Anaerobic benzene degradation by Gram-positive sulfate-reducing bacteria

Authors


  • Editor: Max Häggblom

Correspondence: Draženka Selesi, Helmholtz Zentrum München-German Research Center for Environmental Health, Institute of Groundwater Ecology, Ingolstädter Landstraße 1, D-85764, Neuherberg, Germany. Tel.: +49 89 3187 3122; fax: +49 89 3187 3361; e-mail: drazenka.selesi@helmholtz-muenchen.de

Abstract

Despite its high chemical stability, benzene is known to be biodegradable with various electron acceptors under anaerobic conditions. However, our understanding of the initial activation reaction and the responsible prokaryotes is limited. In the present study, we enriched a bacterial culture that oxidizes benzene to carbon dioxide under sulfate-reducing conditions. Community analysis using terminal restriction fragment length polymorphism, 16S rRNA gene sequencing and FISH revealed 95% dominance of one phylotype that is affiliated to the Gram-positive bacterial genus Pelotomaculum showing that sulfate-reducing Gram-positive bacteria are involved in anaerobic benzene degradation. In order to get indications of the initial activation mechanism, we tested the substrate utilization, performed cometabolism tests and screened for putative metabolites. Phenol, toluene, and benzoate could not be utilized as alternative carbon sources by the benzene-degrading culture. Cometabolic degradation experiments resulted in retarded rates of benzene degradation in the presence of phenol whereas toluene had no effect on benzene metabolism. Phenol, 2-hydroxybenzoate, 4-hydroxybenzoate, and benzoate were identified as putative metabolites in the enrichment culture. However, hydroxylated aromatics were shown to be formed abiotically. Thus, the finding of benzoate as an intermediate compound supports a direct carboxylation of benzene as the initial activation mechanism but additional reactions leading to its formation cannot be excluded definitely.

Introduction

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are frequent pollutants in contaminated groundwater systems. Compared with other aromatic hydrocarbons (Anderson & Lovley, 1997), BTEX compounds are highly water soluble and carcinogenic (Dean, 1978) and consequently are described as hazardous organic substances that endanger the quality of drinking water resources (USEPA, 1998). Benzene is rapidly biodegraded under aerobic conditions in soils and aquifers (Gibson & Parales, 2000). However, the input of heavy organic loads into saturated sediments results in the rapid depletion of available oxygen, turning aquifers anoxic (Christensen et al., 1994). Based on field and batch studies, it could be shown that benzene can be effectively degraded under iron-reducing (Anderson et al., 1998; Rooney-Varga et al., 1999; Kunapuli et al., 2007), sulfate-reducing (Lovley et al., 1995; Kazumi et al., 1997; Phelps et al., 1998; Caldwell & Suflita, 2000), denitrifying (Burland & Edwards, 1999; Coates et al., 2001; Ulrich & Edwards, 2003), and methanogenic conditions (Grbic-Galic & Vogel, 1987; Ulrich & Edwards, 2003; Chang et al., 2005).

Previous studies dealing with 16S rRNA gene based fingerprinting analysis of benzene-degrading enrichment cultures revealed the presence of phylogenetically diverse microorganisms (Ulrich & Edwards, 2003; Chang et al., 2005). Under iron-reducing conditions, bacterial members of the family Geobacteraceae were determined as dominant organisms (Rooney-Varga et al., 1999). Recently, Gram-positive bacteria related to Clostridia were discovered to play an essential role in benzene degradation with iron or sulfate as electron acceptor (Kunapuli et al., 2007; Kleinsteuber et al., 2008). In addition, members of the family Desulfobacteraceae have been identified as dominant organisms in several benzene-degrading cultures under sulfate-reducing and methanogenic conditions (Phelps et al., 1998; Ulrich & Edwards, 2003; Chang et al., 2005; Da Silva & Alvarez, 2007; Musat & Widdel, 2008; Oka et al., 2008). In a highly enriched benzene-degrading culture, Desulfobacterium was identified as the prominent phylotype (Musat & Widdel, 2008). So far, only two denitrifying benzene-degrading strains of the genera Dechloromonas and Azoarcus have been described as pure cultures (Coates et al., 2001; Kasai et al., 2006). However, despite the availability of several enrichment cultures, repeated attempts to isolate obligate anaerobic pure cultures failed and consequently the physiological properties of these microorganisms are still largely unknown.

