Strain Ti was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar ... [a]anthracene even though it is not carcinogenic itself (3).
Vol. 57, No. 4
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1991, p. 1139-1145 0099-2240/91/041139-07$02.00/0 Copyright C) 1991, American Society for Microbiology
Anaerobic Degradation of Toluene by
a
Denitrifying Bacterium
PATRICK J. EVANS,' DZUNG T. MANG,' KWANG SHIN KIM,' AND L. Y. YOUNGl12* Departments of Microbiology' and Environmental Medicine,2 New York University
Medical Center, New York, New York 10016 Received 15 November 1990/Accepted
5
February 1991
A denitrifying bacterium, designated strain Ti, that grew with toluene as the sole source of carbon under anaerobic conditions was isolated. The type of agar used in solid media and the toxicity of toluene were determinative factors in the successful isolation of strain Ti. Greater than 50% of the toluene carbon was oxidized to C02, and 29% was assimilated into biomass. The oxidation of toluene to CO2 was stoichiometrically coupled to nitrate reduction and denitrification. Strain Ti was tolerant of and grew on 3 mM toluene after a lag phase. The rate of toluene degradation was 1.8 ,umol min-' liter-' (56 nmol min-' mg of protein-) in a cell suspension. Strain Ti was distinct from other bacteria that oxidize toluene anaerobically, but it may utilize a similar biochemical pathway of oxidation. In addition, o-xylene was transformed to a metabolite in the presence of toluene but did not serve as the sole source of carbon for growth of strain Ti. This transformation was dependent on the degradation of toluene.
Drinking water contaminated with toluene is a potential hazard in light of the following facts. Toluene is a depressant of the central nervous system (22), and it is an enhancing agent in skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene even though it is not carcinogenic itself (3). Most studies of the health effects of toluene have been concerned with respiratory and dermal exposure as opposed to oral exposure, for which the effects are relatively unknown (22). Nonetheless, toluene is a priority pollutant, and the Environmental Protection Agency has recommended that drinking water contain not more than 2 mg of toluene per liter for lifetime exposure (22). In 1988, the Environmental Protection Agency found toluene in 54% of groundwater samples and 28% of surface water samples near chemical waste sites (22). Significant contamination of groundwater also arises from spillage of gasoline or other petroleum-based fuels and from leakage of gasoline from underground storage tanks. This is not unexpected, since toluene is present in gasoline at 5 to 7% (wt/wt) and has a solubility of 515 mg/liter in water at 20°C (22). Microbial degradation of toluene has the potential to be rapid, provided that a suitable terminal electron acceptor is available for its oxidation. The aerobic biooxidation of toluene has been known for many years (2). However, groundwater is often depleted of oxygen or the amount of dissolved oxygen that is present is insufficient for the complete oxidation of organic contaminants. The anaerobic oxidation of toluene has recently been discovered to occur in the presence of alternative terminal electron acceptors. These compounds, which have been shown to include nitrate, iron, bicarbonate (10, 16, 18, 23, 24, 27), and perhaps sulfate (21a), either are indigenous to groundwater and soil (e.g., iron) or are attractive as possible additives, with certain limitations, because of their high solubility and diffusivity in water (e.g., nitrate). Most of these studies were completed in the presence of sediments either in batch microcosms or in continuous-flow soil columns. However, the isolation of a dissimilatory iron-reducing bacterium (16, 17) and the isolation of a denitrifier (4, 26) that both anaerobically oxidize toluene have been previously reported. *
In a comparative study, complete losses of toluene were observed in seven different enrichment cultures under denitrifying conditions (5). Partial losses of o-xylene that were concomitant to the losses of toluene were also observed in these cultures. Further enrichment led to the isolation of a bacterium that was responsible for toluene degradation. This paper describes the isolation of the pure culture and the characterization of its anaerobic degradation of toluene and transformation of o-xylene. MATERIALS AND METHODS Growth medium composition and preparation. The mineral salts medium used was prepared as described previously, except that yeast extract was not included (5). The medium was added to either serum bottles or crimp tubes (Bellco Glass, Vineland, N.J.) which were sealed with Teflon-coated butyl rubber stoppers (West Co., Lancaster, Pa.) and aluminum crimps. Anaerobic conditions were attained prior to sterilization by evacuation of the headspace and then pressurization with 67 kPa of argon that had passed through a reduced column of R3-11 catalyst (Chemical Dynamics Corp., South Plainfield, N.J.) to remove traces of oxygen. The bottle was then shaken vigorously to ensure adequate liquid-to-gas mass transfer of dissolved oxygen. This procedure was then repeated three times. Aromatic hydrocarbons were added neat with microliter syringes after the medium was deoxygenated and prior to sterilization. The bottles were shaken to dissolve the hydrocarbon, and the pierced stoppers were replaced anaerobically with new ones prior to sterilization. The replacement of the stoppers was completed while the headspace was gently flushed with argon. This procedure was tested for the maintenance of anaerobic conditions and the minimization of hydrocarbon losses. Pierced stoppers were also replaced aseptically after each sampling in time studies. Culture isolation and storage. A denitrifying enrichment culture that degraded toluene was initially developed as described elsewhere (5). This culture (referred to as CA2) was serially diluted into the minimal salts medium plus 300 ,uM toluene and incubated anaerobically. All incubations were at 30°C. The 10-8 dilution was the highest dilution for which growth was observed. An aliquot of the 10-8 culture
Corresponding author. 1139
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EVANS ET AL.
