Biodegradation of Trichloroethylene and Toluene by Indigenous ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p. 1911-1918 0099-2240/93/061911-08$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 59, No. 6

Biodegradation of Trichloroethylene and Toluene by Indigenous Microbial Populations in Soil SHIFANG FAN AND KATE M. SCOW*

Soils and Biogeochemistry Section, Department of Land, Air and Water Resources, University of California, Davis, California 95616 Received 29 October 1992/Accepted 17 March 1993

The biodegradation of trichloroethylene (TCE) and toluene, incubated separately and in combination, by indigenous microbial populations was measured in three unsaturated soils incubated under aerobic conditions. Sorption and desorption of TCE (0.1 to 10 tig ml-') and toluene (1.0 to 20 pg ml-') were measured in two soils and followed a reversible linear isotherm. At a concentration of 1 pg ml-', TCE was not degraded in the absence of toluene in any of the soils. In combination, both 1 pg of TCE ml-l and 20 ,ug of toluene ml-l were degraded simultaneously after a lag period of approximately 60 to 80 h, and the period of degradation lasted from 70 to 90 h. Usually 60 to 75% of the initial 1 ,ug of TCE ml-1 was degraded, whereas 100%o of the toluene disappeared. A second addition of 20 pg of toluene ml-' to a flask with residual TCE resulted in another 10 to 20% removal of the chemical. Initial rates of degradation of toluene and TCE were similar at 32, 25, and 18°C; however, the lag period increased with decreasing temperature. There was little difference in degradation of toluene and TCE at soil moisture contents of 16, 25, and 30%o, whereas there was no detectable degradation at 5 and 2.5% moisture. The addition of phenol, but not benzoate, stimulated the degradation of TCE in Rindge and Yolo silt loam soils, methanol and ethylene slightly stimulated TCE degradation in Rindge soil, glucose had no effect in either soil, and dissolved organic carbon extracted from soil strongly sorbed TCE but did not affect its rate of biodegradation.

addition of toluene, an aromatic cosubstrate supporting TCE degradation, and to test for the potential of other cosubstrates to support TCE degradation by soil microbial communities. Physical and chemical interactions between the volatile organic compounds (VOCs) and the soil matrix were also considered with respect to their impact on biodegradation.

A little over a decade ago, it was believed that trichloroethylene (TCE) could not be biodegraded (4). It is now well established that TCE can be degraded by methanogens (3), methanotrophs (10), and certain species of bacteria able to degrade aromatic compounds (15, 16, 25). The last group includes Pseudomonas cepacia (15), Pseudomonas mendocina (6), and Pseudomonas putida (25). The mono- or dioxygenase enzymes that catalyze the first step in toluene or phenol metabolism are implicated in the breakdown of TCE (6). Aerobic conditions do not appear to support the formation of the undesirable metabolites, such as dichloroethylene and vinyl chloride, that are dehalogenation products of anaerobic degradation of TCE (24). Given the diversity of microorganisms now known to degrade TCE under laboratory conditions, it is surprising that TCE is one of the most prevalent organic groundwater contaminants in the United States (22). The ubiquity of TCE contamination suggests that environmental conditions frequently are not conducive to TCE degradation. An impressive body of work on aromatic-induced TCE degradation has developed; however, most studies have been conducted with isolates of bacteria in pure culture. Although some of the TCE-degrading pure cultures were originally isolated from soil, little information on the potential for and rates of degradation of TCE by indigenous microbial populations in soil under environmental conditions exists. An understanding of the physical, as well as biological, factors controlling rates of degradation of TCE in soil and the vadose zone is critical to the development of successful in situ bioremediation methods and may provide insight into the traits important for survival of introduced organisms in soil. The purpose of this study was to compare the kinetics of biodegradation of TCE in several soils with and without the *

