TEXT

Top Abstract Text References

Alkylbenzene sulfonates are the most commonly used surfactants in domestic detergent formulations (8). In the United States and Europe, linear alkylbenzene sulfonates (LAS) have been used since the early 1960s, when the low rate of biodegradation of branched-chain alkylbenzene sulfonates (BAS) was recognized (2, 4, 5, 8). In some Latin American countries, BAS are currently used in different detergent formulations due to their low costs. Water pollution by BAS is a significant environmental problem in these countries.

A Pseudomonas aeruginosa strain (W51D) which is able to mineralize at least 70% of a BAS commercial mixture and completely degrade LAS has been isolated (17). This strain is resistant to high concentrations of these surfactants (17). P. aeruginosa W51D is the only reported bacterium able to mineralize BAS at a significant rate. LAS-degrading Pseudomonas strain C12B barely degrades BAS (4, 11, 19).

LAS are completely degraded in wastewater treatment plants, and different organisms participate in their mineralization, each degrading a part of the molecule. A four-member consortium was identified as responsible for LAS mineralization (9), and a larger consortium was found to be involved in mineralization in a marine environment (15). In these consortia, some members attacked the side chain, while others degraded the aromatic moiety. So far, no consortium that is able to efficiently degrade BAS has been described. The low rate of BAS biodegradation is due to the presence of highly branched alkyl groups (2, 18). Branched alkanes are generally less susceptible to biodegradation than n-alkanes and certain methyl-branched alkanes. Special 3-methyl-branched and quaternary-substituted alkyl chains can result in environmental recalcitrance (2, 7, 12-14, 18).

Some Pseudomonas strains degrade branched alkanes and alkenes. Citronellol (3,7-dimethyl-6-octen-1-ol) has been used as a model compound to study the route of degradation of branched alkenes (7, 13, 14). It has previously been reported that P. aeruginosa W51D is able to use citronellol as a sole carbon source (17). The characterization of a ctrA mutant derived from strain W51D, which has a low citronellal dehydrogenase activity (6), has also been previously reported.

The aim of this work was to contribute to the elucidation of the branched-chain dodecylbenzene sulfonate (B-DBS) degradation pathway of P. aeruginosa W51D and to evaluate the contribution of the reported route to citronellol degradation. We present evidence showing that the citronellol pathway is not the only route involved in the degradation of the surfactant alkyl lateral chain. Identification of the main B-DBS isomer structures and the direct identification of some of their degradation intermediates suggest that strain W51D completely assimilates the surfactant lateral chain prior to the aromatic ring cleavage.

Identification of the isomers present in the B-DBS mixture. The substrate used for P. aeruginosa W51D degradation studies, B-DBS, was purified by high-performance liquid chromatography (HPLC) from a commercial BAS preparation as a single peak on a semipreparative (250- by 22-mm) Econosil C₁₈ column (Alltech Associates Inc.) by treatment with 60:40 H₂O-acetonitrile (ACN) for 10 min, followed by a linear gradient reaching pure ACN in 5 min; this solvent was kept for an additional 2 min. The flow rate used was 5 ml/min, and the elution profile was monitored as described above. The B-DBS mixture obtained from HPLC was desulfonated by reflux treatment in the presence of phosphoric acid as previously described (18) and was injected onto a gas chromatograph coupled with mass spectrometry (MS). The desulfonated B-DBS mixture showed the existence of multiple isomers. The gas chromatography-MS (GC-MS) analysis of 11 of the branched-chain isomers showed that all of them had a molecular weight (M⁺, molecular ion) of 246 m/z (Table 1). The mass spectrum analysis showed an important ion peak of 91 m/z, indicating the C₆H₅CH₂⁺ ion. The electron impact fractionation also showed a branched nature of the alkyl moiety. Upon electron impact, saturated hydrocarbons fragment preferentially at the branching points; the positive charge remains on the more highly substituted carbon atom, and elimination of the longest carbon chain is favored. The absence of an (M-15)⁺ ion in the mass spectrum of the alkylbenzenes is not surprising, although there are many methyl groups present. The methyl radical is the least stable of the alkyl radicals and will not be eliminated readily if other fragmentations are facile. The analytical techniques used in this work do not allow the determination of the positions of both the methyl groups and phenyl moiety. The suggested structures of the alkyl chains of the five most abundant B-DBS isomers are shown in Fig. 1.

