEX losses never exceeded 5% throughout the duration of any experiment. Another set of controls, containing cells, but no aromatic substrates, was used to measure CO2 production from cell decay or other processes. Each experiment was repeated at least twice to ensure reproducibility of results. (b) Experiments testing the biodegradation of BTEX mixtures were conducted following a similar experimental procedure to the one just described. Each of the aromatics was present in a BTEX mixture at equal initial aqueous phase concentrations.
Substrate Interactions in Mixtures Experiments aimed at characterizing the inhibition effects of BTEX compounds in mixtures were performed in 250mL glass batch reactors similar to the ones just described. Lineweaver–Burk-type plots were produced using initial biodegradation rates of one BTEX compound in a binary mixture with an inhibitor. Initial biodegradation rates were determined from the linear regression of the first six to eight data points from BTEX disappearance plots within the first hour of the experiment. The concentrations of the substrate
and the inhibitor were varied to get a range of kinetic rates while maintaining a fixed cell density. A set of reactors containing the aromatic substrate, but no inhibitor, served as inhibitor-free controls. Cell-free controls as well as BTEXfree controls were also used. Inhibition characterization experiments were repeated three to six times to ensure reproducibility of results. Cometabolism was evaluated by measuring culture growth on each BTEX compound, alone and in the presence of a growth substrate, while monitoring BTEX disappearance.
Culture Growth on BTEX Compounds In experiments in which cell growth on each of the BTEX components was evaluated, the batch reactors consisted of 385-mL side-armed Nephelo culture flasks (Bellco), which allow absorbance measurements without liquid culture removal. The Nephelo flasks were equipped with 38-mm Teflon-lined screw caps (Bellco) and gas-tight Mininert valves on the sidearm (Alltech). They were also equipped with baffles to promote mixing and to prevent cell aggregation. The liquid volume in these reactors was set at 150 mL and consisted of the mineral salts medium and the liquid culture sample. The growth reactors were incubated at 35°C throughout the duration of the experiment. Absorbance readings of liquid culture samples were taken at 600 nm and correlated to gravitametrically measured dry cell mass as described earlier. A sample containing mineral salts medium (but no cells or substrate) was used as a reference for the light spectrophotometer. Cell-free controls were maintained throughout the duration of the experiment; negligible abiotic losses of BTEX were noted. RESULTS Evaluating Temperature Effects on Microbial Activity A microbial consortium, derived from a gasoline-contaminated aquifer, was enriched on toluene and grown in a chemostat at 20°C. Growth rate studies conducted over a
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range of temperatures to optimize microbial growth and degradation activity of this culture revealed that cell growth on toluene increased with temperature from 7° to 35°C, decreased sharply at incubations of 36° to 40°C, and was inhibited entirely at temperatures above 45°C (Deeb and Alvarez-Cohen, 1994a). A summary of the temperature effect studies is presented here in the form of a temperature growth curve (Fig. 1). The exponential growth phase data in this graph can be represented by the Arrhenius equation (rg ⳱ A ⭈ e−E/RT); linear regression of this data on a semilogarithmic scale (logarithm of the growth rate plotted against the reciprocal of the temperature in degrees K) yielded a straight line with a slope of −E/R and a y-intercept of lnA. The Arrhenius constant parameter, A, was calculated to be 2.1 × 1016 g/L per day, while the activation energy E was found to be 97 kJ/mol. The optimum temperature for culture growth and toluene degradation activity was determined to be 35°C. Because toluene oxidation was optimized at 35°C, cells from the 20°C-enriched consortium were transferred to a continuous flow bioreactor maintained at 35°C, and were grown under conditions analogous to those of the parent culture. A comparison was then made of the degradation kinetics of BTEX compounds by both of these consortia to determine whether the higher temperature caused fundamental changes in the overall degradation activity of the microbial culture. Results indicated that for the degradation of BTEX compounds, both individually and in representative mixtures, cells that were enriched and maintained at 35°C had increased degradation rates in most cases, compared with cells that were enriched and maintained at 20°C (Table II). In addition, the 35°C-enriched culture exhibited shorter BTEX degradation lag times than the 20°C-enriched culture. The only exception to these two observations was in the case of ethylbenzene; in a BTEX mixture, the rate of
TABLE IIa. Degradation rates of individual BTEX compounds by the 20°C- and 35°C-enriched cultures (initial B, T, E, and o-X aqueous phase concentrations ⳱ 80 mg/L). Individual BTEX compound
20°C-Enriched culture degradation rate (mg/mg per day)
35° C-Enriched culture degradation rate (mg/mg per day)
Benzene Toluene Ethylbenzene o-Xylene
0.49 0.79 0.41 NAa
1.2 1.4 1.0 0.64
a
Not available. The degradation of o-xylene by the 20°C-enriched culture was inhibited at concentrations >60 mg/L.
