Isolation and Characterization of a Novel Toluene-Degrading, Sulfate ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1996, p. 1188–1196 0099-2240/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 62, No. 4

Isolation and Characterization of a Novel Toluene-Degrading, Sulfate-Reducing Bacterium HARRY R. BELLER,1* ALFRED M. SPORMANN,1 PRAMOD K. SHARMA,1 JAMES R. COLE,2 AND MARTIN REINHARD1 Environmental Engineering and Science, Department of Civil Engineering, Stanford University, Stanford, California 94305-4020,1 and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 488242 Received 27 November 1995/Accepted 31 January 1996

A novel sulfate-reducing bacterium isolated from fuel-contaminated subsurface soil, strain PRTOL1, mineralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. The mineralization of 80% of toluene carbon to CO2 was demonstrated in experiments with [ring-U-14C]toluene; 15% of toluene carbon was converted to biomass and nonvolatile metabolic by-products, primarily the former. The observed stoichiometric ratio of moles of sulfate consumed per mole of toluene consumed was consistent with the theoretical ratio for mineralization of toluene coupled with the reduction of sulfate to hydrogen sulfide. Strain PRTOL1 also transforms o- and p-xylene to metabolic products when grown with toluene. However, xylene transformation by PRTOL1 is slow relative to toluene degradation and cannot be sustained over time. Stable isotope-labeled substrates were used in conjunction with gas chromatography-mass spectrometry to investigate the by-products of toluene and xylene metabolism. The predominant by-products from toluene, o-xylene, and p-xylene were benzylsuccinic acid, (2-methylbenzyl)succinic acid, and 4-methylbenzoic acid (or p-toluic acid), respectively. Metabolic by-products accounted for nearly all of the o-xylene consumed. Enzyme assays indicated that acetyl coenzyme A oxidation proceeded via the carbon monoxide dehydrogenase pathway. Compared with the only other reported toluene-degrading, sulfate-reducing bacterium, strain PRTOL1 is distinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marine environment.

Leakage of gasoline from underground fuel storage tanks is a pervasive source of groundwater contamination in the United States (41). Bioremediation is one of a limited number of options for restoring aquifers contaminated with the hazardous, relatively water-soluble aromatic hydrocarbons that occur in unleaded gasoline, such as benzene, toluene, and xylenes. Because many contaminated aquifers are anaerobic as a result of oxygen depletion by indigenous aerobic microorganisms, hydrocarbon degradation by indigenous anaerobic bacteria merits serious consideration at some sites as a method of groundwater restoration. Partially in response to such environmental concerns, knowledge of the metabolic capabilities of anaerobic bacteria has expanded dramatically in the past decade, particularly with respect to single-ring aromatic hydrocarbons and closely related oxygenated compounds. For example, 18 pure cultures capable of anaerobic toluene degradation have been reported since 1989, including 16 denitrifying cultures (10, 14, 19, 34, 36, 48), one ferric iron-reducing culture (25, 26), and one marine sulfate-reducing culture (33); fermentative-methanogenic mixed enrichment cultures that degrade toluene have also been reported (12, 21, 43). More than 10 sulfate-reducing cultures that can grow on aromatic substrates have been reported since 1980 (3, 4, 8, 9, 11, 22, 23, 32, 33, 35, 39, 40, 46); however, only one of these can metabolize an aromatic hydrocarbon (Desulfobacula toluolica) (33). In this article, we report the isolation of strain PRTOL1, the second sulfate-reducing bacterium known to degrade an aromatic hydrocarbon and the first such organism from a freshwater environment.

MATERIALS AND METHODS Chemicals. The chemicals used in this study were of the highest purity available (generally $99%) and were used as received. Most organic chemicals were purchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.), and most inorganic chemicals were purchased from J. T. Baker, Inc. (Phillipsburg, N.J.). Stable isotope-labeled chemicals included toluene-d8 (.99 atom%; Aldrich Chemical Co.); o-xylene-d10 and p-xylene-d10 (991 atom%; Aldrich Chemical Co.); and benzaldehyde-a-13C,d1 (98% purity [D] and 99% purity [13C]; Isotec, Inc., Miamisburg, Ohio). Gas chromatography-mass spectrometry (GC-MS) analyses of deuterium-labeled alkylbenzenes demonstrated that they were of high purity and contained none of the metabolites reported in this study. For radiolabeled assays, [ring-U-14C]toluene (.98% purity, 10.2 mCi/mmol; Sigma Chemical Co., St. Louis, Mo.) was diluted into unlabeled toluene (.99.9% purity; Aldrich Chemical Co.) to a final specific activity of 75.1 mCi/mmol. Only a few of the compounds reported as metabolic by-products in this study were commercially available: benzylsuccinic acid (Sigma Chemical Co.), 3-benzoylpropionic acid (Aldrich Chemical Co.), and p-toluic acid (Aldrich Chemical Co.). The basis for identification of other by-products without authentic standards is presented in later sections. However, the identifications of benzylfumaric acid and (2-methylbenzyl)fumaric acid require additional discussion. The first report of benzylfumaric acid from anaerobic toluene metabolism was based on the use of a geometric isomer, benzylmaleic acid, as a standard (13). A recent study in which benzylfumaric acid and three structurally similar isomers [benzylmaleic acid and (E)- and (Z)-phenylitaconic acid] were synthesized indicated that the four isomers could be distinguished by their GC retention times but not by their mass spectra (20). Thus, all four isomers would have to be available to make definitive identifications of such metabolic by-products. An analogous situation probably applies to the compound previously identified as (2-methylbenzyl)fumaric acid (13). For the sake of consistency with previous studies and in the absence of the applicable standards, we will retain the names of the compounds previously reported as benzylfumaric acid and (2-methylbenzyl)fumaric acid with the understanding that the closely related phenylitaconic or benzylmaleic acid isomers may actually apply. Source of bacteria. The organism described in this article was isolated from soil collected at the Naval Air Station, Patuxent River, Md. The site was extensively contaminated with aviation fuel. The sample was collected from a Quaternary stratum of an outcrop through which hydrocarbon-contaminated groundwater was seeping. Microcosms inoculated with Patuxent River soil and