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the proposed degradation route of B-DBS (A) and 4-hydroxyphenylpropionate (B) by P. aeruginosa W51D. The numbers in parentheses correspond to the compound numbers in Tables 1 and 2, where the MS results are given. The structures of the alkyl lateral chains of the most abundant B-DBS isomers are shown as R groups. TCA, tricarboxylic acid cycle.

Participation of the citronellol pathway in B-DBS degradation. Pseudomonas degradation of citronellol (12) is the only enzymatic pathway characterized in bacteria for the assimilation of methyl-ramified alkanes so far. The isolation and characterization of a P. aeruginosa W51D mutant (W51M1) unable to degrade citronellol due to the reduced activity of the enzyme citronellal dehydrogenase have previously been described (6). To determine whether the citronellol pathway was used by strain W51D to degrade the B-DBS alkyl chain, we studied the ability of mutant W51M1 to assimilate the surfactants. We found that even though mutant W51M1 is unable to use citronellol as a carbon source, it is still able to grow on M9 plus B-DBS (0.2% [wt/vol]). Quantitative determination of B-DBS degradation by strain W51M1 was obtained from HPLC analysis of culture supernatants, in which the extent of B-DBS consumed by this mutant on M9 plus glucose (0.2% [wt/vol]) plus B-DBS was estimated after 48 h of growth. The consumption by strain W51M1 was reduced by 40% compared to that by the wild-type strain W51D. Even though the total amount of surfactant consumed on this medium was reduced, we could not detect differences in the relative proportion of the accumulated intermediates. These results show that the enzymes involved in citronellol degradation could also be involved in B-DBS degradation, presumably at the first steps of the catabolism, but it is apparent that this pathway is not the main route of B-DBS degradation by strain W51D.

Analysis of the W51D pathway for B-DBS degradation. To obtain information on the main W51D route for B-DBS degradation, we determined the structure of the degradation intermediates by analyzing the 72-h culture supernatants of W51D cells grown on M9 plus B-DBS by GC-MS. These supernatants were acidified with HCl to pH 3 to eliminate the extracellular matrix of the biofilm formed in this medium. The acidified supernatant was centrifuged, the supernatant was extracted three times with ethylacetate, the organic fraction was concentrated by evaporation, and the extracted product was dried with N₂. The dried product was resuspended in methanol at a final concentration of 1 mg/ml. Samples were silylated prior to GC-MS analysis with ClSi(CH₃)₃ (Sigma) according to the manufacturer's instructions. One microliter of the compound solutions separated by HPLC or directly silylated after being concentrated was injected onto a gas chromatograph coupled with an MS detector (Hewlett-Packard HP6890 GC-MS system). Table 2 shows the MS data of these identified metabolites.

W51D pathway for the degradation of 4-hydroxyphenylpropionate. Considering that 4-hydroxyphenylpropionate is a B-DBS catabolic intermediate, we studied the W51D route for the degradation of this compound as a way to analyze further B-DBS catabolism by strain W51D. The proposed route of W51D degradation of 4-hydroxyphenylpropionate (Fig. 1) was elucidated by using this compound as a carbon source and by identifying the intermediates (Table 2) by GC-MS analysis, as described above. The degradation intermediates were purified by HPLC as follows: 50 µl of the concentrated supernatant or of the supernatant of strain W51D grown for 48 h on 4-hydroxyphenylpropionate was injected on a Nova-Pak C₁₈ 3.9- by 150-mm reverse-phase column (Waters), and the elution solvent was H₂O-ACN-acetic acid 75/4/1, vol/vol/vol for 3 min, followed by a linear gradient to reach 100% ACN in 6 min. The flux used was 1 ml/min, and the elution was monitored at 254 nm with a Hewlett-Packard 1050 diode array UV detector. We found, in accordance with the results obtained by studying B-DBS degradation, that the propionate moiety of this molecule was completely oxidized and degraded to 4-hydroxybenzoate prior to the aromatic ring cleavage. This is unusual, since most bacteria cleave the aromatic ring of short-chain alkylbenzenes without previous oxidation of the alkyl chain (16).