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Previous Article | Next Article 
Applied and Environmental Microbiology, January 2002, p. 31-36, Vol. 68, No. 1
0099-2240/02/$04.00+0 DOI: 10.1128/AEM.68.1.31-36.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
School of Biosciences, Cardiff University, Cardiff CF10 3US,1 SEAC Environment Centre, Unilever Research Port Sunlight, Bebington, Wirral, Merseyside L63 3JW, United Kingdom2
Received 18 April 2001/ Accepted 2 October 2001
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Anionic surfactants are discharged after use in large quantities to
waterways, wastewater treatment works, and marine and estuarine
environments (10). The
biodegradability of alkyl sulfate surfactants in such environments is
well established (26). All commercially important alkyl sulfate
structures so far studied are readily biodegradable. Studies on the
mechanisms underlying the biodegradability of alkyl sulfate
surfactants so far have focused mainly on the LPAS. As a result, the
metabolic pathways and enzymologic characteristics in the bacteria
involved are well understood. For example, the biodegradation of LPAS
in Pseudomonas sp. strain C12B is initiated by two
alkylsulfatases, designated P1 and P2 on the basis of electrophoretic
mobility. The liberated long-chain primary alcohols are subsequently
oxidized by alcohol dehydrogenase; for a review, see reference
9. The P1 and P2 enzymes
have been purified and characterized in detail. While they have the
same exclusive specificity for LPAS, they are distinguishable on the
basis of the mechanism of C
OS ester bond cleavage
(OS and CO for P1 and P2, respectively) and the regulation
of synthesis (P1 is constitutive and P2 is inducible) (2, 6, 7). Alkylsulfatase activity has also been found
in Pseudomonas aeruginosa (11) and Aerobacter aerogenes
(24). Comamonas
terrigena produces two secondary alkylsulfatases (CS1 and CS2)
active on various secondary alkyl sulfates (19).Pseudomonas putida FLA produces
six alkylsulfatases, three of which are primary alkylsulfatases (FP1
to FP3) and three of which are secondary alkylsulfatases (FS1 to FS3)
(17).
In contrast to that of LPAS, the biochemistry of the biodegradation of BPAS is less well documented. During the period from 1950 to 1970, poor biodegradability of highly branched tetrapropylene benzene sulfonate surfactants caused significant problems of foaming in receiving waters and wastewater treatment plants (26). Elimination of the problem by switching to linear alkylbenzene sulfonates led to the tacit assumption that any branching in the hydrophobic part would result in poorly degraded and therefore environmentally unacceptable products. Recently, however, this assumption has been successfully challenged in studies which showed that alkyl sulfates with either a small number of methyl branches along the hydrophobic chain (25) or 2-alkyl branches (4) were as rapidly and completely biodegraded as their linear counterparts. However, nothing is known about the biochemical basis of the biodegradation of this group of anionic surfactants, in particular, whether they serve as substrates for existing known alkylsulfatases or whether novel enzymes are involved.
This article describes the first isolation of bacteria capable of utilizing 2-butyloctyl sulfate as a sole source of carbon and the discovery in cell extracts of new alkylsulfatases with novel substrate specificities.
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MATERIALS AND METHODS |
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Pure 2-butyloctanol and an aqueous saturated solution-crystal suspension of 2-butyloctyl sulfate were kindly provided by Condea Chemicals. Based on the smanufacturer’s analysis, the latter contained 2-butyloctyl sulfate (28.8% [wt/vol]), sodium chloride (0.6% [wt/vol]), sodium sulfate (0.8% [wt/vol]), and unsulfated organic matter (0.8% [wt/vol]).
Purification and analysis of 2-butyloctyl sulfate.
The commercial preparation of 2-butyloctyl sulfate was thoroughly mixed and diluted with an equal volume of distilled water. The suspension was then heated to 45°C with stirring until a clear solution was obtained. Subsequent slow cooling to 5°C yielded crystals that were filtered on a Buchner filter (no. 1 paper; Whatman International, Maidstone, United Kingdom) under reduced pressure (water pump) at 5°C. The "sludge" remaining after filtration was washed with acetone and petroleum ether and dried in a rotary evaporator to yield alkyl sulfate crystals.