ethylbenzene degradation was faster for the 20°C-enriched culture (Table IIb). All kinetic rate experiments were repeated several times and the results were reproducible over time. Figure 2 shows representative comparisons of benzene and o-xylene degradation rates by the 35°C- and 20°Cenriched consortia. Substrate Utilization Patterns During BTEX Degradation by the Toluene-Enriched Consortia Both toluene-enriched consortia were capable of degrading all BTEX compounds, individually and in mixtures, over a broad range of hydrocarbon concentrations (up to 80 mg/L for the majority of BTEX compounds). Substrate degradation patterns observed for individual aromatics were compared with patterns for aromatics in mixtures and found to differ significantly. Toluene was repeatedly degraded faster by the toluene-enriched cultures than benzene, ethylbenzene, or the xylenes when each was present individually; this is shown in Figure 3a, and illustrates degradation patterns by the 35°C-enriched culture for each of the BTEX compounds in single-substrate experiments. This pattern was manifested in an even more distinct manner at higher BTEX concentrations, especially in the case of o-xylene, which had a degradation rate that decreased considerably at concentrations >60 mg/L due to substrate toxicity (Deeb and Alvarez-Cohen, 1994b). When cells were exposed to mixtures of aromatics, substrate degradation patterns were altered; ethylbenzene was degraded fastest, followed by toluene, benzene, and the xylenes. Fig-
Table IIb. Degradation rates of BTEX compounds in a mixture (total initial BTEX aqueous phase concentration ⳱ 80 mg/L; B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 20°C- and 35°C-enriched cultures
Figure 1. Growth curve depicting the effect of temperature on the growth rates of a toluene-oxidizing consortium enriched at 20°C. Linear regression of the exponential growth phase data on a semilogarithmic scale (logarithm of the growth rate plotted against the temperature) yielded a straight line.
530
BTEX compounds in a BTE(o-)X mixture
20°C-Enriched culture degradation rate (mg/mg per day)
35°C-Enriched culture degradation rate (mg/mg per day)
Benzene Toluene Ethylbenzene o-Xylene
0.90 1.2 2.7 0.71
1.5 1.4 1.2 0.93
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Figure 2. Representative data comparing the degradation rates of (a) benzene and (b) o-xylene (initial concentration of B ⳱ o-X ⳱ 80 mg/L) by two toluene-enriched consortia grown at 20°C and 35°C, respectively.
ure 3b shows representative results of substrate degradation patterns within a BTEX mixture with o-xylene exemplifying the behavior of xylenes. Similar gas chromatograph retention times for ethylbenzene and two of the xylenes made it difficult to differentiate individual compound degradation rates in comprehensive mixtures. Studies to evaluate substrate degradation patterns in all possible BTEX combinations revealed that, in each of these mixtures, the three isomers of xylene were always the last to be degraded, whereas ethylbenzene was unfailingly the first to be degraded (Table III). These substrate degradation patterns were analogous for both the 20°C- and 35°C-enriched consortia, and were reproducible over time. These patterns were also manifested in the presence of other common gasoline monoaromatics such as n-propylbenzene and 1,2,4trimethylbenzene.
