Anaerobic Toluene Research Articles

Anaerobic degradation of 2-methylnaphthalene by a sulfate-reducing enrichment culture.

Anaerobic degradation of 2-methylnaphthalene was investigated with a sulfate-reducing enrichment culture. Metabolite analyses revealed two groups of degradation products. The first group comprised two succinic acid adducts which were identified as naphthyl-2-methyl-succinic acid and naphthyl-2-methylene-succinic acid by comparison with chemically synthesized reference compounds. Naphthyl-2-methyl-succinic acid accumulated to 0.5 microM in culture supernatants. Production of naphthyl-2-methyl-succinic acid was analyzed in enzyme assays with dense cell suspensions. The conversion of 2-methylnaphthalene to naphthyl-2-methyl-succinic acid was detected at a specific activity of 0.020 +/- 0.003 nmol min(-1) mg of protein(-1) only in the presence of cells and fumarate. We conclude that under anaerobic conditions 2-methylnaphthalene is activated by fumarate addition to the methyl group, as is the case in anaerobic toluene degradation. The second group of metabolites comprised 2-naphthoic acid and reduced 2-naphthoic acid derivatives, including 5,6,7,8-tetrahydro-2-naphthoic acid, octahydro-2-naphthoic acid, and decahydro-2-naphthoic acid. These compounds were also identified in an earlier study as products of anaerobic naphthalene degradation with the same enrichment culture. A pathway for anaerobic degradation of 2-methylnaphthalene analogous to that for anaerobic toluene degradation is proposed.

Anaerobic toluene activation by benzylsuccinate synthase in a highly enriched methanogenic culture.

Permeabilized cells of a highly enriched, toluene-mineralizing, methanogenic culture catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. The specific in vitro rate of benzylsuccinate formation was >85% of the specific in vivo rate of toluene consumption. This is the first report of benzylsuccinate synthase activity in a methanogenic culture; the activity has previously been reported to occur in denitrifying, sulfate-reducing, and anoxygenic phototrophic bacteria.

Anaerobic toluene catabolism of Thauera aromatica: the bbs operon codes for enzymes of beta oxidation of the intermediate benzylsuccinate.

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent beta-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in beta oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of bbs (beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the beta-oxidation pathway of benzylsuccinate. Two of the genes, bbsE and bbsF, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown Thauera aromatica. The bbsG gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in Escherichia coli and detection of enzyme activity. The further enzymes of the pathway are probably encoded by bbsH (enoyl-CoA hydratase), bbsCD (3-hydroxyacyl-CoA dehydrogenase), and bbsB (3-oxoacyl-CoA thiolase). The operon contains two additional genes, bbsA and bbsI, for which no obvious function could be derived. The bbs operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the bbsA gene. This represents the first known promoter site in Thauera spp.

Metabolism of alkylbenzenes, alkanes, and other hydrocarbons in anaerobic bacteria.

Aromatic and aliphatic hydrocarbons are the main constituents of petroleum and its refined products. Whereas degradation of hydrocarbons by oxygen-respiring microorganisms has been known for about a century, utilization of hydrocarbons under anoxic conditions has been investigated only during the past decade. Diverse strains of anaerobic bacteria have been isolated that degrade toluene anaerobically, using nitrate, iron(III), or sulfate as electron acceptors. Also, other alkylbenzenes such as m-xylene or ethylbenzene are utilized by a number of strains. The capacity for anaerobic utilization of alkylbenzenes has been observed in members of the alpha-, beta-, gamma- and delta-subclasses of the Proteobacteria. Furthermore, denitrifying bacteria and sulfate-reducing bacteria with the capacity for anaerobic alkane degradation have been isolated, which are members of the beta- and delta-subclass, respectively. The mechanism of the activation of hydrocarbons as apolar molecules in the absence of oxygen is of particular interest. The biochemistry of anaerobic toluene degradation has been studied in detail. Toluene is activated by addition to fumarate to yield benzylsuccinate, which is then further metabolized via benzoyl-CoA. The toluene-activating enzyme presents a novel type of glycine radical protein. Another principle of anaerobic alkylbenzene activation has been observed in the anaerobic degradation of ethylbenzene. Ethylbenzene in denitrifying bacteria is dehydrogenated to 1-phenylethanol and further to acetophenone; the latter is also metabolized to benzoyl-CoA. Naphthalene is presumably activated under anoxic conditions by a carboxylation reaction. Investigations into the pathway of anaerobic alkane degradation are only at the beginning. The saturated hydrocarbons are most likely activated by addition of a carbon compound rather than by desaturation and hydration, as speculated about in some early studies. An anaerobic oxidation of methane with sulfate as electron acceptor has been documented in aquatic sediments. The process is assumed to involve a reversal of methanogenesis catalyzed by Archaea, and scavenge of an electron-carrying metabolite by sulfate-reducing bacteria. Among unsaturated non-aromatic hydrocarbons, anaerobic bacterial degradation has been demonstrated and investigated with n-alkenes, alkenoic terpenes and the alkyne, acetylene.

