Metabolites and Biodegradation Pathways of Fatty Alcohol Ethoxylates in Microbial Biocenoses of Sewage Treatment Plants

Home , Stearyl alcohol

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1985, p. 530-537 Vol. 49, No. 3 0099-2240/85/030530-08$02.00/0 Copyright C) 1985, American Society for Microbiology Metabolites and Biodegradation Pathways of Fatty Alcohol Ethoxylates in Microbial Biocenoses of Sewage Treatment Plants JOSEF STEBER* AND PETER WIERICH Department of Ecology, Henkel KGaA, D4000 Dusseldorf 1, Federal Republic of Germany Received 26 September 1984/Accepted 3 December 1984

The biodegradation of fatty alcohol polyglycol ethers was studied by analyzing the 14C-labeled intermediates isolated from the effluent of a model continuous-flow sewage treatment plant after dosage of either alkyl- or heptaglycol-labeled stearyl alcohol ethoxylate (SA-7EO). In each case, uncharged and carboxylated (mainly dicarboxylated) polyethylene glycols constituted the most prominent metabolites. The results indicate that there is a faster degradation of the alkyl than the polyethylene glycol moiety and that there are two distinct primary degradation mechanisms acting simultaneously in microbial biocenoses: intramolecular scission of the surfactant as well as w- and I-oxidation of the alkyl chain. Characterization of the bulk of 14C-labeled metabolites as a homologous series of neutral and acidic polyglycol units and identification of several C2-fragments accounted for the depolymerization of the hydrophilic part of the surfactant by stepwise cleavage of ether-bound EO units; from additional degradation studies employing either neutral or carboxylated 14C-labeled polyethylene glycols as model metabolites, it was concluded that hydrolytic as well as oxidative cleavage of C2-units is involved. Most of the identified low-molecular-weight 14C-labeled acids suggest an ultimate degradation of EO monomers by the oxidative dicarbonic acid cycle or the glycerate pathway or both. In addition, the finding of considerable amounts of oxalic and formic acids allow consideration of an additional mineralization route via glyoxylic, oxalic, and formic acids. The simultaneous action of different degradation mechanisms indicates the involvement of several distinct bacterial groups in the biodegradation of fatty alcohol ethoxylates under environmental conditions.