Our knowledge of the biochemical mechanism of anaerobic benzene degradation is still scarce. The analysis of metabolites in diverse benzene-degrading cultures led to the identification of phenol (Caldwell & Suflita, 2000; Chakraborty & Coates, 2005; Kunapuli et al., 2008), toluene (Ulrich et al., 2005), and benzoate (Caldwell & Suflita, 2000; Phelps et al., 2001; Kunapuli et al., 2008) as putative intermediates. Consequently, three different biochemical mechanisms were hypothesized for the initial activation reaction of benzene, including carboxylation to benzoate (Caldwell & Suflita, 2000; Phelps et al., 2001), hydroxylation to phenol (Vogel & Grbic-Galic, 1986; Grbic-Galic & Vogel, 1987; Caldwell & Suflita, 2000; Chakraborty & Coates, 2005), and methylation to toluene (Ulrich et al., 2005).

The present study delivers insights into physiological, phylogenetic, and biochemical characteristics of an enrichment culture able to metabolize benzene under sulfate-reducing conditions. Here, the participation of Gram-positive bacteria in anaerobic benzene metabolism and carboxylation as the putative benzene activation mechanism are highlighted.

Materials and methods

Enrichment of the benzene-degrading culture and growth conditions

The anaerobic benzene-degrading culture BPL was enriched from soil at a former coal gasification site in Gliwice, Poland, with benzene as growth substrate in the presence of the adsorber resin Amberlite XAD-7 (Morasch et al., 2001) and 5 mM Na2SO4 as electron acceptor. The enrichment culture was transferred 15 times with 10% inoculum (v/v) into a new 50 mL sediment-free medium. The enrichment culture was cultivated in bicarbonate-buffered (30 mM) freshwater medium (pH 7.2) under an anaerobic atmosphere of N2/CO2 (80 : 20 v/v) (Widdel & Bak, 1992). The freshwater medium was reduced with 1 mM Na2S, and 3 mM FeCl2 was added to scavenge produced sulfide. After repeated transfers (10% inoculum, v/v) into fresh medium, a stable anaerobic benzene-degrading enrichment culture was obtained. All culture bottles were incubated at 30 °C in the dark.

Alternative substrate and electron acceptor utilization

The benzene-degrading enrichment culture BPL was tested for growth on different organic substrates. A 1 mM total concentration of the aromatic substrates biphenyl, toluene, phenol, 4-hydroxybenzoate, and benzoate was added separately as liquids with a glass syringe or as solid crystals to 120-mL serum bottles containing 50 mL of bicarbonate-buffered mineral medium and 0.3 g of Amberlite XAD-7 (Morasch et al., 2001). Bacterial growth on different substrates was checked visually by turbidity formation of the medium in triplicate incubations over a period of 3 months. For cometabolism experiments, 500 μM of the respective aromatic substrates without amberlite XAD-7 were added to the culture bottles. Formate, pyruvate, and lactate were added to the medium from autoclaved or filter-sterilized aqueous stock solutions to a final concentration of 10 mM. H2/CO2 gas (30 : 70 v/v) was directly injected to the culture bottles. The tested electron acceptors included Na2SO3 (10 mM), Na2S2O3 (10 mM), S0 (1 g L−1), NaNO3 (5 mM), and Fe(OH)3 (50 mM), respectively. Electron acceptors were autoclaved and added to the culture bottles from anoxic stock solutions. Fe(OH)3 was prepared as described elsewhere (Lovley & Phillips, 1986).

Analytical procedures

For quantification of benzene and CO2 in the electron and carbon balance experiments, the culture was cultivated with 350 μM 13C6-benzene (Sigma Aldrich, Steinheim, Germany; 99% purity) without Amberlite XAD-7 in 250-mL serum bottles and sealed with Viton rubber stoppers (Mag Technik, Dübendorf, Switzerland).

13C6-benzene, 12C6-benzene, and toluene concentrations were determined by headspace analysis with GC/MS (GC, Trace-DSQ; MS, Thermo Finnigan, San Jose, CA) in selective ion monitoring mode with a fused-silica capillary column DB-5 [30 m length (L), 0.25 mm inside diameter (ID), 0.25 μm film thickness (T); Agilent, Palo Alto]. The injector temperature was 220 °C, the carrier gas was helium (grade 5.0) at a flow rate of 1 mL min−1 and the split ratio was split : splitless 1 : 10. The temperature was held at 40 °C for 1 min, raised to 200 °C at 15 °C min−1, to 300 °C at 25 °C min−1, and held for 1 min. The total molar mass of benzene in the culture was calculated from benzene in the liquid phase and Henry's constant of benzene in the headspace as given by Peng & Wan (1997).