Metabolic indicators for detecting in situ anaerobic alkylbenzene degradation.

Monitoring programs for intrinsic bioremediation of fuel hydrocarbons require indicators that can convincingly demonstrate in situ metabolism. In this evaluation of potential indicators of in situ anaerobic alkylbenzene metabolism, laboratory and field data are reviewed for two classes of aromatic acids: (i) benzylsuccinate, E-phenylitaconate, and their methyl homologs, and (ii) benzoate, and methyl-, dimethyl-, and trimethylbenzoates. The review includes previously unpublished field data from a hydrocarbon-contaminated site in Fallon (Nevada), at which both classes of metabolites were detected in groundwater. The two classes of compounds were evaluated with respect to specificity (i.e., unique biochemical relationship to a specific alkylbenzene), stability, and generation as degradation intermediates versus dead-end products; recent developments in the biochemistry of anaerobic toluene and xylene degradation were incorporated in this evaluation. In general, benzylsuccinates/E-phenylitaconates are superior to benzoates in terms of their very high specificity to their parent hydrocarbons and their lack of commercial and industrial sources. They are also uniquely indicative of anaerobic conditions. All of the benzoates, benzylsuccinates, and E-phenylitaconates are relatively stable chemically and (with the exception of benzoate) biologically under anaerobic conditions, based on the limited data available. Although benzoate, benzylsuccinate, and E-phenylitaconate are intermediates of anaerobic toluene mineralization to carbon dioxide, their methyl homologs can be either mineralization intermediates or cometabolic dead-end products of alkylbenzenes, depending on the bacteria involved. Benzoates are far more commonly reported in field studies of hydrocarbon-contaminated aquifers than are benzylsuccinates and E-phenylitaconates, although it is not clear whether this is an accurate representation of the relative occurrence of these metabolites at contaminated sites, or whether it instead reflects the limited range of target analytes used in most field studies to date.

Initial reactions in anaerobic oxidation of m-xylene by the denitrifying bacterium Azoarcus sp. strain T.

The initial enzymatic steps in anaerobic m-xylene oxidation were studied in Azoarcus sp. strain T, a denitrifying bacterium capable of mineralizing m-xylene via 3-methylbenzoate. Permeabilized cells of m-xylene-grown Azoarcus sp. strain T catalyzed the addition of m-xylene to fumarate to form (3-methylbenzyl)succinate. In the presence of succinyl coenzyme A (CoA) and nitrate, (3-methylbenzyl)succinate was oxidized to E-(3-methylphenyl)itaconate (or a closely related isomer) and 3-methylbenzoate. Kinetic studies conducted with permeabilized cells and whole-cell suspensions of m-xylene-grown Azoarcus sp. strain T demonstrated that the specific rate of in vitro (3-methylbenzyl)succinate formation accounts for at least 15% of the specific rate of in vivo m-xylene consumption. Based on these findings, we propose that Azoarcus sp. strain T anaerobically oxidizes m-xylene to 3-methylbenzoate (or its CoA thioester) via (3-methylbenzyl)succinate and E-(3-methylphenyl)itaconate (or its CoA thioester) in a series of reactions that are analogous to those recently proposed for anaerobic toluene oxidation to benzoyl-CoA. A deuterium kinetic isotope effect was observed in the (3-methylbenzyl)succinate synthase reaction (and the benzylsuccinate synthase reaction), suggesting that a rate-determining step in this novel fumarate addition reaction involves breaking a C-H bond.

