sugar ester surfactants ;biodegradation

ABSTRACT: In previous work, we found that the presence ofa sulfonyl or alkyl group adjacent to the ester bond of sugarester surfactants is associated with a dramatic reduction in therate of biodegradation relative to that of unsubstituted esters. Inthis study, we investigated the pathways followed during thebiodegradation of sucrose laurate, sucrose α-sulfonyl laurate,and sucrose α-ethyl laurate to determine the reasons for theirdifferent biodegradation rates. Through the use of high-perfor-mance liquid chromatography and proton nuclear magnetic res-onance spectroscopy, the nature of the intermediates formedduring the biodegradation of these three key sugar esters wasdetermined. It was found that sucrose laurate biodegradationoccurs via initial ester hydrolysis. In contrast, sucrose α-sulfonyllaurate degrades by initial alkyl chain oxidation. This indicatesthat the ester hydrolysis pathway is blocked by the sulfonylgroup adjacent to the ester bond so that biodegradation isforced to proceed via the slower alkyl chain oxidation pathway.Sucrose α-ethyl laurate was degraded at least in part by alkylchain oxidation, indicating that ester hydrolysis was also inhib-ited by the presence of an ethyl group. It is therefore concludedthat previously observed relationships between structure andbiodegradability arise because of the influence that differentstructural elements have on the pathways followed duringbiodegradation.

Paper no. S1141 in JSD 3, 13–27 (January 2000).

KEY WORDS: Biodegradation, ester, fatty acid, high-perfor-mance liquid chromatography, laurate, nuclear magnetic reso-nance, pathway, sucrose, sulfonyl, surfactant.

As discussed in our previous paper (1), sucrose fatty acidesters are known to be rapidly biodegradable (2–8). How-ever, relationships between biodegradability and thechemical structure of these surfactants have not been stud-ied. Understanding these relationships would facilitate atargeted design of sugar ester surfactants for use in con-sumer products, while retaining a high degree of bio-degradability.

In our previous paper, we reported the ultimatebiodegradabilities of an array of sugar ester surfactantswhose structures were systematically varied. This revealedthat a sulfonyl or alkyl group adjacent to the ester bond isassociated with a reduction in the biodegradation rate. Thebiodegradation of sucrose laurate was complete within 12h, while that of sucrose α-sulfonyl laurate occurred moreslowly, reaching about 85% in 25 d. Biodegradation of su-crose α-ethyl laurate occurred at a rate that was betweenthose of sucrose laurate and sucrose α-sulfonyl laurate,reaching completion in 4 d. In contrast, other structuralmodifications, including variations in the sugar headgroup size and the length and number of alkyl chains, didnot have a significant effect on biodegradability. In orderto establish reasons behind these relationships betweenstructure and biodegradability, the chemical pathways fol-lowed during biodegradation of three key sugar esterswere investigated. The three surfactants chosen for this in-vestigation were sucrose laurate, sucrose α-sulfonyl lau-rate, and sucrose α-ethyl laurate.

Pathways that are likely to be followed during sugarester surfactant biodegradation can be deduced from pre-viously established biodegradation pathways of othercompounds. It is known that alkylbenzene sulfonates aredegraded by initial terminal oxidation of the alkyl chain(ω-oxidation) followed by sequential cleavage of two car-bon units from the alkyl chain (β-oxidation) (9). In contrast,alcohol ethoxylates are degraded by initial fission into al-cohol and ethoxylate portions and subsequent oxidation ofthese portions independently (9). By analogy, there are twolikely pathways that could be followed during biodegra-dation of sugar esters. One involves initial oxidation of thealkyl chain, as occurs in the biodegradation of alkylben-zene sulfonates. Another pathway, similar to that of alco-hol ethoxylates, initiates with hydrolysis of the ester bond,after which the hydrolysis products are degraded indepen-dently. These alternative pathways are shown in Scheme 1,where (1) represents initial hydrolysis of the ester bondand (2) is initial ω-oxidation of the alkyl chain and subse-quent β oxidation; R = H in the case of sucrose laurate, SO3-Na+ in the case of sucrose α-sulfonyl laurate and CH2CH3in the case of sucrose α-ethyl laurate.

Copyright © 2000 by AOCS Press Journal of Surfactants and Detergents, Vol. 3, No. 1 (January 2000) 13

*To whom correspondence should be addressed at CSIRO MolecularScience, Bag 10, Clayton South MDC, Victoria 3169, Australia.E-mail: [email protected] address: R&D Division, RMIT University, P.O. Box 71,Bundoora 3083, Australia.

Sugar Fatty Acid Ester Surfactants:Biodegradation Pathways

Irene J.A. Bakera, R. Ian Willinga, D. Neil Furlonga,1,Franz Grieserb, and Calum J. Drummonda,*

aCSIRO Molecular Science, Clayton, Victoria 3169, Australia, and bSchool of Chemistry, The University of Melbourne, Parkville, Victoria 3052, Australia

It was suggested from previous studies that biodegra-dation of sugar esters proceeds by initial hydrolysis of theester bond (8). It was also reported that α-sulfonyl methylesters of fatty acids are degraded by initial oxidation of thealkyl chain followed by desulfonation, ester hydrolysis,and bioassimilation (10). Thus, it may be expected that un-derivatized sugar esters investigated in this study wouldbe degraded by initial hydrolysis. In contrast, by analogywith α-sulfonyl methyl esters studied previously, the sugarα-sulfonyl fatty acid esters could be expected to be de-graded by initial alkyl chain oxidation.

In this study, three key sugar esters were submitted tobiodegradation tests as described in the previous paper. Inaddition to the usual dissolved organic carbon (DOC)analyses used to follow ultimate biodegradation, culturesamples were also analyzed by high-performance liquidchromatography (HPLC) and nuclear magnetic resonancespectroscopy (NMR). By means of these analysis methods,the disappearance of intact surfactants from the cultureswas followed and the chemical nature of biodegradationintermediates was elucidated. This enabled pathways bywhich these surfactants were degraded, under the condi-tions of the test, to be proposed.

