1996, 62(4):1188. Appl. Environ. Microbiol. 

H R Beller, A M Spormann, P K Sharma, J R Cole and M Reinhard toluene-degrading, sulfate-reducing bacterium.Isolation and characterization of a novel

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1996, p. 1188–1196 Vol. 62, No. 40099-2240/96/$04.0010Copyright q 1996, American Society for Microbiology

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, min-eralizes toluene as the sole electron donor and carbon source under strictly anaerobic conditions. Themineralization 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 consistentwith 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 toinvestigate 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 (orp-toluic acid), respectively. Metabolic by-products accounted for nearly all of the o-xylene consumed. Enzymeassays 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 isdistinct in that it has a novel 16S rRNA gene sequence and was derived from a freshwater rather than marineenvironment.

Leakage of gasoline from underground fuel storage tanks isa pervasive source of groundwater contamination in the UnitedStates (41). Bioremediation is one of a limited number of op-tions for restoring aquifers contaminated with the hazardous,relatively water-soluble aromatic hydrocarbons that occur inunleaded gasoline, such as benzene, toluene, and xylenes. Be-cause many contaminated aquifers are anaerobic as a result ofoxygen depletion by indigenous aerobic microorganisms, hy-drocarbon degradation by indigenous anaerobic bacteria mer-its serious consideration at some sites as a method of ground-water restoration. Partially in response to such environmentalconcerns, knowledge of the metabolic capabilities of anaerobicbacteria has expanded dramatically in the past decade, partic-ularly with respect to single-ring aromatic hydrocarbons andclosely related oxygenated compounds. For example, 18 purecultures capable of anaerobic toluene degradation have beenreported since 1989, including 16 denitrifying cultures (10, 14,19, 34, 36, 48), one ferric iron-reducing culture (25, 26), andone marine sulfate-reducing culture (33); fermentative-metha-nogenic mixed enrichment cultures that degrade toluene havealso been reported (12, 21, 43). More than 10 sulfate-reducingcultures that can grow on aromatic substrates have been re-ported since 1980 (3, 4, 8, 9, 11, 22, 23, 32, 33, 35, 39, 40, 46);however, only one of these can metabolize an aromatic hydro-carbon (Desulfobacula toluolica) (33). In this article, we reportthe isolation of strain PRTOL1, the second sulfate-reducingbacterium known to degrade an aromatic hydrocarbon and thefirst such organism from a freshwater environment.

MATERIALS AND METHODS

Chemicals. The chemicals used in this study were of the highest purity avail-able (generally $99%) and were used as received. Most organic chemicals werepurchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.), and most inor-ganic chemicals were purchased from J. T. Baker, Inc. (Phillipsburg, N.J.). Stableisotope-labeled chemicals included toluene-d8 (.99 atom%; Aldrich ChemicalCo.); o-xylene-d10 and p-xylene-d10 (991 atom%; Aldrich Chemical Co.); andbenzaldehyde-a-13C,d1 (98% purity [D] and 99% purity [13C]; Isotec, Inc., Mi-amisburg, Ohio). Gas chromatography-mass spectrometry (GC-MS) analyses ofdeuterium-labeled alkylbenzenes demonstrated that they were of high purity andcontained 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 Chem-ical 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-ben-zoylpropionic acid (Aldrich Chemical Co.), and p-toluic acid (Aldrich ChemicalCo.). The basis for identification of other by-products without authentic stan-dards is presented in later sections. However, the identifications of benzylfumaricacid and (2-methylbenzyl)fumaric acid require additional discussion. The firstreport of benzylfumaric acid from anaerobic toluene metabolism was based onthe use of a geometric isomer, benzylmaleic acid, as a standard (13). A recentstudy in which benzylfumaric acid and three structurally similar isomers [benzyl-maleic acid and (E)- and (Z)-phenylitaconic acid] were synthesized indicated thatthe four isomers could be distinguished by their GC retention times but not bytheir mass spectra (20). Thus, all four isomers would have to be available to makedefinitive identifications of such metabolic by-products. An analogous situationprobably applies to the compound previously identified as (2-methylbenzyl)fu-maric acid (13). For the sake of consistency with previous studies and in theabsence of the applicable standards, we will retain the names of the compoundspreviously reported as benzylfumaric acid and (2-methylbenzyl)fumaric acid withthe understanding that the closely related phenylitaconic or benzylmaleic acidisomers 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 exten-sively contaminated with aviation fuel. The sample was collected from a Qua-ternary stratum of an outcrop through which hydrocarbon-contaminated ground-water was seeping. Microcosms inoculated with Patuxent River soil and

* Corresponding author. Phone: (415) 723-0315. Fax: (415) 725-3162.