prepared with minimal medium and 2% (wt/vol) purified agar (Difco, Detroit, Mich.). The agar was autoclaved separately from the mineral salts medium. The plates were incubated anaerobically in the presence of vapor from a 1% (vol/vol) solution of toluene in hexadecane. An anaerobic atmosphere was produced with a C02-H2 generator and palladium catalyst and was assessed by reduction of methylene blue (13BL Microbiology Systems, Div. Becton Dickinson and Co., Cockeysville, Md.). Single colonies formed on this plate within 3 days, and distinctive short, fat, gram-negative rods were observed microscopically. Equivalent growth was also observed on Noble agar (Difco). Growth was inhibited with Bitek and Bacto-Agars (Difco) and Sigma agar (St. Louis, Mo). Growth was also inhibited by increasing the toluene concentration from 1 to 10% (vol/vol) in hexadecane. Single colonies were picked and streaked onto new plates twice in succession. The organism was further purified by picking and streaking a single colony three times in succession onto brain heart infusion (BHI) medium (BBL Microbiology Systems) solidified with purified agar (2% [wt/vol]) and by incubating aerobically at 30°C. Again, the substitution of Noble agar for purified agar did not affect the growth of the isolate on BHI plates, but the substitution of Bitek, Bacto, or Sigma agar inhibited growth on BHI plates. A single colony from the final BHI plate was picked and transferred into 1 ml of the minimal medium in a sterile culture tube with a vented sterile closure. This covered but unsealed tube was incubated in a sealed anaerobic atmosphere in the presence of vapor from a solution of 1% (vol/vol) toluene in hexadecane. The grown culture, which appeared microscopically as short, fat, gram-negative rods similar to those seen previously, was used as an inoculum for 150 ml of minimal medium with 1 mM toluene in a 160-ml serum bottle. After growth was complete, the culture was centrifuged aerobically (10,400 x g, 30 min, 4°C) and the supernatant was discarded. The pellet was resuspended in 5 ml of 30% (vol/vol) glycerol-1% (wt/vol) peptone (Polypeptone; BBL Microbiology Systems)-41 mM phosphate buffer at a pH of 7.5. This cell suspension was frozen in 1-ml aliquots in a dry ice-ethanol bath and preserved at -70°C. Inocula for all experiments were prepared by inoculating 1 ml of mineral salts medium in a vented culture tube with a sterile wooden applicator that was scraped against the frozen cell suspension kept in a dry ice-plus-ethanol bath. This method minimized the introduction of glycerol into the medium. The 1-ml culture was incubated anaerobically in a sealed container in the presence of 1% (vol/vol) toluene in hexadecane for 3 days. This culture was aseptically transferred to a 160-ml serum bottle with 150 ml of deoxygenated mineral salts medium plus 1 mM toluene and incubated for 2 was streaked onto plates
days. Culture conditions. Media were prepared anaerobically and inoculated with 1 to 10% (vol/vol) of an inoculum culture that was grown in the presence of 1 mM toluene. Incubation was in the dark at 30°C. Samples were removed anaerobically with sterile, plastic syringes. No significant difference in the concentration of aromatic hydrocarbon was observed in samples taken with plastic syringes compared with glass
syringes.
The rate of toluene degradation was measured in a cell suspension. Mineral salts medium containing 1 mM toluene
was inoculated with strain Ti and incubated for 2 days after which growth was evident and toluene was depleted. The culture was centrifuged aerobically (10,400 x g, 20 min, 4°C), and the supernatant was discarded. The pellet was
APPL. ENVIRON. MICROBIOL.