EXPERIMENTAL PROCEDURES

Materials. Analytical grade reagents from commercial sup-pliers were used unless stated otherwise. Aerobicallygrowing activated sludge was obtained from the CSIROpilot scale sewage treatment plant located at Lower Plenty,Melbourne, Australia. Sugar ester surfactants, sucrose lau-rate, sucrose α-sulfonyl laurate and sucrose α-ethyl lauratewere synthesized as described in a previous paper (1).

Methods. The method used to perform biodegradationtests [ISO method 7827 (11)] was also described in a previ-ous paper (1). In this method, the ultimate biodegradationof each test compound was followed by DOC analysis atintervals during a period of up to 30 d. This procedure wasexpanded to include additional sample collection to allowmore specific analyses by HPLC and NMR.

Sample preparation. At each sampling time, two 100-mLsamples were withdrawn from the biodegradation cul-tures, in addition to those collected for DOC analysis, andplaced in round-bottom flasks. These samples were freeze-dried and the residues were stored in the dark until theywere analyzed. Compounds of interest were extractedfrom the freeze-dried residues using 1 mL of solvent. Thevolume of solvent was determined accurately by mass dif-ference assuming the appropriate solvent density. The op-timal solvent mixture to achieve maximal simultaneous re-coveries of all compounds of interest was found to bemethanol/water (80:20, vol/vol) in the case of the sucroselaurate and sucrose α-ethyl laurate culture samples. Purewater was found to be the optimal solvent in the case of su-crose α-sulfonyl laurate biodegradation samples.

Prior to HPLC analysis, sucrose laurate and sucrose α-sulfonyl laurate samples were filtered through prerinsed0.2-µm filters (MILLEX-GS SLGO25OS; Millipore, Bedford,MA). However, incomplete recoveries were obtained usingthis method of sample preparation, possibly due to loss ofmaterials during sample filtration. For this reason, the su-crose α-ethyl laurate samples were not filtered, but simplyallowed to settle prior to HPLC analysis.

Samples to be analyzed by NMR were transferred into10-mL round-bottom flasks and freeze-dried again. Theresidues were stored over freshly dried silica gel undervacuum until they were analyzed. They were then dis-solved in the appropriate deuterated solvent mixture[D4methanol/D2O (80:20, vol/vol) for sucrose laurate andsucrose α-ethyl laurate biodegradation samples, and D2Ofor sucrose α-sulfonyl laurate biodegradation samples], fil-tered through dry cotton wool, and transferred into anNMR sample tube. This sample preparation procedure wasperformed under nitrogen to minimize the amount ofwater in the samples, which produces an interfering peakin the NMR spectra.

HPLC. The HPLC system consisted of an Altex (SanRamon, CA) model 110 A pump; a Rheodyne (RohnertPark, CA) model 7120 syringe loading injector with a 50-µL loop attached; and a refractive index detector (model

14 I.J.A. BAKER ET AL.

Journal of Surfactants and Detergents, Vol. 3, No. 1 (January 2000)

SCHEME 1

401 R differential refractometer; Waters Associates, Mil-ford, MA). Data collection and analysis were performedwith a Beckman (San Ramon, CA) System Gold Analog In-terface module 406, and System Gold ChromatographicSoftware. A 25-µL sample injection volume was used.

Analysis of sugar fatty acid esters, their derivatives, andthe fatty acids produced by hydrolysis was performed byreversed-phase HPLC. An octadecylsilica column (Econo-sphere C18 5 µ cartridge 250 × 4.6 mm i.d.; Alltech, Deer-field, IL) and guard column (Adsorbosphere C18 5 µ guardcolumn cartridge; Alltech) were used. All analyses wereperformed by isocratic elution with methanol/water mix-tures containing 0.1 M sodium perchlorate (BDH, Dorset,England; general purpose reagent), pH 6, at room tempera-ture. Proportions of methanol and water in the eluant wereadjusted as needed to achieve the required separations, toallow for day-to-day variations in room temperature.

Simultaneous determination of sucrose laurate and lau-ric acid was done using methanol/water (80:20, vol/vol)with a 1.2 mL/min flow rate and a pressure of 2900 psi at21°C, or methanol/water (70:30, vol/vol) with a 1.0mL/min flow rate and a pressure of 2700 psi at 26°C. Thesame conditions were used for simultaneous determina-tion of sucrose α-ethyl laurate and α-ethyl lauric acid. Forsimultaneous determination of sucrose α-sulfonyl laurateand α-sulfonyl lauric acid, the eluant was methanol/water(65:35, vol/vol) with a 1.2 mL/min flow rate and a pres-sure of 3400 psi at 21°C, or methanol/water (55:45,vol/vol) with a 1.0 mL/min flow rate and a pressure of3100 psi at 26°C. Before each series of analyses, the columnwas reconditioned by first back flushing the guard column,then pumping 30 mL each of water, 0.1 M sulfuric acid,water, methanol, chloroform, and methanol again throughthe guard column and reversed main column. The injec-tion port and injection loop were rinsed with solvent andeluant between each sample injection.

Sucrose analysis was achieved by size-exclusion chro-matography using a Benson organic acid column (300 × 7.8mm i.d.; Alltech), with a guard column of the same mater-ial. Isocratic elution was performed using 3 mM sulfuricacid at 0.5 mL/min and 300 psi at room temperature.

During calibration of the HPLC methods, peak areaswere found to be linearly dependent on analyte concentra-tion over the concentration ranges of interest. The responsefactors were, however, found to vary considerably each timea new batch of eluant was used. Methods were calibrated atthe beginning of each set of analyses using three or morestandard solutions of reference materials. A single averagecalibration factor was calculated for each analyte and usedto determine the concentration in unknown samples.

For these HPLC separations, the methanol used wasBDH HiPerSolv for HPLC, and the water was obtainedfrom a Millipore Ultra-Pure Water System, model ZAMQ050 01.

Recoveries, blanks, and reproducibility. To establish the re-coveries achieved by this HPLC procedure, samples of the

test medium used to prepare the biodegradation cultureswere spiked with known amounts of analytes and submit-ted to sample preparation and analysis. After HPLC analy-sis, the recovery factors were determined as follows: su-crose laurate, 73%; lauric acid, 76%; sucrose, 61%; sucrosesulfonyl laurate, 76%; and α-sulfonyl lauric acid, 99%.These factors were used to correct for incomplete recoverywhen determining concentrations of analytes in biodegra-dation cultures by HPLC. They were determined at singleanalyte loadings, which were at the upper limit of expectedconcentrations.