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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 PRTOL1was similar to the medium used for Patuxent River microcosms and enrichmentcultures (5); however, vitamins and trace elements were added for isolation andmaintenance of the culture. The basal mineral component included the followingcompounds 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), andNa2MoO4 z 2H2O (0.004). The medium (excluding FeSO4 and NaHCO3) wasautoclaved at 1218C for 20 min and then aseptically purged with an oxygen-freemixture of 79% N2–21% CO2 for 45 min. After purging, bicarbonate was addedto the medium as a sterile, anaerobic 1.0 M solution (45) in addition to anaer-obic, filtered ferrous sulfate. Also added were anaerobic and sterile vitamin,trace element, and selenite-tungstate solutions that were prepared as describedby 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 afiltered 0.1 M stock solution. Highly purified water (Milli-Q; Millipore Corp.,Marlborough, Mass.) was used to prepare all the aqueous solutions used in thisstudy; all stock solutions were prepared with Milli-Q water that had been auto-claved at 1218C for 20 min and then aseptically purged with oxygen-free N2. Thefinal 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 con-ditions 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 thecultures were sterile (either autoclaved or purchased sterile) and were allowed todegas in the anaerobic glove box before use. Preparation of enrichment cultureshas been described previously (5). At the time that serial dilutions were initiated,the enrichment cultures had been maintained in the laboratory with toluene andsulfate 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 by150 mm; Bellco Glass, Inc., Vineland, N.J.) that were sealed with polytetrafluo-roethylene (PTFE)-coated butyl rubber liners (Alltech Associates, Inc., Deer-field, Ill.). Toluene (.99.9% purity, glass-distilled, filtered [0.5-mm-pore-sizefilter]; Aldrich Chemical Co.) was added into the medium as a pure liquid witha 10-ml syringe. The syringe was sterilized with ethanol and allowed to dry beforebeing used for toluene. Toluene concentrations were kept at or below 50 mMduring serial dilution to preclude toxicity. Because Patuxent River enrichmentcultures were found to be highly sensitive to sulfide (with inhibition in the rangeof 1 to 3 mM) (6), FeSO4 rather than MgSO4 was used as a sulfate source tominimize the dissolved sulfide concentration. The 1026-diluted culture from thefirst dilution series was grown with toluene for several months before being usedas the inoculum for a second dilution series. The 1028-diluted culture from thissecond series, which appeared homogenous by microscopic examination, wasgrown with toluene for several months before serving as the inoculum for furtherdilution 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 theagar dilution series to expedite growth. Colonies of PRTOL1 were brown andlens shaped. Isolated colonies were transferred to liquid medium with toluene asthe sole carbon source.Cultures were maintained in amber glass, screw-cap bottles that were sealed

with PTFE Mininert valves (Alltech Associates, Inc.). Toluene was monitoredand added when depleted; filter-sterilized, anaerobic FeSO4 was added via sy-ringe when sulfate was depleted (as indicated by cessation of toluene consump-tion). Cultures growing on toluene were examined microscopically for purity ona regular basis. To independently test the purity of the cultures used in this study,complex medium that included 0.5% nutrient broth was inoculated withPRTOL1 (5 to 10% inoculum) and was examined microscopically after 2 to 3weeks of incubation. No microbial contaminants were observed in these tests.Catabolic studies. For catabolic studies, PRTOL1 cells were grown with tol-

uene and sulfate in a 2-liter glass reactor sealed with Mininert valves. Cells wereharvested anaerobically by centrifugation (5,000 3 g for 40 min at 208C) andwere resuspended in the anaerobic growth medium described previously. Allexperiments with volatile aromatic compounds, such as toluene, were conductedin screw-cap glass containers sealed with Mininert valves; for all other com-pounds, PTFE-faced silicone septa inside open-hole screw caps were used to sealbottles. Aromatic compounds that are liquids at room temperature (e.g., ben-zene, toluene, ethylbenzene, xylene isomers, benzyl alcohol, benzaldehyde, andcresol isomers) were added as pure liquids with a 10-ml syringe. All othercompounds were added as anaerobic, filtered aqueous stock solutions; no or-ganic carrier phases were used for amending the cultures. Catabolic studies wereperformed 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) wereharvested by centrifugation, washed in an anaerobic MOPS (morpholinepro-panesulfonic acid) buffer (50 mM, pH 7.0) containing 5 mM dithiothreitol and 5mM MgCl2, and then resuspended in 1.5 ml of the MOPS buffer. Assays were