resuspended in an equal volume of fresh mineral salts medium without substrate. This suspension was deoxygenated and preincubated at 30°C for 30 min to ensure temperature equilibration. The rate study was initiated by the addition of 200 ,uM toluene (neat). Calculation of gaseous toluene concentrations. By using a binary toluene-hexadecane system, a low but constant gaseous concentration of toluene could be provided to the microorganisms. The vapor-liquid equilibrium of the toluene-hexadecane binary system has been investigated at 60 and 80°C by Messow and Engel (19). They observed a satisfactory correlation between the data and both the nonrandom two-liquid and Wilson equations (20). These equations are semitheoretical, and they present relationships between the activity coefficients and the liquid composition. = 32.84 Their published Wilson parameters (60°C: X12 cal moP-1 and X21 -22 = 188.7 cal mol-1; 80°C: X,12 -A = 253.16 cal mol-1 and X21 -22 =-415.03 cal mol-1 [1 cal = 4.184 J]) were used to estimate the activity coefficients for toluene in hexadecane at concentrations of 1 and 10% (vol/vol) at 60 and 80'C. This binary system is not thermodynamically ideal, as indicated by the activity coefficient of 0.745 for 1% (vol/vol) toluene at 80°C. Activity coefficients for toluene at 30°C were extrapolated from these data by use of an established linear relationship between ln y and T', where y is the activity coefficient and T is the absolute temperature. The activity coefficients were then used to calculate the partial pressure of toluene in the gas phase (yP) at equilibrium by the following equation, which states that the component fugacities of each phase are equal at equilibrium (20): yP = yXPsat, where y is the mole fraction of toluene in the gas phase, P is the total pressure in the gas phase, -y is the activity coefficient of toluene, x is the mole fraction of toluene in the liquid phase, and pS"t is the saturation vapor pressure of pure toluene at 30°C. psat was interpolated from data of Perry and Chilton (20) at other temperatures with the Clapeyron equation (20). The concentration of toluene in the gas phase was then calculated from yP with the ideal gas law. The concentrations of toluene in the gas phase in equilibrium with 1 and 10% (vol/vol) toluene in hexadecane and 100% toluene at 30°C were calculated to be 5.3, 45, and 170 mg liter-', respectively. The gas-phase concentrations, calculated assuming that the liquid phase is thermodynamically ideal, are 5.9, 48, and 170 mg liter-', respectively. The gas-phase concentration of pure hexadecane at 30°C is negligible; it was extrapolated from available vapor pressure data (20) to be 0.085 mg liter-. Analytical procedures. Cells were observed by transmission electron microscopy after aerobic growth in BHI medium. Growth was observed after 3 days, and samples were prepared for electron microscopy by placing a drop of the culture fluid on a carbon-coated Formvar grid and staining with 0.5% (wt/vol) phosphotungstic acid in distilled water. Samples were observed with a Seimens Elmiskop I. Pyruvic acid was assayed by flame ionization gas chromatography on a glass column (2 m by 2 mm) packed with 100/120 mesh 10% SP1000-1% H3PO4 on Supelcoport W/AW (Supelco, Bellefonte, Pa.). The injector and detector temperatures were 160 and 200°C, respectively. The column was used isothermally at 150°C. Aqueous standards and samples were acidified by the addition (10% [vol/vol]) of 10 N H3PO4 and injected (1 pJl). All aromatic compounds other than the hydrocarbons and metabolites were quantified by isocratic high-performance liquid chromatography (HPLC) with UV detection at 254 nm. A liquid-gradient chromatograph (model 332; Beckman,
VOL. 57, 1991
San Ramon, Calif.) with a variable-wavelength UV-visible light detector (model 155, Beckman) was used. The compounds were separated by a Supelcosil LC-18 column (25 cm by 4.6 mm; particle size, 5 ,um; Supelco). The compounds were eluted with 40, 50, or 60% methanol, 2% acetic acid, and the balance as water at 1 ml min-'. Samples (1 ml) were centrifuged for 2 min, and 10- to 30-ptl samples of the supernatants were injected automatically (model 231-401, Auto-Sampling Injector, Gilson Medical Electronics, Middleton, Wis.). Peak areas were quantified with an integrator (Chromjet; Spectra Physics, San Jose, Calif.). The cell concentration was measured as protein, optical density, or dry cell weight. Cellular protein was quantified with the bicinchoninic acid assay (Pierce, Rockford, Ill.) of lysed cells. Cells were lysed'at 100°C in 0.1 N NaOH for 10 min. The standard was bovine serum albumin, and the sensitivity of the assay was 10 ,ug ml-'. Optical density was measured with a spectrophotometer (Spectronic 20; Bausch & Lomb, Rochester, N.Y.) at 660 nm. Dry cell weight was determined by centrifuging a culture and washing the sedimented cells with distilled water. After a second centrifugation, the cells were transferred to a tared aluminum weighing dish with a minimal amount of distilled water and dried at 1200C overnight. The dish that contained the cells was allowed to cool in a desiccator prior to measurement of the gross weight. Aromatic hydrocarbons, N20, N2, and CO2 were measured by gas chromatography as described previously (5). Nitrate was measured with a nitrate electrode (model 93-07), double junction reference electrode (model 90-02), and digital pH meter (model 701) (all three instruments from Orion Research, Boston, Mass.). Nitrite was measured colorimetrically with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride (9). The A543 was measured with a spectrophotometer (model
UV-240; Shimadzu, Columbia, Md.). RESULTS Electron microscopy and taxonomy. Figure 1 shows an electron photomicrograph of a negatively stained preparation of the isolate, designated strain Ti. The surface shows invaginations typical of gram-negative bacteria. A gramnegative cell wall structure was evident in electron photomicrographs of thin sections of strain Ti. Flagellation is peritrichous, with zero to four flagella per cell. Strain Ti thus does not appear to be a Pseudomonas sp. (13). Additionally, endospores were not evident. The API Rapid NFT test (Analytab Products, Plainview, N.Y.) indicated that strain Ti was able to grow on L-malate and phenylacetate but not on D-glucose, L-arabinose, D-mannose, D-mannitol, N-acetyl-D-glucosamine, maltose, D-gluconate, caprate, adipate, and citrate. Glucose was not fermented, and tryptophanase, arginine dihydrolase, urease, esculin hydrolysis, gelatinase, and P-galactosidase were all negative. Strain Ti had positive oxidase and catalase reactions. There was no significant correlation with any of 18 Pseudomonas species or with 38 additional non-Enterobacteriaceae that are detectable by the Rapid NFT test. Additionally, strain Ti grew aerobically on toluene in the mineral salts medium without nitrate (data not shown). Carbon, nitrogen, and electron balances. Eight 150-ml batches of mineral salts medium with 5 mM KNO3 in 160-ml serum bottles were inoculated with strain Ti (1% [vol/vol]) and incubated for 6 days. Six of these batches contained 1 mM toluene, and two were controls without substrate that
ANAEROBIC TOLUENE DEGRADATION
1141
1u.am
FIG. 1. Transmission electron photomicrograph of strain Ti negatively stained with phosphotungstic acid.