Substrate Interactions During the Degradation of BTEX Mixtures by the 35°C-Enriched Consortium Substrate interactions were investigated in all possible BTEX combinations. Both positive and negative substrate interactions were observed during the biodegradation of BTEX mixtures by the 35°C-enriched consortium. These interactions were found to be different from those previously reported in the literature. In general, the presence of xylenes in most mixtures had little to no effect on the degradation of benzene, toluene, and ethylbenzene. However, in some cases, the presence of o-xylene may have enhanced the degradation of benzene to some extent and that of toluene to a lesser extent. For example, when benzene was present alone at a concentration of 20 mg/L, it took 1.6 h to degrade 50% of the initial aqueous phase concentration (10 mg/L) compared with 1.3 h when benzene was in a mixture with o-xylene. Similarly, when toluene was present alone, it took 0.8 h to degrade 10 mg/L compared with 0.68 h when present with o-xylene (Fig. 4). The presence of either toluene, benzene or ethylbenzene with one of the xylene isomers in binary mixtures consistently had a negative effect on xylene degradation rates; although the degradation of the xylene isomers was not entirely inhibited, a retardation in xylene degradation rates was observed. Figure 5 portrays these observed effects using the ortho isomer as a representative xylene. As can be seen, when o-xylene was present alone at a concentration of 40 mg/L, it was degraded the fastest. When o-xylene (40 mg/L) was present in a mixture with either benzene or toluene (40 mg/L), degradation was delayed by 2 h. The effect of ethylbenzene in a mixture with o-xylene was the strongest; the degradation of o-xylene was retarded by over 4 h in the presence of ethylbenzene. Further studies revealed that ethylbenzene was the most potent inhibitor of BTX in all mixtures tested. Figure 6a illustrates this inhibition by showing that the negative effect of ethylbenzene on toluene degradation was much more significant than that of either benzene or o-xylene. On the other hand, the presence of benzene, toluene, or xylenes in mixtures with ethylbenzene seemed to exert little or no effect on the degradation rate of this compound (Fig. 6b). Other inhibition effects observed in BTEX mixtures included the retardation of toluene degradation in the presence of benzene and the retardation of benzene degradation in the presence of toluene (Fig. 4). Because ethylbenzene was found to be the most potent inhibitor of BTX biodegradation in mixtures, kinetic rate studies were performed using the 35°C-enriched consortium in an effort to identify and quantify the observed inhibition effects. Representative data shown in Figure 7 illustrate the retardation of toluene degradation with increasing concentrations of the inhibitor, ethylbenzene. Double reciprocal (Lineweaver–Burk) plots prepared using observed initial biodegradation rates of toluene in binary mixtures with varied concentrations of ethylbenzene indicated no specific inhibition pattern, but rather suggested a combination of in-
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Figure 3. (a) Degradation of individual BTEX compounds (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ m-X ⳱ p-X ⳱ 20 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.27 g/L). (b) Degradation of a representative BTEX mixture (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.37 g/L).
hibition patterns, including both competitive and noncompetitive inhibition (data not shown). Substrate Interactions During the Degradation of BTEX Mixtures by R. rhodochrous R. rhodochrous was able to degrade all six BTEX compounds, individually and in mixtures, over a broad range of
hydrocarbon concentrations (0 to 40 mg/L). In BTEX mixtures, both positive and negative substrate interactions were observed. In studies similar to the ones performed using the consortium, ethylbenzene was shown to be the most potent inhibitor of BTX degradation in binary mixtures; representative data from such studies is shown in Figure 8. Figure 9
Table III. Order of biodegradation of mixtures of benzene (B), toluene (T), ethylbenzene (E), o-xylene (o-X), m-xylene (m-X), and p-xylene (p-X) by the 35°C-enriched toluene-grown culture. BTEX mixture
B, T B, E B, o-X T, E T, o-X T, m-X T, p-X E, o-X E, m-X B, T, E B, T, o-X B, E, o-X T, E, o-X B, T, E, o-X
532
T
B
E
1
2 2 1
1
2 1 1 1
2 1 2 2
o-X
m-X
p-X
2 1 2 2 2
3 2 2 3
1 1 1 1 1 1
2 2 3 3 3 4
Figure 4. Time in hours to degrade 50% of each of the BTEX compounds (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium alone or in binary mixtures with one other BTEX component.
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tion results are consistent with the limited data that has been reported previously for enhanced degradation rates of BTEX with increased temperatures. For example, in one study, toluene mineralization rates were shown to triple due to an increase in temperature from 11° to 25°C (Armstrong et al., 1991). When a consortium was enriched in a continuous growth reactor at 35°C, BTEX degradation rates increased and lag times decreased relative to the 20°C-enriched consortium. Comparisons of the degradation kinetics of BTEX compounds by the consortia enriched at 20° and 35°C were made in order to determine whether the increased degradation rates observed at the elevated temperature were due solely to temperature-induced increases in enzymatic reaction rates, or whether enrichment at the elevated temperature caused fundamental changes in the microbial consorFigure 5. o-Xylene degradation alone and in binary mixtures with B, T, and E (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 40 mg/L) by the 35°C-enriched consortium (cell density ⳱ 0.27 g/L).