Culture samples for phenol quantification were taken inside an anoxic chamber and 25 μL of sample was analyzed directly using the auto injector (SIL-10Ai, Shimadzu) of a Shimadzu HPLC equipped with Shimadzu RF-10A XL fluorescence and Shimadzu SPD–10AVP UV diode array detectors at 30oC. The column was a C18 Prontosil Eurobond (250 mm L, 4 mm ID, 5 μm particle size; Bischoff, Leonberg, Germany). The mobile phase was (1) 1% acetic acid in water pH 2.7 and (2) 1% acetic acid in methanol at a flow rate of 0.19 and 0.20 mL min−1, respectively. UV absorption was measured at 270 nm; fluorescence excitation was at 265 nm and the emission wavelength 315 nm.

The total amount of 13CO2 produced from 13C6 benzene degradation was determined by measuring the 13CO2/12CO2 isotope ratio in the headspace. The headspace sample (1 mL) was diluted in a 10-mL serum vial filled with helium and stored until analysis. The diluted gas samples (100 mL) were injected to a GC/C/IRMS system consisting of a TRACE GC Ultra gas chromatograph with split/splitless injector (GC) (Thermo Fisher Scientific Corporation, Milan, Italy) coupled to a Finnigan MAT 253 isotope ratio mass spectrometer (IRMS) via Finnigan GC combustion III interface (Thermo Fisher Scientific Corporation, Bremen, Germany). The GC was equipped with a DB-5 column (30 mL, 0.25 mm ID, 0.25 μm T). The temperature of the injector was held at 180 °C isothermally with a split ratio 1 : 10 and column flow of 1.4 mL. Helium (grade 5.0) was used as carrier gas with a constant flow rate of 1.4 mL min−1. The initial oven temperature was 50 °C, ramped at a rate of 45 °C min−1 to 100 °C, and held for 1.4 min. The 13CO2-concentration was calculated from carbon isotope compositions of CO2 expressed in δ-notation (‰) relative to the Vienna Pee Dee Belemnite standard (V-PDB):

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The initial 12CO2-concentration was calculated as the sum of 12CO2 from the NaHCO3 (30 mM) buffer in the liquid phase and 20% of 12CO2 gas in the headspace of the culture bottles.

Sulfate concentrations were analyzed by ion chromatography using a Dionex 300 ion chromatograph (Dionex Corporation, Sunnyvale) equipped with an IonPac AS14 analytical column (4 × 250 mm) (Dionex Corporation). The eluent was Na2CO3 (3.5 mM) and NaHCO3 (1 mM) and the flow rate was 1.2 mL min−1.

Cell counts by FISH and 4,6-diamidino-2-phenylindole (DAPI) staining

Oligonucleotide probes used for FISH were (1) EUB338 I-III mix specifically labeling all Bacteria (Amann et al., 1990; Daims et al., 1999), (2) DEM1164r targeting all Desulfotomaculum cluster I bacteria (Stubner & Meuser, 2000), (3) BET42a in combination with the unlabeled GAM42a oligonucleotide specifically labeling Betaproteobacteria (Manz et al., 1992), and (4) GAM42a in combination with the unlabeled BET42a oligonucleotide used to detect Gammaproteobacteria (Manz et al., 1992). For the determination of cell numbers via FISH and DAPI staining, 1 mL of the benzene-degrading enrichment culture BPL and the control bacterium Thiobacillus thiophilus D24TNT (Kellermann & Griebler, 2009) were fixed for 1–3 h with 1 mL 100% ethanol. The cells were harvested by centrifugation at 6000 g for 5 min and the cells were stored in phosphate-buffered saline (PBS)-ethanol (1 : 1) at −20 °C. The cells were filtered onto black 0.2 μm Nucleopore polycarbonate filters (Whatman, Brentford, UK) under vacuum pressure and dehydrated in 50%, 80%, and 100% ethanol for 3 min each. Hybridization of the filters was performed at 46 °C for 1.5 h with 40 μL hybridization buffer [0.9 M NaCl, 20 mM Tris/HCl, pH 8, 10% formamide, 0.1% sodium dodecyl sulfate (SDS)], 5 μL of Cy3-labelled probe (30 ng μL−1) and 5 μL of fluorescein-labelled probe (50 ng μL−1). The filters were treated for 20 min with washing buffer (0.45 M NaCl, 20 mM Tris/HCl, pH 8, 0.01% SDS) at 48 °C, washed with distilled water and air dried. For staining with the DNA-binding dye DAPI, the hybridized polycarbonate filters were covered with 5 μL DAPI solution (1 mg mL−1). Counting was performed with an Epifluorescence microscope (Zeiss Axioplan 2, Oberkochen, Germany) equipped with the filters Zeiss49 for DAPI, Zeiss9 for fluorescein and Zeiss43 for Cy3. For cell counting, two separate filters were prepared from duplicate incubations and 20 randomly selected fields were counted for each filter.