were used to calculate background nitrate reduction not related to oxidation of added toluene. Background nitrate consumption and dinitrogen formation was less than 2% of that associated with toluene oxidation. The six bottles were separated into pairs, each pair being combined, to increase the accuracy of the dry cell weight data. Table 1 presents the average balance data for carbon, nitrogen, and electrons, these data being representative of 290 ml of culture (i.e., 300 ml minus 10 ml for initial samples). Cellular carbon was determined from the dry cell weight assuming 50% of the dry cell weight was carbon. This was based on data for Escherichia coli (1) and an elemental formula for denitrifying bacterial cells, namely, C5H702N (18a). The carbon content based on this formula is 53% (wt/wt). The carbon balance shows that 51% of the toluene carbon was mineralized to carbon dioxide and 29% was assimilated into biomass. Losses of toluene due to sorption by the Teflon-coated stopper were nil, and losses during stopper replacement were negligible (i.e., 2% based on Henry's law calculations). The remaining 20% was unaccounted for at this point. Nitrate was reduced to nitrite, nitrous oxide, and dinitrogen. The electron balance was based on the following stoichiometric equation: C7H8 + 4.5NO3- + 4.5H+ + 0.68NH3 -* 3.6CO2 + 0.68C5H702N + 2.25N2 + 4.9H20. The stoichiometry of conversion of C7H8 to 3.6C02 was based on the carbon balance in Table 1. The millimoles of electrons yielded by the oxidation of toluene was calculated from the toluene loss data and the relationship 22.4 mmol of electrons per mmol of toluene oxidized given by the above equation. The millimoles of electrons used for the reduction of N03 to NO27, N20, and N2 were calculated from the nitrogen balance data in Table 1 and the following stoichiometric factors: 2 mmol of electrons per mmol of N02 formed, 8 mmol of electrons per mmol of N20 formed, and 10 mmol of electrons per mmol of N2 formed. The reduction of N03 to N2 accounted for 69% of the available electrons from toluene
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EVANS ET AL.
APPL. ENVIRON. MICROBIOL.
TABLE 1. Balances on carbon, nitrogen, and electrons for strain Ti grown on 1 mM toluene Balance
Carbon
Nitrogen
Electron
Source and amt
% Difference
Sink and amt
Toluene, 25.0 mg of C
CO2 (mg of C), Cells (mg of C), Total,
N03- 1.54 mmol of N
12.8 7.25 20.1
N02- (mmol of N),
Toluene -- CO2 + cells, 6.64 mmol of electrons
oxidation, thus confirming the occurrence of denitrification (Table 1). Rate of toluene degradation. The rate of toluene degradation was measured in a cell suspension in which the initial toluene concentration was 200 p.M. A linear period of toluene loss was observed for 60 min, and toluene was depleted in 100 min. The volumetric rate of toluene loss was 1.8 p.mol min-' liter-', and the specific rate of toluene loss was determined to be 56 nmol min-' mg of protein-' based on the initial protein concentration. Toluene tolerance. Mineral salts media that contained different concentrations of toluene were inoculated (10% [vol/vol]) with strain Ti. The greatest toluene concentration at which growth was observed was 3 mM (Table 2). Both the lag phase and the cell density increased as a function of the toluene concentration. Growth substrates. Aromatic hydrocarbons were added to mineral salts medium neat at 300 ,uM, and the media were inoculated with strain Ti (1% [vol/vol]). Other aromatic compounds were added to the medium at 500 ,uM and inoculated (5% [vol/vol]). Toluene was the positive control substrate. Evidence of growth and substrate loss in duplicates were used to determine whether aromatic compounds were growth substrates for strain Ti. No tested aromatic hydrocarbon other than toluene served as a growth substrate for strain Ti, including benzene, ethyl benzene, p-xylene, m-xylene, and o-xylene. Aromatic compounds that did support growth included p-cresol, p-hydroxybenzyl alcohol, m-hydroxybenzaldehyde, p-hydroxybenzaldehyde, benzoic acid, m-toluic acid, m-hydroxybenzoic acid, and p-hydroxybenzoic acid. Complete losses, as determined by HPLC, were observed for all of these compounds with the exception of p-hydroxybenzyl alcohol which incurred a 40% loss in 28 days. Accumulation of p-hydroxybenzoic acid was observed (identified by HPLC coelution
-20
N20 (mmol of N), N2 (mmol of N), Total,
0.326 0.363 0.917 1.61
N03- N02- (mmol of electrons), N03-* N20 (mmol of electrons), N03-* N2 (mmol of electrons), Total,
0.653 1.45 4.58 6.68
0.60
with a p-hydroxybenzoic acid standard) in the cultures with p-cresol and p-hydroxybenzyl alcohol. Although complete loss of benzaldehyde was observed, growth or accumulation of transformation products such as benzoic acid was not observed at the time of sampling, suggesting an abiotic loss. Neither growth nor significant loss of substrate was observed with the following compounds during 18 days of incubation: phenol, m-cresol, o-cresol, benzyl alcohol, o-hydroxybenzyl alcohol, m-hydroxybenzyl alcohol, o-toluic acid, p-toluic acid, and o-hydroxybenzoic acid. Transformation of o-xylene. The dependence of o-xylene metabolism on the concentration of toluene was investigated. Mineral salts media with 100 ,uM o-xylene and different concentrations of toluene in triplicate were inoculated (5% [vol/vol]) with strain Ti and incubated for 6 days. Toluene was depleted in all cultures at the end of the incubation period. Figure 2 shows that growth, measured by cell protein concentration, increased approximately as a linear function of the initial toluene concentration. The assay for protein was sensitive to 10 ,ug ml-' and thus did not detect differences in growth below 400 ,uM toluene. The loss of o-xylene increased as a function of the initial toluene concentration from 0 to 500 ,uM and was independent of the initial toluene concentration greater than 500 ,uM. The loss of 5 ,uM o-xylene in the absence of toluene was attributed to evaporation. The metabolism of o-xylene was investigated further with respect to cosubstrate specificity and identification of metabolites. Media were prepared with various combinations of
60 _" 0 0
U
50
_/
C-,)
40
TABLE 2. Tolerance of strain Toluene concn (mM)
0.5
1.0 1.5 2.0 3.0 4.0 a
-, No growth.