performed with permeabilized cells (2% Triton X-100 [vol/vol]) under anaerobicconditions in stoppered glass cuvettes at 358C. Carbon monoxide dehydrogenaseand formate dehydrogenase activities were assayed by following the reduction ofmethyl viologen at 578 nm, as described elsewhere (38). 2-Oxoglutarate dehy-drogenase activity was also assayed with methyl viologen as the electron accep-tor; a tricine buffer (100 mM, pH 8.5) containing 5 mM dithiothreitol and 1 mMmethyl viologen was used. The protein concentration in the permeabilized cellpreparation was estimated by using the measured cell density and estimatedvalues for cell mass and composition taken from Neidhardt (29); the proteincontent of the cell preparation could not be measured directly because of inter-ferences 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 tech-nique using a model 5890A gas chromatograph (Hewlett-Packard Company,Palo Alto, Calif.) with a model PI 52-02A photoionization detector (10.2 eVlamp; HNU Systems, Inc., Newton, Mass.) and a DB-624 fused silica capillarycolumn (30-m length, 0.53-mm inner diameter, 3.0-mm film thickness; J & WScientific, Folsom, Calif.). Analyses were isothermal (808C) with splitless injec-tion (the split was turned on after 0.5 min). The method is described in detailelsewhere (5). The detachable stainless steel needles for the gas-tight syringesused for headspace sampling were autoclaved before each use. Peak integrationfor aromatic hydrocarbons (as well as for sulfate and oxygenated aromatic com-pounds [described below]) was performed with a Nelson analytical chromatog-raphy 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 studieswith PRTOL1 (phenylpropionate, phenylacetate, benzylsuccinate, benzyl alco-hol, benzaldehyde, benzoate, o- and p-cresol, p-hydroxybenzoate, and p-toluate)were determined in the supernatant of centrifuged samples by reverse-phasehigh-performance liquid chromatography (HPLC) with a Perkin-Elmer series400 liquid chromatograph connected to a Hewlett-Packard model 1050 variablewavelength detector. The mobile phase was a 65:35 (vol/vol) mixture of methanoland 50 mM acetate buffer (pH 3.5) flowing isocratically at 1 ml/min through anAdsorbosphere HS C18 column (5-mm particle size, 250 mm by 4.6 mm innerdiameter; Alltech). Compounds were quantified with a wavelength of either 265nm (for the first four compounds listed above) or 280 nm. For the aqueousHPLC standards, benzyl alcohol, benzaldehyde, and cresols were added as pureliquids; benzoate and p-hydroxybenzoate were added as sodium salts; and theremaining four compounds were added from methanolic stock solutions. Themaximum 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 Cor-poration, Sunnyvale, Calif.) equipped with an HPIC-AS4A column, an anionmicromembrane suppressor, and a conductivity detector. Analyses were iso-cratic, with a 0.75 mM sodium bicarbonate–2.2 mM sodium carbonate eluantflowing at a rate of 2 ml/min.(iv) 14C analysis. 14C-labeled toluene was used in PRTOL1 cultures to inves-

tigate the extent of toluene mineralization and incorporation into biomass. Anal-ysis of 14C activity in culture liquid was performed with a Tri-Carb model 2500TR/AB liquid scintillation analyzer (Packard Instruments Co., Inc., DownersGrove, Ill.). All samples were automatically blank corrected and were correctedfor sample-specific quenching by using an external standard method (with a133Ba gamma source) and a quenching curve developed from a series ofquenched standards.Sample-processing methods were very similar to those described previously

(5), except that headspace was not sampled in the present study because exper-iments were designed to have negligible headspace (,1.5% of the total volume).By this method, three fractions of 14C activity were determined in culture sam-ples: 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 aseparatory funnel with diethyl ether (Ultra Resi-Analyzed, distilled-in-glass; J. T.Baker). The extracts were dried with anhydrous sodium sulfate, derivatized withethereal diazomethane (15), concentrated under a gentle stream of high-puritynitrogen at room temperature, exchanged into dichloromethane (Ultra Resi-Analyzed, distilled-in-glass; J. T. Baker, Inc.), and analyzed by GC-MS. GC-MSanalyses 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 massselective detector; data analysis was performed with Hewlett-Packard G1034Csoftware designed for the MS ChemStation. The GC oven was programmed from458C (held for 2 min) to 1108C at 88C/min and then from 1108C to 2508C at48C/min (held for 5 min); the injection port temperature was 2758C, and thetransfer line temperature was 2808C. Injections were splitless, with the splitturned on after 0.5 min. For data acquisition, the MS scanned from 50 to 350atomic mass units at a rate of two scans per s.Determination of growth. Growth was determined by two methods: micro-

scopic cell counts and optical density. Growth experiments were conducted in thedark with clear glass tubes (13 by 100 mm) that were sealed with PTFE-facedsilicone septa inside open-hole screw caps. The medium used in these experi-ments contained MgSO4 rather than FeSO4 to preclude the formation of FeCO3