Infrared spectra of 2-butyloctyl sulfate (as a Nujol mull) and 2-butyloctanol (pure liquid) were determined by using a Perkin-Elmer 257 grating infrared spectrometer. The peak at 1,060 to 1,110 cm–1 in the parent alcohol, corresponding to C·OH vibrations, was absent in the alkyl sulfate and was replaced with O·S and C·O·S vibrations at 1,200 to 1,250 cm –1, 1,065 cm–1, and 810 cm–1. The water-recrystallized alkyl sulfate, after drying, showed a broad peak centered at 3,560 cm–1, indicating water of crystallization. Carbon and hydrogen analysis (performed at Warwick Analytical Service, Department of Chemistry, Warwick University) indicated a carbon content of 46.82% and a hydrogen content of 8.49% (compared to theoretical contents of 47.06 and 8.82%, respectively, based on the structure C12H25OSO3–Na+· H2O). Gravimetric analysis of duplicate samples for sulfate as BaSO4 indicated the sulfate contents of the ester to be 35.7 and 34.0% (theoretical content, 31.4%). 13C and1H nuclear magnetic resonance (NMR) spectra were consistent with literature values for particular resonance signals (20) and the spectra predicted for the 2-butyloctyl sulfate structure with ChemDraw C NMR software (CambridgeSoft Corporation, Cambridge, Mass.). For the 13C spectra, the peak at 71.7 ppm, the peak at 37.3 ppm, two peaks at 13.67 to 13.72 ppm, and eight peaks at 30.48 to 22.67 ppm were assigned to the ester CH2·O·S, the branch point C-H, two CH3 units, and eight CH2 units, respectively. For the1H spectra, the peaks at 3.7, 1.5, 1.1, and 0.7 ppm were assigned to the ester-linked CH2·O·S hydrogen atoms, the branch point C·H hydrogen atom, 16 hydrogen atoms from the eight CH2 methylene groups in the carbon chain, and 6 hydrogen atoms from the two terminal CH3 groups.
The critical micelle concentration for 2-butyloctyl sulfate in 50 mM Tris-HCl (pH 7.5) was determined by using a modification of the method of Carey and Small (5) which exploits spectral changes of rhodamine 6G upon its incorporation into micelles. The critical micelle concentration was determined to be 100 µg/ml (0.32 mM).
Isolation of bacteria.
Bacteria capable of utilizing 2-butyloctyl sulfate as a sole source of carbon were isolated from activated sewage sludge (from Coslech Sewage Works, Cardiff, Wales) by enrichment in basal salts (BS) medium containing (grams per liter) the following: K2HPO4, 3.5; KH2PO4, 1.5; NH4Cl, 0.5; MgCl2 · 6H2O, 0.15; NaCl, 0.5; and Na2SO4, 0.14. The medium also contained the following trace elements (1 ml of stock) (grams per liter): FeCl3 · 6H2O, 0.24; CoCl2 · 6H2O, 0.04; CuSO4 · 5H2O, 0.06; MnCl2 · 4H2O, 0.03; ZnSO4 · 7H2O, 0.31; and Na2MoO4 · 2H2O, 0.03. The medium also was supplemented with 1 g of 2-butyloctyl sulfate/liter to give a final carbon concentration of 40 mM (3.3 mM substrate). The cultures were incubated at 30°C with shaking at 150 rpm. After 6 days, 1 ml of culture was transferred to 100 ml of fresh medium. After five serial subcultures, a 1-ml sample was taken from the final enrichment flask, serially diluted, and plated on nutrient agar plates. The plates were incubated for several days at 30°C until colonies were visible. Single colonies were plated to purity and characterized by their morphology and Gram classification (23). Pure strains were tested for the ability to grow axenically on 2-butyloctyl sulfate.
16S rRNA analysis.