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VOL. 62, 1996 enrichment cultures developed from these microcosms were described previously (5, 7). Growth medium and conditions of isolation and cultivation. The basal mineral component of the medium used for isolation and maintenance of strain PRTOL1 was similar to the medium used for Patuxent River microcosms and enrichment cultures (5); however, vitamins and trace elements were added for isolation and maintenance of the culture. The basal mineral component included the following compounds added at the concentrations (mM) specified in parentheses: NaHCO3 (30), NH4Cl (28), NaH2PO4 z H2O (4.4), FeSO4 z 7H2O (3), NaCl (1.7), KCl (1.3), CaCl2 z 2H2O (0.68), MgCl2 z 6H2O (0.49), MnCl2 z 4H2O (0.025), and Na2MoO4 z 2H2O (0.004). The medium (excluding FeSO4 and NaHCO3) was autoclaved at 1218C for 20 min and then aseptically purged with an oxygen-free mixture of 79% N2–21% CO2 for 45 min. After purging, bicarbonate was added to the medium as a sterile, anaerobic 1.0 M solution (45) in addition to anaerobic, filtered ferrous sulfate. Also added were anaerobic and sterile vitamin, trace element, and selenite-tungstate solutions that were prepared as described by Widdel and Bak (45) (stock solutions 1, 4, 6, 7, and 8). Prior to inoculation, the medium was prereduced with 150 to 200 mM sodium sulfide added from a filtered 0.1 M stock solution. Highly purified water (Milli-Q; Millipore Corp., Marlborough, Mass.) was used to prepare all the aqueous solutions used in this study; all stock solutions were prepared with Milli-Q water that had been autoclaved at 1218C for 20 min and then aseptically purged with oxygen-free N2. The final pH of the medium was approximately 7. All preparation and incubation of enrichment cultures, serial dilutions, and pure culture suspensions were performed at 358C under strictly anaerobic conditions in an anaerobic glove box (Coy Laboratory Products, Inc., Ann Arbor, Mich.) with a gas composition of approximately 90% N2–7.5% CO2–2.5% H2. Glass, plastic, and stainless steel materials used to contain or manipulate the cultures were sterile (either autoclaved or purchased sterile) and were allowed to degas in the anaerobic glove box before use. Preparation of enrichment cultures has been described previously (5). At the time that serial dilutions were initiated, the enrichment cultures had been maintained in the laboratory with toluene and sulfate as the sole electron donor and acceptor, respectively, for over 3 years. Microscopically pure cultures were obtained by repeated serial dilution of enrichment cultures with liquid medium in crimp-top serum culture tubes (18 by 150 mm; Bellco Glass, Inc., Vineland, N.J.) that were sealed with polytetrafluoroethylene (PTFE)-coated butyl rubber liners (Alltech Associates, Inc., Deerfield, Ill.). Toluene (.99.9% purity, glass-distilled, filtered [0.5-mm-pore-size filter]; Aldrich Chemical Co.) was added into the medium as a pure liquid with a 10-ml syringe. The syringe was sterilized with ethanol and allowed to dry before being used for toluene. Toluene concentrations were kept at or below 50 mM during serial dilution to preclude toxicity. Because Patuxent River enrichment cultures were found to be highly sensitive to sulfide (with inhibition in the range of 1 to 3 mM) (6), FeSO4 rather than MgSO4 was used as a sulfate source to minimize the dissolved sulfide concentration. The 1026-diluted culture from the first dilution series was grown with toluene for several months before being used as the inoculum for a second dilution series. The 1028-diluted culture from this second series, which appeared homogenous by microscopic examination, was grown with toluene for several months before serving as the inoculum for further dilution series. Agar dilution series (45) were performed by using the serially diluted liquid culture as an inoculum. Benzoate (1 mM) was used rather than toluene in the agar dilution series to expedite growth. Colonies of PRTOL1 were brown and lens shaped. Isolated colonies were transferred to liquid medium with toluene as the sole carbon source. Cultures were maintained in amber glass, screw-cap bottles that were sealed with PTFE Mininert valves (Alltech Associates, Inc.). Toluene was monitored and added when depleted; filter-sterilized, anaerobic FeSO4 was added via syringe when sulfate was depleted (as indicated by cessation of toluene consumption). Cultures growing on toluene were examined microscopically for purity on a regular basis. To independently test the purity of the cultures used in this study, complex medium that included 0.5% nutrient broth was inoculated with PRTOL1 (5 to 10% inoculum) and was examined microscopically after 2 to 3 weeks of incubation. No microbial contaminants were observed in these tests. Catabolic studies. For catabolic studies, PRTOL1 cells were grown with toluene and sulfate in a 2-liter glass reactor sealed with Mininert valves. Cells were harvested anaerobically by centrifugation (5,000 3 g for 40 min at 208C) and were resuspended in the anaerobic growth medium described previously. All experiments with volatile aromatic compounds, such as toluene, were conducted in screw-cap glass containers sealed with Mininert valves; for all other compounds, PTFE-faced silicone septa inside open-hole screw caps were used to seal bottles. Aromatic compounds that are liquids at room temperature (e.g., benzene, toluene, ethylbenzene, xylene isomers, benzyl alcohol, benzaldehyde, and cresol isomers) were added as pure liquids with a 10-ml syringe. All other compounds were added as anaerobic, filtered aqueous stock solutions; no organic carrier phases were used for amending the cultures. Catabolic studies were performed in duplicate with appropriate controls, as described in later sections. Enzyme assays. All harvesting and manipulation of cells for enzyme assays were conducted under anaerobic conditions. Benzoate-grown cells (400 ml) were harvested by centrifugation, washed in an anaerobic MOPS (morpholinepropanesulfonic acid) buffer (50 mM, pH 7.0) containing 5 mM dithiothreitol and 5 mM MgCl2, and then resuspended in 1.5 ml of the MOPS buffer. Assays were