MATERIALS AND METHODS

Soils. Yolo silt loam, Reiff fine sandy loam, and Rindge mucky silt loam were collected from the top 20 cm of agricultural fields in Yolo County, Calif., passed through a 2-mm sieve, and stored in double polyethylene bags in the dark at 4°C. Selected soil properties, including the moisture level at which the experiments were conducted, unless otherwise noted, are summarized in Table 1. Organic matter was determined by a Walkley-Black wet combustion method, moisture was determined gravimetrically, and bulk density was determined volumetrically. Chemicals. The radioisotopes k1,2-14C]TCE (specific activity, 4.6 mCi mmol-1) and [ring-1 C]toluene (specific activity, 51.5 mCi mmol-1) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Certified toluene and TCE were obtained from Fisher Scientific (Fair Lawn, N.J.). All solutions were made up with NANOpure water (Barnstead NANOpure Series 550; Barnstead/Thermolyne Corp., Dubuque, Iowa). Experimental system. Fifty grams (dry weight) of soil was incubated with 0.1 or 1 jig of TCE ml of soil solution-1 and/or 5 or 20 ,ug of toluene ml of soil solution-l at room temperature (23 + 2°C) in 254-ml screw-cap glass bottles capped with TFE Mininert screw caps (Dynatech, Baton Rouge, La.). For the 14C02 evolution studies, a center well containing NaOH was also present. Measurement of degradation. Biodegradation was quantified by gas chromatograph analysis of headspace concentra-

Corresponding author. 1911

APPL. ENVIRON. MICROBIOL.

FAN AND SCOW

1912

TABLE 1. Selected properties of soils used in this study Soil

Organic matter (%)

Moisture (%)

Bulk density (g cm-3)

Rindge Yolo Reiff

11.2 2.2 1.4

35.4 11.7 13.9

2.0 2.4 2.5

tions over time. Nonsterile samples were compared with sterile controls to differentiate between losses due to sorption or other abiotic processes and those due to biodegradation. All treatments were in duplicate. To sample, 50 ,ul of headspace was removed with a gastight syringe (Hamilton; 1705RN) and injected into a 30-m megabore column (RTX502.2) on a Shimadzu GC-14A gas chromatograph equipped with both a photoionization detector and an electrolytic conductivity detector. The injector temperature was 200°C. The column temperature was maintained at 90°C for 6 min for isotherm measurement and then raised at a rate of 40°C min-1 to 150°C for 0.5 min to burn residues in the column. The carrier gas was ultrapure He (99.995%) with a flow rate of 15 ml min-1 and a linear velocity of 30 cm s-1. The makeup gas was ultrapure helium at a rate of 30 ml min-'. The reaction gas for the electrolytic conductivity detector was ultrapure hydrogen (99.99%) at a flow rate of 25 ml min-'. The detector temperatures were 205°C for the photoionization detector and 850°C for the electrolytic conductivity detector. In the radioisotope studies, 100,000 dpm of either radiolabeled toluene or TCE was added to flasks with sufficient unlabeled compound to make up concentrations of 20 or 1.0 ,g of toluene or TCE ml of soil solution-', respectively. The compound that was not being monitored was also added only in nonlabeled form. Two milliliters of 0.5 N NaOH was placed in a center well to trap the 14C02 evolved. The alkali was removed periodically with a syringe through the septum of the Mininert cap and replaced with fresh NaOH. For toluene, the sample was placed directly into 5 ml of Aquasol cocktail (NEN, Boston, Mass.), incubated overnight to