Anaerobic degradation of m-cresol by Desulfobacterium cetonicum is initiated by formation of 3-hydroxybenzylsuccinate.

The anaerobic bacterium Desulfobacterium cetonicum oxidized m-cresol completely with sulfate as electron acceptor. During growth, 3-hydroxybenzylsuccinate (identified by gas chromatography/mass spectroscopy and by comparison of high-performance liquid chromatography retention time and UV spectrum with a chemically synthesized reference compound) accumulated in the medium. This finding indicates that the methyl group of m-cresol is activated by addition to fumarate as in the case of anaerobic toluene metabolism. In cell-free extracts of D. cetonicum, the formation of 3-hydroxybenzylsuccinate from m-cresol and fumarate was detected at an activity of 0.5 nmol min(-1) (mg protein)(-1). This reaction depended strictly on anoxic assay conditions. Treatment with air resulted in a complete loss of activity; however, some activity could be recovered after restoring anoxic conditions. The activity was slightly membrane-associated. 3-Hydroxybenzylsuccinate was degraded via CoA thioesterification and further oxidation to 3-hydroxybenzoyl-CoA as subsequent steps in the degradation pathway.

Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product.

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(R)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.

Initial reactions of anaerobic metabolism of alkylbenzenes in denitrifying and sulfate-reducing bacteria

The initial activation reactions of anaerobic oxidation of the aromatic hydrocarbons toluene and ethylbenzene were investigated in cell extracts of a toluene-degrading, sulfate-reducing bacterium, Desulfobacula toluolica, and in cell extracts of strain EbN1, a denitrifying bacterium capable of degrading toluene and ethylbenzene. Extracts of toluene-grown cells of both species catalysed the addition of fumarate to the methyl group of [phenyl-14C]-toluene and formed [14C]-labeled benzylsuccinate. Extracts of ethylbenzene-grown cells of strain EbN1 did not catalyse this reaction, but catalysed the formation of 1-phenylethanol and acetophenone from [methylene-14C]-ethylbenzene. Toluene-grown cells of D. toluolica and strain EbN1 synthesised highly induced polypeptides corresponding to the large subunits of benzylsuccinate synthase from Thauera aromatica. These polypeptides were absent in strain EbN1 after growth on ethylbenzene, although a number of different polypeptides were highly induced. Thus, formation of benzylsuccinate from toluene and fumarate appears to be the general initiating step in anaerobic toluene degradation by bacteria affiliated with the phylogenetically distinct β-subclass (strain EbN1 and T. aromatica) and δ-subclass (D. toluolica) of the Proteobacteria. Anaerobic ethylbenzene oxidation proceeds via a different pathway involving a two-step oxidation of the methylene group to an alcohol and an oxo group; these steps are most probably followed by a biotin-independent carboxylation reaction and thiolytic cleavage.

A two-component system involved in regulation of anaerobic toluene metabolism in Thauera aromatica

The genes for a two-component regulatory system of the denitrifying toluene-degrading bacterium Thauera aromatica were identified immediately upstream of the genes for benzylsuccinate synthase ( bssDCAB), the first enzyme involved in anaerobic toluene metabolism. The genes apparently encode the regulators of toluene catabolic enzymes and were therefore termed tdiSR (for t oluene d egradation i nducing s ensor and r egulator). The tdiR gene product was overproduced in Escherichia coli and assayed for binding to a DNA fragment containing the 5′ region of the bss operon. We observed specific DNA binding with cell extracts containing overproduced TdiR, but not with control extracts. The tdiSR genes are almost identical to two genes of Thauera strain T1, which have not been assigned a function so far. In addition, the derived gene products share similarity with regulators of toluene and styrene catabolic pathways in aerobic Pseudomonas species, and with the tutCB gene products of Thauera strain T1. The latter have previously been implicated in regulating anaerobic toluene metabolism. Our data suggest that toluene catabolism under aerobic and anaerobic conditions is regulated by similar, but distinct two-component systems.