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and FeS precipitates. Cell counting was performed by phase-contrast microscopyat 400-fold total magnification with a Petroff-Hausser counting chamber(Hausser Scientific, Blue Bell, Pa.). Because the cell densities being countedwere generally between 106 and 5 3 107 cells per ml, samples were concentratedfivefold 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 spec-trophotometer (Milton Roy, Rochester, N.Y.). When measurements were made,the sealed tubes were removed from the glove box, analyzed within 10 min, andreturned 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. Morespecifically, the two criteria for growth were (i) an optical density value greaterthan or equal to that of the positive control and (ii) a cell count greater than orequal to four times that of the negative control. In experiments testing potentialelectron donors, the positive controls included benzoate and the negative con-trols had no added electron donor. In experiments testing potential electronacceptors, the positive controls included sulfate and the negative controls had noadded electron acceptor. Growth was not determined for most of the experi-ments that assessed potential electron donors for PRTOL1. In these experi-ments, only metabolism (as indicated by the consumption of the electron donorand/or acceptor) was assessed because the relatively long generation time andlow 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 portionof this DNA (ca. 0.1 mg) was used in the PCR to amplify most of the 16S rRNAgene. The primers used to amplify near full-length 16S rRNA gene sequences(59-AGAGTTTGATCCTGGCTCAG-39 and 59-AAGGAGGTGATCCAGCC-39) 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 deoxynucleo-side triphosphate (dNTP), 0.25 mM each primer, 13 Taq polymerase buffer, and0.75 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 ThermalCycler (Perkin-Elmer Corp., Norwalk, Conn.) with a program consisting of aninitial denaturation at 928C for 130 s; 30 cycles of 948C for 15 s, 558C for 30 s, and728C 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 tothe manufacturer’s suggestions (Bio 101, Inc., La Jolla, Calif.). The purified PCRproduct was cloned in the vector pCRII by using a TA Cloning Kit (Invitrogen,San Diego, Calif.) according to the manufacturer’s directions. Plasmid DNAcontaining the 16S rRNA gene insert was isolated from one clone by using theQiagen 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 ABI373A sequencer (Applied Biosystems, Foster City, Calif.). The primers that were

used corresponded to conserved regions of the 16S rRNA sequence (47). Ap-proximately 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 fast-DNAml (30), by using a weighting mask to include only unambiguously alignedpositions with all other program options at their default values. This analysis wasrepeated on 100 bootstrap samples to obtain confidence estimates of the branch-ing order (16). The program CONSENSE from the PHYLIP program package(17) was used to determine the number of times that each group shown in thefinal 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.

RESULTSMorphology. Cells of PRTOL1 are oval, 2 to 3 mm long, and

1.2 to 1.7 mm in diameter and stain gram negative. A phase-contrast photomicrograph of PRTOL1 cells growing on tolu-ene is presented in Fig. 1. Microscopic examination did notreveal any evidence of spores or of swimming motility.Phylogenetic classification based on 16S rRNA. Phyloge-

netic relationships based on 16S rRNA gene sequences aredepicted in Fig. 2. Strain PRTOL1 is classified in the delta

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

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

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subclass of the Proteobacteria, as are all other known gram-negative mesophilic sulfate-reducing bacteria (45). Desulfo-rhabdus amnigenus (31) is the closest known relative of strainPRTOL1 (96% sequence similarity). Desulforhabdus amnige-nus, which was isolated from anaerobic granular sludge withacetate as the sole carbon and energy source, does not metab-olize benzoate or other aromatic compounds that have beentested (31). As shown in Fig. 2, three bacteria capable ofanaerobic toluene degradation are classified among the deltaProteobacteria: the sulfidogenic Desulfobacula toluolica (33)and PRTOL1 and the iron-reducing Geobacter metallireducens(26).Carbon and electron balance for sulfidogenic toluene deg-

radation. In PRTOL1 cultures, toluene degradation and sul-fate reduction were strongly correlated over time and had aconsistent 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 consump-tion for the cultures had a slope of 3.89 mol of sulfate per molof toluene (r2 5 0.996), which is consistent with theoreticalratios ranging from 4.09 (toluene oxidation to CO2, sulfatereduction to hydrogen sulfide, and estimated cell growth; equa-tion 1) to 4.5 (toluene oxidation to CO2, sulfate reduction tohydrogen sulfide, no cell growth; equation 2). The estimationof cell growth in equation 1 was derived by using the calcula-tion methods described by McCarty (27, 28).