Samples of blank biodegradation cultures containedmaterial with a broad peak that sometimes interfered withthe determination of sucrose laurate and lauric acid (de-pending on the exact chromatographic conditions). Wherethis was the case, it was subtracted from the area of thesepeaks. Test medium and biodegradation culture blanksalso contained a significant amount of sucrose, or a peakwith the same elution time as sucrose. This was subtractedfrom the area of the sucrose peaks in all determinations in-cluding recovery samples.

Reproducibilty of the HPLC determinations was esti-mated from the differences between duplicate injections ofthe same sample. This showed that the HPLC proceduresgave results that were reproducible to within approxi-mately ± 0.005 mM (equivalent to approximately 0.8 ppmDOC). The variability between two independent sets of de-terminations was greater, however, commonly ± 0.01 mM(approximately 1.4 ppm DOC) and as much as 0.03 mM inthe worst case (approximately 4 ppm DOC).

NMR spectroscopy. One-dimensional proton NMR (1HNMR) spectra were measured in D2O/D4methanol for su-crose laurate and sucrose α-ethyl laurate and in D2O forsucrose α-sulfonyl laurate culture samples. Measurementswere recorded on a Bruker AC 250 spectrometer (Karls-ruhe, Germany) operating at 250.1 MHz. Spectra weremeasured with 2500 Hz spectral width over 16,384 datapoints, with a repetition rate of 3.277 s and with a pulseangle of 0.2 × 90° for sucrose laurate and sucrose α-sulfonyllaurate and 0.5 × 90° for sucrose α-ethyl laurate. Data wereprocessed with an exponential multiplication windowwith 0.3 Hz line broadening. The samples were placed inthe spectrometer and allowed to reach thermal equilibriumbefore the spectra were measured. Chemical shifts werereferenced to the D3methanol signal taken to be at 3.47ppm, or the HOD signal taken to be 4.8 ppm.

Two-dimensional proton NMR (2D 1H NMR) spectrawere recorded in D2O using a correlation spectroscopy(COSY) experiment with double-quantum filtering andphase-sensitive mode selection. The spectra were acquiredwith spectral widths F1 ± 700.280 Hz, F2 1400.56 Hz, quad-rature detection in both dimensions, a 90 × 1024 matrix, arecycle time of 2.37 s, and a total acquisition time of 52 h.Data were processed as a 2,048 × 2,048 data matrix using asinebell window function.

The double-quantum filtered COSY experiments have

SUGAR ESTER SURFACTANTS: BIODEGRADATION 15


distinct advantages over the standard COSY experiments.First, partial suppression of diagonal peak intensity makesit easier to observe off-diagonal peaks between signals thatare close together in chemical shift. Second, the double-quantum filter eliminates strong signals from peaks thatdo not experience homonuclear J-coupling (e.g., fromHOD present in the D2O solvent). An additional advantagewas obtained by using the phase-sensitive version of thisexperiment. This has a higher resolution than the other-wise equivalent magnitude spectrum because the magni-tude lineshape is broader than the pure absorption line-shape with the consequence that a slightly better signal-to-noise ratio is achieved.

RESULTS AND DISCUSSION

Sucrose laurate biodegradation pathway. Samples taken froma sucrose laurate biodegradation culture at intervalsthroughout the biodegradation process were analyzed byHPLC for intact sucrose laurate and its hydrolysis prod-ucts, sucrose and lauric acid. A sequential set of HPLCchromatograms is shown in Figure 1 as well as chromato-

grams corresponding to the blank culture. Chromatogramsfor a reference sample containing sucrose laurate and lau-ric acid and one containing sucrose are also shown at thebottom of Figure 1. These chromatograms show that thesucrose laurate concentration in the biodegradation culturedecreased to zero within 6 h of incubation. At the sametime, the concentrations of free lauric acid and sucrose inthe culture increased, reaching maxima after 6 h of incuba-tion. This confirms that the biodegradation of sucrose lau-rate occurs by initial ester hydrolysis releasing sucrose andlauric acid.

It could be argued that the initial hydrolysis observedin this biodegradation culture may not be a result of bacte-rial action, but may be purely due to the chemical instabil-ity of the ester bond. A simple test was performed to esti-mate the rate at which sucrose laurate is hydrolyzed in asolution of the biodegradation test medium in the absenceof bacterial inoculum. It was found that the foam heightabove the solution did not decrease and no precipitatedlauric acid was observed for 2 d. Thus it was concludedthat the rapid hydrolysis of sucrose laurate in the activebiodegradation culture, which is complete within 6 h, is abacterial process.

The sucrose laurate, lauric acid, and sucrose concentra-tion profiles determined from the HPLC chromatogramsare displayed together in Figure 2. A significant amount ofsucrose and lauric acid were determined in the “zero time”sample. This was due to the unavoidable delay betweenthe time when the cultures were inoculated and the timewhen the samples were frozen. The concentration profilesin Figure 2 show the progression of biodegradation stepsoccurring in the sucrose laurate culture; that is, the initialconversion of sucrose laurate to sucrose and lauric acidand subsequent removal of these hydrolysis products. Thelevel of DOC in the culture accounted for by these concen-trations of sucrose laurate, lauric acid, and sucrose as de-termined by HPLC analysis was calculated for each sam-



FIG. 1. Representative chromatograms from high-performance liquidchromatography (HPLC) analysis of sucrose laurate biodegradation sam-ples at between zero and 12 h of incubation (above) and reference sam-ples containing sucrose laurate, lauric acid, and sucrose (below).