SULFIDOGENIC TOLUENE-DEGRADING BACTERIUM

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performed with permeabilized cells (2% Triton X-100 [vol/vol]) under anaerobic conditions in stoppered glass cuvettes at 358C. Carbon monoxide dehydrogenase and formate dehydrogenase activities were assayed by following the reduction of methyl viologen at 578 nm, as described elsewhere (38). 2-Oxoglutarate dehydrogenase activity was also assayed with methyl viologen as the electron acceptor; a tricine buffer (100 mM, pH 8.5) containing 5 mM dithiothreitol and 1 mM methyl viologen was used. The protein concentration in the permeabilized cell preparation was estimated by using the measured cell density and estimated values for cell mass and composition taken from Neidhardt (29); the protein content of the cell preparation could not be measured directly because of interferences caused by the presence of FeS. Analytical methods. (i) Aromatic hydrocarbon analysis. Toluene and other single-ring aromatic hydrocarbons were measured by a static headspace technique using a model 5890A gas chromatograph (Hewlett-Packard Company, Palo Alto, Calif.) with a model PI 52-02A photoionization detector (10.2 eV lamp; HNU Systems, Inc., Newton, Mass.) and a DB-624 fused silica capillary column (30-m length, 0.53-mm inner diameter, 3.0-mm film thickness; J & W Scientific, Folsom, Calif.). Analyses were isothermal (808C) with splitless injection (the split was turned on after 0.5 min). The method is described in detail elsewhere (5). The detachable stainless steel needles for the gas-tight syringes used for headspace sampling were autoclaved before each use. Peak integration for aromatic hydrocarbons (as well as for sulfate and oxygenated aromatic compounds [described below]) was performed with a Nelson analytical chromatography software system (model 2600; Perkin-Elmer, Cupertino, Calif.). (ii) Oxygenated aromatic compound analysis. Concentrations of oxygenated aromatic compounds tested individually as electron donors in catabolic studies with PRTOL1 (phenylpropionate, phenylacetate, benzylsuccinate, benzyl alcohol, benzaldehyde, benzoate, o- and p-cresol, p-hydroxybenzoate, and p-toluate) were determined in the supernatant of centrifuged samples by reverse-phase high-performance liquid chromatography (HPLC) with a Perkin-Elmer series 400 liquid chromatograph connected to a Hewlett-Packard model 1050 variable wavelength detector. The mobile phase was a 65:35 (vol/vol) mixture of methanol and 50 mM acetate buffer (pH 3.5) flowing isocratically at 1 ml/min through an Adsorbosphere HS C18 column (5-mm particle size, 250 mm by 4.6 mm inner diameter; Alltech). Compounds were quantified with a wavelength of either 265 nm (for the first four compounds listed above) or 280 nm. For the aqueous HPLC standards, benzyl alcohol, benzaldehyde, and cresols were added as pure liquids; benzoate and p-hydroxybenzoate were added as sodium salts; and the remaining four compounds were added from methanolic stock solutions. The maximum methanol concentration in the HPLC standards was 0.06% (vol/vol). (iii) Sulfate analysis. Sulfate in filtered samples (0.2-mm-pore-size syringe filters) was determined with an ion chromatograph (series 4000i, Dionex Corporation, Sunnyvale, Calif.) equipped with an HPIC-AS4A column, an anion micromembrane suppressor, and a conductivity detector. Analyses were isocratic, with a 0.75 mM sodium bicarbonate–2.2 mM sodium carbonate eluant flowing at a rate of 2 ml/min. (iv) 14C analysis. 14C-labeled toluene was used in PRTOL1 cultures to investigate the extent of toluene mineralization and incorporation into biomass. Analysis of 14C activity in culture liquid was performed with a Tri-Carb model 2500 TR/AB liquid scintillation analyzer (Packard Instruments Co., Inc., Downers Grove, Ill.). All samples were automatically blank corrected and were corrected for sample-specific quenching by using an external standard method (with a 133 Ba gamma source) and a quenching curve developed from a series of quenched standards. Sample-processing methods were very similar to those described previously (5), except that headspace was not sampled in the present study because experiments were designed to have negligible headspace (,1.5% of the total volume). By this method, three fractions of 14C activity were determined in culture samples: 14CO2, nonvolatile carbon (including biomass and nonvolatile metabolites), and volatile carbon (including toluene and volatile metabolites). (v) Nonvolatile metabolite analysis. Samples of culture (20 to 50 ml) were acidified to a pH of #2 with solvent-cleaned HCl and extracted three times in a separatory funnel with diethyl ether (Ultra Resi-Analyzed, distilled-in-glass; J. T. Baker). The extracts were dried with anhydrous sodium sulfate, derivatized with ethereal diazomethane (15), concentrated under a gentle stream of high-purity nitrogen at room temperature, exchanged into dichloromethane (Ultra ResiAnalyzed, distilled-in-glass; J. T. Baker, Inc.), and analyzed by GC-MS. GC-MS analyses were performed with a model 5890A GC (Hewlett-Packard Company) with a DB-5 fused silica capillary column (30-m length, 0.32-mm inner diameter, 0.25-mm film thickness; J & W Scientific) coupled to an HP 5970 series mass selective detector; data analysis was performed with Hewlett-Packard G1034C software designed for the MS ChemStation. The GC oven was programmed from 458C (held for 2 min) to 1108C at 88C/min and then from 1108C to 2508C at 48C/min (held for 5 min); the injection port temperature was 2758C, and the transfer line temperature was 2808C. Injections were splitless, with the split turned on after 0.5 min. For data acquisition, the MS scanned from 50 to 350 atomic mass units at a rate of two scans per s. Determination of growth. Growth was determined by two methods: microscopic cell counts and optical density. Growth experiments were conducted in the dark with clear glass tubes (13 by 100 mm) that were sealed with PTFE-faced silicone septa inside open-hole screw caps. The medium used in these experiments contained MgSO4 rather than FeSO4 to preclude the formation of FeCO3