Identification and analysis of genes involved in anaerobic toluene metabolism by strain T1: putative role of a glycine free radical.

The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. The tutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism

Toluene is anoxically degraded to CO2 by the denitrifying bacterium Thauera aromatica. The initial reaction in this pathway is the addition of fumarate to the methyl group of toluene, yielding benzylsuccinate as the first intermediate. We purified the enzyme catalysing this reaction, benzylsuccinate synthase (EC 4.1.99-), and studied its properties. The enzyme was highly oxygen sensitive and contained a redox-active flavin cofactor, but no iron centres. The native molecular mass was 220 kDa; four subunits of 94 (alpha), 90 (alpha'), 12 (beta) and 10 kDa (gamma) were detected on sodium dodecyl sulphate (SDS) gels. The N-terminal sequences of the alpha- and alpha'-subunits were identical, suggesting a C-terminal degradation of half of the alpha-subunits to give the alpha'-subunit. The composition of native enzyme therefore appears to be alpha2beta2gamma2. A 5 kb segment of DNA containing the genes for the three subunits of benzylsuccinate synthase was cloned and sequenced. The masses of the predicted gene products correlated exactly with those of the subunits, as determined by electrospray mass spectrometry. Analysis of the derived amino acid sequences revealed that the large subunit of the enzyme shares homology to glycyl radical enzymes, particularly near the predicted radical site. The highest similarity was observed with pyruvate formate lyases and related proteins. The radical-containing subunit of benzylsuccinate synthase is oxygenolytically cleaved at the site of the glycyl radical, producing the alpha'-subunit. The predicted cleavage site was verified using electrospray mass spectrometry. In addition, a gene coding for an activating protein catalysing glycyl radical formation was found. The four genes for benzylsuccinate synthase and the activating enzyme are organized as a single operon; their transcription is induced by toluene. Synthesis of the predicted gene products was achieved in Escherichia coli in a T7-promotor/polymerase system.

Benzylsuccinate Formation as a Means of Anaerobic Toluene Activation by Sulfate-Reducing Strain PRTOL1

Permeabilized cells of toluene-mineralizing, sulfate-reducing strain PRTOL1 catalyzed the addition of toluene to fumarate to form benzylsuccinate under anaerobic conditions. Recent in vitro studies with two toluene-mineralizing, denitrifying bacteria demonstrated the same fumarate addition reaction and indicated that it may be the first step of anaerobic toluene degradation. This study with strain PRTOL1 shows that anaerobic toluene activation by fumarate addition occurs in bacteria as disparate as sulfate-reducing and denitrifying species (members of the delta and beta subclasses of the Proteobacteria, respectively).

Sensitive detection of a novel class of toluene-degrading denitrifiers, Azoarcus tolulyticus, with small-subunit rRNA primers and probes.

Azoarcus tolulyticus is a new class of widely distributed toluene-degrading denitrifiers of potential importance in remediating benzene, toluene, ethylbenzene, and xylene (BTEX)-contaminated environments. To detect these organisms in the environment, 16S rRNA gene-based phylogenetic probes were developed. Two sets of specific PCR amplification primers and two oligonucleotide hybridization probes were designed and tested against both closely and distantly related environmental isolates. All of these primers and probes were specific to the species A. tolulyticus. The sensitivity of the PCR amplification primer sets was evaluated with DNA isolated from A. tolulyticus Tol-4 pure culture and from sterile soils seeded with a known number of Tol-4 and Escherichia coli cells. These primer sets were able to detect 1 fg to 1 pg of template DNA from the pure culture and 1.11 x 10(2) to 1.1 x 10(8) Tol-4 cells per g of soil in the presence of 1.56 x 10(10) E. coli cells. These two PCR amplification primers were also successfully tested at two field sites. The primers identified the A. tolulyticus strains among the toluene-degrading bacteria isolated from a low-O2-high-NO(3)- aquifer at Moffett Field, Calif. Also, the presence of A. tolulyticus was detected in the groundwater samples from a BTEX-contaminated aquifer at an industrial site in Detroit, Mich., which showed anaerobic toluene degradation.

Anaerobic activation of toluene and o-xylene by addition to fumarate in denitrifying strain T.