Lag time (days)
1 1
2-5 14 >30 a
Effi
C
Ti to toluene Optical density at 660 nm
0.18
0.24 0.30 0.36 >0.35
30 ,
a)0) 3%
C
20 %0o
0 u
10
3L 0
200
400
600
800
Initial toluene conc.
10)00
00 12CDO
(AM)
FIG. 2. Loss of o-xylene (O) and growth of strain Ti (-) in the presence of different concentrations of toluene.
ANAEROBIC TOLUENE DEGRADATION
VOL. 57, 1991
TABLE 3. Growth of strain Ti and transformation of o-xylene Substrate and initial concn Toluene o-Xylene Pyruvate (mM) (>M) (>M)
0 0 300 300 300
100 100 100 100 0
Optical density at
o-Xylene
loss (-lM)
660 nm
0.047 (0.010)a 0.073 (0.004) 0.085 (0.007) 0.110 (0) 0.083 (0.004)
0 1 0 1 0
9.1-mm peak area,
p0e
0 0.69 0.27 (1.8) 0 15 (3.2) 4.30 (0.88) 13 (2.0) 3.90 (0.31) 0 NAb
a Each number within parentheses is 1 standard deviation. NA, Not applicable.
b
toluene, o-xylene, and pyruvate as detailed in Table 3. These media were inoculated (1% [vol/vol]) with strain Ti and incubated for 10 days. Nitrate was not limiting in any case. Complete losses of toluene and pyruvate were observed (data not shown). No significant growth was observed in the presence of 100 ,uM o-xylene alone, since the optical density was not greater than that of the control culture (no carbon or nitrate), 0.040. The addition of 300 pFM toluene or 1 mM pyruvate resulted in growth of strain Ti. o-Xylene (100 ,uM) did not enhance the growth of strain Ti on toluene. Thus, o-xylene does not serve as a growth substrate. Loss of o-xylene in the presence of toluene was significant, as opposed to its loss in the presence of pyruvate, even though growth was evident with each substrate. Additionally, pyruvate did not enhance the toluene-mediated loss of o-xylene. The loss of o-xylene was found to be due to its transformation to a metabolite that was detected by HPLC at 9.1 min. This metabolite was specific to o-xylene transformation (Table 3); the loss of o-xylene correlated with the appearance of the 9.1-min metabolite. This peak did not appear in the presence of any other substrate, including the aromatic compounds that were found to serve as growth substrates for strain Ti or were transformed by strain Ti. Additionally, Fig. 3 shows that the production of the 9.1-min metabolite was correlated with the temporal decrease in o-xylene concentration. Production of this metabolite and loss of o-xylene occurred during toluene degradation and for 1 day after toluene was depleted from the medium. A slow decrease in o-xylene concentration after 2 days was due to
1.0~
U
0.8
a 0
o0.6
~50.4
0
z
0.2
0.0
-
0
/ 1
2 Time
3
4
(d)
FIG. 3. Normalized time profiles of toluene (0) and o-xylene (O) degradation and production of the 6.4-min (0) and 9.1-min (U) metabolites. Because of normalization, the illustrated concentrations of the substrates and the metabolites bear no quantitative relationship to each other. d, Days.
