INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Anaerobic metabolism of benzoate to acetate, CO₂, and hydrogen or formate in the absence of light or terminal electron acceptors is thermodynamically unfavorable. Degradation proceeds only if the concentration of hydrogen produced during benzoate oxidation is continuously maintained at a low level by a hydrogen-using microorganism (12, 19, 24, 26). This kind of mutual cooperation between two species to degrade a single substrate via interspecies hydrogen transfer is called syntrophism. In syntrophic cocultures, hydrogen-utilizing bacteria are needed to maintain low hydrogen levels and are not directly involved in the metabolism of the original substrate. Also, many syntrophic microorganisms can grow in the absence of hydrogen-utilizing partners on unsaturated substrate analogues by using dismutation reactions in which part of the original substrate is used as an electron acceptor (24).

The syntrophic benzoate degrader Syntrophus aciditrophicus metabolizes benzoate to acetate, hydrogen, and CO₂ in cocultures with a hydrogen-using methanogen or a sulfate reducer. S. aciditrophicus can also grow in pure culture on crotonate (11, 12). Recent studies of S. aciditrophicus-Methanospirillum hungatei cocultures showed that cyclohexane carboxylate transiently accumulated at a level that was 18% of the original benzoate concentration (8). The amount of methane produced per mole of benzoate consumed by S. aciditrophicus-M. hungatei cocultures was less than the theoretically predicted ratio (0.75) during the initial stages of benzoate metabolism. These two observations led us to hypothesize that a portion of the electrons produced during benzoate oxidation to acetate and CO₂ is used to reduce benzoate to cyclohexane carboxylate (8). Benzoate reduction to cyclohexane carboxylate could provide an alternative to interspecies hydrogen transfer for the disposal of reducing equivalents produced during benzoate oxidation. Thermodynamic calculations indicate that benzoate oxidation to acetate, carbon dioxide, and hydrogen is thermodynamically unfavorable (Table 1, equation 1). However, benzoate reduction to cyclohexane carboxylate is an exergonic reaction (Table 1, equation 2), which makes the overall fermentation of benzoate to acetate, HCO₃, and cyclohexane carboxylate exergonic (G⁰', 12.0 kJ/mol) (Table 1, equation 3). We present evidence in this report that S. aciditrophicus is able to grow and metabolize benzoate in the absence of a hydrogen-utilizing partner. It grows in a monoculture by oxidizing about one half the benzoate to acetate and CO₂ and reducing the other half to cyclohexane carboxylate (Table 1, equation 3).

View this table: [in this window] [in a new window]   TABLE 1.   G0' values for different oxidation-reduction reactions involved in benzoate, cyclohexane carboxylate, and cyclohex-1-ene carboxylate metabolisma

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Microorganisms and media. S. aciditrophicus SB^T (= ATCC 700169^T) (12) and M. hungatei JF1 were obtained from our culture collection. All media and stock solutions were prepared anaerobically by using the techniques described by Balch and Wolfe (3). Pure cultures of S. aciditrophicus and M. hungatei were maintained in the basal medium lacking rumen fluid as described previously (8, 18). Cocultures of S. aciditrophicus and M. hungatei were established and grown in sulfate-free (<5 µM) benzoate basal medium as described previously (8). When S. aciditrophicus was tested for its ability to grow with aromatic or alicyclic substrates, basal medium containing the test substrate at a concentration of 1.2 to 1.7 mM was inoculated with crotonate-grown pure cultures in the stationary phase (inoculum size, 15 to 20%). All cultures were incubated at 37°C. Hydrogen-grown cultures of M. hungatei were incubated with shaking (100 rpm), while monocultures and cocultures of S. aciditrophicus and M. hungatei were incubated without shaking except when the effect of hydrogen on benzoate metabolism was studied. The latter cultures were incubated with shaking (100 rpm). The cultures were routinely checked for purity by microscopic observation, as well as by inoculation of thioglycolate medium. Methane production was routinely checked in pure cultures of S. aciditrophicus growing with benzoate, cyclohexane carboxylate, and cyclohex-1-ene carboxylate to ensure the absence of any methanogenic activity.

