The monoaromatic hydrocarbons benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are mutagenic and putatively carcinogenic substances. Due to their abundance and relatively high solubility in water, aromatics are prominent groundwater contaminants. Among the BTEX compounds, anaerobic degradation pathways of toluene, ethylbenzene, and m-xylene have been well investigated [8, 24, 27]. In all cases investigated so far, under anoxic conditions methylated aromatic hydrocarbons were activated by a reaction of the benzylsuccinate synthase type, where fumarate is added to the methyl side chain [3, 27]. A similar reaction takes place in anaerobic degradation of ethylbenzene by sulfate-reducing bacteria where fumarate is added to the benzyl carbon atom of the ethyl side chain producing (1-phenylethyl)succinate [17]. In contrast, the denitrifying organism EBN1 activates ethylbenzene by a direct hydroxylation by the enzyme ethylbenzene dehydrogenase generating 1-phenylethanol [16].

All three isomers of xylene are known to be readily degradable under oxic conditions [8], whereas degradation under anoxic conditions is not as well studied. So far, two anaerobic o-xylene-degrading pure bacterial cultures have been documented, both of them sulfate reducers. Strain oXyS1 was enriched from marine sludge [12], whereas strain OX39 was isolated from contaminated sediment and is a freshwater organism [21]. Strain OX39 was also able to grow with m-xylene, but did not use the third isomer, p-xylene. Isolation efforts with m-xylene as organic substrate led to some denitrifying [14, 22] and to one marine sulfate-reducing culture, strain mXyS1 [12]. Several denitrifying strains isolated with toluene were later found to grow as well with m-xylene, for example, Azoarcus strain T and Azoarcus tolulyticus strain Td15 [7, 23]. Under anoxic conditions, p-xylene has not yet been found to be degraded by pure cultures. However, removal of p-xylene was found in sediment column experiments under anoxic—but not well-defined redox—conditions, and another study recorded degradation of p-xylene by bacterial enrichment with nitrate provided as the electron acceptor [11, 15]. Until now, this is the only known enrichment culture that degraded p-xylene anaerobically and further attempts to isolate the bacteria responsible for removal of p-xylene failed.

Concentration analysis in the environment provided indications for p-xylene utilization under anoxic conditions in situ [29]. These attempts generally were based on a combined quantification of m- and p-xylene concentrations as the two isomers coelute in standard gas and liquid chromatographic analysis. Thus, assessment of m-/p-xylene degradation in the environment by a decrease in concentration does not necessarily account specifically for degradation of p-xylene. Furthermore, it has to be considered that physicochemical parameters such as adsorption or dilution can also lead to declining concentrations without sustainable removal of the compounds from the site. Evidence for p-xylene degradation in the environment was given by the analysis of putative signature metabolites 4-methylbenzylsuccinic acid and p-toluic acid, extracted from a contaminated groundwater site [6]. Recently, Griebler et al. were able to identify p-toluic acid in several environmental samples taken from monitoring wells of a BTEX- and PAH-contaminated site [10]. However, it is unclear whether p-xylene degradation was initiated by fumarate addition to the methyl group analogously to toluene, o- and m-xylene degradation.

We report here on p-xylene degradation under sulfate-reducing conditions. The bacterial culture presented was enriched from material of a site where Griebler et al. [10] attested bacterial potential of p-xylene degradation.

Materials and Methods

Purification and culture conditions

Bacteria were enriched from anoxic mud from the bottom of a monitoring well at a former gasworks site near Stuttgart, Germany. The area was contaminated with mono- and polycyclic aromatic hydrocarbons, as well as with heterocyclic compounds. Bacteria were cultivated at 30°C in bicarbonate-buffered freshwater mineral medium, pH 7.4, with sulfate as electron acceptor (10 mM) [20]. The medium was prepared under an atmosphere of N2/CO2 (80:20 v/v) and reduced with Na2S (1 mM) [26]. Sterile, anoxic FeCl2 solution was added to a final concentration of 3 mM and led to precipitation of black ferrous sulfide.

For first enrichments, 10 mL of mud per 50 mL of mineral medium were used. The culture was subsequently transferred 1:5. In the meantime, bacteria were growing in the sixth transfer. p-Xylene was supplied from anoxic stock solutions with concentrations of 2–―3 mM. In order to keep concentrations of p-xylene in the water phase low but to simultaneously provide a substrate reservoir, 0.3 g adsorber resin Amberlite-XAD7 (Fluka, Buchs, Switzerland) was added per bottle. As a result, the p-xylene concentration stabilized at a level of 30–60 μm as described previously [20].

Growth was indirectly monitored as the increase in sulfide concentrations over time [4]. This method also determines sulfide from precipitated FeS which was completely dissolved by zinc acetate treatment prior to colorimetric analysis. p-Xylene concentrations were determined by high-performance liquid chromatography (HPLC; Bischoff Chromatography, Leonberg, Germany) [20].