illustrates the retardation of toluene degradation in the presence of increasing concentrations of ethylbenzene. The presence of o-xylene had no significant effect on the degradation of toluene in binary mixtures, whereas benzene had a slight inhibitory effect on toluene degradation (Fig. 8). Culture Growth on BTEX Compounds Studies were performed to evaluate the growth of the mixed and pure cultures on each of the BTEX aromatics. The 20°C- and the 35°C-enriched consortia, as well as R. rhodochrous, were able to effectively utilize toluene, benzene, and ethylbenzene as primary carbon and energy sources. Toluene, benzene, and ethylbenzene biodegradation corresponded to increases in cell mass, CO2 production and O2 consumption. Cell growth was evidenced by increases in optical density at 600 nm; for example, in the case of the growth of the 20°C-enriched consortium using benzene, a change in absorbance from 0.27 to 0.60 corresponded to an increase in cell dry weight from 0.14 to 0.35 g/L. Although initial additions of each of the xylene isomers to the batch reactors were degraded in the absence a growth substrate, the cultures were not able to grow using the xylenes. In fact, significant decreases in cell density were observed after repeated additions of o-xylene and m-xylene. DISCUSSION In batch studies conducted to quantify the effect of temperature on the microbial activity of the 20°C-enriched consortium, cell growth on toluene increased approximately fourfold due to an increase in the incubation temperature from 20° to 35°C. A temperature optimum of 35°C was determined from a range of temperatures tested (7° to 65°C). Although this is the first study to report the effect of temperature on microbial growth on toluene, our degrada-
Figure 6. (a) Toluene degradation alone and in binary mixtures with B, E, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium. (b) Ethylbenzene degradation alone and in binary mixtures with B, T, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by the 35°C-enriched consortium.
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Figure 7. Toluene degradation alone (initial concentration ⳱ 15 mg/L) and in the presence of ethylbenzene at a range of concentrations by the 35°C-enriched consortium (cell density ⳱ 0.21 g/L).
tium. Although degradation rates for individual BTEX compounds were higher for the 35°C-grown cells relative to 20°C-grown cells, the rate of ethylbenzene degradation in a BTEX mixture by the 35°C-enriched consortium was consistently lower than that by the 20°C-enriched consortium. These results suggest that fundamental changes in the composition of the consortium were promoted by enrichment at a higher temperature. Both the 20°C- and the 35°C-enriched consortia were able to degrade all BTEX compounds at higher concentrations than those commonly present in gasoline-contaminated subsurface environments. Additionally, the 35°Cenriched consortium was shown to mineralize all the BTEX compounds (14C-labeled) to 14CO2 (Deeb and Alvarez-
Figure 8. Toluene degradation alone and in binary mixtures with B, E, or o-X (initial concentration of B ⳱ T ⳱ E ⳱ o-X ⳱ 20 mg/L) by R. rhodochrous (cell density ⳱ 28 mg/L). The plotted data is the average of duplicates samples and the error bars represent the range.
534
Figure 9. Toluene degradation alone (initial concentration ⳱ 15 mg/L) and in the presence of ethylbenzene at a range of concentrations by R. rhodochrous (cell density ⳱ 35 mg/L). The plotted data is the average of duplicates samples and the error bars represent the range.
Cohen, 1998). As expected, both consortia degraded the enrichment substrate toluene the fastest when BTEX compounds were supplied individually. The fact that the xylenes were degraded the slowest among the BTEX compounds is consistent with what has been reported in the literature previously (Alvarez and Vogel, 1991). When cells were exposed to mixtures of aromatics present at equal concentrations, degradation patterns were modified substantially from those of individual BTEX compounds with disappearance in the order of ethylbenzene, toluene, benzene, and the xylenes. Surprisingly, ethylbenzene underwent preferential degradation over the enrichment substrate toluene. This pattern was also maintained in the presence of other monoaromatics such as n-propylbenzene and 1,2,4-trimethylbenzene, and even in a complex mixture such as gasoline (Deeb and Alvarez-Cohen, 1994b). In view of the fact that the bioremediation of gasoline contaminated sites requires the microbial degradation of complex waste mixtures, it is important to understand the potential enhancement or inhibition effects caused by the concurrent presence of multiple BTEX compounds in the subsurface. In this study, our major objective was to identify both negative (i.e., inhibition) and positive (i.e., stimulation, cometabolism) substrate interactions in comprehensive BTEX mixtures; to our knowledge, no previously reported work focusing on substrate interactions during the bacterial degradation of BTEX has included mixtures that incorporated ethylbenzene and all three xylene isomers. In the case of the 35°C-enriched consortium, the enhancement of BTEX degradation in mixtures was observed to a small extent for benzene and toluene when each was present in a binary mixture with o-xylene. This was an interesting observation, but is difficult to explain from the available information, especially because the cells were incapable of sustained o-xylene degradation in the absence of