Anaerobic assays conducted with strain T, a denitrifying bacterium capable of mineralizing toluene to carbon dioxide, demonstrated that toluene-grown, permeabilized cells catalyzed the addition of toluene to fumarate to form benzylsuccinate. This reaction was not dependent on the presence of coenzyme A (CoA) or ATP. In the presence of CoA, formation of E-phenylitaconate from benzylsuccinate was also observed. Kinetic studies demonstrated that the specific rate of benzylsuccinate formation from toluene and fumarate in assays with permeabilized cells was >30% of the specific rate of toluene consumption in whole-cell suspensions with nitrate; this observation suggests that benzylsuccinate formation may be the first reaction in anaerobic toluene degradation by strain T. Use of deuterium-labeled toluene and gas chromatography-mass spectrometry indicated that the H atom abstracted from the toluene methyl group during addition to fumarate was retained in the succinyl moiety of benzylsuccinate. In this study, no evidence was found to support previously proposed reactions of toluene with acetyl-CoA or succinyl-CoA. Toluene-grown, permeabilized cells of strain T also catalyzed the addition of o-xylene to fumarate to form (2-methylbenzyl)succinate. o-Xylene is not a growth substrate for strain T, and its transformation was probably cometabolic. With the exception of specific reaction rates, the observed characteristics of the toluene-fumarate addition reaction (i.e., retention of a methyl H atom and independence from CoA and ATP) also apply to the o-xylene-fumarate addition reaction. Thus, addition to fumarate may be a biochemical strategy to anaerobically activate a range of methylbenzenes.

Evidence for acetyl coenzyme A and cinnamoyl coenzyme A in the anaerobic toluene mineralization pathway in Azoarcus tolulyticus Tol-4.

A toluene-degrading denitrifier, Azoarcus tolulyticus Tol-4, was one of eight similar strains isolated from three petroleum-contaminated aquifer sediments. When the strain was grown anaerobically on toluene, 68% of the carbon from toluene was found as CO2 and 30% was found as biomass. Strain Tol-4 had a doubling time of 4.3 h, a Vmax of 50 micromol x min-1 x g of protein-1, and a cellular yield of 49.6 g x mol of toluene-1. Benzoate appeared to be an intermediate, since F-benzoates accumulated from F-toluenes and [14C]benzoate was produced from [14C]toluene in the presence of excess benzoate. Two metabolites, E-phenylitaconic acid (1 to 2%) and benzylsuccinic acid (<1%), accumulated from anaerobic toluene metabolism. These same products were also produced when cells were grown on hydrocinnamic acid and trans-cinnamic acid but were not produced from benzylalcohol, benzaldehyde, benzoate, p-cresol, or their hydroxylated analogs. The evidence supports an anaerobic toluene degradation pathway involving an initial acetyl coenzyme A (acetyl-CoA) attack in strain Tol-4, as proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for another toluene-degrading denitrifier, strain T1. Our findings support a modification of the proposed pathway in which cinnamoyl-CoA follows the oxidation of hydrocinnamoyl-CoA, analogous to the presumed oxidation of benzylsuccinic acid to form E-phenylitaconic acid. Cinnamic acid was detected in Tol-4 cultures growing in the presence of toluene and [14C]acetate. We further propose a second acetyl-CoA addition to cinnamoyl-CoA as the source of benzylsuccinic acid and E-phenylitaconic acid. This pathway is supported by the finding that monofluoroacetate added to toluene-growing cultures resulted in a significant increase in production of benzylsuccinic acid and E-phenylitaconic acid and by the finding that [14C]benzylsuccinic acid was detected after incubation of cells with toluene, [14C]acetate, and cinnamic acid. Evidence for anaerobic toluene metabolism by methyl group oxidation was not found, since benzylsuccinic acid and E-phenylitaconic acid were not detected after incubation with benzylalcohol and benzaldehyde, nor were benzylalcohol and benzaldehyde detected even in 14C trapping experiments.

Benzylfumaric, benzylmaleic, and Z- and E-phenylitaconic acids: synthesis, characterization, and correlation with a metabolite generated by Azoarcus tolulyticus Tol-4 during anaerobic toluene degradation.

