Environ. Sci. Technol. 2000, 34, 1737-1741
Biodegradation Mechanisms of Linear Alcohol Ethoxylates under Anaerobic Conditions MARTIN HUBER, ULRICH MEYER,* AND PAUL RYS Laboratory of Industrial Chemistry and Chemical Engineering, Swiss Federal Institute of Technology (ETH), CH-8092 Zu ¨ rich
The anaerobic biodegradation mechanisms of linear alcohol ethoxylates (LAE) were studied in incubation experiments with anoxic sewage sludge. Sophisticated analytical techniques were applied, such as solid-phase extraction (SPE) followed by reversed phase high performance liquid chromatography (HPLC) procedures based on the derivatization of LAE and poly(ethylene glycol) (PEG). During the degradation of LAE C12(EO)˜9, a technical dodecanol ethoxylate with an average of nine ethoxy (EO) units, and LAE C12(EO)8, a single ethoxymer, alcohol ethoxylates with shortened EO chains were released as the first identifiable metabolites, but no PEG products were observed. From our results it was concluded that the first step of anaerobic microbial attack on the LAE molecule is the cleavage of the terminal EO unit, releasing acetaldehyde stepwise, and shortening the ethoxy chain until the lipophilic moiety is reached. In contrast to the aerobic degradation pathway, where central scission prevails (the cleavage of the ether bond between alkyl and ethoxy chains), such a primary attack on the surfactant molecule is very unlikely in an anaerobic community of fermenting bacteria.
Introduction A knowledge of the anaerobic biodegradability of surfactants is necessary for a complete assessment of their environmental fates. Due to their amphiphilic properties, surfactants adsorb readily onto solid particles such as those in primary sludges in aerobic sewage treatment plants and can often reach anaerobic conditions quickly without a preceding aerobic treatment. Moreover, direct anaerobic treatment processes for highly loaded wastewaters in food or textile manufacturing industries have become the focus of interest because of their positive energy balance and the significantly lower costs of sludge disposal in comparison with aerobic treatments (1). Especially in textile wastewaters, a good anaerobic biodegradability of the surfactants which are present is a prerequisite for such a treatment, because the toxicity of higher surfactant concentrations is likely to endanger the sensitive acetogenic and methanogenic bacterial activity (2-4). Linear alcohol ethoxylates (LAE) of the common structural formula CH3(CH2)mO(CH2CH2O)nH (m ) 7-17, n ) 1-25) represent the economically most important group of nonionic surfactants and are widely used as detergents, solubilizers, emulsifiers, and wetting and dispersing agents in household and industrial chemicals. Although the ultimate anaerobic biodegradability of linear alcohol ethoxylates has been demonstrated in several studies * Corresponding author phone: +41 1 632 30 36; fax: +41 1 632 10 74; e-mail:
[email protected]. 10.1021/es9903680 CCC: $19.00 Published on Web 03/30/2000
2000 American Chemical Society
(5-7), the primary degradation mechanism has not yet been fully elucidated because of the lack of appropriate and sensitive analytical methods for the characterization of the degradation metabolites. There is a current belief that the major primary attack on the LAE molecule by anaerobic microorganisms occurs at the central ether bridge linking the alkyl chain with the ethoxy chain (central scission), as is well-known under aerobic conditions (8, 9), and has clearly been shown recently by Marcomini and Zanette (10). However, in contrast to extensive aerobic studies, firm evidence of anaerobic degradation by central scission has been scarce, and therefore the discussion of the primary mechanism has still remained controversial. Steber and Wierich (5) concluded from their work with two stearyl alcohol ethoxylates 14C-labeled either in the alkyl or in the ethoxy chain that the surfactants were cleaved by central scission, because small amounts of neutral PEG-like materials were found in the experiment with the EO chain labeled surfactant in contrast to the one with the alkyl chain labeled LAE. Wagener and Schink (6), however, proposed that the degradation of LAE proceeded in analogy to that of poly(ethylene glycol)s from the free PEG end, releasing C2 units stepwise until the lipophilic alkyl moiety is reached. Data from anaerobic PEG degradation showed acetaldehyde as the first identifiable metabolite (11-13), and the authors suggested that the initial degradation step is a hydroxyl group exchange reaction, followed by a shortening of the ethoxy chain by stepwise cleavage of acetaldehyde. However, both studies carried out lacked the elucidation potential of sophisticated analytical techniques, such as recently developed chromatographic procedures based on the derivatization of LAE and PEG followed by reversedphase liquid chromatography (HPLC) (14). The object of this work has been that of elucidating the primary anaerobic degradation mechanism by applying specific HPLC analytical methods in order to characterize and quantify all relevant degradation metabolites. For this purpose, the progression of the biodegradation of two different LAE, a pure C12(EO)8 (single ethoxymer) and a technical C12(EO)˜9 with an average of nine ethoxy units, has been investigated in detail, separating and identifying all relevant alcohol ethoxymers and poly(ethylene glycol)s as possible metabolites.