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FIG. 1. Phase contrast micrograph of PRTOL1 cells growing on toluene. Bar, 5 mm.

and FeS precipitates. Cell counting was performed by phase-contrast microscopy at 400-fold total magnification with a Petroff-Hausser counting chamber (Hausser Scientific, Blue Bell, Pa.). Because the cell densities being counted were generally between 106 and 5 3 107 cells per ml, samples were concentrated fivefold by centrifugation before being counted. A more rapid method used to assess growth was determining the optical density (absorbance) at 600 nm with a Spectronic model 20D single-beam spectrophotometer (Milton Roy, Rochester, N.Y.). When measurements were made, the sealed tubes were removed from the glove box, analyzed within 10 min, and returned to the glove box. A determination that growth had occurred in a given sample was based on positive results relative to controls for both optical density and cell counts. More specifically, the two criteria for growth were (i) an optical density value greater than or equal to that of the positive control and (ii) a cell count greater than or equal to four times that of the negative control. In experiments testing potential electron donors, the positive controls included benzoate and the negative controls had no added electron donor. In experiments testing potential electron acceptors, the positive controls included sulfate and the negative controls had no added electron acceptor. Growth was not determined for most of the experiments that assessed potential electron donors for PRTOL1. In these experiments, only metabolism (as indicated by the consumption of the electron donor and/or acceptor) was assessed because the relatively long generation time and low growth yield of PRTOL1 made growth difficult to quantify reliably. 16S rRNA gene isolation, sequencing, and analysis. Total DNA was isolated from PRTOL1 by a method shown to work for diverse bacteria (42). A portion of this DNA (ca. 0.1 mg) was used in the PCR to amplify most of the 16S rRNA gene. The primers used to amplify near full-length 16S rRNA gene sequences (59-AGAGTTTGATCCTGGCTCAG-39 and 59-AAGGAGGTGATCCAGCC39) were modified versions of the primers fD1 and rD1 used by Weisburg et al. (44). The PCR mixture consisted of 1.5 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate (dNTP), 0.25 mM each primer, 13 Taq polymerase buffer, and 0.75 U of Taq polymerase (Promega Corp., Madison, Wis.) in a volume of 30 ml. Amplification was carried out by using a GeneAmp PCR System 9600 Thermal Cycler (Perkin-Elmer Corp., Norwalk, Conn.) with a program consisting of an initial denaturation at 928C for 130 s; 30 cycles of 948C for 15 s, 558C for 30 s, and 728C for 130 s; and a final elongation cycle at 728C for 370 s. The resulting PCR product was purified by gel electrophoresis with a 1% agarose gel and was recovered using Gene Clean purification resin according to the manufacturer’s suggestions (Bio 101, Inc., La Jolla, Calif.). The purified PCR product was cloned in the vector pCRII by using a TA Cloning Kit (Invitrogen, San Diego, Calif.) according to the manufacturer’s directions. Plasmid DNA containing the 16S rRNA gene insert was isolated from one clone by using the Qiagen plasmid mini kit according to the manufacturer’s directions (Qiagen, Chatsworth, Calif.). The DNA sequence of the insert was determined by automated fluorescent dye terminator sequencing with an ABI Catalyst 800 laboratory robot and ABI 373A sequencer (Applied Biosystems, Foster City, Calif.). The primers that were

used corresponded to conserved regions of the 16S rRNA sequence (47). Approximately 95% of the insert sequence was determined in both directions. Related sequences were obtained from the Ribosomal Database Project (24). A maximum-likelihood phylogenetic tree was created with the program fastDNAml (30), by using a weighting mask to include only unambiguously aligned positions with all other program options at their default values. This analysis was repeated on 100 bootstrap samples to obtain confidence estimates of the branching order (16). The program CONSENSE from the PHYLIP program package (17) was used to determine the number of times that each group shown in the final tree was monophyletic in the bootstrap analysis. Nucleotide sequence accession number. The 16S rRNA gene sequence of strain PRTOL1 has been assigned GenBank accession number U49429.

RESULTS Morphology. Cells of PRTOL1 are oval, 2 to 3 mm long, and 1.2 to 1.7 mm in diameter and stain gram negative. A phasecontrast photomicrograph of PRTOL1 cells growing on toluene is presented in Fig. 1. Microscopic examination did not reveal any evidence of spores or of swimming motility. Phylogenetic classification based on 16S rRNA. Phylogenetic relationships based on 16S rRNA gene sequences are depicted in Fig. 2. Strain PRTOL1 is classified in the delta

FIG. 2. Maximum-likelihood phylogenetic tree of PRTOL1 and representative delta Proteobacteria. The bar represents nucleotide changes per position. Numbers at internal nodes are the percentage of 100 bootstrap samples in which the organisms to the right of the node were monophyletic.

