INTRODUCTION

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One of the major xenobiotic compounds degraded in sewage works worldwide is linear alkylbenzenesulfonate (LAS) surfactant, which has an annual production of about 2,500,000 tons (40). Whereas the degradation of LAS is well established (16, 26, 27, 35, 39, 43), the mechanisms of its degradation are not (4).

Commercial LAS (Fig. 1) is, ideally, a mixture of linear alkanes (C₁₀ to C₁₃) subterminally substituted with a single 4-sulfophenyl moiety (31): this means a mixture of 20 compounds, 18 of which are racemic mixtures. Although many different degradative reactions have been deduced from the intermediates which have been observed (38, 43), the general opinion is that the major degradative pathway involves oxygenation of one or both of the terminal methyl groups by an unknown oxygenase (see also references 19 and 20) followed by rounds of -oxidation to yield a large set of transient sulfophenyl(di)carboxylates (Fig. 1) (9, 14, 17, 38, 43, 47, 49). On paper, seven sulfophenyl(di)carboxylates, representing a maximum number of -oxidation steps, can be proposed: 2-(4-sulfophenyl)butyrate (SPB) (which is often observed during the degradation of commercial LAS [e.g., see reference 14]), 2-(4-sulfophenyl)propionate, 2-(4-sulfophenyl)malonate, 3-(4-sulfophenyl)butyrate, 2-(4-sulfophenyl)succinate, 3-(4-sulfophenyl)valerate and 3-(4-sulfophenyl)glutarate.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Presumed major pathway for the conversion of racemic, commercial LAS to racemic sulfophenylcarboxylates, with an example yielding the two enantiomers of SPB, which are converted to 4SC and subject to ortho ring cleavage and desulfonation by D. acidivorans SPB1. The square brackets represent metabolism in another portion(s) of the putative community which degrades commercial LAS (21, 36a, 41). SPB represents one transient intermediate found extracellularly in sewage works, receiving waters, and soil (9, 14, 17). It is then sensible to anticipate the transport of SPB into the cell, because all sulfonates require transport systems (7). The mechanism of conversion of SPB to 4SC is still unknown. SPB is one of the seven smallest sulfophenyl(di)carboxylates which can be derived from LAS.

Many of these sulfophenyl(di)carboxylates are optically active (24), which can be assumed to have consequences for their further degradation (25, 50). SPB is quantitatively converted to cell material, CO₂, water, and sulfate by activated sludge (27), but none of the degradative mechanisms are known (4).

We now describe the isolation of Delftia acidovorans SPB1, which degrades both enantiomers of SPB via 4-sulfocatechol (4SC) and ortho ring cleavage (7, 13).

	MATERIALS AND METHODS

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Materials. Racemic, R- and S-SPB were synthesized by reacting sulfuric acid with the corresponding phenylcarboxylic acid (45). Yields of about 30% were obtained at about 97% purity; the major by-product, 2-(2-sulfophenyl)butyrate, was not totally removed on recrystallization (retention time, about 4 min [Fig. 2]). The identity of the product was confirmed by electrospray mass spectrometry (MS) and nuclear magnetic resonance. The following mass spectral data were collected in the negative ion mode: [M  2H]², m/z 121; [M  H], m/z 243; [M + Na⁺  2H], m/z 265. ¹H nuclear magnetic resonance measurements gave four sets of peaks with chemical shifts downfield of tetramethylsilane: 0.87 ppm (t, 3H), CH₃ (C-4); 1.85 ppm (m, 2H), CH₂ (C-3); 3.47 ppm (t, 1H), CH (C-2); 7.6 ppm (pq, 4H), aromatic protons indicating para substitution of the sulfonate group (34). Synthetic R-SPB was obtained at 96% purity and an enantiomer excess of 96%; S-SPB was 96% pure with an enantiomer excess of 94%.

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Separation of R- and S-SPB from one another, from traces of the impurity, racemic 2-(2-sulfophenyl)butyrate, and from 4SC.

