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Keywords:

  • anaerobic benzene mineralisation;
  • syntrophy;
  • interspecies metabolite transfer;
  • Pelotomaculum

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Syntrophic mineralisation of benzene, as recently proposed for a sulphate-reducing enrichment culture, was tested in product inhibition experiments with acetate and hydrogen, both putative intermediates of anaerobic benzene fermentation. Using [13C6]-benzene enabled tracking the inhibition of benzene mineralisation sensitively by analysis of 13CO2. In noninhibited cultures, hydrogen was detected at partial pressures of 2.4 × 10−6± 1.5 × 10−6 atm. Acetate was detected at concentrations of 17 ± 2 μM. Spiking with 0.1 atm hydrogen produced a transient inhibitory effect on 13CO2 formation. In cultures spiked with higher amounts of hydrogen, benzene mineralisation did not restart after hydrogen consumption, possibly due to the toxic effects of the sulphide produced. An inhibitory effect was also observed when acetate was added to the cultures (0.3, 3.5 and 30 mM). Benzene mineralisation resumed after acetate was degraded to concentrations found in noninhibited cultures, indicating that acetate is another key intermediate in anaerobic benzene mineralisation. Although benzene mineralisation by a single sulphate reducer cannot be ruled out, our results strongly point to an involvement of syntrophic interactions in the process. Thermodynamic calculations revealed that, under in situ conditions, benzene fermentation to hydrogen and acetate yielded a free energy change of ΔG′=−83.1 ± 5.6 kJ mol−1. Benzene mineralisation ceased when ΔG′ values declined below −61.3 ± 5.3 kJ mol−1 in the presence of acetate, indicating that ATP-consuming reactions are involved in the pathway.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Benzene is toxic and among the most abundant groundwater pollutants worldwide. Under anoxic conditions, it is considered the most recalcitrant one of all BTEX (benzene, toluene, ethylbenzene, xylene) compounds, as the majority of studies failed to demonstrate its breakdown. However, mineralisation was shown for some cultures using different electron acceptors (for reviews, see Weelink et al., 2010; Vogt et al., 2011). The underlying degradation mechanisms are not yet understood and little is known about the organisms involved. On the one hand, pure cultures of nitrate-reducing benzene-degrading bacterial strains have been reported (Kasai et al., 2006) and single microorganisms were proposed to be responsible for benzene breakdown in highly enriched strictly anaerobic consortia (Musat & Widdel, 2008; Oka et al., 2008; Abu Laban et al., 2009). On the other hand, benzene degradation accomplished by a syntrophic cooperation between metabolically different organisms was suggested (Ulrich & Edwards, 2003; Kunapuli et al., 2007). In several independent studies, evidence accumulated indicating that members of the family Peptococcaceae may play an important role in this process: in a methanogenic benzene-degrading enrichment culture, the most abundant bacterial community members belonged to the genera Desulfosporosinus (Peptococcaceae) and Desulfobacterium (Ulrich & Edwards, 2003). In an iron-reducing benzene-mineralizing enrichment culture, Peptococcaceae- and Desulfobulbaceae-related organisms incorporated most of the 13C-labelled benzene and were therefore suggested to be responsible for syntrophic benzene degradation (Kunapuli et al., 2007).

Based on research in our lab, a functional model was developed for syntrophic benzene mineralisation in a sulphate-reducing enrichment culture. The culture is dominated by three phylotypes affiliated to Cryptanaerobacter/Pelotomaculum (Peptococcaceae), Epsilonproteobacteria and the genus Desulfovibrio (Deltaproteobacteria) (Kleinsteuber et al., 2008). The Cryptanaerobacter/Pelotomaculum phylotype was suggested to mediate the breakdown of benzene to acetate and hydrogen. These central intermediates were proposed to be consumed by Delta- and Epsilonproteobacteria. The Cryptanaerobacter/Pelotomaculum phylotype and the epsilonproteobacterium, but not the putative hydrogen-scavenging Deltaproteobacteria assimilated 13C-benzene in subsequent DNA-stable isotope probing (SIP) experiments (Herrmann et al., 2010).