Linear alcohol polyglycol ethers (alcohol ethoxylates) of Data on the ultimate biodegradation of stearyl alcohol the common structural formula R(OCH2CH2),OH represent ethoxylates (SA7-EO) (average number of seven EO units in the economically most important group of nonionic surfac- the hydrophilic part of the surfactant showing a typical tants. As a consequence of their use in detergent formula- homologue distribution with numbers of 1 - nEO c ca. 14), tions, they enter the environment in considerable amounts. separately labeled in the alkyl (1-_4C) as well as in the EO The question of their biodegradability and, ultimately, their chain (U-14C), have been reported previously (29). Employ- environmental fate constitutes the decisive criterion in their ing a continuous activated sludge model plant (3-h retention environmental compatibility assessment. time), the 14C-distribution among carbon dioxide, activated Experimental results from many studies in biodegradation sludge, and plant effluent was determined. The degree of discontinuous tests (13, 14, 17, 35), as well as in continuous ultimate biodegradation resulting from mineralization and model sewage treatment plants (12, 27, 29), suggest that biomass production amounted to more than 75% in the case there is a high extent of ultimate biodegradation of these surfactants in the environment. The polyethylene glycol of [1-_4C]SA7-EO and 65% in the case of the analogous (PEG) chain of linear alcohol ethoxylates has been found to EO-labeled model surfactant. be the slower degradable moiety of the surfactant molecule In this report will be described the characterization and (3, 12, 17, 18, 27, 29, 34). Therefore, a number of investiga- identification of radiolabeled metabolites isolated from the tions have dealt with the biodegradation of the PEG struc- effluent of the model plant. With the data from supplemen- ture and short chain homologues (4, 36), mainly employing tary degradation studies, as well as taking pertinent work of isolates from mixed bacterial cultures (10, 11, 26). A com- other investigators into account, a picture of the microbial prehensive picture of the microbial degradation routes lead- pathways that bring about ultimate biodegradation of fatty ing to the mineralization and assimilation of the surfactant alcohol ethoxylates (FAEs) in the environment will be made. molecule through metabolic intermediates is still lacking. The processes involved in the biodegradation of xenobiotic compounds can be well simulated in a model continu- MATERIALS AND METHODS ous-flow-activated sludge plant (6) such as the OECD Con- firmatory Test (19). Employment of radiolabeled model Test compounds. Stearyl alcohol-[U-14C]SA7-EO (specific compounds in such tests facilitates not only the acquisition activity, 20.5 mCi/g; radiochemical purity, 97%), [1- of information of the extent of ultimate biodegradation under 14C]stearyl alcohol-7 EO (specific activity, 19.2 mCi/g; radio- environmentally realistic conditions, i.e., in the simultane- chemical purity, 98%), and [U-14C]PEG 400 (average molec- ous presence of a surplus of other readily biodegradable ular weight, 400; specific activity, 8.3 mCi/g; radiochemical substances, but offers also the opportunity to analyze all purity, 97%) were purchased from NEN Chemicals, Drei- relevant degradation products and to get a deeper insight into the catabolic pathways involved. eich, Federal Republic of Germany (FRG). Carboxylated [14C]PEG 400 (ca. 10% monocarboxylated, 90% dicarboxylated) was prepared by adding 1 ml of aque- * Corresponding author. ous [14C]PEG 400 solution (7 mg/ml) to 0.8 ml of potassium 530 VOL. 49, 1985 MICROBIAL DEGRADATION OF FATTY ALCOHOL ETHOXYLATES 531 dichromate solution (40 mg/ml in 2 N sulfuric acid) and was performed with an Automatic Linear Analyzer LB 2828 reacting at 60°C for 2 h (23). Then, 3 ml of a saturated (Berthold, Wildbad, FRG). magnesium sulfate solution was added, and the mixture was Low-molecular-weight acids were analyzed by high-pres- extracted three times with 5 ml of chloroform each time (20). sure liquid chromatography (HPLC) with a Siemens S-100 After combining the organic extracts, evaporation, and unit equipped with a Rheodyne model 7125 syringe loading redissolving in water, the 14C-labeled sample was applied, sample injector (Latek, Heidelberg, FRG). Elution of refer- together with 1 mg of unlabeled PEG 400, on a small ence compounds was monitored continuously with a UV (volume, 3 ml) Dowex 21 K (OH-) column. After elution detector at 200 to 210 nm, and the eluate was sampled with with water, the carboxylated 14C-labeled material was recov- a fraction collector (model FOXY, ISCO Lincoln, Nebr.). ered by washing the column with 10 ml of 0.5 M sodium HPLC runs were made under the following conditions: (i) acetate solution (yield, 87% of original 14C-radioactivity). Column (250 by 4.6 mm) filled with RP 18 (Knauer, Berlin, Biodegradation test system. The OECD model sewage FRG); mobile phase, aqueous phosphoric acid, pH 2.6; treatment plant (19), which has a 3-h mean retention time temperature, 34°C; flow rate, 1.5 mlmin; pressure, 4 MPa; and employs an organic base nutrient medium (correspond- injection volume, 50 pA; fractionation volume, 150 [L. (ii) All ing to about 130 mg C/liter), was modified for investigations conditions as in (i), except that the mobile phase was as with 14C-labeled compounds as described in detail previ- follows: aqueous solution of 5.05 g of NaH2PO4 * H20 per ously (28, 29). During