Cells from overnight nutrient broth cultures were harvested from 1 ml of culture by centrifugation at 17,900 x g for 3 min. The cells were washed in 1 ml of sterile distilled water, resuspended in 100 µl of sterile distilled water, and boiled for 5 min. The supernatant (0.2 µl) containing template DNA was mixed with 2.5 µl of 10x PCR buffer, (0.5 M KCl, 0.1 M Tris HCl, pH9, at 25°C, 1% [vol/vol] Triton X-100), 1.5µl of MgCl2 (25 mM), 18.25 µl of ultrapure water (MilliQ), and 0.25 µl of Taq polymerase. PCR primers 63f (5'-CAG GCC TAA CAC ATG CAA GTC-3') and 1387r (5'-GGG CGG WGT GTA CAA GGC), designed and evaluated by Marchesi et al. (18), were used for the amplification of 16S rRNA genes. The PCR program, performed by using an MWG-Biotech Primus thermal cycler, consisted of 5 min at 96°C for 5 min; 30 cycles of 96°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and an additional extension step of 5 min at 72°C. The PCR products were purified by using a QIAquick PCR purification kit (Qiagen) in accordance with the manufacturer’s instructions and were separated on agarose gels. The DNA sequence was determined by using an ABI PRISM dRhodamine terminator cycle sequencing ready reaction kit (Perkin-Elmer Applied Biosystems, Warrington, Cheshire, United Kingdom) in accordance with the manufacturer’s instructions. Sequences were compared to known 16S rRNA gene sequences in the composite nonredundant database (includes GenBank, EMBL, DDBJ, and PDB) by using the BLAST search program (1).
Maintenance and growth of bacterial strains.
Bacterial strains were maintained on nutrient agar slopes at room temperature for routine use or stored at –20°C in PROTECT vials (beads in a cryopreservative fluid; Technical Service Consultants Ltd., Heywood, Lancs, United Kingdom) for long-term storage. To initiate batch cultures, single colonies from nutrient agar plates were inoculated into nutrient broth, and culture flasks were incubated aerobically at 30°C with shaking (150 rpm). After 18 h, cells were harvested from 1 ml of culture, washed by centrifugation, and resuspended in 1 ml of BS medium. The cell suspensions were used to inoculate (1% [vol/vol]) flasks containing BS medium and an appropriate quantity of alkyl sulfate, and the flasks were incubated as before. Growth was measured as the optical density at 540 nm by use of a Thermomax Maxline microplate reader (Molecular Devices, Wokingham, United Kingdom). In anticipation that biodegradation might produce the accumulation of insoluble intermediates (e.g., 2-butyloctanol), samples removed from flasks for growth measurements were each mixed with an equal volume of methanol to solubilize such compounds before optical densities were measured. Controls with cells grown in nutrient broth (i.e., no insoluble alcohol production) indicated that this dilution had no effect on cells other than the expected twofold reduction in cell density, and this factor has been taken into account in the data presented.
Preparation of enzyme extracts.
Bacterial cells were harvested by centrifugation of the cultures at 10,000 xg for 20 min at 4°C in a Sorvall RC-5B Superspeed centrifuge (DuPont Company, Wilmington, Del.). Cell pellets were resuspended in 10 mM Tris-HCl (pH 7.5) (1/50 the original volume). The cells were ruptured by sonication for a total duration of 3 min, consisting of intermittent sonication for 30 s on and 30 s off, by using a Lucas Dawe sonicator with a Micro-tip probe (Branson Technology) operated at level 5 and a 25% duty cycle. To minimize heat inactivation of enzymes, samples were sonicated in thin-glass vials kept on ice. Cell debris was removed by centrifugation at 12,000 x g for 40 min at 4°C in a Sorvall RC-5B Superspeed centrifuge. The cell extracts were stored at –20°C prior to being assayed for alkylsulfatase activity.
Polyacrylamide gel zymography.