Materials and Methods Chemicals. Linear C12(EO)8 as the original surfactant for the degradation experiment as well as C12(EO)9, C12(EO)7, C12(EO)6, C12(EO)5, C12(EO)4, C12(EO)3, C12(EO)2, C12(EO)1, C12OH, and C10(EO)8 as calibration standards (all purities higher than 98%) were purchased from Fluka AG, Buchs, Switzerland. The technical linear C12(EO)˜9, a dodecanol ethoxylate with an average of nine ethoxy units, was kindly supplied by Dr. W. Kolb AG, Hedingen, Switzerland. Poly(ethylene glycol) 400 was purchased from Fluka AG, Switzerland. Biodegradation Assay. A 350 mL batch bottle was connected by a steel capillary to two glass cylinders filled with NaCl saturated 1 M sulfuric acid as a gas blocking solution, allowing for simple volumetric biogas measurement. Three hundred milliliters of RAMM (revised anaerobic mineral medium by Shelton and Tiedje (15)) to which 1 g of dry substance from an anaerobic sludge had been added, were transferred under nitrogen pressure into the batch bottle. The sludge came from the anaerobic sewage treatment reactor of the potato chip factory 11er in Frastanz, Austria. At the beginning of the experiment the surfactant solution VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Reversed-phase separation of ethoxymers of C12(EO)˜9 (A) and poly(ethylene glycol) 400 (B). was injected through a rubber/Teflon septum, and the two gas cylinders were leveled. The biodegradation assay was performed under continuous stirring and buffered at pH 7.2 and at a constant water bath temperature of 37 °C. All experiments were conducted in two identical bottles: In the first one only the biogas production was measured. Liquid samples (2 mL) were drawn from the second one for immediate analysis. Additional samples were stored at -20 °C after addition of Formalin (8% final concentration), to inhibit bacterial activity. Sample Preparation. The samples were centrifuged, and the biomass was extracted twice with methanol. The supernatant aqueous phase and the two extraction phases were combined, and the analytes were enriched and further extracted from the bioassay solution by solid phase extraction (SPE), using a polystyrene-divinylbenzene solid phase (Chromabond HR-P, 200 mg/3 mL cartridges, Macherey-Nagel, Oensingen, Switzerland), extracting specifically LAE and PEG. The cartridges were sequentially conditioned with 6 mL of methylene chloride/methanol 80:20 (v/v), 2 mL of methanol, and 20 mL of HCl 10 mM. Then the sample was spiked with 7.8 µmol/L of C10(EO)8 as internal standard, and the cartridge was rinsed with 5 mL of water. The analytes were desorbed from the solid phase with 2 mL of methanol and 7 mL of methylene chloride/methanol 80:20 (v/v), and this extract was then evaporated and transferred with 1.5 mL of acetone into a 2 mL screw cap vial. After evaporation 200 µL of acetonitrile, 15 µL of pyridine, and 20 µL of 1-naphthoyl chloride (NC) were added, and the LAE and PEG were derivatized into the corresponding fluorescent ester in 20 min at 80 °C. The excess of NC was hydrolyzed with 1 mL of acetonitrile/water 90:10 (v/v), sonicated, and centrifuged, and the liquid phase was further diluted for HPLC analysis. 1738
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Similar LAE analytical methods have been described elsewhere (14, 10, 16). Analysis. The HPLC apparatus consisted of a Merck L-7100 binary pump, an L-7200 autosampler, a L-7480 fluorescence detector, and a Merck HPLC System-Manager data processing software. LAE and PEG were chromatographed on a 250 × 4 mm LiChrospher 100, 5 µm, C18 reversed-phase column, and a 40 mm precolumn of the same sorbing material (Merck, Dietikon, Switzerland). The ethoxymer separation of alcohol ethoxylates was attained with a linear gradient elution, starting with 70:30 (v/v) acetonitrile/water, which was increased to 94:6 in 66 min. The flow rate of the mobile phase was 1.5 mL/min. The ethoxymer separation of poly(ethylene glycol)s was achieved with a 50:50 acetonitrile/water gradient, increasing to 72:28 in 30 min and to 86:14 in 36 min with the same flow rate of 1.5 mL/min. The fluorescence detector (FD) was operated at λecc ) 228 nm and λem ) 365 nm.
Results and Discussion Surfactant and Intermediate Quantitation. Analyte quantitation was accomplished by the internal standard quantification procedure. The pure LAE with ethoxylation units ranging from 9 to 0 were calibrated at four concentration standard levels, the molar responses of C12(EO)n species with n > 9 were considered to be equal to that of C12(EO)9. Poly(ethylene glycol) ethoxymers were calibrated using a mixture of PEG 400. Figure 1 shows the attained reversed-phase separation of individual LAE- and PEG-ethoxymers in two selected LC-FD chromatograms. The application of this analytical procedure allowed a safe and sensitive determination of analytes down to concentrations of at least 0.5 µmol/L.