a 2-week working-in period, the base liter and 1.7 g of tetrabutyl ammonium hydrogensulfate medium, as well as 10 mg of unlabeled stearyl alcohol-8 EO (Merck, Darmstadt, FRG) per liter adjusted to pH 2.0 with per liter was continuously administered to the activated phosphoric acid (16). (iii) Column, lonpak Shodex C 811 (500 sludge of the plant that originated from a municipal sewage by 8 mm; Macherey-Nagel, Duren, FRG); mobile phase, as treatment plant. In the actual test, 14C-labeled model sub- in (i); temperature, 40°C; flow rate, 1 ml/min; pressure, 1.5 stance was employed. Within a 14C-feeding period of about 1 MPa. week, the radioactivity of evolved carbon dioxide, collected For the isolation of distinct metabolite species, the follow- effluents and, eventually, the sludge was determined. ing extraction procedures were employed: (i) Neutral PEGs Analytical methods. Neutral (uncharged) and anionic 14C- ( -4 EO) were recovered from a sodium chloride-saturated labeled metabolites were separated on a Dowex 1 x 2 (OH-) solution at pH 7 by threefold extraction with 1 volume of column after isolation of the residual surfactants from the chloroform (1); after acidification (hydrochloric acid, ca. pH plant effluent (29) and subsequent concentration of the 1) and extraction, the corresponding carboxylated PEGs surfactant-free solution with a rotatory evaporator. Then, were obtained. (ii) Uncharged and acidic PEGs (2 2 EO) the column was eluted consecutively with water and 1 N were separately extracted after the neutral solution was hydrochloric acid. evaporated and the solid residue was triturated with chloro- Gel chromatography was carried out with a Biogel-P 2 form to obtain the neutral oligomers (10). The acidic oligo- column (85 by 1.5 cm; Biorad, Munich, FRG). The solution mers were gained after acidification of the redissolved resi- to be analyzed (0.1 to 0.6 ml) was applied on the column due and evaporation and extraction as described above. (iii) together with standards (mono-, di-, tri-, and tetraethylene Isolation of oxalic, glycolic, and several other organic acids glycol and PEG 400; 2 mg each) and was eluted with water. was possible by extracting the acidic aqueous solution with Elution of reference compounds was monitored with a diethyl ether in a perforator for 24 h (15). refractometer (Knauer, Berlin, FRG) and the eluate sampled Chemical reduction of carboxylated PEGs to the corre- with a fraction collector. sponding neutral compounds was achieved by treating the Mono- and dicarboxylated PEGs were preparatively sep- sample by the method of Taylor and Conrad (32). After arated by anion exchange chromatography. Desalted (by gel purification by gel filtration, the acidic 14C-labeled material filtration) samples were applied, together with unlabeled and 7 mg of glycolic acid were dissolved in 10 ml of water, PEG 400 and glycolic and diglycolic acids (1 mg each) on a and the solution was adjusted to pH 4.75. Then, 200 mg of Dowex 1 x 2 (OH-) column (bed volume, 2.5 ml). After 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (Sigma elution with water, the acidic material was recovered from Chemical Co., Munich, FRG) was added in small portions, the column by linear gradient elution with 0.05 to 1 M with the pH maintained by the addition of 1.0 N hydrochlo- sodium acetate solution (20 ml each). ric acid. When the proton uptake was complete (ca. 2 h), 3 For the differentiation by thin-layer chromatography (TLC) ml of the solution was mixed dropwise with 6 ml of 2 M of neutral and carboxylated PEGs, the following systems sodium borohydride solution at room temperature. After were used: (i) silanized silica gel 60 (Merck, Darmstadt, stirring for several hours, the mixture was cooled in ice, and FRG), methanol-water-pyridine (50:50:5 [vol/vol]) (36); (ii) excess borohydride was destroyed by the dropwise addition silica gel 60, 2-butanone-methanol-water-ammonia of acetic acid. The carboxyl-reduced material was recovered (13:4:1:2); (iii) silica gel, diethyl ether-methanol-water from the concentrated solution by extraction as described (12:4:2); mono- and dicarboxylated PEGs were separated on above. cellulose plates in solvent (ii) and in the system; (iv) ethylace- Measurement of 14C-labeled compounds in solution was tate-pyridine-acetic acid-water (5:5:1:3). Amino acid compo- performed by liquid scintillation counting (28). sition of 14C-labeled protein hydrolysates (6 N hydrochloric acid, 100°C, 12 h) was assayed by TLC on silica gel plates RESULTS with authentic standards and the following solvents: (v) Analysis of degradation intermediates from model plant methanol-chloroform-17% ammonia (41:41:18); (vi) phenol- effluents. In the biodegradation test with the FAE radiolab- water (75:25); a ninhydrin spray reagent was used to detect eled in the EO chain, about 25% of the total radioactivity reference amino acids. For thin-layer electrophoresis on added (AO) was found in the plant effluent as soluble com- cellulose plates, the following buffers were employed: (vii) pounds, whereas the radioactivity of the effluent from the pyridine-acetic acid-formic acid-water (1:10:1.5:90), pH 2.8; alkyl-labeled model surfactant amounted to 9% of AO. Only (viii) acetic acid-pyridine-water (10:5:85), pH 4.3. Quantita- small amounts (1% of AO) of each could be attributed to tive determination of relative 14C-distribution on TLC plates intact parent surfactants (29). The main portion of effluent 532 STEBER AND WIERICH APPL. ENVIRON. MICROBIOL.