So far, evidence in support of syntrophic benzene degradation in this culture has been obtained by analysing the community composition using molecular biological techniques. Here, we used a physiological approach and investigated the effects of hydrogen or acetate additions on benzene mineralisation. If benzene is degraded syntrophically with hydrogen and acetate as central intermediates, then the addition of sufficient amounts of hydrogen or acetate is expected to inhibit benzene mineralisation for thermodynamic reasons. However, these thermodynamic effects have to be discriminated from inhibitory effects that hydrogen or acetate additions could exert on a potential single sulphate reducer mineralizing benzene.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Chemicals

Chemicals were either purchased from Sigma-Aldrich (Germany) or Merck (Germany) and were of analytical-grade quality. [13C6]-benzene (≥99 atom%) was obtained from Campro Scientific (Veenendaal, the Netherlands).

Source of enrichment culture and experimental set-up

Benzene-degrading sulphate-reducing cultures were derived from a column system described elsewhere (Vogt et al., 2007). Microcosms were set up at room temperature with 100 g of column sand and 40 mL anoxic mineral salt medium containing 20 mM sulphate (for a detailed description, see Kleinsteuber et al., 2008). Additional replicate cultures were set up for analysis of in situ concentrations of hydrogen and acetate; six and four individual actively benzene-degrading cultures were analysed, respectively. Mean values ± SD (n=5 for hydrogen, one replicate was below the detection limit and was excluded from the calculation; n=4 for acetate) represent the concentration range applied to thermodynamic calculations and were taken into account by calculation of error propagation (see Thermodynamic calculations). Two additional replicate microcosms were autoclaved (20 min, 121 °C) on three consecutive days and served as abiotic controls. For benzene degradation under Fe(III)-reducing or methanogenic conditions, sulphate was replaced by 50 mM Fe(III)-citrate or excluded, respectively.

For the experiment under methanogenic conditions in which methanogens were added, Methanospirillum hungatii DSM 864 was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), and Methanosarcina barkeri DSM 800 was kindly provided by Michael Siegert and Dr Martin Krueger (Federal Institute for Geosciences and Natural Resources, Hannover, Germany). Both were pregrown in an anoxic mineral salt medium described by Hutten et al. (1981), which was slightly modified by adding anoxic stock solutions of vitamins, thiamine, tungsten-selenite, nonchelated trace elements and vitamin B12 (each 1 mL L−1) according to Widdel & Bak (1992). (NH4)2SO4 and MgSO4× 7H2O were replaced by NH4Cl (0.1 g L−1) and MgCl2 (0.04 g L−1), respectively. Methanospirillum hungatii and M. barkeri were cultivated at 37 and 30 °C, respectively, with hydrogen (M. hungatii) or acetate (M. barkeri) as an electron donor. A volume of 5 mL of each methanogenic culture was injected into each benzene-degrading enrichment culture. In this experiment, the enrichment culture was also cultivated using the modified anoxic mineral salt medium according to Hutten et al. (1981).

In order to sensitively track benzene mineralisation, approximately 800 μM of [13C6]-benzene was added to the microcosms and autoclaved controls, allowing the analysis of 13CO2 that evolved. Sodium acetate or sodium molybdate was added to the cultures from a 1 M anoxic stock solution. To apply 0.1, 0.25 or 0.5 atm hydrogen, 4, 10 or 20 mL of headspace gas was replaced with the same volume of hydrogen (99.99% purity).

Analytical methods

Benzene concentrations were determined by GC as described previously (Kleinsteuber et al., 2008).

For measurements of hydrogen concentration, a 50-μL gas sample was separated on a 1.8 m × 2 mm stainless-steel column with a molecular sieve (5 Å). The oven was set to room temperature; the carrier gas was nitrogen at a flow rate of 12 mL min−1. Hydrogen was measured using an RGD2 reduction gas detector (Trace Analytical Inc.). The detection limit was 5 × 10−9 atm.