To determine the multiplicity of alkylsulfatase activities in cell extracts, polyacrylamide gel electrophoresis (PAGE) was carried out by using 7% polyacrylamide rod gels prepared in 0.378 M Tris-glycine buffer (pH 8.3) and cast in glass tubes (80 mm by 20 mm [internal diameter]) (22). Per rod gel, 150 µl of sample was applied, and electrophoresis was conducted with a current of 2 mA. Stock running buffer containing 0.25 M Tris and 14.4 M glycine was diluted 1:50 (vol/vol) prior to use. Extruded gels were incubated in developing solution containing 20 mM appropriate alkyl sulfate (either 2-butyloctyl sulfate or SDS) in 0.1 M Tris-HCl (pH 7.5). Locations of active alkylsulfatases were revealed by the formation of white bands of insoluble alcohol droplets.
MBAS assay.
The concentrations of anionic surfactant (2-butyloctyl sulfate and SDS) were determined by the methylene blue active substance (MBAS) assay as described by Hayashi (13) with minor modifications. The method is based on the formation of a complex between the anionic surfactant and an excess of the cationic dye methylene blue, followed by extraction of the complex (but not excess dye) into chloroform and measurement of the absorbance of the blue chloroform layer. A stock solution of methylene blue (0.0625 g in 100 ml of distilled water) was underlaid with 10 ml of chloroform to remove any chloroform-extractable impurities and was stored in a brown bottle to minimize photochemical degradation. Aliquots of methylene blue solution (0.1 ml), 0.4 ml of 0.825 mM phosphate buffer (pH 7.2), and an appropriate volume of sample (up to 1 ml, in triplicate) were mixed in acid-washed, optically matched glass tubes (12 cm by 1 cm [internal diameter]) and vortexed intermittently five times for 3 s each time. Chloroform was added to each tube, and the contents were vigorously vortexed for 5 s. The tubes were allowed to stand at 4°C for 5 min, followed by centrifugation in an MSE Centaur 2 centrifuge at 2,000 rpm for 4 min. The tubes were allowed to warm to room temperature, and the absorbance of the chloroform layer was measured at 655 nm, against an appropriate blank, by using an LKB Novaspec spectrophotometer. Calibration curves were prepared by using standard solutions (based on weight) of pure SDS or pure 2-butyloctyl sulfate.
Gas chromatographic detection of 2-butyloctanol.
Native proteins in cell extracts of the selected isolate,Pseudomonas strain AE-D, were separated by PAGE on duplicate rod gels. The gels were incubated in a 20 mM solution of 2-butyloctyl sulfate (in 50 mM Tris-HCl [pH 7.5]) which previously had been extracted repeatedly with redistilled petroleum ether (60 to 80°C bp) in order to remove any traces of 2-butyloctanol. From each rod, the section of gel containing the single opaque white band indicative of DP1 alkylsulfatase activity was excised and washed three times with distilled water. The duplicate pieces were crushed together in 1 ml of ethanol by using a glass rod, and the mash was vortexed repeatedly for 3 min. Petroleum ether (2 ml) was added, and the mixture was intermittently vortexed for 4 min and then centrifuged (4,000x g, 5 min) to separate the layers. The top layer (containing petroleum ether and extracted material) was removed and treated with anhydrous sodium sulfate to remove residual water. For the control, the above procedure was repeated, except that no protein was included in the tracker dye sample loaded onto the gel prior to electrophoresis, incubation with substrate, and extraction of the corresponding section of the gel. Samples were analyzed for 2-butyloctanol by using a Perkin-Elmer 8410 gas chromatography system fitted with a flame ionization detector and a glass column (2 m by 2 mm) packed with 10% SP-2330. Operating conditions were as follows: oven temperature, 140°C; injection temperature, 250°C; detector temperature, 250°C; and N2 carrier gas flow rate, 20 ml/min.
Alkylsulfatase assay.
Cell extracts (up to 0.5 ml, in duplicate) were incubated (30°C) with 0.5 ml of 2-butyloctyl sulfate at a final concentration of 50 µg/ml (0.16 mM) in 50 mM Tris-HCl (pH 7.5) and with an appropriate volume of 10 mM Tris-HCl (pH 7.5) buffer to bring the initial assay volume to 1 ml. Samples (100 µl) were removed at intervals and assayed for residual alkyl sulfate by using the MBAS method. Total enzyme activity was determined from initial rates of surfactant disappearance. One unit of enzyme activity was defined as the amount of enzyme which converted 1 µmol of substrate per minute under the assay conditions.