Metabolite analysis

Metabolite analysis was performed using a LC-ESI-MS-MS system with a method developed to measure 2-hydroxybenzoate, 4-hydroxybenzoate, benzoate, and benzylsuccinate. Samples of 15 mL from the cultures were spiked with 1 mL of a 100 μg L−1 fluorinated benzoate solution as internal standard, filtered (0.45 μm pore size; Millipore, Eschborn, Germany), acidified to pH 1 and stored at 4 °C. Metabolites were extracted from the samples and preconcentrated by solid phase extraction with ISOLUTE ENV+ (200 mg adsorbent; Biotage, Uppsala, Sweden). The analysis was performed using an HPLC Agilent 1100 (Agilent Technologies, Santa Clara) equipped with a binary pump, a degasser, a column oven, and an auto sampler, which was coupled with an electrospray ionization source (TurboIon Spray, Applied Biosystems MDS/SCIEX, Foster City) to the triple quadrupole mass spectrometer ABI 3000 (Applied Biosystems MDS/SCIEX) (Ohlenbusch et al., 2002). The separation column was a Purospher RP-18e (125 × 2 mm L, 5 μm particle size; Merck, Darmstadt, Germany). The column oven was set to 35±2 °C. The eluent consisted of (A) water with 0.1% acetic acid (v/v) and (B) 100% acetonitrile. The eluent B was raised from 20% to 90% within 10 min. The flow rate was 0.3 mL min−1 and the sample injection volume was 50 μL. At the ESI interface the nebulizer gas flow was set to 1.5 L min−1, the curtain gas flow to 1.6 L min−1, and the dry gas flow to 6 mL min−1. The dry gas temperature was 450 °C and the ion spray voltage of the ESI system was set to −5500 V. MS/MS measurements were performed in the negative multiple reaction mode and the most abundant fragment ion was recorded after collision induced dissociation. For benzylsuccinate, a second, but much less sensitive, fragment (m/z 91) was used to confirm the detection of this analyte. The ratio of m/z 91 to m/z 163 is 0.07 (±20%).

Molecular and phylogenetic analysis

Cells of 50 mL enrichment culture were harvested by centrifugation at 3345 g for 15 min at 4 °C. The pellet was washed twice with 1 mL of 1 × PBS. Genomic DNA was extracted and purified using the FastDNA Spin Kit for Soil according to the manufacturer's protocol (MP Biomedicals, Illkirch, France).

Terminal restriction fragment length polymorphism (T-RFLP) was performed using the 16S rRNA gene primer set Ba27f-FAM/907r (Lane et al., 1985). T-RFLP analysis of benzene-grown cells was performed from two separate incubations. T-RFLP analyses of cells grown on other substrates were performed once. The T-RFLP analysis was carried out with 20 ng of the amplicon as described previously (Lueders et al., 2006).

16S rRNA gene sequences for construction of the clone library were amplified using the universal primers Ba27f/Ba1492r (Weisburg et al., 1991) resulting in almost full-length products. The PCR reaction, cloning, and sequencing were conducted as described previously (Winderl et al., 2007). The check_chimera program of the ribosomal database project II (Biomedical and Physical Sciences Building, Michigan State University) was used to check for chimeric sequences. The aligned sequences were compared with the closely related sequences available in the public database using blastn program (http://ncbi.nlm.nih.gov/BLAST). The 16S rRNA gene sequences were added into a database existing of about 25 000 small-subunit rRNA gene sequences (http://arb-home.de) (Ludwig et al., 2004). Phylogenetic analysis was performed by parsimony, maximum-likelihood, and verified by the neighbour-joining as method implemented in the arb software package. The sequences determined in this study are available at GenBank under accession nos. EU523065EU523097.

Results

Physiological properties of the benzene-degrading culture

Enrichment of benzene-degrading, sulfate-reducing bacteria was performed with contaminated soil of a former coal gasification site as inoculum and was since then transferred 15 times into new sterile, soil-free medium. The enrichment culture was able to tolerate benzene up to 1 mM without Amberlite XAD-7. Carbon and electron balances of the culture showed that the complete degradation of 350 μM 13C6-benzene was coupled to the production of 1.8 mM 13CO2 (Eqn. 1–3; Fig. 1) and to the reduction of 1.2 mM sulfate (Fig. 1). These values correspond to an electron recovery of 88.8±10.7% in the reduced sulfate for total reduction to HS and to the production of 83.6%±2.6%13CO2 from 13C6-benzene mineralization. These experimental results were close to the theoretically stoichiometric values for the complete degradation of benzene to carbon dioxide (Eqn. 4):

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Figure 1.