C7H8 1 4.09 SO422 1 0.17 NH41 1 2.49 H2O3 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 H2O3 2.25 H2S 1 2.25 HS2 1 (2)

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

Toluene mineralization to CO2 was confirmed by usingPRTOL1 suspensions supplied with [ring-U-14C]toluene as thesole electron donor. The results of a 4-day incubation withapproximately 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 ofbiomass and nonvolatile metabolites). All of the added toluenewas consumed in this experiment, as indicated by an interme-diate sampling that demonstrated that 80% mineralization of

toluene had already occurred by day 2 (data not shown). Inuninoculated 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 dem-onstrated in the enrichment cultures from which PRTOL1 wasisolated (7), the transformation of toluene to benzylsuccinicacid can be verified by using stable isotope-labeled toluene(e.g., toluene-d8) and GC-MS analysis. Extraction and GC-MSanalysis of the replicates shown in Fig. 3, which were giventoluene-d8, revealed that 2.70 to 2.75% of toluene carbon hadbeen converted to deuterium-labeled benzylsuccinic acid, witha trace amount of labeled benzylfumaric acid (or a closelyrelated isomer) also detectable (,0.1% of toluene carbon).However, higher benzylsuccinic acid yields of approximately7% of toluene carbon have been observed in similar experi-ments (data not shown) and in the mixed enrichment culturefrom which PRTOL1 was isolated (7); therefore, it appearsthat the benzylsuccinic acid yield may not be constant.The cell yield of PRTOL1 growing on toluene can be esti-

mated from the data for radiolabeled toluene and benzylsuc-cinic acid; these data were collected from experiments thatwere run concurrently. Fifteen percent of toluene carbon wasconverted to nonvolatile carbon (Table 1), and approximately3% was converted to benzylsuccinic acid, a nonvolatile metab-olite. By difference, approximately 12% of toluene carbon wasconverted to cells. By using an average cell composition ofC5H7O2N (27), the cell yield is estimated as 19 g of cells (dryweight) per mol of toluene. Notably, this value is virtuallyidentical to the theoretical prediction given in equation 1. Adirect gravimetric determination of cell yield was not practicalbecause of the relatively low yield and long doubling timeinvolved and because of FeS and FeCO3 precipitates thatformed in the medium. The doubling time of PRTOL1 growingon 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 thantoluene, including single-ring aromatic hydrocarbons, short-chain aliphatic acids, and potential intermediates of toluenemetabolism, was tested (Table 2). All five of the short-chainaliphatic acids tested (formate, acetate, pyruvate, succinate,and fumarate) were metabolized, as indicated by sulfate con-sumption. Growth was observed for the latter three acids butwas not confirmed for formate and acetate during a 40-dayincubation period. Strain PRTOL1 oxidized phenylpropionate,phenylacetate, benzaldehyde, benzoate, p-cresol, and p-hy-

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

TABLE 1. Carbon balance for toluene degradationby strain PRTOL1

14C fraction% of total dpma

PRTOL1 Control

Initial, volatile 14C 98.2 6 0.5 99.6Final (day 4)Volatile 14C 4.7 6 0.2 99.614CO2 80.3 6 0.2 0.2Nonvolatile 14Cb 15.0 6 0.4 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 balancesfor strain PRTOL1 and the control were 106 and 99%, respectively, defined as(the total number of final dpm [disintegrations per minute] divided by the totalnumber of initial dpm)3 100. The results are expressed as the means6 standarddeviations. Of the PRTOL1 replicates prepared on day 0, two were analyzed onday 0 and three were analyzed on day 4. Each replicate bottle was sampled onlyonce.b Nonvolatile carbon represents the sum of biomass and nonvolatile metabo-

lites.

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droxybenzoate but not benzyl alcohol, o-cresol, p-toluate, andbenzylsuccinate during a 15-day incubation period (Table 2).Among these oxygenated aromatic compounds, benzaldehydewas the most rapidly consumed. Among the single-ring aro-matic hydrocarbons tested, no discernible consumption of ben-zene, ethylbenzene, or m-xylene was observed in a 3-weekperiod. The results for o- and p-xylene merit detailed elabora-tion.In experiments testing the metabolism of o- and p-xylene, as

well as the other three single-ring aromatic hydrocarbons listedin Table 2, toluene was initially added at approximately thesame concentration as the test substrate but was not replacedafter it was depleted. This design was intended to demonstratethat the cultures were active and to investigate cometabolismwith toluene. In PRTOL1 suspensions amended with o-xylene(Fig. 4), both the toluene and o-xylene concentrations de-