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RESULTS |
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Analysis of 16S rRNA gene sequences in isolate AE-A showed 99% sequence similarity to five sequences in the database, three of which were unidentified or uncultured strains and two of which were Pseudomonas spp. Of the 100 BLAST sequence matches, all of which showed between 96 and 99% sequence similarity, 94 were of the genus Pseudomonas. Analysis of the 16S rRNA gene in isolate AE-D showed 95% sequence similarity to five different sequences, each of which was of the genus Pseudomonas and two of which were Pseudomonas putida. As for isolate AE-A, a high proportion (94 of the 100) of the BLAST sequence matches were Pseudomonas spp. Isolates AE-A and AE-D were hereafter designated Pseudomonas sp. strain AE-A and Pseudomonas sp. strain AE-D, respectively.
Growth on surfactants.
Figures 1, 2, and 3 depict bacterial growth and surfactant disappearance in batch cultures for Pseudomonas sp. strain AE-A utilizing SDS, Pseudomonas sp. strain AE-A utilizing 2-butyloctyl sulfate, and Pseudomonas sp. strain AE-D utilizing 2-butyloctyl sulfate, respectively. For each, bacterial growth coincided with surfactant disappearance. Pseudomonas sp. strain AE-D produced a flocculated culture after 20 h, making measurements of growth unreliable for older cultures.Pseudomonas sp. strain C12B, originally isolated for its ability to grow on SDS (21), did not grow in batch cultures with 2-butyloctyl sulfate as the sole carbon source under the conditions used.
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Depending on growth substrate and culture age, Pseudomonas sp. strain AE-A produced up to three primary alkylsulfatases (AP1, AP2, and AP3; Fig. 4 and Table 1). The AP1 band was revealed by incubating gels with either SDS or 2-butyloctyl sulfate. AP1 was observed throughout batch culture growth in nutrient broth as a well-defined band with an electrophoretic mobility of 0.27 relative to that of bromophenol blue. When strain AE-A was grown at the expense of SDS, the enzyme was detectable only in cells in stationary phase (Fig. 1). In contrast, when strain AE-A was grown on 2-butyloctyl sulfate, it produced the enzyme throughout batch culture growth (Fig. 2).
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A third, SDS-inducible alkylsulfatase (AP3) was observed in zymograms of extracts of Pseudomonas sp. strain AE-A throughout batch culture growth on SDS as the sole carbon source (Fig. 1). The enzyme was observed as a band with a relative mobility of approximately 0.55. It was not detected when isolate AE-A was grown at the expense of either nutrient broth or 2-butyloctyl sulfate. The AP3 band was observed when gels were incubated with SDS but not with 2-butyloctyl sulfate and thus appeared to be inducible by and specific for SDS.
Pseudomonas sp. strain AE-D grown on 2-butyloctyl sulfate produced a single alkylsulfatase which was present on gels as a band with a relative mobility of approximately 0.27 (DP1; Fig. 4) in samples taken throughout batch culture growth (Fig. 3). DP1 was not detected when isolate AE-D was grown in nutrient broth. The DP1 band appeared when the strain was incubated with 2-butyloctyl sulfate but not with SDS. This enzyme thus appeared analogous to AP2 in being inducible by and specific for 2-butyloctyl sulfate.
Extracts of Pseudomonas sp. strain C12B cells grown to late exponential phase on SDS-supplemented BS medium in batch cultures produced the anticipated (8) P1 and P2 bands when gels were incubated with SDS. When the same cell extracts were separated on gels and the gels were incubated with 2-butyloctyl sulfate instead of SDS, no alkylsulfatase bands were detectable.
Alkylsulfatase activity in cell extracts.