In situ concentrations of acetate were measured by injecting a 5-μL liquid sample onto a Dionex DX500 ion chromatograph system equipped with a IonPac®AS18 column (4 × 250 mm) and a CD20 conductivity detector (Dionex, Canada). Elution was achieved under the following conditions: 5 mM KOH for 5 min and increasing to 40 mM over 14 min. The flow rate of the eluent was set to 1 mL min−1. The detection limit was 0.5 μM. Acetate concentrations above 100 μM were measured using a high-performance liquid chromatograph (Shimadzu, Germany) with a Rezex ROA-Organic Acid H+ (8%) column (3 m × 7.8 mm; Phenomenex). Elution was achieved with 0.005 N H2SO4 at a flow rate of 0.6 mL min−1 at 40 °C. Acetate was detected using the refractive index detector RID-10A (Shimadzu).

The carbon isotope composition (13CO2/12CO2) of carbon dioxide (CO2) was determined using a GC-combustion-isotope ratio mass spectrometer as described elsewhere (Herrmann et al., 2010). Carbon isotope signatures are given in δ-notation (‰) relative to the Vienna Pee Dee Belemnite standard (V-PDB) and were converted to molar concentrations of CO2 as described recently (Kunapuli et al., 2007).

Thermodynamic calculations

Standard Gibbs free energy change (ΔG0′) was calculated from Gibbs free energy of formation (ΔGf0) (Thauer et al., 1977) at T=298 K. In the case of a single organism, the ΔG0′ values were subsequently corrected for the actual concentrations of benzene, acetate and hydrogen:

  • image(1)

R is the gas constant (R=8.31 J K−1 mol−1) and T is the temperature (T=293 K).

Uncertainties of ΔG′ in Eqn. (2) were determined by error propagation:

  • image(2)

s is the error of each parameter.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Mineralisation of [13C6]-benzene in noninhibited cultures

In all microcosms, benzene degradation and 13CO2 formation started without a lag phase. Noninhibited cultures consumed around 0.8 mM benzene within 91 days, corresponding to a shift in the isotope ratio of 13CO2/12CO2 produced of about +14 000‰, equalling 14.7 atom%13C (Fig. 1a and b), and indicating that 87% of the added benzene was released as CO2. This degree of mineralisation is similar to the values previously observed for this (Herrmann et al., 2010) and other benzene-mineralizing cultures (Kunapuli et al., 2007; Abu Laban et al., 2009). In autoclaved control bottles, the stable isotope signatures of CO2 were always between −20‰ and −16‰ within 133 days, demonstrating that the increase in 13CO2 was a truly biotic process. In situ concentrations of hydrogen and acetate were analysed from several actively benzene-degrading cultures set up in replicates. Hydrogen was detected at low partial pressures of 2.4 × 10−6± 1.5 × 10−6 atm (n=5) (Table 1), corresponding to a concentration of 1.9 ± 1.2 nM in the aqueous phase. Acetate was detected at concentrations of 17 ± 2 μM (n=4). Environments like sewage sludge or anoxic freshwater sediments typically display low acetate concentrations of 2–7 μM and hydrogen partial pressures of 1 × 10−6–5 × 10−5 atm (Lovley & Phillips, 1987; de Graaf et al., 1996; Hoehler et al., 1998; Schink & Stams, 2006). The acetate concentrations detected here are somewhat higher, but still comparable. Notably, the detected hydrogen partial pressure is in the same range as that described previously for sulphate-reducing conditions, where, due to the sulphate reducer's high affinity to hydrogen, partial pressures are reduced to values of 1 × 10−6–2 × 10−6 atm (Lovley & Phillips, 1987; Hoehler et al., 1998).

image

Figure 1.  Degradation of [13C6]-benzene (a, c, e) and concomitant formation of 13CO2 (b, d, f) in a sulphate-reducing enrichment culture (plotted in triplicate). Noninhibited cultures (a, b) were compared with cultures spiked with 0.3 atm hydrogen (c, d) or 30 mM acetate (e, f). Arrows indicate the addition of hydrogen or acetate on day 35, when approximately 25% of the benzene was degraded, and an additional spiking with acetate on day 52. Note the different axis scale in graph (f). Graphs (g) and (h) show autoclaved control cultures (plotted in duplicate).

