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. 11881196 Vol. 62, No. 4
0099-2240/96/$04.000
Copyright 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 REINHARD
1
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 48824
2
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. The
mineralization of 80% of toluene carbon to CO
2
was demonstrated in experiments with [ring-U-
14
C]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 sulde.
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 op-
tions for restoring aquifers contaminated with the hazardous,
relatively water-soluble aromatic hydrocarbons that occur in
unleaded gasoline, such as benzene, toluene, and xylenes. Be-
cause many contaminated aquifers are anaerobic as a result of
oxygen 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 environmental
concerns, knowledge of the metabolic capabilities of anaerobic
bacteria has expanded dramatically in the past decade, partic-
ularly 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-metha-
nogenic 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 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 report
the isolation of strain PRTOL1, the second sulfate-reducing
bacterium known to degrade an aromatic hydrocarbon and the
rst 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 were
purchased from Aldrich Chemical Co., Inc. (Milwaukee, Wis.), and most inor-
ganic chemicals were purchased from J. T. Baker, Inc. (Phillipsburg, N.J.). Stable
isotope-labeled chemicals included toluene-d
8
(99 atom%; Aldrich Chemical
Co.); o-xylene-d
10
and p-xylene-d
10
(99 atom%; Aldrich Chemical Co.); and
benzaldehyde--
13
C,d
1
(98% purity [D] and 99% purity [
13
C]; Isotec, Inc., Mi-
amisburg, 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-
14
C]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 nal specic activity of 75.1 Ci/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 Chemical
Co.). The basis for identication of other by-products without authentic stan-
dards is presented in later sections. However, the identications of benzylfumaric
acid and (2-methylbenzyl)fumaric acid require additional discussion. The rst
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 [benzyl-
maleic 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
denitive identications of such metabolic by-products. An analogous situation
probably applies to the compound previously identied as (2-methylbenzyl)fu-
maric 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 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.
1188