FIGURE 2. Concentration vs time plots of the individual dodecanol ethoxymers during anaerobic biodegradation of 40 mg/L of C12(EO)˜9.
FIGURE 3. Concentration vs time plots of the individual dodecanol ethoxymers during anaerobic biodegradation of 5 mg/L of C12(EO)8. Biodegradation of Technical LAE C12(EO)˜9. C12(EO)˜9, a dodecanol ethoxylate with an average of nine ethoxy units, was incubated up to 40 days at a starting concentration of about 40 mg/L. Within 25 days (600 h) the surfactant was completely mineralized with a dissolved organic carbon (DOC) elimination amounting to 97.5% and a degree of mineralization (biogas plus dissolved inorganic carbon) of 85.1%. Figure 2 shows the biodegradation time profile of each individual C12-ethoxymer (in µmol/L), ranging from C12(EO)17 down to the unethoxylated dodecanol (C12(EO)0 ) C12OH). During biodegradation the EO-distribution shifted toward LAE of lower degrees of ethoxylation, while the concentrations of C12(EO)4, C12(EO)3, and C12(EO)2 significantly exceeded those of the original ethoxymers after about 45 h of degradation time. Even if these lower ethoxylated LAE were eliminated more slowly compared to the higher homologues (which in fact would contradict results elsewhere (5)), an actual increase in concentration could only be explained by the formation of these compounds as metabolites from the degradation of higher LAE by subsequent shortening of the EO chain. During the biodegradation time span of 40 days examined no poly(ethylene glycol)s in the range from (EO)16 to (EO)5 could be observed, although the analytical method was capable of detecting amounts as low as 0.5 µmol/L. Theo-
retically, it could be assumed that further conversion of PEG metabolites from a possible central scission occurred much faster than their production. However, such an argument is not in line with published data of aerobic LAE and PEG degradation experiments (8, 10), which demonstrated a considerably slower degradation of PEG in comparison with that of LAE. Biodegradation of Pure LAE C12(EO)8. To investigate the shortening of the alcohol ethoxy chain in more detail, a pure dodecanol octaethoxymer (>98%) was incubated at a starting concentration of about 40 mg/L. In addition, because of possible surfactant toxicity at such concentrations, another experiment was performed with a lower concentration of 5 mg/L. Figure 3 depicts the biodegradation profile of 5 mg/L of C12(EO)8 over the time span of approximately 22 days (528 h), in which the original surfactant was completely eliminated (>99%). As a result of the cleavage of EO units, LAE of lower degrees of ethoxylation accumulated as metabolites, which were stepwise shortened, so that every single ethoxymer from C12(EO)7 to C12OH was observed. As in the experiment with the technical LAE C12(EO)˜9, no detectable amounts of PEG were released. The biodegradation time profile of 40 mg/L of C12(EO)8 (Figure 4) showed a similar degradation pattern to that of a 5 mg/L solution, however, due to slight inhibitory surfactant VOL. 34, NO. 9, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Concentration vs time plots of the individual dodecanol ethoxymers during anaerobic biodegradation of 40 mg/L of C12(EO)8. effects the reaction time was considerably extended. Complete removal of all LAE ethoxymers was attained after about 50 days (1200 h). As a consequence of cleavage of EO units considerable amounts of LAE of lower degrees of ethoxylation were released as metabolites in the range between C12(EO)7 and C12(EO)3, whereas the shortest LAE accumulated in trace amounts only. After about 40 days (1000 h) a substantial increase of degradation rate was observed, eliminating all the LAE present very quickly. Once again, no central scission occurred and no PEG metabolites were observed. Primary Anaerobic Biodegradation Pathway of LAE. In all of the three biodegradation assays performed on linear alcohol ethoxylates, the first identifiable degradation products were LAE shortened by a stepwise cleavage of EO units. Subsequent metabolites included dodecanoic acid, acetic acid, and the final products methane and carbon dioxide, whereas no PEG were observed during the whole degradation period. These results support the biodegradation pathway proposed by Wagener and Schink (6) (Figure 5), where the first step of anaerobic microbial attack on the surfactant molecule is the cleavage of the terminal EO unit, releasing C2 units stepwise as acetaldehyde to give the corresponding shortened LAE, until the lipophilic moiety is reached. Although the release of acetaldehyde could not be observed during our degradation