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Table 1.   Influence of different hydrogen or acetate concentrations on the calculated Gibbs free energy changes (ΔG′) associated with benzene degradation
 Benzene (μM)Acetate (mM)H2 (atm)ΔG′ (kJ mol−1)
  1. ΔG′ was calculated from replicate microcosms (n=3 for noninhibited cultures, H2 spiking and 30 mM acetate spiking; n=2 for 3.5 mM acetate and 0.3 mM acetate spiking) according to Eqns (1) and (4), R=8.31 J K−1 mol−1, T=293 K, at the time of acetate or hydrogen addition. The SD of ΔG′ was calculated according to Eqn. (2).

Noninhibited655 ± 510.017 ± 0.0022.37 × 10−6± 1.50 × 10−6−83.1 ± 5.6
H2 spiking629 ± 890.017 ± 0.0020.31 ± 0.04+3.2 ± 2.0
Acetate spiking
 30 mM658 ± 3429.5 ± 2.22.37 × 10−6± 1.50 × 10−6−28.7 ± 5.3
 3.5 mM457 ± 1033.5 ± 0.12.37 × 10−6± 1.50 × 10−6−43.3 ± 5.5
 0.3 mM549 ± 30.32 ± 0.032.37 × 10−6± 1.50 × 10−6−61.3 ± 5.3

Effects of hydrogen spiking on [13C6]-benzene degradation

Blocking substrate turnover by increasing the concentration of hydrogen is an established technique for detecting interspecies hydrogen transfer, and has been applied previously to verify, for example, syntrophic degradation of butyrate (Ahring & Westermann, 1988) or benzoate (Warikoo et al., 1996). The effect of hydrogen spiking on [13C6]-benzene degradation was tested in two separate experiments. In the first experiment, 0.3 atm hydrogen was added to static cultures when 25% of the benzene added (0.8 mM) had been degraded. Benzene degradation and 13CO2 formation ceased 22 days after hydrogen spiking (Fig. 1c and d). The inhibition was a temporary effect and benzene degradation resumed after about 60 days (Fig. 1c and d).

To assess the effects of hydrogen spiking more precisely, a second experiment was carried out using three different hydrogen concentrations (0.1, 0.25 and 0.5 atm). In the first experiment, there had been a considerable delay between hydrogen addition and inhibition of benzene mineralisation. This was thought to have been brought about by the static cultivation of the microcosms, which, in conjunction with the low water solubility of hydrogen and hydrogen consumption by sulphate reducers, hampered the increase of the hydrogen partial pressure in the medium. Therefore, in the second experiment, all cultures were shaken to enhance the hydrogen transfer rate between the gaseous and the liquid phase. Furthermore, to monitor the anticipated increased sulphate reduction rates due to hydrogen addition, sulphide concentrations were analysed. The results of the second hydrogen spiking experiment are shown in Fig. 2. Hydrogen spiking caused an immediate production of sulphide compared with nonspiked control cultures, with the amounts of sulphide produced mirroring the amounts of hydrogen having been added. Hydrogen was likely effectively consumed by the various sulphate-reducing species belonging to the Deltaproteobacteria previously detected in the culture (Kleinsteuber et al., 2008; Herrmann et al., 2010). Coinciding with sulphide production, 13CO2 production stopped in all hydrogen-spiked cultures. Notably, the delay between hydrogen addition and inhibition of benzene degradation was considerably shortened compared with the first experiment, probably due to cultivation on a rotary shaker. In the cultures spiked with 0.1 atm hydrogen, 13CO2 production resumed after approximately 14 days (Fig. 2c). In contrast, cultures spiked with 0.25 or 0.5 atm hydrogen did not evolve 13CO2 for the 42 days (Fig. 2e and g). By day 49 after hydrogen addition, the hydrogen added was completely consumed, as indicated by the low hydrogen partial pressures in all the cultures (Table 2). In conclusion, the results indicate that hydrogen spiking exerted two separate effects on benzene mineralisation: first, spiking with low hydrogen amounts (0.1 atm) inhibited benzene mineralisation temporarily, indicating that benzene degradation proceeded only at very low hydrogen concentrations and suggesting that hydrogen might be a key intermediate in the (syntrophic) metabolism of benzene in this culture. Second, spiking with higher hydrogen amounts (0.25 and 0.5 atm) inhibited benzene mineralisation for an extended period. Mineralisation did not immediately restart when the hydrogen added had been consumed. We cannot exclude the toxic effects of the elevated sulphide concentrations produced after the addition of higher amounts of hydrogen.