E-Phenylitaconic acid has been isolated as a metabolite generated by Azoarcus tolulyticus Tol-4 along with benzylsuccinic acid during anaerobic degradation of toluene. Strain Tol-4 converted 1 to 2% of toluene carbon to E-phenylitaconate and benzylsuccinate (10:1). The identification of E-phenylitaconic acid was based on 1H nuclear magnetic resonance (NMR) characterization of degradation products derived from 13C-labeled toluene followed by comparison of spectroscopic and chromatographic data for the isolated, unlabeled metabolite with those for chemically synthesized benzylfumaric acid, benzylmaleic acid, E-phenylitaconic acid, and Z-phenylitaconic acid. Spectroscopic comparisons included 1H NMR, 13C NMR, and nuclear overhauser effect correlations. High-pressure liquid chromatography (HPLC) retention times and HPLC coinjections with synthetic dioic acids provided another reliable line of evidence for structure assignment. The formation of E-phenylitaconic acid differs from previous reports of benzylfumaric acid generation along with benzylsuccinic acid during anaerobic microbial degradation of toluene. This has important implications relevant to elaboration of the metabolic route for anaerobic toluene degradation by strain Tol-4 and related organisms. Similar amounts of E-phenylitaconic acid were also produced by seven other strains of A. tolulyticus.

Conversion studies with substrate analogues of toluene in a sulfate-reducing bacterium, strain Tol2.

Anaerobic toluene oxidation by the sulfate-reducing bacterium, strain Tol2 (proposed name Desulfobacula toluolica) was specifically inhibited by benzyl alcohol when added at concentrations around 500 microM. Benzyl alcohol added at lower, non-inhibitory concentrations (around 5 microM) was not oxidized by active cells pregrown on toluene, indicating that the alcohol is not a free intermediate of toluene metabolism in the sulfate reducer. Conversion of p-xylene in toluene-metabolizing cells to p-methylbenzoate as dead-end product suggests that the sulfate reducer, like denitrifiers, initiates toluene oxidation at the methyl group.

The role of iron in enhancing anaerobic toluene degradation in sulfate-reducing enrichment cultures

Ferrous iron enhanced the toluene degradation rate of sulfidogenic enrichment cultures inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Ferrous iron had an analogous effect on the degradation rate of benzoic acid, a transient metabolite of anaerobic toluene degradation in these cultures, when benzoic acid was used as a sole carbon and energy source. Two hypotheses were proposed to explain iron's effect: (a) Iron may have prevented sulfide toxicity via precipitation of sulfide as FeS, and (b) iron might have been a limiting nutrient required for degradation (i.e., amendments of iron could have compensated for iron removed from solution by precipitation as FeS). To test these hypotheses, substrate degradation rates were compared in the presence of FeSO4 (a sulfate source that both precipitates sulfide species and precludes iron limitation) versus ZnSO4 (a sulfate source that precipitates sulfide species but does not preclude iron limitation) versus MgSO4 (a sulfate source that neither precipitates sulfide nor precludes iron limitation). For both toluene and benzoic acid, FeSO4 and ZnSO4 were comparable in their enhancement of substrate degradation rates and were superior to MgSO4 in that respect. Thus, iron appears to ameliorate sulfide toxicity, not nutritional iron limitation, in these cultures. The observation that ethylenediaminetetraacetic acid, a chelating agent capable of retaining iron in solution in the presence of sulfide, did not stimulate the cultures is consistent with this conclusion. The implications of these results for bioremediation of fuel-contaminated aquifers that contain sulfate-reducing bacteria are discussed.

A method for detection of aromatic metabolites at very low concentrations: application to detection of metabolites of anaerobic toluene degradation.

Difficulties inherent in working with anaerobic microorganisms and mixed cultures have hampered efforts to detect and identify metabolites of anaerobic degradation of monoaromatic compounds. Isotope-trapping experiments and analysis using a high-performance liquid chromatograph equipped with a flow-through radioactivity detector were used to detect very low concentrations of metabolites. Data obtained by this method suggest that toluene was degraded via methyl hydroxylation by a mixed methanogenic culture.