 Degradation of 13C6-benzene by the sulfate-reducing enrichment culture BPL. Carbon and electron balance between 13C6-benzene and 13CO2-evolution and sulfate reduction. Na2MoO4 5 mM was added after 38 days (arrows). Data are the means of triplicate incubations and error bars represent SDs.

There was no decrease of 13C6-benzene or sulfate in autoclaved control cultures. The addition of 5 mM molybdate (Na2MoO4), a specific inhibitor of sulfate reduction, at day 38 after inoculation, caused a complete inhibition of 13C6-benzene degradation and 13CO2-production (Fig. 1). To prevent a potential toxic effect of sulfide on the microorganisms in the enrichment culture BPL, ferrous chloride was added to the culture medium and consequently a solid iron sulfide mineral (greigite) precipitated.

The enrichment culture BPL was tested for utilization of several monoaromatic, polycyclic, and nonaromatic compounds as carbon source with sulfate as electron acceptor. Besides benzene, the culture was able to grow with biphenyl, whereas benzoate, 4-hydroxybenzoate, phenol, and toluene could not be utilized. The enrichment culture was also tested for its capability to use phenol and toluene as cosubstrates (Fig. 2). Here, neither phenol nor toluene could be degraded cometabolically. Interestingly, the rate of benzene degradation was retarded in the presence of phenol (Fig. 2a) whereas toluene had no effect on the rate of benzene degradation (Fig. 2b). Additionally, growth of microorganisms in the culture BPL could be observed with the organic acids formate, lactate, and pyruvate as well as H2/CO2 (30 : 70 v/v) as evaluated by turbidity formation of the growth medium. None of the tested electron acceptors sulfite, thiosulfate, elemental sulfur, nitrate, or ferrihydrite was utilized with benzene as carbon source.

Figure 2.

 Degradation of benzene by the enrichment culture BPL in the presence of (a) phenol and (b) toluene as co-substrates using sulfate as an electron acceptor. Data are the means of duplicate incubations.

Community fingerprinting and phylogenetic analysis

The bacterial community composition of the enrichment culture BPL grown on different substrates was assessed by T-RFLP analysis of 16S rRNA gene sequences. The T-RFLP profile of benzene-grown cells was clearly dominated by 16S rRNA gene sequences forming a 141-bp T-RF (Fig. 3a). In silico analysis considered the occurrence of an additional T-RF of 152 bp length to be a pseudo T-RF (Fig. 3a), which was verified by T-RFLP analysis of specific 16S rRNA gene clone sequences. Pseudo T-RFs are formed when the restriction enzyme failed to cut at the first recognition site, and thus a longer T-RF is formed by restriction at the second site (Egert & Friedrich, 2003). Thus, the T-RFs of 141 and 152 bp have derived from the same 16S rRNA gene sequence. The 141 bp T-RF could also be identified as a dominant peak in the biphenyl-grown culture together with an additional T-RF of 164 bp in length (Fig. 3b). A 164-bp T-RF was continuously present in all growth cultures (except lactate) tested for alternative electron donors (Fig. 3c–f).

Figure 3.

 T-RFLP fingerprinting profiles of 16S rRNA gene sequences of the enrichment culture BPL grown on (a) benzene, (b) biphenyl, (c) formate, (d) pyruvate, (e) lactate, and (f) H2/CO2 gas. Dominant base pairs are labelled with lengths (bp).

To get insights into the phylogenetic affiliation of the microoorganims in the benzene-grown culture, we performed cloning and sequencing of almost full-length 16S rRNA gene sequences. The analysis clearly showed a dominance of 16S rRNA gene sequences (designated as BpP) that accounted for 30 out of 33 analyzed small-subunit sequences and that could be correlated with the 141-bp T-RF. These highly abundant sequences were affiliated with the genus Pelotomaculum within the Gram-positive family Peptococcaceae (Fig. 4) and grouped to a cluster that was related to Pelotomaculum isophthalicum JI (95% sequence similarity). Moreover, two closely related 16S rRNA gene sequences (BpC52 and BpC43; represented by the 305-bp T-RF) were detected that were affiliated to the family Clostridiaceae. The 16S rRNA gene sequence of the clone BpD108 was affiliated to the deltaproteobacterial family Syntrophaceae, exhibiting a similarity value of 93.3% to the dehalogenating bacterium Desulfomonile tiedjei.

Figure 4.