reduce chemiluminescence, and counted in a liquid scintillation counter (Packard A3000). For TCE, Ba(OH)2 was added to the NaOH to precipitate out the counts present in carbonate form. The precipitate was washed with dilute Ba(OH)2 solution until no counts could be detected in the wash solution. Then the precipitate was resuspended and counted. To test the effect of oxygen on the rate of biodegradation of TCE and toluene, 20 ml of headspace was replaced with the same volume of pure oxygen (99.99%) in flasks containing 50 g of Reiff soil amended with 20 and 1 ,ug of toluene and TCE ml-', respectively. After the oxygen was added, initial concentrations of TCE and toluene were measured. The influence of temperature was tested by incubating Yolo soil in incubators at temperatures of 32, 26, 18, and 10°C. Soil moisture effects were measured in Yolo soil incubated at moisture levels of 2.5, 5, 16, 25, and 30% (wt/wt). Effect of secondary carbon sources. Seven carbon sources, including toluene (22.7 mg), glucose (51.9 mg), phenol (27.1 mg), benzoate (30.1 mg), methanol (55.4 mg), ethylene (24.2 mg), and native dissolved organic carbon, were tested to determine their effect on the degradation of 1 ,ug of TCE ml-1 in Rindge and Yolo soils. With the exception of the native dissolved organic carbon, the mass of each chemical added provided the same amount of carbon as toluene at a concentration of 20 ,g ml of soil solution-'. Ethylene was added as a gas. All other chemicals were added in enough NANOpure water to make up a total volume of 400 ,ul. Native dissolved organic carbon was extracted by autoclaving 50 g (dry weight) of Rindge or Yolo soil with 100 ml of tap water for 1 h. After centrifugation at 9,200 x g for 10 min, the supematant was freeze-dried and then reconstituted with NANOpure water to make 400 pl of solution. Henry's law constant. Sterile water (5, 10, or 15 ml) was added to sterile 25-ml glass vials sealed with TFE Mininert screw caps. An aliquot of 1.5 ml of air was removed from each vial and replaced with 1 ml of saturated TCE or toluene vapor with 0.5 ml of vapor of stock solution of [1,2-14C]TCE

A D

s

O R

|o*

.000P 0000

SOIL AIR

NEW BIOMASS

NI & DISSOLUTION VOLATILIZATION OLLOIDaD SOIL

s 0

II 0

SOLUTIONt

SOIL MICROBES

1C02 FIG. 1. Soil compartments and transfer

processes

affecting the distribution of volatile organic chemicals in soil.

VOL. 59, 1993

or L-[U-ring-'4C]toluene. The vials were incubated for 24 h on a horizontal shaker, after which 1 ml of gas or liquid was removed for 14C counting or 100 ,ul of gas was removed for gas chromatography analysis. The Henry's law constant was calculated from the ratio of the headspace to the solution concentration. Measurement of adsorption and desorption. Adsorption and desorption isotherms were determined in sterile Rindge and Yolo soil under the same conditions under which biodegradation studies were conducted. In a 25-ml glass vial fitted with a Mininert screw cap, 5 g of soil was equilibrated with TCE and/or toluene on a vertical rotator (9-in [ca. 23-cm] diameter) at 72 revolutions per h (3600 rotation). The initial concentrations of TCE and toluene were estimated to be 0.1, 0.5, 1, and 5 ,ug ml of soil solution-1 and 5, 10, 20, and 50 ,ug ml of soil solution ml-', respectively. After rotating for 20 and 34 h, 50 ,ul of headspace was removed for gas chromatography to determine the equilibrium concentrations in the gas phase. The actual concentration in the soil solution phase was calculated with the Henry's law constant. The quantity of adsorbed phase was calculated by subtracting the mass in the gas and liquid phases from the total mass input. Desorption was measured by removing 60 cm3 of headspace from the flasks used in the adsorption assay described above after an equilibrium between the soil and atmosphere was established. The same volume of clean air was added back to the flask, and a headspace sample was taken immediately. The soil was then incubated, and the concentration of chemical in the headspace at 24 and 48 h was measured. This procedure was repeated twice. Mass balance analysis. Both TCE and toluene are subject to leakage; in fact, Teflon Mininert caps were used after preliminary studies showed that crimp-top Teflon seals frequently leaked TCE after the septum had been pierced one to two times by a syringe. Because biodegradation may be erroneously inferred from what is actually leaking of a chemical, mass balance analyses are crucial. At the end of the biodegradation experiments in Rindge and Yolo soils, 20 g of soil from each flask was extracted with 20 ml of methanol. After the soil particles were allowed to settle for 30 min, a 0.01- or 0.1-ml sample of supernatant was removed from the sterile or nonsterile sample, respectively, and mixed with 5 ml of NANOpure water. The mixture was analyzed for TCE and toluene with a gas chromatograph (Perkin-Elmer 8500; column, AT-624 megabore; flame ionization detector; carrier gas, He) equipped with a purge and trap (Tekmar LSC 2000). Approximately 83 to 100% of the added TCE and 80 to 100% of toluene could be recovered from sterile Yolo and Rindge soils after incubation and continuous sampling over a period of 450 h. Statistical analysis. An analysis of variance was performed on all sets of data by using Statview SE+ Graphics (ABACUS Concepts, Berkeley, Calif.). The F test showed that all treatments in which degradation occurred were significantly different from the sterile controls at the 95% confidence

interval. RESULTS Determination of physical and chemical constants for VOCs in soil. VOCs in unsaturated soil are subject to sorption, volatilization, and solubilization and are distributed among the soil solution, atmosphere, and solid phase (e.g. organic matter or clay) composing soil (Fig. 1). Microbial populations in soil are concentrated in the water films that fill pores