processes, acetaldehyde was shown to appear as the first identifiable product during the anaerobic degradation of PEG (11-13). Recently, Frings et al. (17) detected two active enzymes in extracts of strictly anaerobic PEG fermenting bacteria. These enzymes were characterized as a diol dehydratase and a PEG acetaldehyde lyase. This study confirmed Wagener and Schink’s proposed initial step of PEG degradation, i.e., a terminal hydroxyl group shift, analogous to a coenzyme B12-dependent diol dehydratase reaction to form a hemiacetal that can be easily cleaved. The acetaldehyde released is then further disproportionated to acetic acid and ethanol and finally mineralized (18). Analogous to the PEG degradation pathway it can be assumed that the degradation of LAE proceeds via the same enzymatic steps. In the present study no evidence was found for a LAE degradation initiated by central scission, as proposed by Steber and Wierich (5) and which is known to occur predominantly in the aerobic pathway (8, 9). Recently, in extensive aerobic degradation studies with sophisticated HPLC techniques (similar to those applied in the present work), Marcomini and Zanette (10) demonstrated the release 1740
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FIGURE 5. Proposed pathway of LAE degradation by an anaerobic community of fermenting bacteria. of 60-70 mol % of the corresponding PEG simultaneously with the elimination of the LAE investigated. Furthermore, no LAE shortened by EO cleavage were observed. Central scission in anaerobic degradation cannot be completely ruled out, but it is improbable: Enzymes for the direct hydrolysis of ether bonds do exist but are less common than are those for hydrolysis of esters and hemiacetals (8). In the aerobic LAE degradation pathway it is assumed that the mechanism of the central ether scission involves an initial oxygenation at the R-carbon atom of the hydrophobe, forming a hemiacetal or an ester, which can then be easily cleaved by hydrolysis, as shown in the following reaction sequence (8).
This type of oxidation requires molecular oxygen and is therefore not possible under anaerobic conditions. Further-
more, the degradation of secondary alcohol ethoxylates (AE) cannot proceed via this oxidation route without deep-seated preliminary changes to allow acceptance of the oxo oxygen, nor can alkylphenol ethoxylates (APE) do so without prior disruption of the aromatic ring structure. In fact, neither the degradation of secondary AE nor that of APE occurs via central scission. The former proceeds by ω-alkylchain oxidation or shortening of the EO chain, the latter only by EO cleavage. Therefore, a direct hydrolytic ether scission of any AE must be ruled out for the time being. From our results and the above discussion it is clear that the anaerobic degradation of LAE can proceed only via terminal attack on the EO chain.
Literature Cited (1) Stronach, S. M.; Rudd, T.; Lester, J. N. In Anaerobic digestion processes in industrial wastewater treatment; Springer: Berlin, 1986. (2) Jones, J. B.; Bowers, B.; Stadtman, T. C. J. Bacteriol. 1977, 130, 1357. (3) Rinzema, A.; Boone, M.; Knippenberg, K. Water Environ. Res. 1994, 66, 40. (4) Hanaki, K.; Matsuo, T.; Nagase, M. Biotech. Bioeng. 1981, 23, 1591. (5) Steber, J.; Wierich, P. Water Res. 1987, 21, 661. (6) Wagener, S.; Schink, B. Appl. Environ. Microbiol. 1988, 54, 561.
(7) Salanitro, J. P.; Diaz, L. A. Chemosphere 1995, 30, 813. (8) Swisher, R. D. In Surfactant Biodegradation, 2nd ed.; Surfactant Science Series 18; Marcel Dekker: New York, 1987. (9) Holt, M. S.; Mitchell, G. C.; Watkinson, R. J. In Anthropogenic compounds: Detergents; de Oude, N. T., Ed.; The Handbook of Environmental Chemistry; Springer: Berlin, 1992; Vol. 3, Part F. (10) Marcomini, A.; Zanette, M. Riv. Ital. Sostanze Grasse 1996, 73, 213. (11) Dwyer, D. F.; Tiedje, J. M. Appl. Environ. Microbiol. 1983, 46, 185. (12) Dwyer, D. F.; Tiedje, J. M. Appl. Environ. Microbiol. 1986, 52, 852. (13) Strass, A.; Schink, B. Appl. Microbiol. Biotechnol. 1986, 35, 37. (14) Marcomini, A.; Zanette, M. Riv. Ital. Sostanze Grasse 1994, 71, 203. (15) Shelton, D. R.; Tiedje, J. M. Appl. Environ. Microbiol. 1984, 47, 850. (16) Lux, J. A.; Schmitt, M. Proceedings of the 4th World Surfactants Congress; Barcelona, 1996; Vol. 3, p 113. (17) Frings, J.; Schramm, E.; Schink, B. Appl. Environ. Microbiol. 1992, 58, 2164. (18) Schink, B.; Stieb, M. Appl. Environ. Microbiol. 1983, 45, 1905.
Received for review March 31, 1999. Revised manuscript received January 18, 2000. Accepted January 18, 2000. ES9903680
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