Demonstration of 2-butyloctyl sulfate sulfatase activity in cell extracts was achieved by incubating extracts of Pseudomonas sp. strain AE-D (containing the single DP1 enzyme) with 2-butyloctyl sulfate and measuring the rate of loss of the substrate. The disappearance of 2-butyloctyl sulfate was linear with time under the assay conditions until at least 50% of 2-butyloctyl sulfate had been degraded. Rates measured from these linear portions were used to assay relative enzyme concentrations. Preliminary experiments indicated that enzyme activity was proportional to the amount of extract used in the assay. Figure 5 shows the activity of DP1 in extracts at various stages of batch culture growth. Total 2-butyloctyl sulfate sulfatase activity (units per milliliter) increased with increasing culture density up to the onset of stationary phase, at which time the disappearance of 2-butyloctyl sulfate from the growth medium was almost complete (Fig. 3); thereafter, enzyme activity declined.
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DISCUSSION |
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Degraders of LPAS are also known from previous studies to be widely distributed both geographically and across bacterial genera, with a strong representation of Pseudomonas spp. (26). However, the following observations now show that degraders of LPAS are not necessarily degraders of BPAS. First, Pseudomonas sp. strain C12B, well known and extensively studied for its ability to biodegrade SDS and related alkyl sulfates, failed to grow at the expense of 2-butyloctyl sulfate, and its alkylsulfatases, P1 and P2, active on a range of linear primary alkyl sulfates, were inactive on 2-butyloctyl sulfate (Table 1 shows a summary of specificities). One of the new strains, Pseudomonas sp. strain AE-A, was also able to grow on SDS and produced an SDS-inducible and SDS-specific alkylsulfatase (AP3) analogous to P2. However, this enzyme also was neither active on nor induced by growth on 2-butyloctyl sulfate. Moreover, the growth of Pseudomonas sp. strain AE-A on 2-butyloctyl sulfate induced a novel alkylsulfatase (AP2) specific for that substrate and inactive on SDS. An analogous enzyme (DP1) which was specifically induced by and active on 2-butyloctyl sulfate was produced by Pseudomonas sp. strain AE-D. Gas chromatographic analysis of material in opaque white bands on zymograms for this enzyme showed the presence of 2-butyloctanol, thus indicating that the catalytic role of the enzyme is probably as a sulfatase.
Thus, collectively these microorganisms produce two sets of alkylsulfatases with mutually exclusive specificities for either SDS or 2-butyloctyl sulfate. Against the background of exclusive specificities for LPAS (P1, P2, and AP3) and 2-butyloctyl sulfate (AP2 and DP1), alkylsulfatase AP1 was unique in showing activity toward both substrates (Table 1).
In addition, in contrast to AP2, AP3, and DP1, which were all inducible specifically by their respective substrates, AP1 was also the only enzyme from the new strains which was produced constitutively. There were also indications from its late appearance during batch culturing on SDS that its expression may be repressible. However, despite its dual specificity and constitutive expression, it appears that AP1 provides insufficient activity for Pseudomonas sp. strain AE-A to utilize either SDS or 2-butyloctyl sulfate, because cells challenged with either substrate induced the respective specific enzymes AP3 and AP2.
The combined occurrence of constitutive and inducible enzymes with overlapping specificities (AP1 and AP2 during growth on 2-butyloctyl sulfate and AP1 and AP3 on SDS) has a parallel in Pseudomonas sp. strain C12B, in which the constitutive and repressible P1 (2) and inducible P2 (7) are both present during growth on SDS. P1 and P2 have very similar chain-length substrate specificities but are clearly distinguishable by the mechanism of COS ester bond cleavage, i.e., OS in P1 and CO in P2 (3, 6). It will be interesting to determine in closer detail the substrate specificities and mechanisms of ester bond cleavage for the new enzymes AP1, AP2, AP3, and DP1; to this end, attempts are currently under way to purify and characterize DP1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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]) and residual surfactant concentration (mean and standard deviation, n = 3 [
]) were measured with BS medium supplemented with 1 g of surfactant/liter in batch cultures. Samples were removed at the times indicated by the arrows and analyzed by gel zymography for specific alkylsulfatases. Designations above arrows, e.g., AP3, indicate alkylsulfatase bands present in the sample. O.D.540, optical density at 540 nm; concn., concentration.
]) during growth (mean and standard deviation, n = 4 [