FIG. 2. Concentration profiles of (uu) sucrose laurate, (ss) lauric acid,and (ll) sucrose in the sucrose laurate biodegradation culture deter-mined by HPLC. See Figure 1 for abbreviation.

ple time. These are shown in Figure 3. DOC levels in theculture determined by direct measurements are alsoshown for comparison. During the first 6 h of incubation,there was a discrepancy of up to 12 ppm between the totalamount of DOC accounted for by the concentrations of su-crose laurate, lauric acid, and sucrose and the level of DOCdetermined by direct analysis. There are several possibleexplanations for this discrepancy. One possibility is that itarises from the errors in the HPLC and DOC measure-ments. The HPLC measurements are reproducible towithin about ± 1.5 ppm DOC equivalent, and the DOCmeasurements are reproducible to within ± 3 ppm. To-gether these errors are not enough to account for the dis-crepancy. Another source of error is the time delay in-volved in the sampling techniques. This is longer for HPLCsamples (up to 1.5 h for the initial sample) than for DOCsamples (<0.5 h). This means that the true curves shouldbe similar to those shown in Figure 3, but shifted relativeto each other in the direction of the time axis. Even aftersuch a shift, the curves would not be in agreement. A fur-ther possibility is that there are additional metabolitespresent that have not been detected by HPLC, but con-tribute to the DOC observed by direct measurement; forexample, subsequent intermediates formed by biodegra-dation of sucrose and lauric acid or intermediates formedby an alternative biodegradation pathway. In fact, the ob-served levels of lauric acid and sucrose in the culture donot build up in proportion to the amount of intact sucroselaurate consumed. This suggests that other biodegradationprocesses are occurring simultaneously with initial esterhydrolysis, giving rise to DOC in addition to that ac-counted for by sucrose laurate and its hydrolysis products.

Samples of the sucrose laurate biodegradation culturealso were analyzed by 1H NMR spectroscopy. Referencespectra of samples containing sucrose, lauric acid, and su-

crose laurate, recorded under the same conditions as usedfor the culture samples, are shown in Figure 4. Structuresof the reference compounds and peak assignments also areshown in Figure 4. The spectrum of sucrose consists of agroup of peaks with chemical shifts between 3.5 and 4.4ppm and a doublet at 5.6 ppm. The latter is due to theanomeric proton in the glucose ring adjacent to the etherlink between the two sugar rings. The lauric acid spectrumshows distinct peaks due to the terminal CH3 group(triplet peak at 1.0 ppm), the mid-chain CH2 groups (broadpeak at 1.4 ppm), the β-CH2 group two carbons removedfrom the carboxylic group (broad multiplet at 1.7 ppm),and the α-CH2 adjacent to the carboxylic group (tripletpeak at 2.3 ppm). The spectrum of sucrose laurate is a com-bination of these two spectra with the distinction that thepeak due to the α-CH2 group is shifted downfield (tripletpeak at 2.55 ppm), and there are some additional peaks inthe sugar region of the spectrum (peaks at 4.5–4.6 ppm).Peaks due to HOD and D3methanol are also present in allspectra at 5 ppm and 3.47 ppm, respectively.

NMR spectra for sequential samples of the sucrose lau-rate biodegradation culture and for a blank biodegradationculture are shown in Figure 5. The spectrum for the blankculture shows that there are no significant interferingpeaks due to unknown substances in the biodegradationmedium. There are small peaks indicating the presence ofsome alkyl and saccharide materials. Most of these back-ground peaks are too small to be quantified and all haveareas less than 10% of the corresponding peaks in the ref-erence spectra.

Regarding spectra for sequential samples of the bio-degradation culture, a number of observations can bemade. During the first 6 h of incubation, the relative inten-sity of the triplet peak corresponding to the α-CH2 protonsof the intact ester (centered at 2.55 ppm) decreased to zero.There was a concurrent increase in the triplet peak inten-sity corresponding to the α-CH2 protons of free lauric acid(centered at 2.3 ppm). This was also accompanied by thedisappearance of some peaks in the sugar region (4.5–4.6ppm). After 6 h, only those peaks associated with free su-crose and lauric acid remained. These rapidly decreased insubsequent spectra.

Through examination of the relative areas of character-istic peaks in the 1H NMR spectra, it was possible to esti-mate the concentrations of sucrose laurate, sucrose, andlauric acid in the biodegradation culture at each samplingtime. Concentration profiles determined in this way areshown in Figure 6. These profiles are very similar to thosedetermined by HPLC analyses. They show that the concen-tration of intact sucrose laurate decreased to zero duringthe first 6 h of incubation. Simultaneously, the concentra-tions of hydrolysis products (sucrose and lauric acid) roseto a maximum, subsequently decreasing to zero within20 h of incubation.

At all times, the ratios of all peak areas in the NMR spec-tra remained consistent with calculated concentrations of



FIG. 3. Dissolved organic carbon (DOC) content of the sucrose lauratebiodegradation culture determined by (n) direct measurements is com-pared with levels of DOC accounted for by (uu) sucrose laurate, (ss) lau-ric acid, and (ll) sucrose concentrations determined by HPLC measure-ments, and the (nn) sum of these. See Figure 1 for abbreviation.

sucrose laurate, lauric acid, and sucrose, to within 10%.This indicates that any additional compounds present inthe samples must be at low levels such that they do not re-sult in significant peaks (less than about 0.01 mM). Thus, ifthere are additional metabolites present in the culture, aswas suggested from the comparison of the HPLC resultswith DOC measurements, they are not detected by this

NMR procedure. It appears that additional metaboliteshave not been recovered by the procedure used to preparesamples for HPLC and NMR analyses, but were retainedin the DOC samples. This implies that either they are suf-ficiently volatile and lost during freeze-drying, or theyhave low solubility in the methanol/water solvent mixtureused to prepare HPLC and NMR samples.



FIG. 4. Reference 1H nuclear magnetic resonance (NMR) spectra of sucrose, lauric acid andsucrose laurate in D4methanol/D2O (80:20, vol/vol).

1H NMR spectroscopy was thus found to be a usefulmethod for observing transformations that occur duringbiodegradation. The 1H NMR spectra provided further

support for conclusions drawn from HPLC analyses. Theresults from both methods showed that the major sucroselaurate degradation pathway is via initial hydrolysis of theester bond, releasing lauric acid and sucrose. This is con-sistent with findings of previous investigators (8). Hydrol-ysis products were subsequently degraded, resulting in thecomplete removal of organic material from the biodegra-dation culture. This demonstrated the validity and useful-ness of HPLC and 1H NMR methods for biodegradationpathway determination.

Sucrose α-sulfonyl laurate biodegradation pathway. Thesame analysis techniques were used to investigate thepathway followed during sucrose α-sulfonyl laurate bio-degradation. Samples collected from a sucrose α-sulfonyl



FIG. 5. 1H NMR spectra recorded for sequential samples from the su-crose laurate biodegradation culture and a blank biodegradation cul-ture, in D4methanol/D2O (80:20, vol/vol). See Figure 4 for abbrevia-tion.