image

Figure 2.  Formation of 13CO2 (a, c, e, g) and sulphide (b, d, f, h) in a sulphate-reducing enrichment culture (plotted in triplicate). Noninhibited cultures (a, b) were compared with cultures spiked with 0.1 atm (c, d), 0.25 atm (e, f) or 0.5 atm (g, h) hydrogen. The arrow indicates the addition of hydrogen on day 26.

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Table 2.   Hydrogen concentrations determined in spiked and nonspiked cultures
 Time point of samplingH2 concentration (atm)
Nonspiked control culturesAfter 113 days of repeated benzene spiking, containing benzene2.4 × 10−6± 1.5 × 10−6
After 79 days, benzene was fully consumed (see Fig. 2a and b)1.3 × 10−6± 5.1 × 10−7
0.1 atm H249 days after H2 spiking (see Fig. 2c and d)1.3 × 10−6± 1.9 × 10−7
0.25 atm H249 days after H2 spiking (see Fig. 2e and f)1.6 × 10−6± 2.3 × 10−7
0.5 atm H249 days after H2 spiking (see Fig. 2g and h)1.1 × 10−6± 2.7 × 10−7

Effects of acetate spiking on [13C6]-benzene degradation

Besides hydrogen, the effect of acetate on benzene mineralisation was tested. Acetate is another central intermediate in syntrophic degradation pathways, because the conversion of acetyl-CoA, a central metabolic intermediate, to acetate can be coupled to ATP synthesis by the reactions of phosphotransacetylase and acetate kinase. In the present study, acetate addition immediately inhibited benzene mineralisation for at least 30 days (Figs 1e, f and 3). This inhibitory effect was observed in all microcosms, regardless of the acetate concentration added (30, 3.5 and 0.3 mM). Interestingly, benzene mineralisation stopped completely even at low acetate concentrations (0.3 mM). This is in contrast to a previous study (Ahring & Westermann, 1988). There, different concentrations of acetate were added to a syntrophic butyrate-degrading culture, resulting in a gradual decrease in the degradation rate, rather than a complete inhibition of the process. Likewise, syntrophic benzoate degradation continued at low rates in the presence of up to 50 mM acetate (Schöcke & Schink, 1997). These inhibitory effects were reported to be due to thermodynamic constraints and uncoupling effects (Ahring & Westermann, 1988; Schöcke & Schink, 1997; Jackson & McInerney, 2002).

image

Figure 3.  Effect of different acetate concentrations (open symbols) on the formation of 13CO2 (filled symbols) from [13C6]-benzene (plotted in duplicate). Arrows indicate spiking with 0.3 mM acetate (a) or 3.5 mM acetate (b) on day 70.