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FIG. 3. Cumulative sulfate consumed versus cumulative toluene consumed for duplicate suspensions of strain PRTOL1. The 200-ml replicates were incubated for 9 days.

subclass of the Proteobacteria, as are all other known gramnegative mesophilic sulfate-reducing bacteria (45). Desulforhabdus amnigenus (31) is the closest known relative of strain PRTOL1 (96% sequence similarity). Desulforhabdus amnigenus, which was isolated from anaerobic granular sludge with acetate as the sole carbon and energy source, does not metabolize benzoate or other aromatic compounds that have been tested (31). As shown in Fig. 2, three bacteria capable of anaerobic toluene degradation are classified among the delta Proteobacteria: the sulfidogenic Desulfobacula toluolica (33) and PRTOL1 and the iron-reducing Geobacter metallireducens (26). Carbon and electron balance for sulfidogenic toluene degradation. In PRTOL1 cultures, toluene degradation and sulfate reduction were strongly correlated over time and had a consistent stoichiometric relationship (Fig. 3). The data in Fig. 3 represent a 9-day incubation of duplicate 200-ml cultures. The regression equation relating toluene and sulfate consumption for the cultures had a slope of 3.89 mol of sulfate per mol of toluene (r2 5 0.996), which is consistent with theoretical ratios ranging from 4.09 (toluene oxidation to CO2, sulfate reduction to hydrogen sulfide, and estimated cell growth; equation 1) to 4.5 (toluene oxidation to CO2, sulfate reduction to hydrogen sulfide, no cell growth; equation 2). The estimation of cell growth in equation 1 was derived by using the calculation methods described by McCarty (27, 28). C7H8 1 4.09 SO4

22

1

1 0.17 NH4 1 2.49 H2O 3 2.04 H2S 1

(1)

2.04 HS2 1 0.17 C5H7O2N (cells) 1 6.16 HCO32 1 0.2 H1 C7H8 1 4.5 SO422 1 3 H2O 3 2.25 H2S 1 2.25 HS2 1

toluene had already occurred by day 2 (data not shown). In uninoculated controls, toluene was not converted to CO2. PRTOL1 cultures convert a portion of toluene carbon to a metabolic by-product, benzylsuccinic acid. As has been demonstrated in the enrichment cultures from which PRTOL1 was isolated (7), the transformation of toluene to benzylsuccinic acid can be verified by using stable isotope-labeled toluene (e.g., toluene-d8) and GC-MS analysis. Extraction and GC-MS analysis of the replicates shown in Fig. 3, which were given toluene-d8, revealed that 2.70 to 2.75% of toluene carbon had been converted to deuterium-labeled benzylsuccinic acid, with a trace amount of labeled benzylfumaric acid (or a closely related isomer) also detectable (,0.1% of toluene carbon). However, higher benzylsuccinic acid yields of approximately 7% of toluene carbon have been observed in similar experiments (data not shown) and in the mixed enrichment culture from which PRTOL1 was isolated (7); therefore, it appears that the benzylsuccinic acid yield may not be constant. The cell yield of PRTOL1 growing on toluene can be estimated from the data for radiolabeled toluene and benzylsuccinic acid; these data were collected from experiments that were run concurrently. Fifteen percent of toluene carbon was converted to nonvolatile carbon (Table 1), and approximately 3% was converted to benzylsuccinic acid, a nonvolatile metabolite. By difference, approximately 12% of toluene carbon was converted to cells. By using an average cell composition of C5H7O2N (27), the cell yield is estimated as 19 g of cells (dry weight) per mol of toluene. Notably, this value is virtually identical to the theoretical prediction given in equation 1. A direct gravimetric determination of cell yield was not practical because of the relatively low yield and long doubling time involved and because of FeS and FeCO3 precipitates that formed in the medium. The doubling time of PRTOL1 growing on toluene is estimated to be 1.5 to 2 days. Use of electron donors and acceptors other than toluene and sulfate. The use of selected organic compounds other than toluene, including single-ring aromatic hydrocarbons, shortchain aliphatic acids, and potential intermediates of toluene metabolism, was tested (Table 2). All five of the short-chain aliphatic acids tested (formate, acetate, pyruvate, succinate, and fumarate) were metabolized, as indicated by sulfate consumption. Growth was observed for the latter three acids but was not confirmed for formate and acetate during a 40-day incubation period. Strain PRTOL1 oxidized phenylpropionate, phenylacetate, benzaldehyde, benzoate, p-cresol, and p-hy-

TABLE 1. Carbon balance for toluene degradation by strain PRTOL1 % of total dpma

14

C fraction 14

(2)

7 HCO32 1 0.25 H1 (DG89 5 2205 kJ/mol of toluene)

Toluene mineralization to CO2 was confirmed by using PRTOL1 suspensions supplied with [ring-U-14C]toluene as the sole electron donor. The results of a 4-day incubation with approximately 80 mM toluene are summarized in Table 1. Eighty percent of toluene carbon was converted to 14CO2, whereas 15% was converted to nonvolatile 14C (the sum of biomass and nonvolatile metabolites). All of the added toluene was consumed in this experiment, as indicated by an intermediate sampling that demonstrated that 80% mineralization of

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Initial, volatile C Final (day 4) Volatile 14C 14 CO2 Nonvolatile 14Cb

PRTOL1

Control

98.2 6 0.5

99.6

4.7 6 0.2 80.3 6 0.2 15.0 6 0.4

99.6 0.2 0.2

a A total of 4.7 mmol of [ring-U-14C]toluene (75.1 mCi/mmol, specific activity) was added to PRTOL1 cultures and to the uninoculated control. Mass balances for strain PRTOL1 and the control were 106 and 99%, respectively, defined as (the total number of final dpm [disintegrations per minute] divided by the total number of initial dpm) 3 100. The results are expressed as the means 6 standard deviations. Of the PRTOL1 replicates prepared on day 0, two were analyzed on day 0 and three were analyzed on day 4. Each replicate bottle was sampled only once. b Nonvolatile carbon represents the sum of biomass and nonvolatile metabolites.