TABLE 1. 14C distribution of residual surfactants and degradation intermediates from the model plant effluent Radioactivity in effluent (% AO) 14C substrate Residual Degradation surfac- intermedi- tants ates Stearyl alcohol-[14C]7 EO 1.1 24.1 [1-14C]SA7 EO 0.6 8.6 I

radioactivity was due to intermediates of FAE biodegrada- 4 tion (Table 1). The surfactant-free effluent fraction was separated by ion-exchange chromatography into fractions of nonionic (neutral) and acidic "4C-labeled compounds. The radioactive metabolites of the EO-labeled model surfactant were mainly neutral, whereas after degradation of [1-14C]SA7-EO largely acidic 14C-labeled compounds were obtained (Table 2). Neutral metabolites. Neutral fractions originating from the 60 50 40 30 20 10 two differently labeled SA-7EOs with 14C were further traction number separated on a Biogel P-2 column. The resulting elution FIG. 1, Separation of neutral metabolites from the biodegrada- pattern of radioactivity resembled exactly a homologous tion of stearyl alcohol-[14C]7 EO (O) and [1-'4C]stearyl alcohol-7 series of PEG units (Fig. 1); the same characterization EO (A), respectively, on a Biogel P-2 column, The elution pattern of resulted from analyses by TLC. Neutral biodegradation reference nEO compounds (mono-, di-, tri-, and tetraethylene glycols, PEG 400, and PEG 1000) was monitored by refractometry intermediates ofthe EO-labeled model surfactant exhibited a distribution of PEG homologues beginning with the mono- ( ). mer (1 EO, ethylene glycol) and going to the polymers (>10 formerly carboxylated PEG compounds, an average chain EO) (Fig. 1; Table 3). The analogous metabolite fraction length of 6.3 (EO-labeled SA7-EO) and 7.7 (alkyl-labeled originating from the alkyl-labeled surfactant presumably SA7-EO) EO units was calculated (Table 3); these values contained PEGs with a chain length of .4 EOs (Fig. 1; must be corrected to somewhat lower figures because an Tables 2 and 3). Considering the respective specific molar extraction step was necessary before gel filtration, resulting radioactivity of single nEO homologues, an average chain in an incomplete recovery of short-chain PEGs (<4 EO). length of 3.3 EO and 6.7 EO, respectively, was calculated To differentiate between mono- and dicarboxylated PEGs, for the two PEG fractions originating from the EO- and the "4C-labeled acidic nEO compounds were separated by alkyl-labeled surfactants (Table 3). Acidic metabolites. On gel filtration of the two acidic metabolite fractions, the main portion of radioactivity was eluted along with the high-molecular-weight reference com- TABLE 3. Relative molar distribution of the individual 14C- pound PEG 1000 (Fig. 2). The acidic compounds migrated on labeled nEO homologues in the metabolite fractions from the TLC plates like carboxylated PEGs according to the char- biodegradation of the two model surfactantsa acterization by Watson and Jones (36). In addition, the Relative molar distribution (%) of the following electrophoretic mobility of these fractions at pH 4.5 and metabolites: their immobility at pH 2.8 indicated the presence of carboxyl nEO Alkyl-labeled groups. EO-labeled Finally, the main part of acidic 14C-labeled intermediates Neutral Acidicb Neutral AcidiCb originating from the two differently labeled model surfac- 1 14.4 Oc 6.4 1.7c tants was characterized unequivocally as carboxylated PEGs 2 26.6 9.7c 6.1 7.2c by chemical reduction of the acidic "4C-labeled material and 3 22.4 14.3c 5.7 5.4C subsequent gel chromatographic proof of formation of neu- 4 12.9 13.0c 11.6 5.4c tral PEGs (Fig. 2). From these gel filtration analyses of 5 12.0 8.5 7.2 5.4 6 6.1 12.0 11.3 7.7 7 1.7 6.3 8.9 12.0 8 1.3 10.0 10.5 10.5 TABLE 2. Relative distribution of radiolabeled intermediates in 9 0.9 7.2 9.8 19.2 the effluent after differentiation by ion-exchange chromatography 10 0.7 7.2 9.0 13.0 and PEG-specific extraction 11 0.5 3.3 4.0 9.0 Relative distribution of 14C intermediates (%) .12 0.5 8.6 9.5 3.2 4C substrate Neutral Acidic Avg nEO 3.3 <6.3c 6.7 <7.7c nEO com- Nonextrac- nEO com- Nonextrac- poundsa table poundsa table Based on gel chromatography data of "4C distribution and taking the specific activity of the individual homologues into account, the molar distribu- Stearyl alcohol- 35 31 14 20 tion as well as the average EO number was calculated. V4C]7 EO b Determined as neutral nEO after sodium borohydride reduction of the EO 12 <1 31 57 fraction. [1-_4C]SA7 c Low-molecular-weight homologues (nEO <4) were recovered incom-. a Average EO-number n - 4 (by the extraction method of Anthony and pletely, resulting in an excessive average EO number because an extraction Tobin [1]). step was necessary before gel filtration. VOL. 49, 1985 MICROBIAL DEGRADATION OF FATTY ALCOHOL ETHOXYLATES 533