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As observed in the hydrogen inhibition experiment, acetate inhibition was reversible, but only in those cultures with low acetate concentrations added. In two replicate cultures spiked with 0.3 mM acetate, acetate was consumed at rates of 10–14 μM day−1, and benzene mineralisation was detectable again when acetate concentrations decreased to 52 or 4 μM (Fig. 3). A similar observation was recently made in a BTEX- and ethanol-contaminated aquifer, where the transient accumulation of acetate was thought to prevent the anaerobic degradation of benzene for thermodynamic reasons (Corseuil et al., 2011). In contrast, no further benzene mineralisation was observed in the cultures spiked with 3.5 or 30 mM acetate, despite acetate concentrations decreasing slightly over the observation period (2.9 ± 0.1 mM acetate on day 128 and 26.1 ± 1.0 mM acetate on day 80, respectively). Acetate might be consumed by members of different ecophysiological groups detected previously in this enrichment culture, for example Epsilonproteobacteria, sulphate reducers and aceticlastic methanogens (Kleinsteuber et al., 2008; Herrmann et al., 2010).

Dependence of benzene mineralisation on sulphate as an electron acceptor

Despite the biodiversity of the enrichment culture, it appeared to be strongly adapted to sulphate as the main electron acceptor. Benzene mineralisation ceased when cultures were treated with molybdate (Fig. 4), an inhibitor of sulphate reduction, indicating that sulphate acts as the principal electron acceptor during benzene degradation. Benzene mineralisation also ceased in the carbonate-buffered medium under sulphate-limited conditions, and continued after the addition of 200 μM sulphate (data not shown). With carbonate as the sole electron acceptor (methanogenic conditions), benzene was not mineralised over the course of 93 days, despite a previous study reporting the production of small amounts of 13CH4 from 13C-benzene and the presence of Methanosaetaceae in the culture (Herrmann et al., 2010). However, we cannot rule out the possibility that the failure to shift benzene mineralisation to methanogenic conditions was due to the absence or the inactivity of hydrogen- and acetate-consuming methanogens in the enrichment culture. For this reason, a second experiment was set up, in which active cells of hydrogen-consuming (M. hungatii) and acetate-consuming (M. barkeri) methanogens were added to virtually sulphate-free enrichment cultures. However, for unknown reasons, the cultures treated in this way did not produce measurable amounts of methane during a period of 57 days; the δ13C-CO2 values only slightly increased up to +50‰ (see Supporting Information, Fig. S1).

image

Figure 4.  Effect of molybdate addition (a) on the formation of 13CO2 from [13C6]-benzene in a sulphate-reducing culture compared with untreated cultures (b) (plotted in triplicate). The arrow indicates the addition of 15 mM molybdate on day 23.

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Attempts to shift the culture from sulphate to Fe(III) as an alternative electron acceptor were not successful either. The δ13CO2 values remained between −20‰ and +40‰ over a period of 200 days, suggesting no substantial benzene mineralisation (data not shown).

Thermodynamic considerations

In this section, we will discuss whether the inhibitory effects at elevated hydrogen or acetate concentrations can be solely explained by thermodynamic constraints, supporting the hypothesis of syntrophic benzene mineralisation, or whether they are explicable if benzene was mineralised by a single sulphate reducer. Under standard conditions, benzene is mineralised under sulphate-reducing conditions according to the following equation (Herrmann et al., 2010):

  • image(3)

This reaction could be performed by a single sulphate reducer or within a syntrophic consortium of organisms in which sulphate acts as a terminal electron acceptor. A single benzene-mineralising organism is not expected to evolve free hydrogen or acetate; hence, hydrogen or acetate addition would not affect the thermodynamics of the net reaction and a single benzene-mineralising sulphate reducer cannot be thermodynamically inhibited by adding hydrogen or acetate.