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a 1, 70 to 100% decrease in concentration observed in 15 days, with exceptions as noted in other footnotes; 2, no decrease in concentration outside the range of analytical error. b 1, .1 mM sulfate consumed in excess of negative control (no added electron donor); 2, ,0.2 mM sulfate consumed in excess of negative control; ND, not determined. c For these compounds, toluene was originally present and was allowed to be depleted without further addition (see text). d Metabolism was determined by GC-MS detection of distinctive metabolic products (see text) and by some observed decrease in the substrate concentration over time. However, the rate of metabolism was very slow and metabolism could not be sustained over time. Cometabolism with toluene may apply to these compounds (see Discussion). e The metabolism of these electron donors was determined by assessment of growth rather than by the assessment of the decrease in concentration described in footnote a. The metabolism of sulfate was determined as described in footnote b. f The o-cresol concentration did not decrease over the 15-day test period, and sulfate was also not used during this time. However, a minor HPLC peak appeared after 15 days that corresponded to the retention time of p-hydroxybenzoate. It is probable that this peak indicates a minor conversion to o-hydroxybenzoate, but this possibility was not confirmed by GC-MS.

creased within the first 10 days, after which time no further o-xylene decrease was detectable. Although a 55 to 60 mM addition of toluene was rapidly consumed between days 20 and 30, the o-xylene concentration did not decrease during this time. Extraction and GC-MS analysis of the sample shown in Fig. 4 on day 35 indicated a complete conversion of the consumed o-xylene to nonvolatile metabolic products. Based on the strong similarity of the mass spectra of the two observed products (Fig. 5A and C) to published spectra (13), and based on the demonstrated formation of benzylsuccinic and benzylfumaric acid from toluene by PRTOL1, (2-methylbenzyl)succinic acid and (2-methylbenzyl)fumaric acid (or a closely related isomer) are indicated as the predominant products of o-xylene metabolism. Because authentic standards of these compounds were not commercially available, their concentrations were estimated by using total ion current areas and the structurally similar benzylsuccinic acid as a standard. On the basis of this estimation technique, ca. 125 mol% of the o-xylene consumed was transformed to (2-methylbenzyl)succinic acid and ca. 13 mol% was converted to (2-methylbenzyl)fumaric acid. Thus, considering overall experimental error, including the lack of authentic standards, it appears that the consumed o-xylene was quantitatively converted to these products. Notably, 2-methylbenzoic acid was also detected by GC-MS analysis, but its concentration was very low (ca. 0.3 mol% of the o-xylene consumed). Formation of deuterium-labeled (2-methylbenzyl)succinic acid and (2-methylbenzyl)fumaric acid (or a closely related isomer) from o-xylene-d10 was demonstrated in a similar experiment (Fig. 5B and D). Similar patterns of oxylene transformation were observed in a separate experiment during which toluene-grown PRTOL1 cells were supplied with o-xylene in the absence of toluene (data not shown). The results of an experiment testing the metabolism of pxylene by PRTOL1 are depicted in Fig. 6. In the first 10 days, toluene was rapidly depleted and p-xylene was depleted more slowly. Additional p-xylene amended on day 9 was partially consumed, but consumption was not sustained over time. Toluene added to another replicate on day 22 was rapidly consumed, but no further p-xylene consumption was observed (data not shown). Extraction and GC-MS analysis of the sample represented in Fig. 6 on day 34 indicated a partial conversion to metabolic products; the mass spectra of the products are shown in Fig. 7B and D. The major product, constituting approximately 23 mol% of the p-xylene consumed, was confirmed to be 4-methylbenzoic acid (p-toluic acid), for which an authentic standard was available (Fig. 7A). A compound assumed to be (4-methylbenzyl)succinic acid [based on the argu-

droxybenzoate but not benzyl alcohol, o-cresol, p-toluate, and benzylsuccinate during a 15-day incubation period (Table 2). Among these oxygenated aromatic compounds, benzaldehyde was the most rapidly consumed. Among the single-ring aromatic hydrocarbons tested, no discernible consumption of benzene, ethylbenzene, or m-xylene was observed in a 3-week period. The results for o- and p-xylene merit detailed elaboration. In experiments testing the metabolism of o- and p-xylene, as well as the other three single-ring aromatic hydrocarbons listed in Table 2, toluene was initially added at approximately the same concentration as the test substrate but was not replaced after it was depleted. This design was intended to demonstrate that the cultures were active and to investigate cometabolism with toluene. In PRTOL1 suspensions amended with o-xylene (Fig. 4), both the toluene and o-xylene concentrations de-

FIG. 4. Metabolism of o-xylene by strain PRTOL1 in the presence of toluene. Toluene was added again on day 22.

TABLE 2. Use of electron donors by strain PRTOL1 (excluding toluene) Metabolism Compound (concn)

a

Donor

Acceptor (sulfate)b

Aromatic hydrocarbonsc Benzene (50–60 mM) Ethylbenzene (20–40 mM) o-Xylene (20–40 mM) m-Xylene (20–40 mM) p-Xylene (20–40 mM)

2 2 1d 2 1d

ND ND ND ND ND

Short-chain aliphatic acidse Formate (5 mM) Acetate (4 mM) Pyruvate (3 mM) Succinate (2 mM) Fumarate (2 mM)

2 2 1 1 1

1 1 1 1 1

Potential toluene intermediates Phenylpropionate (0.5 mM) Phenylacetate (0.5 mM) Benzaldehyde (0.5 mM) Benzoate (0.5 mM) p-Cresol (0.5 mM) p-Hydroxybenzoate (0.5 mM) Benzyl alcohol (0.125 and 0.25 mM) o-Cresol (0.5 mM)

1 1 1 1 1 1 2 2f

1 1 1 1 1 1 2 2

Alkylbenzene by-products p-Toluate (0.5 mM) Benzylsuccinate (0.5 mM)