Analytical methods. Racemic SPB was initially detected by reversed-phase high-performance liquid chromatography (HPLC) (8, 32). When it became clear that a chiral separation was required, we chose a -pm-Nucleodex column (5-µm diameter particles; 200-mm column of 4-mm internal diameter; Macherey & Nagel, Düren, Germany). We found 100 mM potassium phosphate buffer, pH 6.0, to give optimal separation and operated the column at 0.5 ml/min at room temperature (about 23°C). The sample volume did not exceed 50 µl. The diode array detector was set to give a chromatogram at 220 nm. R-SPB eluted at 6.5 min with baseline separation from S-SPB (8.4 min) (Fig. 2). The ortho analogues, impurities from the synthesis, eluted at about 4.1 min, whereas 4SC eluted at 4.5 min (Fig. 2). The standard curve for R-SPB, S-SPB, or 4SC was linear to about 40, 40, or 10 nmol/injection, and the limits of detection were about 0.25, 0.25, and 0.05 nmol/injection, respectively. The reproducibility of quantitation (peak area) for the SPB enantiomers was 98.8% (n = 6), and that for 4SC was 97.2% (n = 6). The retention times at constant temperature varied by ±0.05 min.

Enrichment cultures and growth of organisms. Aerobic enrichment cultures were set up with inocula from three activated sludges. Cultures (5 ml) in 30-ml screw-cap tubes were incubated at 30°C on a roller in the dark. The substrate, racemic SPB as the sole source of carbon and energy for growth, was present at 6 mM in a salts medium buffered with 50 mM phosphate, pH 7.2 (46). Cultures were streaked on Plate Count Agar (Difco) after at least three passages in the enrichment medium. Individual colonies were picked and removed to selective liquid medium. Positive cultures were restreaked and subcultured in selective liquid medium. A culture was considered pure when three successive plates were homogeneous and identical. Short-term storage of the organisms was on selective medium solidified with 1.5% agar as slants at 4°C. Long-term storage was in 50% glycerol at 80°C. The organism was deposited with the German Culture Collection under the accession no. DSM 12586.

Cell extracts. Cells in the mid-exponential phase of growth with R-SPB in the racemic mixture (or sulfanilate) were harvested, washed twice in 50 mM potassium phosphate buffer, pH 7.2, and resuspended for cell rupture in a French press as described elsewhere (23). Some experiments to monitor the disappearance of 4SC were done in 1-ml reaction mixtures in a 1-cm light path quartz cuvette in a Uvikon 922 spectrophotometer, and UV spectra from 200 to 400 nm were taken at intervals. The reaction mixture (13), which contained 0.3 mg of protein (crude extract, supernatant fluid), was buffered with 50 mM Tris-HCl, pH 8.0, and the reaction was started by the addition of 150 nmol of 4SC. In some experiments, samples were taken at intervals and examined by HPLC.

	RESULTS

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Enrichment cultures, isolation, characterization, and identification of D. acidovorans SPB1. Enrichment cultures for organisms which could utilize racemic SPB (three of five positive) were obtained easily. Growth (turbidity) of the first culture within 1 week was normal, and subcultures usually grew overnight. Three mixed cultures, SPB1 (from Radolfzell), SPB2 (from Konstanz), and SPB3 (from Ludwigshafen), were essentially homogeneous (by light microscopy) and comprised largely a single bacterial morphotype with some protozoan grazers. Strain SPB1 was readily isolated from the mixed culture of the same name. Isolation of a bacterium from cultures SPB2 and SPB3 was not achieved.

Growth characteristics of D. acidovorans SPB1. Preliminary growth experiments in racemic SPB-salts medium were done with suboptimal aeration. The culture developed a transient pink or purple color for several hours, during which time the R enantiomer disappeared. The S enantiomer then largely disappeared without significant growth. The coloration was interpreted as the iron complex of a catechol (12), and chromatography on the chiral column yielded a peak at 4.2 min, which coeluted with authentic 4SC and had the same UV spectrum as the standard (pH 6; see the legend to Fig. 4). Cochromatography of the unknown with 4SC was also observed on a reversed-phase column, where the (different) UV spectrum at pH 2 was identical for the unknown and the standard. A portion of the unknown was separated by HPLC and collected, and its UV spectrum was analyzed at pH 13. We thus have five properties which together identify the unknown as 4SC.