FIG. 6. Concentration profiles of (uu) sucrose laurate, (ss) lauric acid,and (ll) sucrose in the sucrose laurate biodegradation culture, deter-mined from peak ratios in 1H NMR spectra. See Figure 4 for abbrevia-tion.

FIG. 7. Representative chromatograms from HPLC analysis of sucroseα-sulfonyl laurate biodegradation samples at between zero and 50 h ofincubation (above) and a reference sample containing sucrose α-sul-fonyl laurate and α-sulfonyl lauric acid (below). See Figure 1 for abbre-viation.

laurate biodegradation culture were analyzed by HPLC todetermine concentrations of intact sucrose α-sulfonyl lau-rate and the hydrolysis product, α-sulfonyl lauric acid. Arepresentative selection of the sequential chromatogramsrecorded in this way is shown in Figure 7. A chromato-gram for a sample of the blank biodegradation culture isincluded in the same figure. A reference chromatogram ob-tained for a sample containing both sucrose α-sulfonyl lau-rate and α-sulfonyl lauric acid is also shown at the bottomof the figure. Chromatograms of sequential biodegradationsamples clearly indicate the disappearance of intact su-crose α-sulfonyl laurate during the first 44 h of incubation,with no concurrent buildup of the hydrolysis product, α-sulfonyl lauric acid.

The concentration of sucrose α-sulfonyl laurate was cal-culated from chromatograms by reference to results fromstandard samples. From these concentrations, the levels ofDOC in each sample were determined. DOC levels equiv-alent to sucrose α-sulfonyl laurate concentrations deter-mined by HPLC are shown in Figure 8 and compared withthe results of direct DOC analysis. Clearly there was a largeamount of DOC present in the biodegradation culture fromthe third day of incubation onward, which was not ac-counted for by intact starting material or products of its hy-drolysis. The most likely reason for this is that biodegrada-tion does not occur by initial hydrolysis, but by initial alkylchain oxidation. This could result in the presence of an in-termediate chain-shortened sucrose α-sulfonyl car-boxyalkyl ester such as α-sucrose sulfonyl carboxyhexa-noate. The nature of the biodegradation intermediatesformed in the culture was further investigated by 1H NMRanalysis.

Samples of the sucrose α-sulfonyl laurate biodegrada-tion culture were analyzed by 1H NMR. This allowed thebiodegradation processes to be followed in more detailthan was possible using HPLC. 1H NMR spectra of some

reference materials measured under the same conditionsas used for culture samples are shown in Figure 9. Refer-ence materials considered were intact sucrose α-sulfonyllaurate, hydrolysis product α-sulfonyl lauric acid, methylα-sulfonyl palmitate (an α-sulfonyl fatty acid ester whosespectrum does not contain interfering peaks from sucrose),and glucose α-sulfonyl laurate (an analog of a proposedbiodegradation intermediate).

The α-sulfonyl lauric acid spectrum shows distinctpeaks from the terminal CH3 group (triplet peak at 0.85ppm), the mid-chain CH2 groups (broad peak at 1.3 ppm),the β-CH2 group two carbons removed from the carboxylicgroup (broad multiplet at 1.85 ppm), and the α-sulfonylCH adjacent to the carboxylic group (doublet of doubletsat 3.6 ppm). The spectrum of sucrose α-sulfonyl laurate isa combination of the above spectrum and that of sucrose.The β-CH2 peak (broad multiplet at 2.0 ppm) is shiftedslightly downfield relative to its position in the free α-sul-fonyl fatty acid spectrum. Also note that the α-sulfonyl CHgroup in the ester spectrum is partially masked by over-lapping peaks from the sucrose moiety. However, by anal-ogy with the spectrum of methyl α-sulfonyl palmitate de-scribed below, it is expected to give a peak at around 3.8ppm. The glucose α-sulfonyl laurate spectrum is similar tothat of sucrose α-sulfonyl laurate, but the overall shape ofthe group of sugar peaks is distinctly different. The spec-trum of methyl α-sulfonyl palmitate is similar to that of α-sulfonyl lauric acid. It again shows the terminal CH3 group(triplet peak at 0.85 ppm) and the mid-chain CH2 groups(broad peak at 1.3 ppm). The β-CH2 peak is shifted slightlydownfield (broad multiplet at 2.0 ppm), as seen in spectraof other esters. The α-sulfonyl CH group is also shifteddownfield (single peak at 3.75 ppm). There is an additionalpeak due to the esterified methyl group (peak at 3.85 ppm).A peak due to HOD at 4.8 ppm is also present in all thesespectra.

Spectra recorded for sequential samples of the sucroseα-sulfonyl laurate biodegradation culture and for a sampleof the blank biodegradation culture are shown in Figure10. The blank spectrum indicates that there are no signifi-cant interfering peaks due to the presence of unknown ma-terials in the test medium. The following points can bemade concerning spectra of sequential sucrose α-sulfonyllaurate biodegradation samples. Between zero and 32 h ofincubation, there was a reduction in alkyl chain peak arearelative to the sugar group peaks. After 32 h, two newpeaks had appeared in the alkyl chain region at 1.6 and 2.2ppm. After 44.5 h, peaks assigned to terminal and mid-chain alkyl groups had essentially disappeared, while thepeaks assigned to the sugar group remained essentiallyunchanged. The new peaks in the region of the alkyl chainand the peak assigned to β-CH2 protons remained. No dra-matic changes in the spectrum were seen until the 116-h(5 d) sample. At this time, the area of the sugar peaks rela-tive to that of the β-CH2 protons had decreased by half andtheir general shape had changed to resemble that of a glu-



FIG. 8. DOC content of the sucrose α-sulfonyl laurate biodegradationculture determined by (u) direct measurements compared with the lev-els of DOC accounted for by sucrose α-sulfonyl laurate concentrationsfrom (uu) HPLC measurements. See Figures 1 and 3 for abbreviations.