BIODEGRADATION OF TCE AND TOLUENE IN SOIL

1913

A tol only

300

co (I) 0

TCE + tol 250-

c 0

200

± 150 A :

100

I

10

ui (I)

15

20

I 25

30

gg toluene/ml headspace

0 I--

gg TCE/mI headspace FIG. 2. Sorption and desorption isotherms for toluene (tol) in the absence and presence of 1 ,ug of TCE ml-' (A) and TCE in the absence and presence of 20 ,ug of toluene ml-' (B). Solid lines show adsorption isotherms, and dotted lines show desorption isotherms.

and coat soil particles, and only the solution phase of a chemical is considered to be directly available for uptake by microorganisms. It is virtually impossible to determine the initial VOC concentration in the soil solution without measuring or calculating the sorption partition coefficient and the Henry's law constant, as well as knowing the volumes of organic matter, soil solution, and headspace in the flask. Isotherms for the adsorption and desorption of toluene and TCE were measured in sterilized Rindge soil under the conditions of the biodegradation studies. The isotherms for the two chemicals were linear over the concentration range tested, and calculated sorption partition coefficients normalized for organic carbon were 157 and 213 ,ug g of organic carbon-' for TCE and toluene, respectively. In sterile Yolo soil, the values were 88 and 114 ,ug g-1 for TCE and toluene, respectively. These values are similar to other values reported for the chemicals in soil (8, 11). Figure 2 shows the desorption isotherm for two of the concentrations of TCE and toluene measured in Rindge soil, and the results indicate that sorption was reversible after 34 h of exposure of the soil to the chemicals. When both chemicals were mixed together in the soil, 1 jig of TCE ml-' did not greatly influence the sorption of toluene in Rindge soil (Fig. 2A). The addition of 20 ,ug of toluene ml-', however, caused a decrease in the slope of the isotherm for TCE (Fig. 2B), suggesting compe-

10

FAN AND SCOW

1914

120

TABLE 2. Initial percentage of distribution of TCE and toluene in the phases of three soils Toluene (%)

TCE (%) Soil

Rindge Yolo Reiff

Gas

Liquid

Adsorbed

Gas

Liquid

Adsorbed

24 52 57

5 3 4

71 45 39

7 71 74

2 7 8

91 22 18


I

TCE (+Tot) sterle

A

Tol (+TCE) sterile

g\t^ *m 80 80 A

60

-

0o

4040 -~~~~

TCE (+Tol)

20

tition of the aromatic with TCE for sorption sites within the concentration range tested. Henry's law constants of 0.42 and 0.27 (dimensionless) were measured for TCE and toluene, respectively, which agreed well with values calculated from solubility and vapor pressure (18, 21, 27). The measured Henry's law constant and sorption partition coefficients were used to calculate the initial equilibrium concentration of TCE and toluene in the soil solution based on the headspace concentration. The initial percent distribution of TCE and toluene in the three soils is shown in Table 2. Yolo and Reiff soils were similar with respect to the distribution of VOCs; however, the majority of TCE and toluene started out in the sorbed phase in the high-organic Rindge soil. Degradation of TCE and toluene in Rindge, Yolo, and Reiff soils. Biodegradation in Rindge soil of [14C]toluene at concentrations of 5 and 20 p,g ml of soil solution-' began after an 80-h lag period and was completed by 240 h (Fig. 3). The approximately 5% 14C measured over the course of the experiment in the sterile control was due to partitioning of 14C-labeled toluene into NaOH. After incubation of radiolabeled TCE in the presence of unlabeled toluene, 38% of the original counts could be recovered as carbonate, indicating that TCE was mineralized to CO2. Gas chromatographic analysis showed that the headspace concentration of toluene added at an initial concentration of 20 ,ug of toluene ml-' in Rindge soil was reduced (Fig. 4A) over the same time period as shown in the radioisotope study. The TCE concentration was substantially reduced in the presence of toluene over the same time period during which toluene was degraded. In the sterile flasks, however, neither TCE nor toluene was reduced in concentration. The initial addition of toluene to Rindge soil reduced TCE