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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 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) specied in parentheses: NaHCO
3
(30), NH
4
Cl (28), NaH
2
PO
4
H
2
O (4.4), FeSO
4
7H
2
O (3), NaCl (1.7), KCl
(1.3), CaCl
2
2H
2
O (0.68), MgCl
2
6H
2
O (0.49), MnCl
2
4H
2
O (0.025), and
Na
2
MoO
4
2H
2
O (0.004). The medium (excluding FeSO
4
and NaHCO
3
) was
autoclaved at 121C for 20 min and then aseptically purged with an oxygen-free
mixture of 79% N
2
21% CO
2
for 45 min. After purging, bicarbonate was added
to the medium as a sterile, anaerobic 1.0 M solution (45) in addition to anaer-
obic, ltered 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 M sodium sulde added from a
ltered 0.1 M stock solution. Highly puried 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 auto-
claved at 121C for 20 min and then aseptically purged with oxygen-free N
2
. The
nal pH of the medium was approximately 7.
All preparation and incubation of enrichment cultures, serial dilutions, and
pure culture suspensions were performed at 35C under strictly anaerobic con-
ditions in an anaerobic glove box (Coy Laboratory Products, Inc., Ann Arbor,
Mich.) with a gas composition of approximately 90% N
2
7.5% CO
2
2.5% H
2
.
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 polytetrauo-
roethylene (PTFE)-coated butyl rubber liners (Alltech Associates, Inc., Deer-
eld, Ill.). Toluene (99.9% purity, glass-distilled, ltered [0.5-m-pore-size
lter]; Aldrich Chemical Co.) was added into the medium as a pure liquid with
a 10-l syringe. The syringe was sterilized with ethanol and allowed to dry before
being used for toluene. Toluene concentrations were kept at or below 50 M
during serial dilution to preclude toxicity. Because Patuxent River enrichment
cultures were found to be highly sensitive to sulde (with inhibition in the range
of 1 to 3 mM) (6), FeSO
4
rather than MgSO
4
was used as a sulfate source to
minimize the dissolved sulde concentration. The 10
6
-diluted culture from the
rst dilution series was grown with toluene for several months before being used
as the inoculum for a second dilution series. The 10
8
-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; lter-sterilized, anaerobic FeSO
4
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 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 tol-
uene and sulfate in a 2-liter glass reactor sealed with Mininert valves. Cells were
harvested anaerobically by centrifugation (5,000 g for 40 min at 20C) 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 com-
pounds, PTFE-faced silicone septa inside open-hole screw caps were used to seal
bottles. Aromatic compounds that are liquids at room temperature (e.g., ben-
zene, toluene, ethylbenzene, xylene isomers, benzyl alcohol, benzaldehyde, and
cresol isomers) were added as pure liquids with a 10-l syringe. All other
compounds were added as anaerobic, ltered aqueous stock solutions; no or-
ganic 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 (morpholinepro-
panesulfonic acid) buffer (50 mM, pH 7.0) containing 5 mM dithiothreitol and 5
mM MgCl
2
, and then resuspended in 1.5 ml of the MOPS buffer. Assays were
performed with permeabilized cells (2% Triton X-100 [vol/vol]) under anaerobic
conditions in stoppered glass cuvettes at 35C. 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 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 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 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 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-m lm thickness; J & W
Scientic, Folsom, Calif.). Analyses were isothermal (80C) with splitless injec-
tion (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 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 studies
with 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-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) owing isocratically at 1 ml/min through an
Adsorbosphere HS C
18
column (5-m particle size, 250 mm by 4.6 mm inner
diameter; Alltech). Compounds were quantied with a wavelength of either 265
nm (for the rst 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 ltered samples (0.2-m-pore-size syringe
lters) was determined with an ion chromatograph (series 4000i, Dionex Cor-
poration, Sunnyvale, Calif.) equipped with an HPIC-AS4A column, an anion
micromembrane suppressor, and a conductivity detector. Analyses were iso-
cratic, with a 0.75 mM sodium bicarbonate2.2 mM sodium carbonate eluant
owing at a rate of 2 ml/min.
(iv)
14
C analysis.
14
C-labeled toluene was used in PRTOL1 cultures to inves-
tigate the extent of toluene mineralization and incorporation into biomass. Anal-
ysis of
14
C 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-specic 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 exper-
iments were designed to have negligible headspace (1.5% of the total volume).
By this method, three fractions of
14
C activity were determined in culture sam-
ples:
14
CO
2
, 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
acidied 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 Resi-
Analyzed, 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-m lm thickness; J & W Scientic) 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
45C (held for 2 min) to 110C at 8C/min and then from 110C to 250C at
4C/min (held for 5 min); the injection port temperature was 275C, and the
transfer line temperature was 280C. 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: micro-
scopic 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 experi-
ments contained MgSO
4
rather than FeSO
4
to preclude the formation of FeCO
3
VOL. 62, 1996 SULFIDOGENIC TOLUENE-DEGRADING BACTERIUM 1189