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evaporation. Evaporative loss of the aromatic hydrocarbon was observed in this experiment because the stopper was replaced after each sample. The balance experiment (Table 1) was not subject to these losses, because only initial and final samples were taken. No subsequent decrease in the concentration of the 9.1-min metabolite was observed up to 7 days, at which time the experiment was terminated, indicating the lack of further metabolism. An additional metabolite that was observed only in active cultures that degraded toluene was detected at 6.4 min (Fig. 3). This peak also was not formed during the metabolism of any other aromatic or nonaromatic substrate. The production of the 6.4-min metabolite was not affected by the presence of o-xylene. The concentration of this metabolite did not decrease following the depletion of toluene. Thus, this toluene metabolite also does not appear to be further metabolized. The 6.4- and 9.1-min metabolites did not coelute by HPLC with any of the following compounds: benzyl alcohol, benzaldehyde, benzoic acid, hydroxybenzyl alcohol (all isomers), hydroxybenzaldehyde (all isomers), hydroxybenzoic acid (all isomers), catechol, phenol, cresol (all isomers), toluic acid (all isomers), o-methylbenzyl alcohol, o-tolualdehyde, 3,4-dimethylphenol, and 2,3-dimethylphenol. DISCUSSION The successful isolation of strain Ti was dependent on the type of agar used to solidify the mineral salts and complex media. In fact, attempts to reisolate strain Ti from the original enrichment culture were futile when agars other than purified and Noble were used. One possible explanation is that impurities in the other agars may have inhibited the growth of strain Ti. In addition, the concentration of toluene in the anaerobic atmosphere was a determinant in the isolation of strain Ti on solid media. The gas-phase concentration of toluene could be varied easily by varying the concentration of toluene in hexadecane (i.e., the liquid-phase concentration). The concentration of toluene in the gas phase in equilibrium with 10% (vol/vol) toluene in hexadecane, 45 mg liter-', was inhibitory to the growth of strain Ti, whereas that with 1% toluene in hexadecane, 5.3 mg liter-1, was not. This method is applicable to any isolation procedure in which the substrate is volatile and inhibitory at saturation concentrations. The only requirement is a negligibly volatile diluent with which the substrate is miscible. Since the volatility of hexadecane at 30°C is nil and there is no physical contact between hexadecane and the culture, it can be used under aerobic conditions as well without the risk of its utilization as a carbon source. A chemically similar diluent is optimal, since the binary system formed may approximate thermodynamic ideality. In this case, the concentration of the substrate in the gas phase is proportional to its mole fraction in the liquid phase. Thermodynamic vapor-liquid equilibrium data are unnecessary as supported by the small difference (.11%) between the actual concentrations of toluene in the gas phase and those calculated assuming that the liquid phase is ideal. This concept has been previously utilized, in principle, to alleviate the inhibition of toxic growth substrates to yeasts (7) and to alleviate the toxicity of water-immiscible organic solvents to Clostridium acetobutylicum during the extractive fermentation of glucose to butanol (6). Interestingly, the sensitivity of the isolation procedure to the concentration and toxicity of toluene does not mean that
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EVANS ET AL.
strain Ti is extremely sensitive to toluene. Strain TI was able to grow after a lag phase in the presence of 3 mM toluene, which is equivalent to 50% of the aqueous solubility of toluene (Table 2). It should be noted that the isolation technique is quite different from that utilized for the growth of aerobic toluene degraders on solid media in which a small tube of toluene is placed in the petri plate lid as the substrate source (25). However, anaerobic isolations commonly require the use of a sealed container to maintain anaerobic conditions. If toluene were administered as for the aerobic experiments, equilibration would yield an inhibitory vapor concentration (170 mg liter-1) in a sealed anaerobic container. On the other hand, during aerobic incubations, the plates are generally not in sealed containers, which allows toluene to dissipate into the surrounding environment and not reach inhibitory concentrations. Direct comparisons of the toluene tolerance of strain Ti to other aerobic and anaerobic toluene degraders is not straightforward primarily because of the techniques used to administer toluene and because its concentration was usually not measured, especially during aerobic studies. However, unpublished work in our laboratory showed that the oxidation of succinate by Pseudomonas aeruginosa PAO under denitrifying conditions was completely inhibited by 2 mM toluene. On the other hand, a recently described Pseudomonas putida strain is more tolerant of toluene than strain Ti (11). This organism cannot catabolize toluene but was able to grow on glucose in the presence of 50% (vol/vol) toluene. This concentration is approximately 840 times the solubility of toluene in water (i.e., 515 mg/liter [22] or 0.0595%) and, therefore, creates a biphasic system composed of a discrete organic phase and an aqueous phase saturated with toluene. Therefore, this P. putida strain was tolerant of toxicity related to both dissolved toluene and the aqueous-organic interface. The dissimilatory iron-reducer GS-15 grew on toluene at a concentration noted as 10 mM, which is also greater than the solubility limit (17). However, butyl rubber stoppers without a Teflon lining, which are known to readily absorb toluene, were used; thus, the actual concentration of toluene probably was lower. Toluene was degraded by strain Ti at a rate that resulted in complete utilization of 200 ,uM within 2 h and at a specific rate (56 nmol min-1 mg of protein-1) comparable with that reported for a highly enriched denitrifying culture, namely, 45 nmol min-1 mg of protein-1 (12). Volumetric rates that have been reported for mixed and pure anaerobic cultures range from weeks to months for complete loss of 100 to 103 FLM toluene (4, 10, 17, 18, 24, 26). These lower rates may be attributable to lower biomass concentrations. However, unless the cell concentrations are reported, specific rates cannot be determined. Cleavage of the aromatic structure of toluene by strain Ti is proven by the fact that 80% of the toluene carbon is accounted for as either CO2 (51%) or cell carbon (29%) (Table 1). These data support those reported for the denitrifier Pseudomonas sp. strain T (4) and the dissimilatory iron reducer GS-15 (17). The unaccounted fraction may be due to an inaccurate assumption that carbon made up 50% of the dry cell weight or to accumulation of the 6.4-min metabolite, which cannot be quantitated until its structure is elucidated. The nitrogen and electron balances illustrated that strain Ti is a denitrifier and that the growth and oxidation of toluene are coupled stoichiometrically to the reduction of nitrate and denitrification. Strain Ti appears to be distinct from other anaerobic bacteria that have been reported to degrade toluene. This is
APPL. ENVIRON. MICROBIOL.