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1

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Growth of D. acidovorans SPB1 with racemic SPB. Growth is shown in a conventional semilogarithmic plot (OD, optical density) (a) and in a linearized plot of the concentrations of substrates and products as a function of growth (b). Turbidity, ; R-SPB, ; S-SPB, ; 4SC, ; sulfate, .

Ring cleavage of 4SC in crude extracts. Extracts of cells growing with R-SPB degraded 4SC (Fig. 4). No yellow color, which might indicate a meta cleavage, was detected, so we explored the possible presence of the ortho cleavage pathway, which was established by Feigel and Knackmuss (13). The pattern of biotransformation of 4SC (Fig. 4) is identical with that previously observed by Feigel (11). Samples taken during similar reactions and examined by reversed-phase HPLC showed the disappearance of the 4SC peak (at 2.2 min) and the appearance of a novel peak (at 1.9 min). In acidified samples, the novel peak disappeared and was replaced by another peak (at 2.1 min). This resembles the description of Contzen et al. (2), who found the elution of 3-sulfo-cis,cis-muconate prior to that of 4SC during reversed-phase HPLC and, after acidification, the spontaneous formation of the corresponding lactone, which had a longer retention time. The short retention times and interference from material in the enzyme assay made the measurement of UV spectra unsatisfactory, so we repeated the analyses on the chiral column. Our putative 3-sulfo-cis,cis-muconate coeluted (at 3.4 min) with authentic material from extracts of H. palleronii S1 and A. radiobacter S2, and both samples had the same UV spectrum in the diode array detector (a single maximum at 206 nm). Our putative lactone coeluted (at 4.1 min) with authentic material generated from the authentic sulfomuconate, and both samples had the same UV spectrum (with a maximum at 215 nm).

View larger version (18K): [in this window] [in a new window]   FIG. 4.   UV spectra of the transformation of 4SC by crude extract of D. acidovorans SPB1. Spectra were taken at intervals; arrows pointing up or down indicate whether the absorption was increasing or decreasing. These conditions mask some of the spectral properties of 4SC. In our hands, 4SC at pH 2 displayed a maximum at low wavelengths (min, 217 nm; max, 233 nm; min, 255 nm; max, 279 nm). At pH 6 the maximum at low wavelengths was accompanied by values of min, 226; max, 234; min, 267; and max, 282 nm. At pH 13 the maximum at low wavelengths was accompanied by values of min, 243 nm; max, 266 nm; min, 285 nm; and max, 303 nm, similar to published data (48).

Substrate range of D. acidovorans SPB1. Strain SPB1 was able to grow with 4-hydroxybenzoate (6 mM) and 3,4-dihydroxybenzoate (3 mM) but not with 4-methylbenzoate (3 mM). The organism was found to utilize SPB, 4SC (3 mM), and 4-(1-octyl)benzenesulfonate (1 mM). There was no growth with 4-hydroxybenzenesulfonate (3 mM), 4-carboxybenzenesulfonate (3 mM), 4-methylbenzenesulfonate (3 mM), or commercial LAS (0.5 mM). With commercial LAS, the further addition of 4-hydroxybenzoate allowed growth but did not alter the foaming of the LAS, which obviously was not degraded.

	DISCUSSION

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The worldwide degradation of SPB, an intermediate from the degradation of LAS, has been implicitly recognized for many years (e.g., see references 17 and 43) and demonstrated directly in material from sewage works (e.g., see reference 27), so the isolation of a bacterium, D. acidovorans SPB1, able to degrade the compound is not surprising. What has been less widely considered is the importance of the chirality of molecules like SPB (Fig. 1 and 3), as emphasized by Kohler and coworkers (24, 50). Given that the subterminal substitution of alkanes is normal not only in LAS surfactants but also in the widespread secondary alkanesulfonate surfactants (30), the supposedly facile degradation of common household products is more complex than previously realized.