FIG. 9. Reference 1H NMR spectra of α-sulfonyl lauric acid, sucrose α-sulfonyl laurate, glu-cose α-sulfonyl laurate, and methyl α-sulfonyl palmitate in D2O. See Figure 4 for abbrevia-tion.

cose group (the spike at 3.7 ppm was no longer present).In contrast, the β-CH2 peak and the new alkyl chain peakswere little different from those present in the 44.5-h sam-ple. The only significant difference was that a shoulderpeak at 1.9 ppm had appeared next to the β-CH2 peak.Spectra of subsequent samples show a progressive de-crease in the relative area of the sugar group peaks and re-placement of the β-CH2 peak at 2.0 ppm by a new peak at1.9 ppm. After 358 h (15 d), the only significant peaks re-maining were the new alkyl chain peaks at 1.6, 1.9, and 2.2ppm and a doublet of doublets centered at 3.55 ppm. After430 h (18 d) the areas of these remaining peaks were fur-ther reduced but remained at the same ratio as for the 358-h sample.

The dramatic disappearance of peaks assigned to thealkyl chain after 44.5 h of incubation, while peaks assignedto the sugar portion remained unchanged, suggests thatthe α-sucrose sulfonyl laurate was degraded by initial oxi-dation of the alkyl chain to form a short-chain intermedi-ate in which the sugar group was essentially unchanged.This deduction, which is consistent with results obtainedby DOC and HPLC analyses, was further confirmed byobtaining more information about the identity of interme-diate metabolites present in the biodegradation culture be-

tween 44.5 and 116 h of incubation. The technique of 2D 1HNMR was used to observe spin coupling between protonson adjacent carbon atoms, allowing the topographical rela-tionships between groups of protons within the chemicalstructure of the intermediates to be deduced.

A reference 2D spectrum of sucrose α-sulfonyl laurateis shown in Figure 11. The corresponding simple 1H NMRspectrum and peak assignments are shown at the bottomof Figure 11. In the 2D spectrum, peaks are identified as x,y,where x and y are the chemical shift coordinates along thehorizontal and vertical axes. Peaks that are off the main di-agonal of the spectrum indicate coupling between protonsattached to adjacent carbon atoms. The off-diagonal peakat (0.8 ppm, 1.3 ppm) indicates coupling between the ter-minal CH3 and adjacent alkyl chain CH2 protons. The peakat (1.3 ppm, 2.0 ppm) indicates coupling between alkylchain CH2 and β-CH2 protons. The peak at (2.0 ppm, 3.9ppm) indicates coupling between the β-CH2 protons andthe α-CH proton. The α-CH proton peak (at 3.9 ppm) in thesimple 1H NMR spectrum is masked by peaks from thesugar group, but its approximate position can be deducedby reference to the spectrum of methyl α-sulfonyl palmi-tate shown in Figure 9. Couplings between protons in thesugar region of the spectrum have not been considered inthis study. Thus, the chain of couplings between protons inthis spectrum is consistent with the structure of the refer-ence material.

The 2D spectrum of material in the sucrose α-sulfonyllaurate biodegradation culture after 68.5 h of incubation isshown in Figure 12. The simple 1H NMR spectrum (alsoshown in Fig. 12) indicates that the material had been de-graded to the extent that the original alkyl chain was nolonger present, but the sugar part of the molecule had notbeen significantly altered. A possible structure for this in-termediate (sucrose α-sulfonyl carboxyhexanoate) isshown in the lower part of Figure 12 along with the pro-posed peak assignment in the simple 1H NMR spectrum.A spectrum of the intermediate material contained theβ-CH2 proton peak at 2.0 ppm, which was present in thespectrum of the starting material, and a slight shoulderpeak at 1.9 ppm. This shoulder peak replaced the β-CH2peak in the spectra of later samples. From inspection of ref-erence spectra in Figure 9, it is seen that the β-CH2 peak isat 2.0 ppm in the esterified α-sulfonyl alkanates, but is at1.9 ppm in the free acid form, as is α-sulfonyl lauric acid. Itis possible that the peak at 1.9 ppm in spectra of laterbiodegradation intermediates is due to a β-CH2 group in afree α-sulfonyl alkyl acid. The peak at 1.5 ppm in the inter-mediate spectrum is the most shielded and probably cor-responds to the γ-CH2 protons in the proposed structure.The peak at 2.2 ppm is the least shielded of the peaks inthis region and is probably due to the δ-CH2 adjacent to theterminal carboxylate group. By analogy with the peak as-signments for reference spectra in Figure 9, the α-protonsof the esterified or free acid form of this material are ex-pected to produce peaks in the sugar region of the spec-



FIG. 10. 1H NMR spectra recorded for residues of sequential samplesfrom the sucrose α-sulfonyl laurate biodegradation culture in D2O. SeeFigure 4 for abbreviation.



FIG. 11. Two dimensional proton–proton NMR spectrum of sucrose α-sulfonyl laurate in D2O (above) and the corresponding simple 1H NMRspectrum (below). Couplings between protons on adjacent carbon atoms in the 2D spectrum are indicated by arrow brackets on the simple 1HNMR spectrum. See Figure 4 for abbreviation.

trum, at about 3.6 ppm for the free acid and about 3.8 ppmfor the ester.

The 2D NMR spectrum of the intermediate metaboliteprovides some confirmation of this proposed structure. An

off-diagonal peak at (1.6 ppm, 2.2 ppm) indicates couplingbetween the γ-CH2 group and the δ-CH2 group, as wouldbe expected from the proposed structure. The peaks at (1.6ppm, 2.0 ppm) and (1.6 ppm, 1.8 ppm) indicate coupling



FIG. 12. Two-dimensional proton–proton NMR spectrum of the 68.5-h sucrose α-sulfonyl laurate biodegradation sample (above) and the corre-sponding simple 1H NMR spectrum (below). Couplings between protons on adjacent carbon atoms in the 2D spectrum are indicated by arrowbrackets on the simple 1H NMR spectrum. The proposed structure of the intermediate present at this stage is shown below with indications of peakassignments. Peaks labeled with a prime (′) correspond to the ester form of the proposed intermediate, while others correspond to the free acidform. See Figure 4 for abbreviation.

between the γ-CH2 and the β-CH2 groups in the esterifiedand free acid forms, respectively. The peak at (1.8 ppm, 3.6ppm) indicates coupling between free acid β-CH2 protonsand the free acid α-CH proton. The peak at (2.0 ppm, 3.9ppm) indicates coupling between esterified β′-CH2 protonsand the esterified α′-CH proton. These couplings are con-sistent with the proposed structure and indicate the pres-ence of materials in both esterified and free acid forms. Theintensity of peaks in 2D spectra of this type is related to thestrength of the coupling between the protons concernedand not to the concentrations of the species in which thecoupling occurs. Thus, although the simple 1H NMR spec-trum shows the presence of only a small amount of the freeacid material, its presence is clearly seen in the 2D spec-trum.