A

~~~~~Tol(+TCE)

A

0

0 120 c 0 cu

L._

100

C) I._

0 c

0-

400

TCE (+Tol) stenle Tol (+TCE) sterle

A

4-.

300

200 a

100

-

A

80

60-

TCE (+Tol)

40 20-

Tol (+TCE)

0 0

100

200

300

400

0

100

200

300

400

Hour FIG. 4. Degradation of 20 ptg of toluene (Tol) and 1 ,ug of TCE ml-' in Rindge (A), Yolo (B), and Reiff (C) soils.

80

C)

N

-4ffi~ a

60

20

Ftg

ml

~to

200 h (Fig. SB, inset). A second addition of toluene at 600 h further reduced the TCE concentration by another 10 to 20% in soil that originally contained TCE and toluene and stimulated TCE degradation in soil originally containing TCE only (Fig. SB). The first addition of toluene was rapidly removed from the headspace (Fig. 5A, inset), whereas the second addition disappeared at a lower rate (Fig. 5A). With addition of oxygen at 3,000 h, additional TCE was removed

co

b-

.)

C)

approximately 60% of its initial headspace concentration

over

40

0-0

20

/20 pg ml 200

400

~(stenle) 600

(Fig. 5). 800

10 00

Hour FIG. 3. Mineralization of 5 and 20 ,ug of toluene soil.

ml-' in Rindge

The kinetics of biodegradation (again analyzing headspace ,ug of TCE, both with and without 20 ,ug of toluene ml of soil solution-', in Yolo (Fig. 4B) and Reiff (Fig. 4C) soils were similar to data observed in Rindge soil. TCE was not degraded in the absence of toluene in any of the soils (data not shown). For all three soils, the lag period was concentration) of 1

VOL. 59, 1993

BIODEGRADATION OF TCE AND TOLUENE IN SOIL

(U

1915

0

C 0

1000

0

2000

3000

c

CA C.)

140

E

120

0-0 100 100 l

=

15 to 50%; ++, >50% degraded within 1,900 h.

w U I-

C

-0

0-

120

100

._

C.)

0

200

400

600

800

Hour FIG. 8. Effect of soil moisture on the degradation of 20 ,ug of toluene (A) and 1 ,ug of TCE (B) ml-' in Yolo soil.

site (12). In rhizosphere and nonrhizosphere soils collected from a site contaminated with chlorinated solvents, radioisotope studies showed that the percentage of TCE mineralized in 30 days was considerably lower than what was observed in the present study (26). Therefore, only a small amount of cosubstrate would be required to support TCE degradation, and this could easily be present in slurried soil. In the well water study, total bacterial numbers increased to greater than 108 cells ml-' at TCE concentrations of 0.56 and 6.7 mg liter-1 without the addition of a cosubstrate (12). Because the final population density was achieved before TCE began to degrade and was independent of the TCE concentration, growth must have been due to an unidentified carbon source which may have also supported TCE cometabolism. Adsorption of TCE and toluene followed reversible, linear sorption isotherms. Thus, it appears unlikely that desorption is limiting biodegradation in the short-term experiments reported in this study. This is in contrast to observations from longer-term studies in which if the soil is exposed for weeks or months before significant biodegradation occurs, there may be formation of residues that are difficult or impossible to desorb and thus recalcitrant to degradation.

This phenomenon has been reported for TCE, tetrachloroethylene, and other volatile chemicals (2, 17, 19). The lag period preceding degradation was approximately 60 to 80 h in all three soils tested at the concentrations tested. This was surprising, given the differences in the physical properties and, presumably, the microbial populations of Rindge soil and the other two soils. Thus, soil organic matter content, which influences sorption and hence chemical availability and has been shown to influence the lag period for some chemicals (20), did not appear to affect the lag in this study. This may be because the high volatility of TCE and toluene facilitates a more rapid exchange between the solid, solution, and atmospheric components of soil than is possible for a less volatile compound and therefore the rate of desorption is