600 - 0

r--0 0

E z.20 73 - 400- -T .2 0

200-

60 50 40 30 20 10 traction number FIG. 2. Separation of acidic metabolites from the biodegradation of stearyl alcohol-[U4C]7 EO by gel filtration on Biogel P-2 before (A) and after (0) chemical reduction with sodium borohydride. Reference nEO compounds were monitored by refractometry ( ). gradient ion-exchange chromatography (Fig. 3). Irrespective carboxylic acids present only in small amounts were tenta- of the radiolabel position, the main portion (80 to 90%) of tively identified (Table 4). these intermediates turned out to be dicarboxylated. Model studies on the further biodegradation of neutral and In addition to carboxylated nEO homologues, the acidic acidic PEGs. An additional biodegradation test in a continu- metabolite fraction contained other low-molecular-weight ous-flow-activated sludge model plant was carried out to "4C-labeled compounds that were partly eluted very prema- study the biodegradation of the main metabolite fractions, turely from the gel filtration column, i.e., contrary to expec- i.e., neutral and acidic PEGs, in more detail. The plant tations on the grounds of their molecular weight. As shown influent contained artificial sewage, according to the OECD by HPLC methods, the qualitative composition of this Confirmatory Test (19) and, in addition, unlabeled SA8-EO fraction of low-molecular-weight acids was largely compara- (10 mg/liter), as well as either neutral (0.6 mg/l) or carboxyl- ble, in spite of their origin from differently labeled model ated (0.4 mg/l) [14C]PEG 400 (equivalent to a chain length of surfactants. In each case, formic, acetic, oxalic, as well as about 9 EO). The resulting 14C-distribution among carbon glycolic and diglycolic acids, constituted the most prominent dioxide, sludge, and plant effluent after a dosage of 14C-la- "4C-labeled acids; in addition, several other C2 and C3 beled model metabolites for 3 and 6 days, respectively, is