Syntrophic benzene degradation, with initial benzene fermentation to acetate and hydrogen, would proceed according to the following equation:

  • image(4)

Therefore, under standard conditions, benzene fermentation is energetically unfavourable. Obviously, the thermodynamics of the reaction are strongly influenced by the in situ hydrogen or acetate concentrations. To gain further insights into the energetic of benzene degradation, ΔG′ was calculated considering three scenarios: (1) noninhibited syntrophic benzene degradation, (2) spiking with hydrogen and (3) spiking with acetate. The concentration data applied when calculating ΔG′ are given in Table 1. In noninhibited cultures (scenario 1), fermentation of benzene yields a free energy change of −83.1 kJ mol−1, allowing a net conservation of more than 1 mol ATP. Spiking with 0.3 atm hydrogen (scenario 2) altered the Gibbs free energy change of benzene fermentation to a slightly endergonic value (+3.2 kJ mol−1). At 0.1 atm hydrogen, the reaction became exergonic (approximately −5 kJ mol−1), but was still insufficient for ATP conservation. For acetate spiking (scenario 3), 0.3 mM acetate temporarily inhibited benzene mineralisation in two replicate cultures. Adding 0.3 mM acetate to a noninhibited culture containing a hydrogen partial pressure of 2.4 × 10−6 atm would reduce the ΔG′ from −83.1 to −61.3 kJ mol−1. The cultures resumed benzene degradation when acetate concentrations decreased to values of 52 and 4 μM acetate (Fig. 2b). In conclusion, when considering only the thermodynamic constraints pertaining to the culture studied, the results of the spiking experiments favour the hypothesis of benzene being mineralised syntrophically involving interspecies hydrogen and acetate transfer. The results indicate that, compared with syntrophic butyrate-, propionate- or benzoate-degrading cultures, a rather high threshold ΔG′ value for syntrophic benzene mineralisation via hydrogen and acetate exists. Syntrophic butyrate and propionate fermentation yields −22 and −21 kJ mol−1, respectively (Stams & Plugge, 2009). Syntrophic benzoate fermentation yields between −25 and −45 kJ mol−1, depending on the final electron acceptor (Warikoo et al., 1996; Schöcke & Schink, 1997; Jackson & McInerney, 2002). For syntrophic butyrate and benzoate degradation, the slight differences in the ΔG′ values observed at the threshold were suggested to be related to the amount of energy required for butyrate and benzoate activation, respectively (Jackson & McInerney, 2002). In some members of the genus Syntrophus, benzoate is activated in an ATP-dependent reaction catalysed by the enzyme benzoyl-CoA ligase (Harwood et al., 1998). In contrast, butyrate is activated by Syntrophomonas wolfei by the energy-neutral reaction of the enzyme butyryl-acetyl-CoA transferase (Wofford et al., 1986; Jackson & McInerney, 2002). Hence, the high threshold ΔG′ value of syntrophic benzene mineralisation indicates that an ATP-consuming reaction is involved in the activation of the chemically stable aromatic ring of benzene, assuming that benzoyl-CoA is reduced by an ATP-independent benzoyl-CoA reductase, as demonstrated recently for strictly anaerobic aromatics degraders (Kung et al., 2009). Moreover, this ATP dependency might explain why benzene mineralisation is completely blocked and does not gradually continue or recover from inhibition, despite the comparatively high ΔG′ being available.

Finally, we would like to discuss four uncertainties of our hypothesis about syntrophic benzene mineralisation in our culture. (1) A toxic effect resulting from increased sulphide concentrations cannot be ruled out on the basis of the results of this study (Fig. 2). The effects of higher hydrogen additions (>0.25 atm) are not solely explicable by thermodynamics, but probably in part due to the physiological effects of increased sulphide concentrations. It is known that sulphide is toxic to all prokaryotes including sulphate reducers, because it reacts with metal ions of biomolecules (Koschorreck, 2008). (2) It is possible that the consumption of high amounts of hydrogen changed the community composition, favouring specific sulphate-reducing hydrogen consumers and impeding a restart of benzene mineralisation after consumption of the hydrogen added. In this study, community composition was not monitored. (3) It is theoretically conceivable that, upon the addition of acetate, a benzene-mineralising strain might immediately shift from benzene to acetate as the sole source of carbon and energy. However, to our knowledge, such behaviour has never been observed for any aromatics-degrading anaerobic culture. (4) A weakening of the ΔpH component of the proton electrochemical gradient by acetate addition as observed for benzoate degradation of Syntrophus gentianae in syntrophic methanogenic co-cultures (Schöcke & Schink, 1997) should only be of minor importance in our study, as already the addition of low concentrations of acetate (0.3 mM) resulted in strong inhibitory effects.