Analytical procedures. Benzoate, cyclohexane carboxylate, and cyclohex-1-ene carboxylate were analyzed by high-performance liquid chromatography with a reverse-phase C₁₈ Econosphere column (250 by 4.6 mm; particle size, 5 µm; Alltech Inc., Deerfield, Ill.). The isocratic mobile phase consisted of 75% phosphate buffer (25 mM sodium dihydrogen phosphate, pH 2.75) and 25% acetonitrile. A variable-wavelength UV absorbance detector set at 214 nm was used to detect substrates and metabolites. Benzoate was also occasionally analyzed with the same column by using detection at 254 nm and an isocratic mobile phase consisting of 70% 50 mM sodium acetate buffer (pH 4.5) and 30% acetonitrile (11). Gas chromatography-mass spectroscopy was used to identify and quantify metabolites produced at micromolar levels as described previously (8).

Thermodynamic calculations. The G' values for the hydrogen-producing, overall syntrophic substrate degradation, and substrate fermentation reactions under experimental conditions were calculated according to Thauer et al. (29). Equations describing benzoate, cyclohexane carboxylate, and cyclohex-1-ene carboxylate fermentation or syntrophic degradation, as well as the G⁰' values used in these calculations, are given in Table 1. Most values are molar concentrations; the only exceptions are the hydrogen and methane values, which are expressed in atmospheres (1 atm = 101,325 Pa). The HCO₃ concentration was not determined and was assumed to be 0.0365 M in all experiments.

Chemicals. Sodium benzoate was purchased from Sigma Chemical Co. (St. Louis, Mo.), cyclohexane carboxylic acid was purchased from Acros Organics (Fair Lawn, N.J.), and cyclohex-1-ene carboxylic acid was purchased from Aldrich Chemical Co. (Milwaukee, Wis.). All other chemicals used in this study were obtained from Sigma, Aldrich, or Fisher (Pittsburgh, Pa.).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Metabolism of benzoate and cyclohexane carboxylate by S. aciditrophicus in the presence and absence of H₂-utilizing microorganisms. Pure cultures of S. aciditrophicus metabolized benzoate exponentially (Fig 1A) at a rate of 0.41 ± 0.04 day¹. S. aciditrophicus produced 0.53 mol of cyclohexane carboxylate and 1.44 mol of acetate per mol of benzoate degraded (Fig. 1A and Table 2). These values are close to those predicted by the fermentation reaction (Table 1, equation 3). There was a significant correlation (r² = 0.99) between benzoate consumption and cyclohexane carboxylate production during the experiment (Fig. 1A, inset). The benzoate fermentation activity of monocultures of S. aciditrophicus could be maintained by repeated subculturing. No methane was detected during the experiment. Microscopic observation revealed the presence of only the S. aciditrophicus cell morphotype at the end of the experiment. The hydrogen concentration increased from 38.5 ± 9.5 Pa at the start of the experiment to 97.9 ± 7.2 Pa at the end of the experiment (Table 2), indicating that a small portion of the electrons produced during benzoate oxidation was used to reduce protons to hydrogen. The initial crotonate concentration was 120 µM due to carryover with the inoculum. The final crotonate concentration was 85 µM. Butyrate was not produced at detectable levels (<0.5 µM) by the end of the experiment, which indicated that crotonate did not act as a terminal electron acceptor in this experiment. The G' values for benzoate fermentation (Table 1, equation 3) and for hydrogen production from benzoate (Table 1, equation 1) were 15.4 and 3.3 kJ/mol, respectively. Analysis by gas chromatography-mass spectrometry showed that cyclohex-1-ene carboxylate, pimelate, and glutarate transiently accumulated at maximum concentrations of 24.2, 4.5, and 3.67 µM, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Metabolism of benzoate by pure cultures of S. aciditrophicus (A)and by S. aciditrophicus-M. hungatei cocultures (B). Symbols: , benzoate; , cyclohexane carboxylate; , acetate; , methane; , benzoate in autoclaved controls; , cyclohexane carboxylate in autoclaved controls; , acetate in autoclaved controls; , methane in autoclaved controls. (Inset) Correlation between benzoate consumption and cyclohexane carboxylate production in S. aciditrophicus pure cultures. The data are averages ± standard deviations based on triplicate microcosms.