creased within the first 10 days, after which time no furthero-xylene decrease was detectable. Although a 55 to 60 mMaddition of toluene was rapidly consumed between days 20 and30, the o-xylene concentration did not decrease during thistime. Extraction and GC-MS analysis of the sample shown inFig. 4 on day 35 indicated a complete conversion of the con-sumed o-xylene to nonvolatile metabolic products. Based onthe strong similarity of the mass spectra of the two observedproducts (Fig. 5A and C) to published spectra (13), and basedon the demonstrated formation of benzylsuccinic and benzyl-fumaric acid from toluene by PRTOL1, (2-methylbenzyl)suc-cinic acid and (2-methylbenzyl)fumaric acid (or a closely re-lated isomer) are indicated as the predominant products ofo-xylene metabolism. Because authentic standards of thesecompounds were not commercially available, their concentra-tions were estimated by using total ion current areas and thestructurally similar benzylsuccinic acid as a standard. On thebasis of this estimation technique, ca. 125 mol% of the o-xyleneconsumed was transformed to (2-methylbenzyl)succinic acidand ca. 13 mol% was converted to (2-methylbenzyl)fumaricacid. Thus, considering overall experimental error, includingthe lack of authentic standards, it appears that the consumedo-xylene was quantitatively converted to these products. Nota-bly, 2-methylbenzoic acid was also detected by GC-MS analy-sis, but its concentration was very low (ca. 0.3 mol% of theo-xylene consumed). Formation of deuterium-labeled (2-meth-ylbenzyl)succinic acid and (2-methylbenzyl)fumaric acid (or aclosely related isomer) from o-xylene-d10 was demonstrated ina similar experiment (Fig. 5B and D). Similar patterns of o-xylene transformation were observed in a separate experimentduring which toluene-grown PRTOL1 cells were supplied witho-xylene in the absence of toluene (data not shown).The results of an experiment testing the metabolism of p-

xylene by PRTOL1 are depicted in Fig. 6. In the first 10 days,toluene was rapidly depleted and p-xylene was depleted moreslowly. Additional p-xylene amended on day 9 was partiallyconsumed, but consumption was not sustained over time. Tol-uene added to another replicate on day 22 was rapidly con-sumed, but no further p-xylene consumption was observed(data not shown). Extraction and GC-MS analysis of the sam-ple represented in Fig. 6 on day 34 indicated a partial conver-sion to metabolic products; the mass spectra of the productsare shown in Fig. 7B and D. The major product, constitutingapproximately 23 mol% of the p-xylene consumed, was con-firmed to be 4-methylbenzoic acid (p-toluic acid), for which anauthentic standard was available (Fig. 7A). A compound as-sumed to be (4-methylbenzyl)succinic acid [based on the argu-

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

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

Compound (concn)Metabolism

Donora Acceptor (sulfate)b

Aromatic hydrocarbonsc

Benzene (50–60 mM) 2 NDEthylbenzene (20–40 mM) 2 NDo-Xylene (20–40 mM) 1d NDm-Xylene (20–40 mM) 2 NDp-Xylene (20–40 mM) 1d ND

Short-chain aliphatic acidse

Formate (5 mM) 2 1Acetate (4 mM) 2 1Pyruvate (3 mM) 1 1Succinate (2 mM) 1 1Fumarate (2 mM) 1 1

Potential toluene intermediatesPhenylpropionate (0.5 mM) 1 1Phenylacetate (0.5 mM) 1 1Benzaldehyde (0.5 mM) 1 1Benzoate (0.5 mM) 1 1p-Cresol (0.5 mM) 1 1p-Hydroxybenzoate (0.5 mM) 1 1Benzyl alcohol (0.125 and 0.25 mM) 2 2o-Cresol (0.5 mM) 2f 2

Alkylbenzene by-productsp-Toluate (0.5 mM) 2 2Benzylsuccinate (0.5 mM) 2 2

a 1, 70 to 100% decrease in concentration observed in 15 days, with exceptionsas noted in other footnotes;2, no decrease in concentration outside the range ofanalytical 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, notdetermined.c For these compounds, toluene was originally present and was allowed to be

depleted without further addition (see text).dMetabolism was determined by GC-MS detection of distinctive metabolic

products (see text) and by some observed decrease in the substrate concentrationover time. However, the rate of metabolism was very slow and metabolism couldnot be sustained over time. Cometabolism with toluene may apply to thesecompounds (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 describedin footnote a. The metabolism of sulfate was determined as described in footnoteb.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 peakappeared after 15 days that corresponded to the retention time of p-hydroxy-benzoate. It is probable that this peak indicates a minor conversion to o-hydroxy-benzoate, but this possibility was not confirmed by GC-MS.

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ments given for (2-methylbenzyl)succinic acid] accounted forapproximately 3 mol% of the p-xylene consumed. Thus, incontrast to the results for o-xylene, the metabolic productsfound for p-xylene did not account for the total mass of p-xylene consumed, but accounted for approximately 26 mol%.Mineralization of a portion of p-xylene by PRTOL1 is possiblebut could not be confirmed because radiolabeled p-xylene isnot commercially available. An experiment with stable isotope-labeled p-xylene (p-xylene-d10) demonstrated the formation ofdeuterium-labeled metabolites; the spectrum of methyl-p-tolu-ate-d7 is shown in Fig. 7C.Of the other tested electron donors that were used by