supported by the observations that the dissimilatory Fe(III) reducer GS-15 can grow on phenol (17), Pseudomonas sp. strain T can grow on m-xylene (4, 26), and Pseudomonas pickettii PKO1 can grow on solid mineral salts media in the presence of benzene and ethyl benzene vapor (15). Strain Ti, however, was not able to grow on any of these compounds. The biochemical pathway of anaerobic toluene oxidation has been proposed to involve an initial hydroxylation of the benzene ring para to the methyl group to form p-cresol (4, 8, 14, 17, 23, 26). The methyl group would then be oxidized sequentially to form p-hydroxybenzoic acid which is then dehydroxylated to benzoic acid. Although this pathway is not the only one that has been proposed and has yet to be proven, strain Ti utilized all of the intermediates in the pathway as growth substrates. This is consistent with the proposed pathway but is not in itself proof of its existence. Thus far, none of the pure culture studies (strain Ti included) have yielded transiently produced metabolites which are known pathway intermediates. p-Cresol, o-cresol, and benzoic acid have been observed as transient intermediates during the methanogenic degradation of toluene in mixed culture (8, 23); however, whether the mechanism is different in denitrifying bacteria is unclear at present. Strain Ti is also novel in its ability to transform o-xylene. o-Xylene did not serve as a source of carbon for growth and was transformed only in the presence of toluene (Table 3). The only similar biological transformation of o-xylene that has been reported is mediated by Nocardia corallina A-2 in which it is transformed to o-toluic acid during growth on hexadecane (21). A different mechanism was employed by strain Ti, since o-xylene was transformed to a metabolite that did not coelute with o-toluic acid. In a microcosm study under denitrifying conditions, partial loss of o-xylene was observed simultaneous to the complete loss of toluene (10). However, whether a similar or different mechanism was in effect is unknown. Toluene-dependent transformation of o-cresol has been reported in a highly enriched denitrifying culture that degrades toluene (12). An unidentified metabolite of toluene was also formed. This metabolite is likely part or all of the unaccounted 20% of toluene carbon; however, since it is not subsequently degraded by strain Ti, it does not appear to be an intermediate in the oxidation of toluene to CO2. While only hypothetical at this time, strain Ti may have two pathways of toluene metabolism, one for its mineralization and another for transformation of toluene to the 6.4-min metabolite, the latter pathway also being able to transform o-xylene. Strain Ti was isolated from a mixed enrichment culture. This mixed culture and six others, all derived from different sources of inocula that were obtained from various environments and geographical regions, demonstrated an identical relationship of toluene degradation and o-xylene transformation (5). This relationship was also found to exist in a single bacterium, strain Ti, implying that the same organism or, more probably, the same mechanism may be widespread in the environment. Toluene was degraded at an appreciable rate (1.8 ,umol min-' liter-') and was tolerant of concentrations greater than 50% of its aqueous solubility. These characteristics have implications with respect to potential practical applications. However, the narrow range of aromatic hydrocarbons that were utilized by strain Ti is representative of some of the limitations that are faced with the use of pure cultures for degradative applications.
VOL. 57, 1991
ANAEROBIC TOLUENE DEGRADATION ACKNOWLEDGMENTS
This research was partially supported by NIEHS grant ES 04895, the New York State Center for Hazardous Waste Management, and the Hazardous Substances Management Research Center of New Jersey. Partial support for P.J.E. was from an NIH National Research Service award (5 T32 Al-07180) from the NIAID. REFERENCES 1. Bailey, J. E., and D. F. Ollis. 1977. Biochemical engineering fundamentals. McGraw-Hill Book Co., New York. 2. Claus, D., and N. Walker. 1964. The decomposition of toluene by soil bacteria. J. Gen. Microbiol. 36:107-122. 3. Dean, B. J. 1978. Genetic toxicology of benzene, toluene, xylenes and phenols. Mutat. Res. 47:75-97. 4. Dolfing, J., J. Zeyer, P. Binder-Eicher, and R. P. Schwarzenbach. 1990. Isolation and characterization of a bacterium that mineralizes toluene in the absence of molecular oxygen. Arch. Microbiol. 154:336-341. 5. Evans, P. J., D. T. Mang, and L. Y. Young. 1991. Degradation of toluene and m-xylene and transformation of o-xylene by denitrifying enrichment cultures. Appl. Environ. Microbiol. 57:450-454. 6. Evans, P. J., and H. Y. Wang. 1988. Response of Clostridium acetobutylicum to the presence of mixed extractants. AppI. Biochem. Biotechnol. 17:175-192. 7. Gill, C. O., and C. Ratledge. 1972. Toxicity of n-alkanes, n-alk-1-enes, n-alkan-1-ols and n-alkyl-1-bromides towards yeasts. J. Gen. Microbiol. 72:165-172. 8. Grbic-Galic, D., and T. M. Vogel. 1987. Transformation of toluene and benzene by mixed methanogenic cultures. Appl.