1

Effects of hydrogen and acetate on benzoate metabolism by pure cultures of S. aciditrophicus. Benzoate degradation by S. aciditrophicus monoculture was inhibited by high levels of hydrogen (10.1 kPa) (Fig 2A). Benzoate consumption was not inhibited in controls that received an equal amount of nitrogen. Benzoate metabolism in pure cultures of S. aciditrophicus that received 60 mM sodium acetate was inhibited compared to controls that received 60 mM sodium chloride (Fig 2B).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   (A) Effect of hydrogen on benzoate metabolism by S. aciditrophicus. Symbols:  and , benzoate utilization and cyclohexane carboxylate production, respectively, in cultures receiving 10.1 kPa of hydrogen;  and , benzoate production and cyclohexane carboxylate production, respectively, in cultures receiving 10.1 kPa of nitrogen. (B) Effect of acetate on benzoate metabolism by S. aciditrophicus. Symbols:  and , benzoate utilization and cyclohexane carboxylate production, respectively, in cultures receiving 60 mM sodium acetate;  and , benzoate production and cyclohexane carboxylate production, respectively, in cultures receiving 60 mM sodium chloride. The data are averages ± standard deviations based on triplicate microcosms.

Metabolism of cyclohex-1-ene carboxylate by pure cultures of S. aciditrophicus. S. aciditrophicus partially metabolized cyclohex-1-ene carboxylate with concurrent production of cyclohexane carboxylate and small amounts of acetate (Fig 3). Only 63% of the initial amount of substrate was metabolized, and about 1 mol of cyclohexane carboxylate and 0.3 mol of acetate per mol of cyclohex-1-ene carboxylate were produced by S. aciditrophicus pure cultures (Table 2). The hydrogen level in cyclohex-1-ene carboxylate-grown monocultures increased from 51.7 ± 1.6 to 839 ± 68 Pa. The final value was about 320 times the value obtained for S. aciditrophicus-M. hungatei cocultures grown with cyclohex-1-ene carboxylate and 9 times the value obtained for S. aciditrophicus pure cultures grown with benzoate. The G' calculated for cyclohex-1-ene carboxylate oxidation (Table 1, equation 4) at the end of the experiment was 4.7 kJ, while the G' obtained for the overall cyclohex-1-ene carboxylate fermentation reaction (Table 1, equation 6) was 9.2 kJ/mol. S. aciditrophicus-M. hungatei cocultures completely metabolized cyclohex-1-ene carboxylate and produced 2.71 mol of acetate and 1.30 mol of methane per mol of cyclohex-1-ene carboxylate. These values are consistent with the theoretical stoichiometry for cyclohex-1-ene carboxylate metabolism by syntrophic cocultures (Table 1, equation 9).

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Metabolism of cyclohex-1-ene carboxylate by S. aciditrophicus pure cultures. Symbols: , cyclohex-1-ene carboxylate; , cyclohexane carboxylate; , acetate; , cyclohex-1-ene carboxylate in autoclaved controls; , cyclohexane carboxylate in autoclaved controls; , acetate in autoclaved controls. The data are averages ± standard deviations based on triplicate microcosms.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

An S. aciditrophicus monoculture metabolized benzoate to cyclohexane carboxylate, acetate, and CO₂ (Table 1, equation 3). This reaction supported the growth of S. aciditrophicus, as indicated by our ability to successfully transfer the activity and by the results of the growth yield experiments which indicated that 6.9 g (dry weight) of cells was made per mol of benzoate. The redox potential of the benzoate-cyclohexane carboxylate electron pair (244 mV) makes benzoate reduction to cyclohexane carboxylate exergonic if hydrogen (410 mV), NADH (320 mV), or reduced flavin adenine dinucleotide (220 mV) is the electron donor. Our results add benzoate to the growing list of compounds, such as crotonate (2, 5, 27, 28, 31), unsaturated short-chain volatile fatty acids (1), pyruvate, fumarate (10, 30), acetoin, acetaldehyde (7), and acetylene (23), that support the growth of microorganisms once believed to be obligate syntrophs. It is clear that many of these organisms have diverse modes of metabolism. This may make assignment of physiological function based on molecular identification methods difficult.