 Phylogenetic tree showing the affiliation of the 16S rRNA gene sequences amplified from the enrichment culture BPL. 16S rRNA clone sequences from the enrichment culture BPL are shown in bold. Subdivisions Ia–Ih of the Desulfotomaculum cluster I are listed. An encompassing collection of organisms representing all major lineages of the Archaea and Bacteria was used as outgroup for tree calculations. The scale bar represents 10% sequence divergence.

Cell counts determined by FISH and DAPI staining

In order to demonstrate that bacteria with Pelotomaculum-related 16S rRNA gene sequences (BpP) are dominant and their cell numbers are increasing during the full time course of benzene degradation, in situ hybridization with the probes DEM1164r, specific for Desulfotomaculum cluster I bacteria comprising Pelotomaculum sp. and EUB338 I-III mix (Bacteria) was performed (Fig. 5). No hybridization to 16S rRNA gene sequences of the enrichment culture BPL could be observed with the negative control probes BET42a and GAM42a. However, the probe BET42a but not DEM1164r hybridized to the control organism T. thiophilus D24TNT. During the time course of benzene degradation, the average percentage of DEM1164r-labelled cells was 94.5% (n=9 time points) of the EUB338 I-III mix-positive cells (Fig. 5). The portion of the bacterial cells stained with DEM1164r and EUB338 I-III mix constituted 86.6% (n=9) and 91.8% (n=9) of the total DAPI-positive cells (Fig. 6).

Figure 5.

 Micrographs of cells from the culture BPL grown with benzene and sulfate as electron acceptor. (a) Specific labeling of Desulfotomaculum subcluster I cells with the Cy3-labeled probe DEM1164r, (b) general labeling with the Fluos-labeled probe mixture EUB338 I-III, (c) DAPI-staining of all bacteria, and (d) labeling of cells with the specific probe DEM1164r and DAPI resulting in overlays of the probe and DAPI signals; cells targeted by both labels appear in magenta and nontargeted cells in blue.

Figure 6.

 Relative quantification of bacterial cells detected by in situ hybridization with the specific probe DEM1164r (Desulfotomaculum cluster I) or EUB338 I-III mix (bacteria) and DAPI staining during the full time course of benzene degradation by the enrichment culture BPL. Data are the means of duplicate incubations with two separate filter counting each and error bars represent SDs.

Metabolite analysis

Metabolites were screened and quantified with LC-ESI-MS-MS and HPLC/UV fluorescence analysis during benzene degradation (54 days). Benzoate, 4-hydroxybenzoate, and phenol were detected in active as well as in autoclaved control cultures (Fig. 7). The phenol formation increased concomitant to benzene degradation in the active culture reaching a maximum concentration of 1.8 μM at day 28. Interestingly, the phenol concentration in the autoclaved control culture was comparably high, reaching the maximum value of 1.6 μM at day 47. The amount of benzoate in the active culture was highest at day 47 increasing to 0.4 μM, whereas its concentration in the autoclaved culture remained constantly low (0.06 μM). Additionally, at the end of the time course of benzene degradation, 4-hydroxybenzoate was identified in the active culture at 0.14 μM. 2-Hydroxybenzoate was detected at maximum concentration of 2.9 nM from autoclaved control cultures and with a slightly higher concentration (7.9 nM) in the active culture (data not shown). Benzylsuccinate could not be identified.

Figure 7.

 Metabolite analysis from active and autoclaved control cultures under sulfate-reducing conditions during the full time course of benzene degradation. Data are the means of duplicate incubations.

Discussion

In the present study, we report on anaerobic benzene degradation by a sulfate-reducing enrichment culture obtained from soil at a former coal gasification site. Electron and carbon balances with 13C6-benzene indicated a total oxidation of the aromatic hydrocarbon to CO2 coupled to sulfate reduction.