2 2

2 2

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mM) was added to a PRTOL1 suspension to determine whether benzylsuccinic acid was formed from benzaldehyde. Benzaldehyde has been reported as an intermediate of anaerobic toluene degradation (1, 2) and thus is a potential intermediate of anaerobic toluene transformation to benzylsuccinic acid. In the experiment with strain PRTOL1, no labeled benzylsuccinic acid was formed from labeled benzaldehyde. However, labeled 3-benzoylpropionic acid (Fig. 8) was identified at a relatively low concentration (ca. 0.5 mol% of the 490 mM benzaldehyde consumed). Various electron acceptors, including thiosulfate, sulfite, nitrate, and fumarate, were tested for their use by PRTOL1 in the presence of benzoate. Only thiosulfate and sulfite were found to support metabolism (as indicated by benzoate consumption) and growth of PRTOL1. Key enzymes involved in acetyl coenzyme A oxidation. Enzyme assays performed with permeabilized, benzoate-grown cells indicated that specific activities of carbon monoxide dehydrogenase and formate dehydrogenase were on the order of 3.8 and 22 mmol z min21 z mg of protein21, respectively. The activity of 2-oxoglutarate dehydrogenase was no greater than the detection limit (approximately 0.15 mmol z min21 z mg of protein21). DISCUSSION

FIG. 5. Mass spectra of the dimethyl esters of o-xylene metabolites. (A) The predominant metabolite, tentatively identified as (2-methylbenzyl)succinic acid (see text), resulting from o-xylene metabolism by PRTOL1; (B) the predominant metabolite, tentatively identified as (2-methylbenzyl)succinic acid-d10, resulting from o-xylene-d10 metabolism by PRTOL1; (C) a lesser metabolite, tentatively identified as (2-methylbenzyl)fumaric acid (or a closely related isomer; see text), resulting from o-xylene metabolism by PRTOL1; and (D) a lesser metabolite, tentatively identified as (2-methylbenzyl)fumaric acid-d8 (or a closely related isomer), resulting from o-xylene-d10 metabolism by PRTOL1. Spectra A and C were acquired from an extract of the sample represented in Fig. 4.

ments given for (2-methylbenzyl)succinic acid] accounted for approximately 3 mol% of the p-xylene consumed. Thus, in contrast to the results for o-xylene, the metabolic products found for p-xylene did not account for the total mass of pxylene consumed, but accounted for approximately 26 mol%. Mineralization of a portion of p-xylene by PRTOL1 is possible but could not be confirmed because radiolabeled p-xylene is not commercially available. An experiment with stable isotopelabeled p-xylene (p-xylene-d10) demonstrated the formation of deuterium-labeled metabolites; the spectrum of methyl-p-toluate-d7 is shown in Fig. 7C. Of the other tested electron donors that were used by PRTOL1 (Table 2), only benzaldehyde was investigated with respect to metabolic products. Benzaldehyde-a-13C,d1 (490

Strain PRTOL1 is a gram-negative, mesophilic sulfate-reducing bacterium that, unlike its closest known phylogenetic relatives, can degrade a range of aromatic compounds including an aromatic hydrocarbon, toluene. PRTOL1 metabolized six of the oxygenated aromatic compounds tested in this study. These compounds were chosen because they are proposed or demonstrated intermediates of anaerobic toluene degradation, based on studies of a range of toluene-degrading bacteria. As shown in Table 2, PRTOL1 can metabolize phenylpropionate (potentially resulting from the condensation of acetyl coenzyme A with the methyl carbon of toluene, as proposed by Evans et al. [13]), benzaldehyde (potentially formed following the formation of benzyl alcohol by hydroxylation of the methyl carbon of toluene, as described for denitrifying Thauera sp. strain K172 [1, 2]), p-cresol (potentially resulting from the hydroxylation of the aromatic ring of toluene, as shown for a mixed methanogenic culture [21, 43]), phenylacetate (potentially resulting from carboxylation of the methyl carbon of toluene, as suggested by Altenschmidt and Fuchs [1]), and benzoate (an intermediate common to all proposed anaerobic toluene degradation pathways [e.g., 13, 26]). As PRTOL1 metabolizes intermediates from several different potential toluene

FIG. 6. Metabolism of p-xylene by strain PRTOL1 in the presence and absence of toluene. p-Xylene was added again on day 9.

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mechanisms that are currently unknown. For toluene, and possibly for other aromatic substrates such as benzaldehyde, this transformation occurs in conjunction with mineralization. The formation of benzylsuccinic acid and a much smaller amount of benzylfumaric acid (or a closely related isomer) from toluene was reported previously for the enrichment cultures from which PRTOL1 was isolated (7). The observation that benzylsuccinic acid was not metabolized by PRTOL1 (Table 2) is consistent with the previous finding that benzylsuccinic acid was a dead-end product in those enrichment cultures (7). The formation of benzylsuccinic acid from toluene by PRTOL1 does not proceed via benzaldehyde, as indicated by catabolic studies in which benzaldehyde was a sole carbon source; similar results were reported for a denitrifying bacterium that metabolizes toluene to dead-end products (18). However, a trace amount (0.5%) of benzaldehyde was converted by PRTOL1 to 3-benzoylpropionic acid (Fig. 8). In other catabolic studies, PRTOL1 transformed o-xylene quantitatively to (2-methylbenzyl)succinic acid and (2-methylbenzyl)fumaric acid (or a closely related isomer). In accordance with toluene transformation, the benzylsuccinic acid analog of o-xylene was formed at a much higher concentration than the benzylfumaric acid analog. Notably, transformation of o-xylene to (2-methylbenzyl)succinic and (2-methylbenzyl)fumaric acids without concurrent mineralization was also observed by Evans and coworkers for denitrifying strain T1 (13). PRTOL1 also transformed p-xylene to p-toluic acid (an apparent dead-end product; Table 2) and a lesser amount of (4-methylbenzyl)succinic acid. An additional compound detected at a low concentration in the p-xylene culture (data not shown) suggested that p-toluic acid was formed via p-tolualdehyde. This compound was likely 3-(p-toluyl)propionic acid, a methyl homolog of 3-benzoylpropionic acid. The mass spectral fragmentation pattern of the methyl ester of this p-xylene metabolite was analogous to the methyl ester of 3-benzoylpropionic acid (Fig. 8A) but with the m/z 51, 77, 105, 161, and 192 fragments all replaced by fragments that were 14 atomic mass units higher. Thus, by analogy with 3-benzoylpropionic acid formation from benzaldehyde, it