rapid relative to the rate of biodegradation. A long lag period for TCE under unsaturated conditions could be problematic in bioremediation efforts if diffusion is so rapid that retardation of the chemical in a biologically active location is too brief for significant degradation to occur. Because the exposure of contaminated soil to pollutants may be intermittent, the duration of enzymatic activity on TCE after toluene has disappeared from the system is important. Pure cultures of phenol- or toluene-degrading organisms have been shown to have activity on TCE after induction on, but in the absence of, the cosubstrate (7). In soil, on the other hand, degradation of TCE usually terminated abruptly when toluene was no longer detectable. Additional removal of residual TCE required a second addition of toluene. The disappearance of toluene and TCE was simultaneous, and the period of active degradation lasted from 70 to 90 h in all three soils. Population densities of microorganisms able to degrade specific compounds are difficult to determine in soil, and these measurements were not attempted in this study. Therefore, an exact comparison of rates measured in soil and rates measured in pure cultures (usually reported on a per-cell or per-unit of biomass basis) is not possible. Nevertheless, it was evident that degradation in soil was considerably slower, e.g., on the order of days, than in induced solution cultures of bacteria where similar concentrations are degraded within a few hours with no lag period (7, 9, 25). Mass transfer limitations in unsaturated soil may contribute partially to lower rates, as suggested by data showing that microbial populations in shaken suspensions of Yolo soil degraded TCE and toluene at considerably higher rates than in unsaturated soil (13). Despite significant differences in rates of metabolism, there were similarities between the behavior of pure cultures of bacteria and that of indigenous soil microbial populations

1918

FAN AND SCOW

that cometabolize TCE in the presence of toluene. Questions remain regarding the specific populations of microorganisms responsible for TCE degradation in the different soils, whether toxicity or inhibition may ultimately decrease rates of TCE degradation in soil as observed in pure cultures, and the factors responsible for the observed lag phase. In order to extend this information to practical applications, it will be important to investigate the interactions between convective and diffusive mass transfer and biodegradation of TCE and toluene to predict the potential and rates of degradation of these chemicals in different environments. ACKNOWLEDGMENTS This work was supported by National Institute of Environmental Health Science's Superfund Basic Research program P42 ESO 4699; the U.S. Department of Energy (DOE) Office of Environmental Restoration and Waste Management Young Faculty Awards Program administered by Oak Ridge Associated Universities for DOE; the U.S. Department of Interior Geological Survey, through the State Water Resources Research Institute of California, Water Resources project UCAL-WRC-W-759; the EPA Center for Ecological Health Research (EPA-CR819659010); the U.C. Toxic Substances Program in Ecotoxicology; and Hatch Experiment Station project 5108-H. We are grateful to Blythe Hoyle for reviewing the manuscript. REFERENCES 1. Abril, M.-A., C. Michan, K. N. Timmis, and J. L. Ramos. 1989. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J. Bacteriol. 171:6782-6790. 2. Ball, W. P., and P. V. Roberts. 1991. Long-term sorption of halogenated organic chemicals by aquifer material. 1. Equilibrium. Environ. Sci. Technol. 25:1223-1236. 3. Bouwer, E. J., and P. L. McCarty. 1984. Modeling of trace organics biotransformation in the subsurface. Ground Water 22:433-440. 4. Bouwer, E. J., B. E. Rittman, and P. L. McCarty. 1981. Anaerobic degradation of halogenated 1- and 2-carbon organic compounds. Environ. Sci. Technol. 