To identify putative intermediates of p-xylene degradation, cultures were adjusted to pH < 1.5 and subsequently extracted with diethyl ether. Metabolites were converted to methyl esters with a mixture of trimethylchlorosilane (Fluka, Buchs, Switzerland) and methanol (1:8) at 75°C for 1 h, or carboxylic groups were silylated with N,O-bistrimethylsilyltrifluoro-acetamide (Merck, Darmstadt, Germany) for 6 h at 60°C. For mass spectroscopic analysis, the reaction mixture was dried under a stream of N2 and then dissolved in ethyl acetate (for methylated compounds) or cyclohexane (for silylated compounds). Samples were analyzed by gas chromatography/mass spectrometry (GC/MS) as described previously [20]. Identity of substances was confirmed by published chromatograms or reference compounds.

All chemicals used were of analytical grade (Merck, Darmstadt, Germany, or Fluka, Buchs, Switzerland). Gases were purchased from Messer Griesheim (Krefeld, Germany).

Results and Discussion

A p-xylene-degrading bacterial culture was enriched from contaminated aquifer material with p-xylene as organic substrate and sulfate as electron acceptor. Adsorber resin Amberlite-XAD7 was added to the culture bottles to keep the concentration of the aromatic substrate constantly low and to avoid growth inhibition or poisoning. After several 1:5 transfer steps, sediment particles with hydrocarbons that might act as additional carbon sources were diluted out. Also, bacterial growth with p-xylene became more reproducible and significantly faster. Degradation of p-xylene was stoichiometrically coupled to sulfate reduction. The recovery of electrons from the substrate in the sulfide produced was 95.2%, and verified that the enrichment culture mineralized p-xylene completely to CO2 (Fig. 1). The maximum toluene degradation rate obtained was 3.2 μM/day.

Fig 1
figure 1

Substrate utilization by strain OX39 in a batch experiment. Degradation of p-xylene (▪) and concomitant production of sulfide (•) were monitored over time.

Full size image

In order to elucidate the pathway of p-xylene degradation under sulfate-reducing conditions, metabolites were extracted from the enrichment cultures with organic solvents. 4-Methylbenzylsuccinate could be tentatively identified by GC-MS analysis as the mass fragmentation pattern was in agreement with published spectra (Fig. 2A) [18]. Another compound extracted from supernatants of our enrichment culture was 4-methylphenylitaconic acid (Fig. 2B). p-Toluic acid or benzoic acid could not be detected as metabolites, although we tried two different derivatization techniques. 4-Methylbenzylsuccinate and 4-methylphenylitaconic acid detected in the cultures are both probably specific for a degradation pathway initiated by fumarate addition to one of the methyl groups [25]. Therefore, we concluded that p-xylene is degraded analogously to the well-investigated benzylsuccinate synthase pathway of toluene [3]. Other authors showed that fumarate adducts of o-xylene were detected in experiments with permeabilized cells of the denitrifying strain T grown on toluene [2]. In these experiments, o-xylene was probably converted because the toluene-activating enzyme benzylsuccinate synthase has rather broad substrate specificity [25]. The benzylsuccinate synthase gene was found to encode for the initial enzyme of toluene and also m-xylene degradation in the denitrifying bacterium Azoarcus sp. strain T, indicating that both compounds are activated by the same enzyme [1, 5, 19]. Metabolites of o- and m-xylene degradation were also investigated for the sulfate-reducing strain OX39 [21]. However, in strain OX39, toluene, o-, and m-xylene were not activated by the identical benzylsuccinate synthase, but by an individual enzyme degrading each substrate [21]. Induction times lasted up to several weeks when strain OX39 was transferred from one of the three compounds to another. This implies that a specific activating enzyme exists for each of the three compounds. However, although it is very unlikely, one can not exclude that one of the consecutive enzymes in the pathway exhibits the substrate specificity and toluene and the xylenes are activated by the same benzylsuccinate synthase type enzyme. The concept of specific degradation pathways for methylated benzenes under sulfate-reducing conditions is supported by the fact that the isolates strain OxyS1 and strain MxyS1 were capable of degrading either o-isomers or m-isomers of methylated aromatic compounds but not both [12].

Fig 2
figure 2

GC-MS fragmentation patterns of tentatively identified intermediates. (A) 4-Methylbenzylsuccinic acid methyl ester and (B) 4-methylphenylitaconic acid methyl ester both extracted from cultures grown with p-xylene.

Full size image

Summarizing all these findings, the upper pathway of o-, m-, and also p-xylene degradation under anoxic conditions very likely starts with activation by fumarate addition and further proceeds via stepwise β-oxidation to methyl-benzoyl-CoA, as is known for toluene.

It has to be mentioned that so far nothing is known about the lower pathway and the further degradation of the 4-methylbenzoyl-CoA. 4-Methylbenzoyl-CoA might be demethylated and the following steps could be identical to the metabolization of benzoyl-CoA [9, 13]. Also, in the course of β-oxidation, the methyl group at position 4 of the aromatic ring might be rearranged into meta-position, because then the methyl group would not interfere with regular β-oxidation [27]. Comparable rearrangement reactions have been found for stepwise oxidation of n-hexane by a denitrifying strain [28]. Another possibility might be a hydroxylation of methylbenzoyl-CoA prior to ring reduction facilitating the following reduction steps. Respective substances were discovered in supernatants of sulfate-reducing strain OX39 grown either with m-xylene or with toluene [21].