Having determined that the major intermediate in theculture after 68.5 h is likely to have been sucrose α-sulfonylcarboxyhexanoate, it is possible to speculate about trans-formations that occurred. A possible biodegradation se-quence for sucrose α-sulfonyl laurate is represented inScheme 2 and is rationalized in the following way: Subse-quent to formation of sucrose α-sulfonyl carboxyhexanoate

in the culture, the next major changes in the 1H NMR spec-tra occurred after 116 h when the relative area of the sugargroup was half the original value and its general shape re-sembled that of a glucose group. Most of the ester bondswere still intact as indicated by the relative areas of thepeaks due to the β-CH2 groups of the esterified (2.0 ppm)and free acid forms (1.8 ppm). At this time, the DOC con-tent of the culture, shown in Figure 8, was about 50% theoriginal value. This shows that about 12 out of every 24original carbons remained. These observations are consis-tent with the presence of glucose α-sulfonyl carboxyhexa-noate, as shown in Scheme 2.

Between 116 and 430 h of incubation, the major changesin the 1H NMR spectra were a further decrease in the rela-tive area of the sugar peaks, replacement of the esterβ′-CH2 peak by the free acid β-CH2 peak, and increased rel-ative intensity of a doublet of doublets at 3.55 ppm. Thisdoublet of doublets was in approximately the same posi-tion as the α-CH group in the spectrum of α-sulfonyl lau-ric acid (Fig. 9). The splitting of this doublet of doubletswas J = 10 and 4 Hz, which is the same as observed for theα-sulfonyl lauric acid spectrum. This suggests that theα-sulfonyl CH group present in the original sucrose α-sul-fonyl laurate ester had been retained, leading to an analo-gous α-sulfonyl CH group adjacent to a free carboxylicacid group. These observations indicate that the final ma-terial present in the culture after 430 h was an α-sulfonylcarboxyhexanoic acid (Scheme 2).

Thus, by a combination of one- and two-dimensional 1HNMR analyses, it has been possible to confirm the conclu-sion of the HPLC study, namely, that the degradation of su-crose α-sulfonyl laurate does not occur by initial hydroly-sis. Additional information concerning the processes thatoccur during biodegradation was obtained using theseanalysis techniques, allowing the biodegradation pathwayof sucrose α-sulfonyl laurate to be proposed as shown inScheme 2. The proposed pathway is analogous to path-ways previously reported for linear alkylbenzene sul-fonates (9) and α-sulfonyl methyl esters (10). Similar ulti-mate biodegradation rates of sucrose α-sulfonyl laurateand linear alkylbenzene sulfonate, reported previously (1),provide further support for the proposal that these twocompounds are degraded by similar pathways involvinginitial alkyl chain oxidation.

The fact that sucrose α-sulfonyl laurate is not biode-graded via initial ester hydrolysis, as in the case of sucroselaurate, indicates that the sulfonyl group adjacent to theester inhibits ester hydrolysis. Consequently the α-sulfonylester cannot be degraded by initial ester hydrolysis, butfollows an alternative pathway involving initial alkyl chainoxidation. Because the latter process occurs more slowlythan the former, this has the effect that the two compoundsshow different ultimate biodegradation rates as measuredby DOC analyses.

The α-sucrose ethyl laurate biodegradation pathway. Finally,the biodegradation pathway of sucrose α-ethyl laurate was



SCHEME 2

investigated using the same analysis techniques. Sucroseα-ethyl laurate, which contains an ethyl group adjacent tothe ester bond, showed a biodegradation rate betweenthose of the unsubstituted sucrose esters and the sul-fonated sucrose esters in our previous study. Concen-trations of sucrose α-ethyl laurate and its hydrolysis prod-ucts, sucrose and α-ethyl lauric acid, in samples of asucrose α-ethyl laurate biodegradation culture were deter-mined by HPLC analysis. The sequential chromatogramsshowed the disappearance of sucrose α-ethyl laurate withno concurrent buildup of its hydrolysis products, α-ethyllauric acid and sucrose. The sucrose concentration wasonly slightly raised on the second and third days of incu-bation.

From these HPLC results, the concentrations of eachspecies in the culture samples were calculated. Levels ofDOC accounted for by each species were then deduced.Results by direct DOC analysis were compared with DOClevels calculated from the HPLC results as shown in Fig-ure 13. This indicates that there was about 8.5 ppm of resid-ual organic material present in the culture after 3 d of incu-bation that was not accounted for by sucrose α-ethyl lau-rate, sucrose, or α-ethyl lauric acid. The additional DOCwas possibly due to one or more unknown organic inter-mediates arising from an alternative biodegradation path-way or from subsequent biodegradation of hydrolysisproducts.

1H NMR spectra indicated that after 2 d of incubation,some alkyl chain degradation had occurred. By day 3, therelative amount of sugar was reduced by half. At this time,the amount of intact ester was also reduced by half. In con-trast, the relative amount of alkyl chain CH2 groups haddiminished by a greater extent, to one-fifth the originalvalue. Thus, the alkyl chain degraded more rapidly thanthe sugar group and the intact ester link. These findings

suggest that sucrose α-ethyl laurate is degraded, at least inpart, by initial alkyl chain oxidation. DOC and HPLCanalyses showed the presence of residual organic matterin the culture, after 3 d of incubation, which was not ac-counted for by levels of intact sucrose α-ethyl laurate orthe products of its hydrolysis. The residual material cannow be attributed to a biodegradation intermediateformed by alkyl chain oxidation. Once again, the differentpathway followed during biodegradation of sucroseα-ethyl laurate explains why it is degraded more slowlythan underivatized sucrose laurate. The fact that it is de-graded at a faster rate than the ester containing a sulfonylgroup adjacent to the ester could be due to some simulta-neous degradation occurring by ester hydrolysis or to afaster rate of alkyl chain oxidation in the presence of theless bulky and uncharged ethyl group.