FIG. 7. Mass spectra. (A) A methyl-p-toluate standard; (B) the methylated predominant product resulting from p-xylene metabolism by PRTOL1 (sample represented in Fig. 6); (C) the methylated predominant product, tentatively identified as methyl-p-toluate-d7, resulting from p-xylene-d10 metabolism by PRTOL1; and (D) the methylated lesser product, tentatively identified as (4methylbenzyl)succinic acid (see text), resulting from p-xylene metabolism by PRTOL1 (sample represented in Fig. 6).

degradation pathways, no preliminary indication of a specific mineralization pathway used by PRTOL1 is evident from this study. The inability to metabolize benzyl alcohol noted for PRTOL1 (Table 2) has been observed for other anaerobic toluene degraders, including Desulfobacula toluolica (33) and three denitrifying bacteria (strains T [10], mXyN1 [34], and T1 [14]). In contrast, a denitrifying bacterium shown to degrade toluene via benzyl alcohol, Thauera sp. strain K172, metabolized benzyl alcohol as a sole carbon source after growth on toluene (1, 2). Nonetheless, a pathway involving benzyl alcohol as an intermediate cannot be excluded based on the lack of benzyl alcohol metabolism. PRTOL1 transforms a number of aromatic substrates, including toluene, o- and p-xylene, and benzaldehyde, to metabolic by-products; most of these products result from the bonding of a short-chain aliphatic acid to a benzylic carbon atom by

FIG. 8. Mass spectra. (A) A methyl 3-benzoylpropionate standard and (B) a methylated product resulting from metabolism of benzaldehyde-a-13C,d1.

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is likely that 3-(p-toluyl)propionic acid was formed from ptolualdehyde, a likely intermediate to p-toluic acid formation from p-xylene. Notably, direct evidence of the formation of p-toluic acid via p-tolualdehyde was recently demonstrated with cell suspensions of a denitrifying bacterium amended with p-xylene; analogous findings were made for the meta isomers of these compounds (37). The nature of o- and p-xylene transformation by toluenegrown PRTOL1 cells is complex (Fig. 4 and 6) and not well understood. Transformation of o-xylene to by-products could be considered cometabolic because (i) it appears to involve an incomplete oxidation that provides no carbon and little or no energy to the cell and (ii) it may require the activity of an enzyme involved in the metabolism of a growth substrate (toluene). For p-xylene, these criteria may be less applicable because mineralization of a portion of p-xylene to CO2 cannot be ruled out. For both xylene isomers, a confounding observation was that consumption ceased over time and was not stimulated by the addition and rapid consumption of toluene. One possible explanation for this observation is that a product formed during xylene metabolism (e.g., a methylbenzylsuccinic acid isomer) specifically inhibited further xylene metabolism but did not affect toluene metabolism. Further studies to examine the cessation of xylene metabolism are warranted and may have considerable relevance for anaerobic bioremediation of gasoline-contaminated sites. As more anaerobic bacteria capable of aromatic hydrocarbon degradation are isolated and studied, the potential for anaerobic bioremediation of gasoline-contaminated aquifers will be easier to assess. However, a better understanding of the details of anaerobic hydrocarbon metabolism (e.g., formation of by-products and substrate interactions) will be required to optimize anaerobic bioremediation and to reliably predict its consequences. ACKNOWLEDGMENTS Funding for this study was provided by the Office of Research and Development, U.S. Environmental Protection Agency, under grant R-815738 through the Western Region Hazardous Substance Research Center. Additional funding for 16S rRNA analysis performed at Michigan State University was provided by NSF grant BIR-9120006 to the Center for Microbial Ecology.

ADDENDUM IN PROOF In a recently published study of the sulfate-reducing Desulfobacula toluolica (Arch. Microbiol. 164:448–451, 1995), Rabus and Widdel reported the transformation of p-xylene to 4-methylbenzoate by dense suspensions of toluene-grown cells that were supplied with a mixture of p-xylene and toluene. In addition, a relatively low yield (0.1%) of benzylsuccinate was observed in toluene-metabolizing cell suspensions. REFERENCES 1. Altenschmidt, U., and G. Fuchs. 1991. Anaerobic degradation of toluene in denitrifying Pseudomonas sp.: indication for toluene methylhydroxylation and benzoyl-CoA as central aromatic intermediate. Arch. Microbiol. 156: 152–158. 2. Altenschmidt, U., and G. Fuchs. 1992. Anaerobic toluene oxidation to benzyl alcohol and benzaldehyde in a denitrifying Pseudomonas strain. J. Bacteriol. 174:4860–4862. 3. Bak, F., and F. Widdel. 1986. Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures, and description of Desulfobacterium indolicum gen. nov., sp. nov. Arch. Microbiol. 146:170–176. 4. Bak, F., and F. Widdel. 1986. Anaerobic degradation of phenol and phenol derivatives by Desulfobacterium phenolicum sp. nov. Arch. Microbiol. 146: 177–180. 5. Beller, H. R., D. Grbic´-Galic´, and M. Reinhard. 1992. Microbial degradation of toluene under sulfate-reducing conditions and the influence of iron on the

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