E .0 to

5 10 15 20 25 fraction number FIG. 3. Separation of mono- and dicarboxylated nEO metabolites from the biodegradation of stearyl alcohol-[14C]7 EO (0) and [1-_4C]stearyl alcohol-7 EO (O), respectively, by gradient elution from a Dowex 1 x 2, OH- column. 534 STEBER AND WIERICH APPL. ENVIRON. MICROBIOL. shown in Table 5. There were no substantial quantitative TABLE 5. Balance and characterization of metabolites after differences between the results f6r the two 14C-labeled biodegradation of neutral and carboxylated ["4C]PEG 400, compounds, showing a considerable rate of ultimate biodeg- respectively, in the continuous activated sludge model plant radation. However, the composition of '4C-labeled com- Metabolites in pounds in the plant effluent was quite different. Biodegrada- '4C"Cbalancebalane(% Ao) the effluent (% tion of carboxylated [14C]PEG 400 yielded virtually only SA8 EO (unlabeled) test Aeffluent) acidic 14C-labeled products, whereas the neutral '4C-labeled substrate plus: substrate was degraded to neutral (average EO number, 4.9) Plant efflu- vatedActi- Neutral CO2Pan,ffuent sludge Acidic and carboxylated metabolites to about the same extent (Table 5). [14C]PEG 400a 52.0 3.9 + 3.7b 40.5 48.7 51.3 Carboxylated [14C]PEG 56.3 2.5 + 2.2b 39.0 5.6 94.4 DISCUSSION 400C Characterization of '4C-labeled metabolites from plant a Continuous addition to the model plant for 3 days. effluent revealed unequivocally that the PEG chain, as the b Radioactivity in the supernatant of the model plant at completion of the hydrophilic part of the surfactant molecule, is degraded test run. I Continuous addition to the model for 6 more slowly than the fatty alcohol moiety. This is in good plant days. agreement with the higher mineralization rates of the alkyl- labeled model surfactant (29) and confirms corresponding conclusions from several degradation studies conducted in static (3, 17, 18, 24, 34) and continuous (12, 27) test systems. Nevertheless, there is ample evidence that the PEG moiety yielded after biodegradation a much higher radioactivity in of detergent-range FAEs (with EO numbers of 5 to 15) is the lipid fraction of the produced biomass than did the ultimately also biodegradable (3, 14, 17, 18, 24, 29). Apart EO-labeled surfactant (29). This can be explained as a from that, the results of metabolite analyses and additional consequence of alkyl chain degradation via P-oxidation, experimental work presented here allow the discussion of because the resulting products represent the elementary the individual microbial degradation steps that result in the precursors of fatty acid biosynthesis. (iii) Degradation kinet- complete decomposition of the parent surfactant molecule. ics (17, 18) and characterization of carboxyalkyl-PEG inter- Primary degradation steps. Proof of acidic and, to a minor mediates (21, 27, 37) in other studies also indicate that extent, neutral PEGs as the main radiolabeled intermediates primary degradation of linear alcohol ethoxylates by micro- of the [1-_4C]SA7-EO showed that these polyglycol struc- bial biocenoses can proceed by this w- and p-oxidation tures still contain small residual fragments of the radiolab- scheme. eled alkyl chain which do not affect the characterization of From the formation of mainly dicarboxylated [alkyl-14C] was the metabolites as oxidized or neutral PEGs. This can be polyglycols, it concluded that the alkyl chain degrada- easily explained by a fast degradation of the fatty alcohol tion is accompanied by a slower, probably time-delayed, moiety of the surfactant, beginning with the terminal methyl microbial attack on the PEG chain end. This idea is sup- group and slowing down before radiolabeled C-1 is reached ported by the finding of smaller amounts of alkyl-labeled so that alkyl-labeled nEO compounds represent the most monocarboxylated nEO-intermediates (Fig. 3). prominent degradation intermediates of this model surfact- No satisfactory explanation exists at present for the ant. formation of the small but significant fraction of alkyl-la- There are several reasons for the existence of such a beled neutral nEO compounds (Table 2). The masking of primary degradation pathway, which is analogous to that of carboxyl groups by ester or amide formation could be linear alkylbenzene sulfonates (31) that encompass the ter- excluded after TLC examination of base- or acid-treated minal oxidation of the alkyl chain (w-oxidation) and subse- material. Therefore, a microbial reduction step, the purpose quent stepwise removal of C2 units at a time by of which is speculative at present, might have to be as- P-oxidation. sumed. (i) The isolated alkyl-labeled nEO metabolites are mainly dicarboxylated and must therefore originate from an oxida- Neutral PEGs representing the main portion of [14C]SA7- tive alkyl degradation. (ii) The alkyl-labeled surfactant EO degradation products (Tables 2 and 3) revealed the existence of a second mechanism of primary surfactant biodegradation because virtually all intermediates of the oxidative pathway would be acidic. This conclusion was TABLE 4. Radiolabeled low-molecular-weight acids identified in confirmed by degradation studies with a model metabolite, the acidic metabolite fractions by HPLC carboxylated [14C]PEG 400, which indicated that more than Low-molecular-weight '4C-labeled acids formed in the biodegradation of: 90% of the '4C-labeled intermediates are carboxylated (Ta- Stearyl alcohol-[14C]7 EO [1-14CJSA7 EO ble 5). In addition, it is evident from quantitative calcula- tions (Table 2) that the neutral PEGs under consideration are Acetate (+a) Acetate (+) largely not identical with the small fraction of uncharged Formate (+) Formate (+) nEO compounds which still contain minor fragments of the Oxalate (+) Oxalate (+) Diglycolate Diglycolate alkyl chain. Glycolate Glycolate These results give evidence for the existence of an addi- (Glyoxylateb) Succinate tional primary degradation mechanism by scission of the (Lactate) (Glyoxylate) surfactant molecule into the alkyl and PEG moieties. Similar (Oxaloacetate) (Malate) indications for such a mechanism have been reported by (Succinate) several authors who employed isolated (9, 25) or mixed (21, test a +, Main peak. 24, 34, 35) bacterial cultures in discontinuous systems. b Parentheses around acid name indicates that small amounts were tenta- Thus, in agreement with Patterson et al. (21), our results tively identified. demonstrate the simultaneous action of two distinct primary VOL. 49, 1985 MICROBIAL DEGRADATION OF FATTY ALCOHOL ETHOXYLATES 535