PRTOL1 (Table 2), only benzaldehyde was investigated withrespect to metabolic products. Benzaldehyde-a-13C,d1 (490

mM) was added to a PRTOL1 suspension to determine whetherbenzylsuccinic acid was formed from benzaldehyde. Benzalde-hyde has been reported as an intermediate of anaerobic tolu-ene degradation (1, 2) and thus is a potential intermediate ofanaerobic toluene transformation to benzylsuccinic acid. In theexperiment with strain PRTOL1, no labeled benzylsuccinicacid was formed from labeled benzaldehyde. However, labeled3-benzoylpropionic acid (Fig. 8) was identified at a relativelylow concentration (ca. 0.5 mol% of the 490 mM benzaldehydeconsumed).Various electron acceptors, including thiosulfate, sulfite, ni-

trate, and fumarate, were tested for their use by PRTOL1 inthe presence of benzoate. Only thiosulfate and sulfite werefound to support metabolism (as indicated by benzoate con-sumption) and growth of PRTOL1.Key enzymes involved in acetyl coenzyme A oxidation. En-

zyme assays performed with permeabilized, benzoate-growncells indicated that specific activities of carbon monoxide de-hydrogenase and formate dehydrogenase were on the order of3.8 and 22 mmol z min21 z mg of protein21, respectively. Theactivity of 2-oxoglutarate dehydrogenase was no greater thanthe detection limit (approximately 0.15 mmol z min21 z mg ofprotein21).

DISCUSSION

Strain PRTOL1 is a gram-negative, mesophilic sulfate-re-ducing bacterium that, unlike its closest known phylogeneticrelatives, can degrade a range of aromatic compounds includ-ing an aromatic hydrocarbon, toluene. PRTOL1 metabolizedsix of the oxygenated aromatic compounds tested in this study.These compounds were chosen because they are proposed ordemonstrated intermediates of anaerobic toluene degradation,based on studies of a range of toluene-degrading bacteria. Asshown in Table 2, PRTOL1 can metabolize phenylpropionate(potentially resulting from the condensation of acetyl coen-zyme A with the methyl carbon of toluene, as proposed byEvans et al. [13]), benzaldehyde (potentially formed followingthe formation of benzyl alcohol by hydroxylation of the methylcarbon of toluene, as described for denitrifying Thauera sp.strain K172 [1, 2]), p-cresol (potentially resulting from thehydroxylation of the aromatic ring of toluene, as shown for amixed methanogenic culture [21, 43]), phenylacetate (poten-tially resulting from carboxylation of the methyl carbon oftoluene, as suggested by Altenschmidt and Fuchs [1]), andbenzoate (an intermediate common to all proposed anaerobictoluene degradation pathways [e.g., 13, 26]). As PRTOL1 me-tabolizes intermediates from several different potential toluene

FIG. 5. Mass spectra of the dimethyl esters of o-xylene metabolites. (A) Thepredominant metabolite, tentatively identified as (2-methylbenzyl)succinic acid(see text), resulting from o-xylene metabolism by PRTOL1; (B) the predominantmetabolite, tentatively identified as (2-methylbenzyl)succinic acid-d10, resultingfrom o-xylene-d10 metabolism by PRTOL1; (C) a lesser metabolite, tentativelyidentified 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 relatedisomer), resulting from o-xylene-d10 metabolism by PRTOL1. Spectra A and Cwere acquired from an extract of the sample represented in Fig. 4.

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

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degradation pathways, no preliminary indication of a specificmineralization pathway used by PRTOL1 is evident from thisstudy. The inability to metabolize benzyl alcohol noted forPRTOL1 (Table 2) has been observed for other anaerobictoluene degraders, including Desulfobacula toluolica (33) andthree denitrifying bacteria (strains T [10], mXyN1 [34], and T1[14]). In contrast, a denitrifying bacterium shown to degradetoluene via benzyl alcohol, Thauera sp. strain K172, metabo-lized benzyl alcohol as a sole carbon source after growth ontoluene (1, 2). Nonetheless, a pathway involving benzyl alcoholas an intermediate cannot be excluded based on the lack ofbenzyl alcohol metabolism.PRTOL1 transforms a number of aromatic substrates, in-

cluding toluene, o- and p-xylene, and benzaldehyde, to meta-bolic by-products; most of these products result from the bond-ing of a short-chain aliphatic acid to a benzylic carbon atom by