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and FeS precipitates. Cell counting was performed by phase-contrast microscopy
at 400-fold total magnication with a Petroff-Hausser counting chamber
(Hausser Scientic, Blue Bell, Pa.). Because the cell densities being counted
were generally between 10
6
and 5 10
7
cells per ml, samples were concentrated
vefold 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, 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
specically, 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 con-
trols 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 experi-
ments that assessed potential electron donors for PRTOL1. In these experi-
ments, 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 difcult 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 g) 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
(5-AGAGTTTGATCCTGGCTCAG-3 and 5-AAGGAGGTGATCCAGCC-
3) were modied versions of the primers fD1 and rD1 used by Weisburg et al.
(44). The PCR mixture consisted of 1.5 mM MgCl
2
, 0.2 mM each deoxynucleo-
side triphosphate (dNTP), 0.25 M each primer, 1 Taq polymerase buffer, and
0.75 U of Taq polymerase (Promega Corp., Madison, Wis.) in a volume of 30 l.
Amplication 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 92C for 130 s; 30 cycles of 94C for 15 s, 55C for 30 s, and
72C for 130 s; and a nal elongation cycle at 72C for 370 s.
The resulting PCR product was puried by gel electrophoresis with a 1%
agarose gel and was recovered using Gene Clean purication resin according to
the manufacturers suggestions (Bio 101, Inc., La Jolla, Calif.). The puried PCR
product was cloned in the vector pCRII by using a TA Cloning Kit (Invitrogen,
San Diego, Calif.) according to the manufacturers directions. Plasmid DNA
containing the 16S rRNA gene insert was isolated from one clone by using the
Qiagen plasmid mini kit according to the manufacturers directions (Qiagen,
Chatsworth, Calif.).
The DNA sequence of the insert was determined by automated uorescent
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). 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 aligned
positions with all other program options at their default values. This analysis was
repeated on 100 bootstrap samples to obtain condence 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 the
nal 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 m long, and
1.2 to 1.7 m 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 not
reveal any evidence of spores or of swimming motility.
Phylogenetic classication based on 16S rRNA. Phyloge-
netic relationships based on 16S rRNA gene sequences are
depicted in Fig. 2. Strain PRTOL1 is classied in the delta
FIG. 1. Phase contrast micrograph of PRTOL1 cells growing on toluene. Bar, 5 m.
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 which
the organisms to the right of the node were monophyletic.
1190 BELLER ET AL. APPL. ENVIRON. MICROBIOL.

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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 strain
PRTOL1 (96% sequence similarity). Desulforhabdus amnige-
nus, which was isolated from anaerobic granular sludge with
acetate as the sole carbon and energy source, does not metab-
olize benzoate or other aromatic compounds that have been
tested (31). As shown in Fig. 2, three bacteria capable of
anaerobic toluene degradation are classied among the delta
Proteobacteria: the suldogenic Desulfobacula toluolica (33)
and PRTOL1 and the iron-reducing Geobacter metallireducens
(26).
Carbon and electron balance for suldogenic toluene deg-
radation. In PRTOL1 cultures, toluene degradation and sul-
fate 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 consump-
tion for the cultures had a slope of 3.89 mol of sulfate per mol
of toluene (r
2
0.996), which is consistent with theoretical
ratios ranging from 4.09 (toluene oxidation to CO
2
, sulfate
reduction to hydrogen sulde, and estimated cell growth; equa-
tion 1) to 4.5 (toluene oxidation to CO
2
, sulfate reduction to
hydrogen sulde, no cell growth; equation 2). The estimation
of cell growth in equation 1 was derived by using the calcula-
tion methods described by McCarty (27, 28).
C
7
H
8
4.09 SO
4
2
0.17 NH
4

2.49 H
2
O 32.04 H
2
S (1)
2.04 HS

0.17 C
5
H
7
O
2
N (cells) 6.16 HCO
3

0.2 H

C
7
H
8
4.5 SO
4
2
3 H
2
O 32.25 H
2
S 2.25 HS

(2)
7 HCO
3

0.25 H

(G 205 kJ/mol of toluene)