9.
10.
11.
12.
13. 14.
15.
Environ. Microbiol. 53:254-260. Greenberg, A. E., R. R. Trussell, L. S. Clesceri, and M. A. H. Franson (ed.). 1985. Standard methods for the examination of water and wastewater, 16th ed. American Public Health Association, Washington, D.C. Hutchins, S. R., G. W. Sewell, D. A. Kovacs, and G. A. Smith. 1991. Biodegradation of aromatic hydrocarbons by aquifer microorganisms under denitrifying conditions. Environ. Sci. Technol. 25:68-76. Inoue, A., and K. Horikoshi. 1989. A Pseudomonas thrives in high concentrations of toluene. Nature (London) 338:264-266. J0rgensen, C., and J. Flyvbjerg. 1990. Toluene metabolism and its effect on o-cresol transformation under nitrate reducing conditions. Abstract from Coopdration Scientifique et Technologique seminar on anaerobic biodegradation of xenobiotic compounds. Copenhagen, 22-23 November 1990, in press. Krieg, N. R., and J. G. Holt (ed.). 1984. Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. Kuhn, E. P., J. Zeyer, P. Eicher, and R. P. Schwarzenbach. 1988. Anaerobic degradation of alkylated benzenes in denitrifying laboratory aquifer columns. Appl. Environ. Microbiol. 54: 490-496. Kukor, J. J., and R. H. Olsen. 1990. Diversity of toluene
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degradation following long term exposure to BTEX in situ, p. 405-421. In D. Kamely, A. Chakrabarty, and G. S. Omenn (ed.), Biotechnology and biodegradation. Gulf Publishing Company, Houston. 16. Lovley, D. R., M. J. Baedecker, D. J. Lonergan, I. M. Cozzarelli, E. J. P. Phillips, and D. I. Siegel. 1989. Oxidation of aromatic contaminants coupled to microbial iron reduction. Nature (London) 339:297-300. 17. Lovley, D. R., and D. J. Lonergan. 1990. Anaerobic oxidation of toluene, phenol, and p-cresol by the dissimilatory iron-reducing organism, GS-15. Appl. Environ. Microbiol. 56:1858-1864. 18. Major, D. W., C. I. Mayfield, and J. F. Barker. 1988. Biotransformation of benzene by denitrification in aquifer sand. Ground Water 26:8-14. 18a.McCarty, P. L., L. Beck, and P. St. Amant. 1969. In Proceedings of the 24th Industrial Waste Conference. Purdue University, Lafayette, Ind. 19. Messow, U., and I. Engel. 1977. Thermodynamische Untersuchungen an Losungsmittel/n-Paraffin-Systemen. Z. Phys. Chem. 258:798-800. 20. Perry, R. H., and C. H. Chilton. 1973. Chemical engineers' handbook, 5th ed. McGraw-Hill Book Company, New York. 21. Raymond, R. L., V. W. Jamison, and J. 0. Hudson. 1967. Microbial hydrocarbon co-oxidation. I. Oxidation of mono- and dicyclic hydrocarbons by soil isolates of the genus Nocardia. Appl. Microbiol. 15:857-865. 21a.Reinhard, M., F. Haag, and P. L. McCarty. 1989. Proceedings of the International Symposium on Processes Governing the Movement and Fate of Contaminants in the Subsurface Environments, Stanford, Calif., 23 to 26 July 1989 (unpublished
abstract). 22. U.S. Public Health Service. 1989. Toxicological profile for toluene. Publication ATSDR/TP-89/23. Agency for Toxic Substances and Disease Registry, U.S. Public Health Service, Atlanta. 23. Vogel, T. M., and D. Grbic-Galic. 1986. Incorporation of oxygen from water into toluene and benzene during anaerobic fermentative transformation. Appl. Environ. Microbiol. 52:200-202. 24. Wilson, B. H., G. B. Smith, and J. F. Rees. 1986. Biotransformations of selected alkylbenzenes and halogenated aliphatic hydrocarbons in methanogenic aquifer material: a microcosm study. Environ. Sci. Technol. 20:997-1002. 25. Worsey, M. J., and P. A. Williams. 1975. Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) [sic] mt-2: evidence for a new function of the TOL plasmid. J. Bacteriol. 124:7-13. 26. Zeyer, J., P. Eicher, J. Dolfing, and R. P. Schwarzenbach. 1990. Anaerobic degradation of aromatic hydrocarbons, p. 33-40. In D. Kamely, A. Chakrabarty, and G. S. Omenn (ed.), Biotechnology and biodegradation. Gulf Publishing Company, Houston. 27. Zeyer, J., E. P. Kuhn, and R. P. Schwarzenbach. 1986. Rapid microbial mineralization of toluene and 1,3-dimethylbenzene in the absence of molecular oxygen. Appl. Environ. Microbiol. 52:944-947.