The combination of HPLC and 1H NMR analysis tech-niques has provided a powerful method for determiningbiodegradation pathways. It has enabled the pathways fol-lowed during the biodegradation of three key sugar estersurfactants to be investigated in detail, revealing reasonsfor different rates at which their ultimate biodegradationoccurs. We have shown that while unsubstituted sucroselaurate is degraded rapidly by initial ester hydrolysis, thispathway is inhibited by the presence of α-sulfonyl or α-ethyl groups. Consequently, sucrose α-sulfonyl laurate isdegraded more slowly via alkyl chain oxidation. Biodegra-dation of sucrose α-ethyl laurate occurs at an intermediaterate, at least in part by alkyl chain oxidation. A kineticstudy of the base-catalyzed hydrolysis of sucrose esterswas performed to investigate the roles played by steric andelectronic interactions in the inhibition of ester hydrolysisby α-subtituents. The results of the kinetic study are de-scribed in a separate publication (12).

ACKNOWLEDGMENTS

We are grateful to the Australian Sugar Research and Develop-ment Corporation and the Australian Research Council Ad-vanced Mineral Products Special Research Centre for financial as-sistance. We thank Dr. Barry Matthews, Hector Suares, and Dr.Irena Krodkiewska who synthesized the sugar ester surfactantsused in this study, and Darrell Wells, Anton Launikonis, and OdiBatistatos for technical assistance.

REFERENCES

1. Baker, I.J.A., B. Matthews, H. Suares, I. Krodkiewska, D.N.Furlong, F. Grieser, and C.J. Drummond, Sugar Fatty AcidEster Surfactants: Structure and Ultimate Aerobic Biodegrad-ability, J. Surfact. Deterg. 3:1 (2000).

2. Isaac, P.C.G., and D. Jenkins, Biological Oxidation of Sugar-Based Detergents, Chem. Ind. Aug. 2:976 (1958).

3. Isaac, P.C.G., and D. Jenkins, A Laboratory Investigation ofthe Breakdown of Some of the Newer Synthetic Detergents inSewage Treatment, J. Proc. Inst. Sewage Purif. 314 (1960).

4. Isaac, P.C.G., and D. Jenkins, The Biological Breakdown ofSome Newer Synthetic Detergents, in Conference of Biological



FIG. 13. DOC content of the sucrose α-ethyl laurate biodegradationculture determined by (l) direct measurements compared with levelsof DOC accounted for by the concentrations of (ss) sucrose α-ethyl lau-rate, and (nn) sucrose determined by HPLC, and the (ll) sum of these.See Figure 3 for abbreviation.

Waste Treatment: Advances in Biological Waste Treatment,Macmillan, London, 1963, p. 61.

5. Wayman, C.H., and J.B. Roberts, Biodegradation of Anionicand Nonionic Surfactants Under Aerobic and Anaerobic Con-ditions, Biotechnol. Bioeng. V:367 (1963).

6. Ruiz Cruz, J., and M.C. Doborganes García, The Pollution ofNatural Waters by Synthetic Detergents XIII: Biodegradationof Nonionic Surface Active Agents in River Water and Deter-mination of Their Biodegradability by Different Test Meth-ods, Grasas Aceites 29:1 (1978).

7. Sturm, R.N., Biodegradability of Nonionic Surfactants:Screening Test for Predicting Rates and Ultimate Biodegrada-tion, J. Am. Oil Chem. Soc. 50:159 (1973).

8. Brebion, G., R. Cabridenc, and A. Lerenard, Evaluation of theBiodegradation of an Ethoxylated Tallow Sucroglyceride, Rev.Fr. Corps Gras 11:191 (1964).

9. Swisher, R.D., Surfactant Biodegradation, Surfactant Science Se-ries Vol. 18, 2nd edn., Marcel and Dekker Inc., New York,1987.

10. Masuda, M., H. Odake, K. Miura, K. Ho, K. Yamada, and K.Oba, Biodegradation of 2-Sulfonatofatty Acid Methyl Ester(α-SFMe), II. Biodegradation Pathways of α-SFMe, J. Jpn. OilChem. Soc. 42:21 (1993).

11. International Standards Organisation, ISO 7827-1984(E),Water Quality—Evaluation in an Aqueous Medium of the Ul-timate Aerobic Biodegradability of Organic Compounds—Method by Analysis of Dissolved Organic Carbon (DOC),Method no. ISO 7827-1984 (E).

12. Baker, J.A., D.N. Furlong, F. Grieser, and C.J. Drummond,Sugar Fatty Acid Ester Surfactants: Base-Catalyzed Hydroly-sis, J. Surfact. Deterg. 3:29 (2000).

[Received May 30, 1999; accepted October 5, 1999]

Irene Baker investigated the biodegradation of surfactants dur-ing her doctoral studies at the University of Melbourne andCSIRO Molecular Science. She performed research in the areasof surfactant chemistry and formulation of cleaning products.She also worked at the Victorian Environment Protection Au-thority, where she undertook air-quality monitoring and assess-ment.

Ian Willing is the director of the NMR facilities at CSIROMolecular Science, Clayton. He has over 30 years’ experience inthe application of multinuclear high-resolution NMR spec-troscopy to wide-ranging chemical problems.

Neil Furlong has worked at CSIRO for 18 years, most re-cently as a Chief Research Scientist and Program Manager ofSpecialty Chemicals and Environmental Technologies. His re-search focused on varied aspects of surface chemistry with par-ticular focus on surfactants, Q-state particles, and biosensors.

Franz Grieser completed his Ph.D. studies at the Universityof Melbourne in 1977 in the Department of Physical Chemistry.He held research fellow positions at the University of NotreDame, Indiana, and at the Hahn-Meitner Institute, Berlin, andis now a Reader in Physical Chemistry at the University of Mel-bourne. His research interests are in interfacial chemistry, sono-chemistry, and the physical properties of nanoparticles.

Calum Drummond studied physical chemistry at The Uni-versity of Melbourne. In 1987 he began working at the CSIRO.He is currently a Senior Principal Research Scientist and Pro-ject Leader for Applied Surface Chemistry in the Division of Mo-lecular Science. His research interests include colloid and sur-face science with an emphasis on surfactants.



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