Toluene mineralization to CO
2
was conrmed by using
PRTOL1 suspensions supplied with [ring-U-
14
C]toluene as the
sole electron donor. The results of a 4-day incubation with
approximately 80 M toluene are summarized in Table 1.
Eighty percent of toluene carbon was converted to
14
CO
2
,
whereas 15% was converted to nonvolatile
14
C (the sum of
biomass and nonvolatile metabolites). All of the added toluene
was 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). In
uninoculated controls, toluene was not converted to CO
2
.
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 was
isolated (7), the transformation of toluene to benzylsuccinic
acid can be veried by using stable isotope-labeled toluene
(e.g., toluene-d
8
) and GC-MS analysis. Extraction and GC-MS
analysis of the replicates shown in Fig. 3, which were given
toluene-d
8
, 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 experi-
ments (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 esti-
mated from the data for radiolabeled toluene and benzylsuc-
cinic 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 metab-
olite. By difference, approximately 12% of toluene carbon was
converted to cells. By using an average cell composition of
C
5
H
7
O
2
N (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 FeCO
3
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, short-
chain aliphatic acids, and potential intermediates of toluene
metabolism, was tested (Table 2). All ve of the short-chain
aliphatic acids tested (formate, acetate, pyruvate, succinate,
and fumarate) were metabolized, as indicated by sulfate con-
sumption. Growth was observed for the latter three acids but
was not conrmed for formate and acetate during a 40-day
incubation period. Strain PRTOL1 oxidized phenylpropionate,
phenylacetate, benzaldehyde, benzoate, p-cresol, and p-hy-
FIG. 3. Cumulative sulfate consumed versus cumulative toluene consumed
for duplicate suspensions of strain PRTOL1. The 200-ml replicates were incu-
bated for 9 days.
TABLE 1. Carbon balance for toluene degradation
by strain PRTOL1
14
C fraction
% of total dpm
a
PRTOL1 Control
Initial, volatile
14
C 98.2 0.5 99.6
Final (day 4)
Volatile
14
C 4.7 0.2 99.6
14
CO
2
80.3 0.2 0.2
Nonvolatile
14
C
b
15.0 0.4 0.2
a
A total of 4.7 mol of [ring-U-
14
C]toluene (75.1 Ci/mmol, specic activity)
was added to PRTOL1 cultures and to the uninoculated control. Mass balances
for strain PRTOL1 and the control were 106 and 99%, respectively, dened as
(the total number of nal dpm [disintegrations per minute] divided by the total
number of initial dpm) 100. The results are expressed as the means 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 metabo-
lites.
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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 aro-
matic hydrocarbons tested, no discernible consumption of ben-
zene, ethylbenzene, or m-xylene was observed in a 3-week
period. 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 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-
creased within the rst 10 days, after which time no further
o-xylene decrease was detectable. Although a 55 to 60 M
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 con-
sumed 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 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 of
o-xylene metabolism. Because authentic standards of these
compounds were not commercially available, their concentra-
tions 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. Nota-
bly, 2-methylbenzoic acid was also detected by GC-MS analy-
sis, but its concentration was very low (ca. 0.3 mol% of the
o-xylene consumed). Formation of deuterium-labeled (2-meth-
ylbenzyl)succinic acid and (2-methylbenzyl)fumaric acid (or a
closely related isomer) from o-xylene-d
10
was demonstrated in
a similar experiment (Fig. 5B and D). Similar patterns of o-
xylene 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 p-
xylene by PRTOL1 are depicted in Fig. 6. In the rst 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. 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 products
are shown in Fig. 7B and D. The major product, constituting
approximately 23 mol% of the p-xylene consumed, was con-
rmed to be 4-methylbenzoic acid (p-toluic acid), for which an
authentic 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
Donor
a
Acceptor (sulfate)
b
Aromatic hydrocarbons
c
Benzene (5060 M) ND
Ethylbenzene (2040 M) ND
o-Xylene (2040 M)
d
ND
m-Xylene (2040 M) ND
p-Xylene (2040 M)
d
ND
Short-chain aliphatic acids
e
Formate (5 mM)
Acetate (4 mM)
Pyruvate (3 mM)
Succinate (2 mM)
Fumarate (2 mM)
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)
f