mechanisms that are currently unknown. For toluene, and pos-sibly for other aromatic substrates such as benzaldehyde, thistransformation occurs in conjunction with mineralization. Theformation of benzylsuccinic acid and a much smaller amount ofbenzylfumaric acid (or a closely related isomer) from toluenewas reported previously for the enrichment cultures fromwhich PRTOL1 was isolated (7). The observation that benzyl-succinic acid was not metabolized by PRTOL1 (Table 2) isconsistent with the previous finding that benzylsuccinic acidwas a dead-end product in those enrichment cultures (7). Theformation of benzylsuccinic acid from toluene by PRTOL1does not proceed via benzaldehyde, as indicated by catabolicstudies in which benzaldehyde was a sole carbon source; sim-ilar results were reported for a denitrifying bacterium thatmetabolizes toluene to dead-end products (18). However, atrace amount (0.5%) of benzaldehyde was converted byPRTOL1 to 3-benzoylpropionic acid (Fig. 8). In other cata-bolic studies, PRTOL1 transformed o-xylene quantitatively to(2-methylbenzyl)succinic acid and (2-methylbenzyl)fumaricacid (or a closely related isomer). In accordance with toluenetransformation, the benzylsuccinic acid analog of o-xylene wasformed at a much higher concentration than the benzylfumaricacid analog. Notably, transformation of o-xylene to (2-methyl-benzyl)succinic and (2-methylbenzyl)fumaric acids withoutconcurrent mineralization was also observed by Evans andcoworkers for denitrifying strain T1 (13). PRTOL1 also trans-formed p-xylene to p-toluic acid (an apparent dead-end prod-uct; Table 2) and a lesser amount of (4-methylbenzyl)succinicacid. An additional compound detected at a low concentrationin the p-xylene culture (data not shown) suggested that p-toluicacid was formed via p-tolualdehyde. This compound was likely3-(p-toluyl)propionic acid, a methyl homolog of 3-benzoylpro-pionic acid. The mass spectral fragmentation pattern of themethyl ester of this p-xylene metabolite was analogous to themethyl ester of 3-benzoylpropionic acid (Fig. 8A) but with them/z 51, 77, 105, 161, and 192 fragments all replaced by frag-ments that were 14 atomic mass units higher. Thus, by analogywith 3-benzoylpropionic acid formation from benzaldehyde, it

FIG. 7. Mass spectra. (A) A methyl-p-toluate standard; (B) the methylatedpredominant product resulting from p-xylene metabolism by PRTOL1 (samplerepresented in Fig. 6); (C) the methylated predominant product, tentativelyidentified as methyl-p-toluate-d7, resulting from p-xylene-d10 metabolism byPRTOL1; and (D) the methylated lesser product, tentatively identified as (4-methylbenzyl)succinic acid (see text), resulting from p-xylene metabolism byPRTOL1 (sample represented in Fig. 6).

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

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is likely that 3-(p-toluyl)propionic acid was formed from p-tolualdehyde, a likely intermediate to p-toluic acid formationfrom p-xylene. Notably, direct evidence of the formation ofp-toluic acid via p-tolualdehyde was recently demonstratedwith cell suspensions of a denitrifying bacterium amended withp-xylene; analogous findings were made for themeta isomers ofthese compounds (37).The nature of o- and p-xylene transformation by toluene-

grown PRTOL1 cells is complex (Fig. 4 and 6) and not wellunderstood. Transformation of o-xylene to by-products couldbe considered cometabolic because (i) it appears to involve anincomplete oxidation that provides no carbon and little or noenergy to the cell and (ii) it may require the activity of anenzyme involved in the metabolism of a growth substrate (tol-uene). For p-xylene, these criteria may be less applicable be-cause mineralization of a portion of p-xylene to CO2 cannot beruled out. For both xylene isomers, a confounding observationwas that consumption ceased over time and was not stimulatedby the addition and rapid consumption of toluene. One possi-ble explanation for this observation is that a product formedduring xylene metabolism (e.g., a methylbenzylsuccinic acidisomer) specifically inhibited further xylene metabolism butdid not affect toluene metabolism. Further studies to examinethe cessation of xylene metabolism are warranted and mayhave considerable relevance for anaerobic bioremediation ofgasoline-contaminated sites.As more anaerobic bacteria capable of aromatic hydrocar-

bon degradation are isolated and studied, the potential foranaerobic bioremediation of gasoline-contaminated aquiferswill be easier to assess. However, a better understanding of thedetails of anaerobic hydrocarbon metabolism (e.g., formationof by-products and substrate interactions) will be required tooptimize anaerobic bioremediation and to reliably predict itsconsequences.

ACKNOWLEDGMENTS

Funding for this study was provided by the Office of Research andDevelopment, U.S. Environmental Protection Agency, under grantR-815738 through the Western Region Hazardous Substance Re-search Center. Additional funding for 16S rRNA analysis performed atMichigan State University was provided by NSF grant BIR-9120006 tothe Center for Microbial Ecology.

ADDENDUM IN PROOF

In a recently published study of the sulfate-reducing Desul-fobacula toluolica (Arch. Microbiol. 164:448–451, 1995), Rabusand Widdel reported the transformation of p-xylene to 4-meth-ylbenzoate by dense suspensions of toluene-grown cells thatwere supplied with a mixture of p-xylene and toluene. In ad-dition, a relatively low yield (0.1%) of benzylsuccinate wasobserved in toluene-metabolizing cell suspensions.

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