Growth of D. acidovorans SPB1, certainly with the R enantiomer, gives a normal value for the molar growth yield, which means a mass balance for carbon (see reference 3) as well as a mass balance for the sulfonate moiety (Fig. 3b). Utilization of the second enantiomer was initially not reproducible, and we attribute this to poor aeration, because improved aeration of flask cultures allowed full utilization of the second enantiomer with mass balance for the sulfonate moiety as sulfate (Fig. 3b). The growth yield with the second enantiomer was lower than that with the first, but as there was negligible residual carbon in the medium at the end of growth we presume the difference in yields to be facile, for example due to the increased contribution of maintenance energy to the energy budget at the lower growth rate (1). We also presume that an oxygenase attacking, e.g., S-SPB has a poor K_m for oxygen.

The growth yield, especially with R-SPB, indicates that all the carbon atoms in the compound are utilized for growth. Thus the side chain, as well as the ring, must be processed through the amphibolic pathways to generate carbon skeletons for biosynthesis and conservation of energy. We have, as yet, no information on the pathway involved in utilization of the side chain.

We presume the degradation of R- and S-SPB to be initiated by transport of these charged compounds across the cell membrane (see reference 7), and we hypothesize one transport system per enantiomer (see reference 50). The metabolism of the enantiomers converges at 4SC, because this compound is observed as a transient intermediate during growth with R-SPB (Fig. 3b) and in suspensions of cells from cultures utilizing S-SPB. We do not yet know how these compounds are converted to 4SC, whether in one novel dioxygenation or in several steps. We do, however, know that 4SC is degraded via the ortho cleavage pathway established for sulfanilate and benzene 1,3-disulfonate (2, 13) (Fig. 4). This new example of 4SC functioning as a point of convergence of metabolic pathways and as the substrate for ring cleavage points out the general importance of the previous work (2, 13), especially if the degradation of LAS is always channeled through this pathway.

This is the first pathway for the degradation of sulfophenylcarboxylate(s) from commercial LAS to be proposed with direct evidence from work with a pure culture. Many hypothetical pathways can be found in the literature (e.g., see references 37, 38, 43, 47, and 49), but none involves 4SC. When more isolates are available, we will be able to see whether any of the old hypotheses are correct.

One major dichotomy found in the literature on LAS is whether pure cultures can degrade LAS (49) or whether communities are needed (21, 41; see also reference 19). We suspect the difference lies in the LAS used. Commercial LAS is complex (see the introduction and Fig. 1) and is used by those who have found communities to be needed for complete degradation (10, 21, 41). Model LAS is commercially available as single compounds [e.g., 1-(4-sulfophenyl)octane], each a terminally substituted alkane, whereas commercial LAS is always subterminally substituted (Fig. 1). The published work which illustrates pure cultures (49) seems to involves model LAS. The isolate used in this paper, D. acidovorans SPB1, utilizes model LAS but not commercial LAS. So, we presume that the metabolism of model LAS need not represent metabolism of commercial LAS. We have now isolated a heterotrophic bacterium which utilizes commercial LAS and concomitantly excretes sulfophenylcarboxylates of intermediate length (36a). Hrsak and Begonja (19), with a methanotroph catalyzing the oxygenation of commercial LAS to a sulfophenylcarboxylate of the same chain length, required a tier of heterotrophic organisms to generate compounds analogous to SPB. We presume that communities are generally required to degrade commercial LAS completely, as indicated elsewhere (21, 41).

The organism we isolated to degrade SPB, D. acidovorans SPB1, represents organisms widespread in soil and water, so the degradative pathway could be widespread in this species, or perhaps it is readily spread by horizontal gene transfer. Strain SPB1 has, however, a narrow substrate range for sulfophenylcarboxylates. Many intermediate mono- and dicarboxylated sulfophenyl moieties can be expected from LAS (9, 14, 17), yielding theoretically seven sulfophenylcarboxylates of minimal length, so if only organisms with a narrow substrate range degrade these compounds, the complexity of communities degrading LAS must be considerable. There are three pathways known to degrade 4-toluenesulfonate (7), so given that a higher tonnage of LAS is degraded annually (see the introduction), it is perfectly possible that other ring cleavage pathways are also involved.