Alkylbenzene by-products
p-Toluate (0.5 mM)
Benzylsuccinate (0.5 mM)
a
, 70 to 100% decrease in concentration observed in 15 days, with exceptions
as noted in other footnotes; , no decrease in concentration outside the range of
analytical error.
b
, 1 mM sulfate consumed in excess of negative control (no added electron
donor); , 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 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 peak
appeared 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 conrmed by GC-MS.
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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 p-
xylene consumed, but accounted for approximately 26 mol%.
Mineralization of a portion of p-xylene by PRTOL1 is possible
but could not be conrmed because radiolabeled p-xylene is
not commercially available. An experiment with stable isotope-
labeled p-xylene (p-xylene-d
10
) demonstrated the formation of
deuterium-labeled metabolites; the spectrum of methyl-p-tolu-
ate-d
7
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--
13
C,d
1
(490
M) was added to a PRTOL1 suspension to determine whether
benzylsuccinic 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 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 identied at a relatively
low concentration (ca. 0.5 mol% of the 490 M benzaldehyde
consumed).
Various electron acceptors, including thiosulfate, sulte, ni-
trate, and fumarate, were tested for their use by PRTOL1 in
the presence of benzoate. Only thiosulfate and sulte were
found 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-grown
cells indicated that specic activities of carbon monoxide de-
hydrogenase and formate dehydrogenase were on the order of
3.8 and 22 mol min
1
mg of protein
1
, respectively. The
activity of 2-oxoglutarate dehydrogenase was no greater than
the detection limit (approximately 0.15 mol min
1
mg of
protein
1
).
DISCUSSION
Strain PRTOL1 is a gram-negative, mesophilic sulfate-re-
ducing bacterium that, unlike its closest known phylogenetic
relatives, can degrade a range of aromatic compounds includ-
ing 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 coen-
zyme 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 (poten-
tially 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 me-
tabolizes intermediates from several different potential toluene
FIG. 5. Mass spectra of the dimethyl esters of o-xylene metabolites. (A) The
predominant metabolite, tentatively identied as (2-methylbenzyl)succinic acid
(see text), resulting from o-xylene metabolism by PRTOL1; (B) the predominant
metabolite, tentatively identied as (2-methylbenzyl)succinic acid-d
10
, resulting
from o-xylene-d
10
metabolism by PRTOL1; (C) a lesser metabolite, tentatively
identied 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 identied as (2-methylbenzyl)fumaric acid-d
8
(or a closely related
isomer), resulting from o-xylene-d
10
metabolism by PRTOL1. Spectra A and C
were 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 specic
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, metabo-
lized 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, 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, 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 benzyl-
succinic acid was not metabolized by PRTOL1 (Table 2) is
consistent with the previous nding 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; sim-
ilar 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 cata-
bolic 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-methyl-
benzyl)succinic and (2-methylbenzyl)fumaric acids without
concurrent mineralization was also observed by Evans and
coworkers 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)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-benzoylpro-
pionic 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 frag-
ments 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
identied as methyl-p-toluate-d
7
, resulting from p-xylene-d
10
metabolism by
PRTOL1; and (D) the methylated lesser product, tentatively identied as (4-
methylbenzyl)succinic acid (see text), resulting from p-xylene metabolism by
PRTOL1 (sample represented in Fig. 6).
FIG. 8. Mass spectra. (A) A methyl 3-benzoylpropionate standard and (B) a
methylated product resulting from metabolism of benzaldehyde--
13
C,d
1
.
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is likely that 3-(p-toluyl)propionic acid was formed from p-
tolualdehyde, 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 ndings were made for the meta isomers of
these compounds (37).
The nature of o- and p-xylene transformation by toluene-
grown 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 (tol-
uene). For p-xylene, these criteria may be less applicable be-
cause mineralization of a portion of p-xylene to CO
2
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 possi-
ble explanation for this observation is that a product formed
during xylene metabolism (e.g., a methylbenzylsuccinic acid
isomer) specically 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 hydrocar-
bon 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 Ofce of Research and
Development, U.S. Environmental Protection Agency, under grant
R-815738 through the Western Region Hazardous Substance Re-
search 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 Desul-
fobacula toluolica (Arch. Microbiol. 164:448451, 1995), Rabus
and Widdel reported the transformation of p-xylene to 4-meth-
ylbenzoate by dense suspensions of toluene-grown cells that
were supplied with a mixture of p-xylene and toluene. In ad-
dition, a relatively low yield (0.1%) of benzylsuccinate was
observed in toluene-metabolizing cell suspensions.
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