Fermentation of other aromatic compounds has also been reported. Sporotomaculum hydroxybenzoicum degrades 3-hydroxybenzoate in the absence of H₂-using microorganisms (20) by using the crotonyl coenzyme A produced during substrate degradation as an electron acceptor, which results in the production of butyrate, acetate, and HCO₃ as end products. Karlsson et al. (16) recently described an enrichment that is capable of fermenting phenol by using the reducing equivalents to reduce phenol to benzoate via reductive elimination of the hydroxyl group. Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum has been reported to ferment benzoate (22). These recent findings suggest that fermentation, in addition to syntrophic metabolism, must be considered a possible mechanism for aromatic compound degradation in methanogenic systems.

The ability of S. aciditrophicus to ferment benzoate may have ecological significance since benzoate (or benzoyl coenzyme A) is a central intermediate in anaerobic degradation of many natural and xenobiotic aromatic compounds. Transient accumulation of cyclohexane carboxylate (maximum concentration, 140 µM [representing 28% of the original substrate concentration]) was observed in methanogenic phenol enrichments from landfill sediments (R. Jones and J. M. Suflita, unpublished data). Also, Kleerebezem et al. (17) observed accumulation of cyclohexane carboxylate in anaerobic sewage sludge enrichments amended with benzoate and one of the three phthalate isomers when methanogenesis in these enrichments was inhibited by bromoethanesulfonic acid. This observation implies that cyclohexane carboxylate production could be a mechanism for hydrogen removal in anaerobic ecosystems. Benzoate reduction to cyclohexane carboxylate may be regarded as a survival mechanism used by syntrophic microorganisms in the absence of interspecies hydrogen transfer.

Pure cultures of S. aciditrophicus grown with benzoate accumulated hydrogen at pressures up to 97.9 Pa, compared to the value of 2.3 Pa observed for S. aciditrophicus-M. hungatei cocultures. The higher hydrogen pressure values observed for S. aciditrophicus pure cultures were probably responsible for the higher final benzoate concentrations in these cultures than in a coculture (Table 2). Previous studies showed that hydrogen levels influence the extent of benzoate degradation in syntrophic cocultures, with benzoate metabolism ceasing at higher thresholds in cultures exposed to higher hydrogen levels (11, 13, 25, 32). High levels of hydrogen inhibited benzoate fermentation by S. aciditrophicus pure cultures growing on benzoate (Fig 3) in a manner similar to that observed in S. aciditrophicus cocultures with a hydrogen user. The inhibition of benzoate degradation by high hydrogen levels and the accumulation of hydrogen during benzoate fermentation suggest that hydrogen is produced as a free intermediate during benzoate oxidation by S. aciditrophicus pure cultures.

S. aciditrophicus monoculture was able to metabolize cyclohex-1-ene carboxylate. While only 1 mol of benzoate was reduced per mol of benzoate oxidized to acetate and CO₂, 5 mol of cyclohex-1-ene carboxylate was needed to account for all of the reducing equivalents produced during oxidation of 1 mol of cyclohex-1-ene carboxylate to acetate and CO₂. Therefore, much less energy was available from cyclohex-1-ene carboxylate fermentation than from benzoate fermentation to support growth. Cyclohex-1-ene carboxylate fermentation stopped after only 62.7% of the initial cyclohex-1-ene carboxylate was degraded. This may have been due to accumulation of high levels of hydrogen (839 Pa), which may have inhibited further oxidation of cyclohex-1-ene carboxylate to acetate and CO₂. The G' values for benzoate and cyclohex-1-ene carboxylate metabolism observed in this study (Table 2) indicate that biological reactions can proceed at values close to thermodynamic equilibrium (G' = 0 kJ/mol) rather than at the previously suggested value, 20 kJ/mol (24).