Bacterial community structure of the benzene-degrading culture

Based on the quantitative determination of the putative benzene degrader by in situ hybridization and the analysis of 16S rRNA gene sequences, we have provided evidence leading to the hypothesis that organisms phylogenetically related to the Gram-positive genus Pelotomaculum play an important role in benzene degradation. However, the 16S rRNA gene-based sequence similarity to the next cultivated representative constitutes only 95%. Thus, these sequences could be clustered between the genera Desulfotomaculum and Pelotomaculum. Pelotomaculum isophthalicum-related 16S rRNA gene sequences were detected in benzene-contaminated groundwater and in situ reactor columns, degrading benzene under sulfate-reducing and denitrifying conditions (Kasai et al., 2007; Kleinsteuber et al., 2008). Kleinsteuber et al. (2008) hypothesized that Pelotomaculum syntrophically ferments benzene to acetate under sulfate-reducing conditions. The similarity of the sequences of the Pelotomaculum-related phylotypes described by Kleinsteuber et al. (2008) to those identified in the present study range from 88.8% to 95.7% indicating that these phylotypes are totally different. Based on the results with our highly enriched culture, where only one dominant organism is present, we propose that bacteria with Pelotomaculum-related 16S rRNA gene sequences oxidize benzene directly with sulfate as electron acceptor. However, members of the genus Pelotomaculum were not reported to carry out dissimilatory sulfate reduction so far (Imachi et al., 2002). Recently, Imachi et al. (2006) proposed that Desulfotomaculum subcluster Ih bacteria have lost their ability to reduce sulfate in an evolutionary process and thus are restricted to methanogenic environments for synthrophic oxidation of organic substrates. Such a synthrophic benzene degradation could be definitely excluded for the presented culture because neither methane nor methanogenic archaea have been detected (data not shown). Additionally, the in situ hybridization and T-RFLP analysis of the benzene-grown enrichment culture clearly showed the presence of only one dominant genotype. Thus, we can show here that bacteria that are phylogenetically affiliated to Pelotomaculum but physiologically strongly resemble Desulfotomaculum are utilizing benzene as sole carbon source with sulfate as electron acceptor. This hypothesis is supported by the identification of the γ subunit of the dissimilatory sulfite reductase (key enzyme of dissimilatory sulfate reduction) by proteomic analysis of the benzene-grown culture (data not shown). The identified peptide is closely related to the dissimilatory sulfite reductase of Pelotomaculum thermopropionicum (Imachi et al., 2006).

Our knowledge of the role and importance of Gram-positive bacteria in degradation of aromatic hydrocarbons is quite limited. Recently, an iron-reducing enrichment culture was described where benzene degradation was performed by Clostridia-related bacteria in association with members of Desulfobulbaceae (Kunapuli et al., 2007). In addition, few Gram-positive sulfate-reducers that degrade aromatics were successfully isolated in pure cultures (Cord-Ruwisch & Garcia, 1985; Tasaki et al., 1991; Kuever et al., 1999; Morasch et al., 2004). Interestingly, the majority of these bacterial strains are phylogenetically restricted to the Desulfotomaculum cluster I. Morasch et al. (2004) isolated the strict anaerobic bacterium Desulfotomaculum sp. Ox39 that metabolizes the aromatic hydrocarbons toluene, m-xylene, and o-xylene. Desulfotomaculum sapomandens (Cord-Ruwisch & Garcia, 1985), Desulfotomaculum thermobenzoicum (Tasaki et al., 1991), and Desulfotomaculum gibsoniae (Kuever et al., 1999) exhibit a great versatility in the kind of aromatic electron donors they can use for growth, including benzoate, phenol, and 4-hydroxybenzoate. Molecular based investigations of benzene-degrading communities at contaminated sites and in enrichment cultures in the presence of different electron acceptors showed that they were mostly affiliated to Beta-, Delta-, and Gammaproteobacteria (Phelps et al., 1998; Rooney-Varga et al., 1999; Coates et al., 2001; Musat & Widdel, 2008; Oka et al., 2008). However, several studies identified Firmicutes-related 16S rRNA gene sequences that have so far not been brought into direct correlation with contaminant degradation (Phelps et al., 1998; Ulrich & Edwards, 2003; Chang et al., 2005). Nevertheless, the large diversity of the microbial communities was not enough to emphasize the key players and their role in benzene degradation. So far, only a limited number of sulfate-reducing benzene-degrading enrichment cultures have been purified to an extent that allowed the identification of the key degraders. Deltaproteobacterial phylotypes related to Desulfobacterium were found to be abundant in a benzene-degrading sulfate-reducing culture (Qiu et al., 2006; Musat & Widdel, 2008). Furthermore, members of the family Desulfobacteraceae have been identified as significant bacteria in a sulfidogenic benzene-degrading culture by DNA stable isotope probing (Oka et al., 2008). More recently, bacteria affiliated to the genera Sulfurovum, Desulfovibrio, and Cryptanaerobacter/Pelotomaculum have been characterized to be the most prominent bacteria in sand-filled columns percolated with groundwater from a benzene-contaminated aquifer whereas the proportion of the latter one increased after repeated benzene-spiking, indicating a role in benzene degradation (Kleinsteuber et al., 2008). The cultivation of our Pelotomaculum-related bacterium further supports our hypothesis that Gram-positive microorganisms have been largely overlooked as being important in degradation of benzene (Kunapuli et al., 2007, 2008). Especially Gram-positive sulfate-reducing bacteria may play an up to date underestimated role, because their ability of spore-formation allows this group to survive periods of environmental fluctuating conditions such as alternating oxic and anoxic conditions (Nielsen et al., 2005).