Although the functions and relations of the different organisms in the enrichment culture are not yet fully understood, for the reasons discussed above, it is unlikely that benzene is mineralised by a single sulphate-reducing organism.

Ecological and environmental implications

The syntrophic lifestyle may explain why the enrichment culture degrades benzene only attached to particles [sand or lava granules (Vogt et al., 2007; Herrmann et al., 2008, 2010; Kleinsteuber et al., 2008)]. Attempts to enrich the consortium in liquid medium have been unsuccessful so far. Possibly, the rate of interspecies hydrogen and acetate transfer is significantly and decisively enhanced when the syntrophic partners involved aggregate on surfaces, as described by Stams & Plugge (2009) for methanogenic bioreactors.

Noteworthy is the differing role of Peptococcaceae phylotypes in this and other consortia. In this consortium, benzene mineralisation is presumed to be initiated by the Cryptanaerobacter/Pelotomaculum phylotype as the primary fermenting organism (Kleinsteuber et al., 2008; Herrmann et al., 2010). Kunapuli et al. (2007) showed by DNA-SIP that members of the Peptococcaceae degraded and assimilated carbon from 13C-benzene in an iron-reducing enrichment culture, involving an unknown electron-sharing process with members of the Desulfobulbaceae. Another Pelotomaculum-related phylotype was described recently to be a dominant member within a sulphidogenic benzene-mineralising freshwater enrichment culture, and was proposed to mineralise benzene completely, using sulphate as an electron acceptor (Abu Laban et al., 2009). Hence, Peptococcaceae phylotypes might be able to consume benzene either syntrophically or individually using sulphate as an electron acceptor. The importance of Peptococcaceae phylotypes for syntrophic aromatics degradation is further underlined by the metagenome analysis of a terephthalate-degrading consortium (Lykidis et al., 2011). There, a Pelotomaculum-related phylotype presumably converts terephthalate into acetate, CO2 and hydrogen, thereby establishing the basis for primary and secondary syntrophic interactions.

Furthermore, our results suggest one possible explanation for the frequently observed slow or nondetectable degradation of benzene in anoxic field sites or enrichment cultures set up with contaminated aquifer material (Langenhoff et al., 1996; Johnson et al., 2003). When benzene degradation occurs, it is often preceded by long lag phases and seems to be blocked by co-contaminants (Edwards et al., 1992; Cunningham et al., 2001; Ruiz-Aguilar et al., 2003; Da Silva & Alvarez, 2004). During the degradation of other, more easily accessible hydrocarbons, acetate and/or hydrogen may be formed, which then could inhibit syntrophic degradation of benzene, even at low concentrations. Therefore, favourable conditions for syntrophic benzene breakdown will be established only if the concentrations of inhibitory intermediates are substantially lowered, i.e. after mineralisation of co-contaminants.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work is integrated into the internal research and development programme of the UFZ as well as the SAFIRA I project. We also acknowledge funding from the German Research Foundation, Priority Program 1319. We thank Bernhard Schink (University of Konstanz) and Gabriele Diekert (University of Jena) for valuable discussions. We are also grateful to Michaela Wunderlich and Sibylle Mothes from the UFZ Department Analytical Chemistry for acetate analysis, and Matthias Gehre and Ursula Günther for assistance in our stable isotope laboratory. Finally, we wish to thank two anonymous reviewers and the editor for helpful comments and suggestions, which significantly improved the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Fig. S1. δ13C-CO2 values of 13C-benzene spiked enrichment cultures after 57-day incubation (n=2 for bottles with sulphate; n=3 for all others).

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