15:596-599. 5. Ensign, S. A., M. R. Hyman, and D. J. Arp. 1992. Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain. Appl. Environ. Microbiol. 58:3038-3046. 6. Ensley, B. D. 1991. Biochemical diversity of trichloroethylene metabolism. Annu. Rev. Microbiol. 45:283-299. 7. Folsom, B. R., P. J. Chapman, and P. H. Pritchard. 1990. Phenol and trichloroethylene degradation by Pseudomonas cepacia G4: kinetics and interactions between substrates. Appl. Environ. Microbiol. 56:1279-1285. 8. Garbarini, D. R., and L. W. Lion. 1986. Influence of the nature of soil organics on the sorption of toluene and trichloroethylene. Environ. Sci. Technol. 20:1263-1269. 9. Harker, A. R., and Y. Kim. 1990. Trichloroethylene degradation by two independent aromatic-degrading pathways in Alcaligenes eutrophus JMP134. Appl. Environ. Microbiol. 56:11791181.


10. Little, C. D., A. V. Palumbo, S. E. Herbes, M. E. Lidstrom, R. L. Tyndall, and P. J. Gilmer. 1988. Trichloroethylene biodegradation by a methane-oxidizing bacterium. Appl. Environ. Microbiol. 54:951-956. 11. Lyman, W. J. 1982. Adsorption coefficient for soils and sediments, p. 4-22. In W. J. Lyman, W. F. Reehl, and D. H. Rosenblatt (ed.), Handbook of chemical property estimation methods. McGraw-Hill Book Co., New York. 12. McClellan, K. L., N. Buras, and R. C. Bales. 1989. Biodegradation of trichloroethylene by bacteria indigenous to a contaminated site. J. Environ. Sci. Health A24:561-570. 13. Mu, D. Y., and K. M. Scow. Unpublished data. 14. Nelson, M. J. K., S. 0. Montgomery, W. R. Mahaffey, and P. H. Pritchard. 1987. Biodegradation of trichloroethylene and involvement of an aromatic biodegradative pathway. Appl. Environ. Microbiol. 53:949-954. 15. Nelson, M. J. K., S. 0. Montgomery, E. J. O'Neill, and P. H. Pritchard. 1986. Aerobic metabolism of trichloroethylene by a bacterial isolate. AppI. Environ. Microbiol. 52:383-384. 16. Nelson, M. J. K., S. 0. Montgomery, and P. H. Pritchard. 1988. Trichloroethylene metabolism by microorganisms that degrade aromatic compounds. Appl. Environ. Microbiol. 54:604-606. 17. Pavlostathis, S. G., and G. N. Mathavan. 1992. Desorption kinetics of selected volatile organic compounds from field contaminated soils. Environ. Sci. Technol. 26:532-538. 18. Pearson, C. R., and G. McConnell. 1975. Chlorinated Cl and C2 hydrocarbons in the marine environment. Proc. R. Soc. Lond. B 189:305-322. 19. Pignatello, J. J. 1990. Slowly reversible sorption of aliphatic halocarbons in soils. I. Formation of residual fractions. Environ. Toxicol. Chem. 9:1107-1115. 20. Scow, K. M. Effect of sorption-desorption and diffusion processes on the kinetics of biodegradation of organic chemicals in soil. In D. Linn and T. Carski (ed.), Sorption and degradation of agricultural chemicals in soil, in press. Soil Science Society of America Special Publication, Madison, Wis. 21. Sutton, C., and J. A. Calder. 1975. Solubility of alkylbenzenes in distilled water and seawater at 25'C. J. Chem. Eng. Data 20:320-322. 22. U.S. Environmental Protection Agency. 1980. Ambient water quality criteria for trichloroethylenes. Publication 440/5-80-073. National Technical Information Services, Springfield, Va. 23. Vance, G. F., D. L. Mokma, and S. A. Boyd. 1986. Phenolic compounds in soils of hydrosequences and developmental sequences of spodosols. Soil Sci. Soc. Am. 50:992-996. 24. Vogel, T. M., and P. L. McCarty. 1985. Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions. Appl. Environ. Microbiol. 49:1080-1083. 25. Wackett, L. P., and D. T. Gibson. 1988. Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida Fl. Appl. Environ. Microbiol. 54:17031708. 26. Walton, B. T., and T. A. Anderson. 1990. Microbial degradation of trichloroethylene in the rhizosphere: potential application to biological remediation of waste sites. Appl. Environ. Microbiol. 56:1012-1016. 27. Weast, R. C. (ed.). 1977. CRC handbook of chemistry and physics. CRC Press, Inc., Cleveland.