Initial reaction mechanism of anaerobic benzene degradation

In previous studies, three possible mechanisms have been proposed for the initial reaction of anaerobic benzene degradation. Hydroxylation of benzene to phenol has been hypothesized as one putative pathway under methanogenic and denitrifying conditions (Vogel & Grbic-Galic, 1986; Weiner & Lovley, 1998; Caldwell & Suflita, 2000; Chakraborty & Coates, 2005). In the present study, phenol and the specific intermediate of anaerobic phenol degradation 4-hydroxybenzoate were detected in the active enrichment culture. These results might indicate that benzene is initially hydroxylated to phenol. If the degradation would proceed via hydroxylation to phenol, the benzene-grown culture should have the ability to utilize phenol immediately. However, phenol as sole carbon source was not utilized by the enrichment culture BPL. In addition, benzene degradation was retarded when phenol was added as a cosubstrate. It seems that phenol decreased the activity of the benzene degraders or that the benzene degraders had to adapt to the presence of phenol, which may exhibit a toxic effect. This observation is congruent with results obtained from a sulfate-reducing culture enriched from marine sediment where the rate of benzene degradation was also slightly slower in the presence of phenol (Musat & Widdel, 2008).

In addition, 2-hydroxybenzoate has been identified as a putative metabolite in the benzene-grown cultures, which is not known to be a specific intermediate compound of anaerobic degradation of monoaromatic hydrocarbons, so far. Thus, it might indicate that the detected hydroxylated aromatic compounds are formed abiotically due to contact of reduced compounds with oxygen during sampling. This is strongly supported by the detection of phenol in our autoclaved control cultures. Kunapuli et al. (2008) showed that benzene is readily autohydroxylated by hydroxyl (OH) radicals formed from oxygen during sampling, which might also have produced the hydroxylated monoaromatic compounds in our culture. Another evidence for this abiotic process is the formation of 4-hydroxybenzoate concomitant to benzoate evolution. The medium was supplemented with 3 mM of FeCl2 to scavenge the produced sulfide. As a result of this reaction, reduced iron is produced that is considered to be a catalyst of Fenton's reaction. During sampling, ferrous ion gets in contact with oxygen, which results in the formation of highly reactive OH radicals (Kavith & Palanivelu, 2004; Mortazavi et al., 2005). These OH radicals readily react with organic compounds such as benzene and benzoate forming hydroxylated products (Kunapuli et al., 2008).

Methylation is proposed as an alternative mechanism for anaerobic degradation of nonsubstituted aromatic compounds (Ulrich et al., 2005; Safinowski & Meckenstock, 2006). Benzylsuccinate as the specific metabolite of anaerobic toluene degradation was not detected in our benzene-grown culture. Similarly to recent studies (Musat & Widdel, 2008; Oka et al., 2008), toluene could not be used as substrate by our culture and the presence of toluene in the medium had no effect on the rate of benzene degradation. This is in contrast to investigations where other authors clearly showed that toluene inhibits the rate of benzene degradation (Da Silva & Alvarez, 2007). Thus, methylation could probably be excluded as initial degradation mechanism in the investigated enrichment culture.

Another proposed pathway of anaerobic benzene degradation involves direct carboxylation of benzene to benzoate (Caldwell & Suflita, 2000). Previous studies with sulfate-reducing enrichment cultures using labeled benzene revealed the presence of 13C-benzoate and d5-benzoate as intermediate compounds and showed that the carboxyl group derived from benzene (Caldwell & Suflita, 2000; Phelps et al., 2001). However, Kunapuli et al. (2008) showed for an iron-reducing culture that the carboxyl group of benzoate stems from the carbonate buffer. In the present study, benzoate was identified as putative intermediate compound, but we could not prove that the benzene-degrading bacteria have the capability to utilize benzoate as substrate. As benzoate cannot freely diffuse into cells, we assume that benzene degraders in our culture lack an active transport system for benzoate as it is reported for Pseudomonas putida (Thayer & Wheelis, 1982). Furthermore, it might be possible that benzoate could not be activated to benzoyl-Co-enzyme A due to the lack of a benzoate-CoA ligase (Barragan et al., 2004). Nevertheless, our findings would tentatively favor a direct carboxylation of benzene as the initial activation reaction in the sulfate-reducing enrichment culture BPL.

Acknowledgements

The authors thank Holger Penning and Martin Elsner for help with GC/C/IRMS measurements. This project was supported by the DAAD-Helmholtz PhD exchange program through an operating grant (A/05/22356) awarded to N.A.L.

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