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toluene, benzene, or ethylbenzene. On the other hand, the presence of toluene, benzene, or ethylbenzene with any of the xylene isomers in binary mixtures had a negative effect on xylene disappearance rates, although the degradation of the xylene isomers was not entirely inhibited. The most potent inhibitor of BTX degradation was ethylbenzene, whereas the effect of benzene, toluene, and xylenes on the degradation of ethylbenzene was found to be inconsequential. This is a significant finding, because, to our knowledge, this is the first report of inhibition in monoaromatic mixtures involving ethylbenzene. The use of Lineweaver–Burk plots for toluene degradation in the presence of ethylbenzene as the inhibitor did not yield conclusive results, but did suggest a mixed inhibition pattern for the 35°C-enriched consortium that may encompass both competitive and noncompetitive inhibition. These mixed inhibition effects are not surprising, because the culture tested is a mixed culture that can be expected to exhibit mixed inhibition patterns due to the presence of multiple species with multiple BTEX degradation pathways. Other inhibition effects that were noted, but not characterized due to lesser degrees of magnitude, included toluene inhibition in the presence of benzene and benzene inhibition in the presence of toluene. Because R. rhodochrous was isolated from the 35°Cenriched consortium, we expected to observe similar substrate interactions during the biodegradation of BTEX mixtures by this isolate. As anticipated, ethylbenzene was found to be the most potent inhibitor of benzene, toluene, and xylene degradation in binary mixtures. Moreover, this inhibition was more pronounced in the case of R. rhodochrous than in the case of the parent consortium; in fact, the degradation of BTX in binary mixtures did not proceed until all the ethylbenzene was degraded, although the interim lag period characteristic of diauxy was not observed. As a result, we were unable to obtain initial biodegradation rates of BTX in the presence of ethylbenzene over a range of ethylbenzene concentrations, which prevented us from generating Lineweaver–Burk plots and from identifying or quantifying the observed inhibition effects. In comparing the results of this work with previously published studies, we found some similarities and some differences. For example, the presence of toluene in previous studies has been shown to inhibit the degradation of benzene in mixtures (Burback and Perry, 1993; Chang et al., 1992; Oh et al., 1994) as was shown here. Although the presence of p-xylene in previous work has been shown to inhibit benzene and toluene degradation (Alvarez and Vogel, 1991; Oh et al., 1994), this was not observed in our work; in fact, we found some of the xylene isomers (i.e., o-xylene) to exhibit a slight stimulatory effect on BTE degradation. In this study, benzene and ethylbenzene were effectively utilized by the three toluene-grown cultures as primary carbon and energy sources, whereas the xylene isomers were not. Although initial additions of the xylenes were degraded by the three toluene-degrading cultures, repeated additions of o- and m-xylene caused marked decreases in cell densi-
ties. The inability of the three cultures to grow on the xylenes, combined with the failure of the cultures to degrade repeated additions of these compounds in the absence of a growth substrate, suggest that the xylenes were cometabolically degraded. This assumption is reasonable, because the observed losses in biomass upon the biotransformation of the xylenes can be explained by the dependency of the toluene oxygenase enzyme systems on cell-reducing power. The cometabolic degradation of the xylenes in BTEX mixtures in this work is consistent with what has been reported in previous mixture studies (Alvarez and Vogel, 1991; Chang et al., 1992; Oh et al., 1994). In conclusion, we have observed several patterns of substrate interactions in comprehensive mixtures of benzene, toluene, ethylbenzene, o-xylene, m-xylene, and p-xylene by two mixed consortia and one pure culture, R. rhodochrous. These patterns included no interaction, stimulation, competitive inhibition, noncompetitive inhibition, and cometabolism. Our results suggest that substrate interactions among monoaromatics in mixtures are complicated despite the similarities in the chemical properties and structures of these compounds. Our understanding of these interactions can contribute to a better approach for modeling BTEX biodegradation within a complex mixture such as gasoline. Nevertheless, it would be equally important to understand whether these interactions affect the mineralization (degradation to CO2) of BTEX mixtures. Most of the studies that have been referenced in this work have focused on the effect of mixtures on the biotransformation rates of BTEX. Studies investigating the effect of mixtures on the mineralization of BTEX using 14C-labeled aromatics have been performed in our laboratory. Manuscripts describing the results of these studies are currently in preparation. We thank Dr. Hsiao-Lung Chang for his assistance in enriching the 20°C-enriched consortium, Ms. Ruth Richardson for isolating R. rhodochrous, Prof. David Jenkins and Ms. Linda Sawyer for their suggestions on the analysis of our temperature study results, and Susan Hou and Harold Tully for their assistance in the laboratory.
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