\\/'oN\oNo oXo-\,OH fatty alcohol + n EO

scission radation (w-/p- oxidation)

+ % [HHO\o',\.oxoxo\>OH] I HOOC- *H .0°\_\°% ° \,OOHI alkyl chain PEG chain carboxyalkyl - PEG

I non-oxidative oxidative and oxidative cleavage of f$- oxidation cleavage of C2- units C2- units

Intermediates e g Intermediates. e g HO\,O\,O,FOH neutral nEO [acetyl-CoA] HOOC.,O \,O\,O'COOH HOOC ON^,O COoH acidic nEO acidic n EO

I [neutral and acidic C2-units] [eg.ethylene glycol,gl c acidj [acidic C2-units]

Co2 + biomass FIG. 4. Biodegradation pathways of FAEs and their metabolites in microbial biocenoses of sewage treatment plants. *, the C-1 of the alkyl chain. biodegradation processes for linear alcohol ethoxylates: well as oxidative mechanisms, yielding neutral and acidic intramolecular scission of the surfactant molecule as well as products (Fig. 4). These findings are consistent with a oxidation of the alkyl chain (Fig. 4). picture of several different depolymerization mechanisms Depolymerization of the PEG chain. The reported findings acting simultaneously in microbial biocenoses. PEG degra- from previous (29) and present work allow us to propose a dation may be a consequence of the cleavage of neutral C2 nearly complete ultimate degradation pathway for the fatty units such as ethylene glycol (31) or acetaldehyde (22). alcohol moiety by well-known biochemical reactions (e.g., Acidic nEO compounds and C2 acids may arise from several 1-oxidation). The further biodegradation of the surfactant's oxidative mechanisms. In a repeated sequence, PEGs may hydrophilic part proceeds by the stepwise depolymerization be depolymerized by oxidation of either the terminal (11, 31) of the neutral or carboxylated PEGs obtained after primary or subterminal (31, 33) carbon(s), which is followed by biodegradation. Successive cleavage of monomeric C2 (i.e., cleavage of the ether or ester bond, respectively, leaving a EO) units seems to be the most probable mechanism be- nEO chain shortened by one oxidized C2 unit. Thus, it cause the isolated [14C]nEO metabolites represented in each appears understandable that single bacterial isolates degrad- case a homologous series of PEG units, ranging from the ing PEG oxidatively do not necessarily need to be able to monomers (ethylene glycol, glycolic acid) to dimers (diethyl- utilize a neutral EO monomer. This might explain why ene glycol, diglycolic acid) and the corresponding polymers certain isolates from enrichment cultures grown on high- or with EO numbers > 10 (Tables 3 and 4). Differences low-molecular-weight PEGs do not grow on ethylene glycol between the average EO numbers of EO- and alkyl-labeled (4). neutral and acidic nEO compounds, respectively (Table 3), Ultimate biodegradation of PEG depolymerization prod- are primarily because of the position of the 14C-label because ucts. According to the scheme in Fig. 4, one can expect detectability of [alkyl-'4C]PEGs becomes more and more formation of characteristic monomeric PEG-derived prod- unlikely with every cleavage of ether-bound EO units (cf. ucts from the degradation of the two differently labeled Fig. 4). As a result of several studies on the biodegradation model surfactants. As a matter of fact, in both cases C2 mechanism of PEGs and the corresponding oligomers, this fragments originating from PEG chain depolymerization, type of chain-shortening has been assumed to be the case by such as 14C-labeled ethylene glycol and glycolic, oxalic, and many other investigators as well (8, 11, 22, 33, 36). No glyoxylic acids were obtained (Tables 3 and 4). This empha- evidence was found for PEG chain degradation by the sizes the conclusion that after w- and 1-oxidation of FAEs, stepwise cleavage of oxidized Cl compounds (carbon diox- the small remaining portion of alkyl-derived carbons still ide, formate) as was recently proposed by Schoberl (26) bound to the PEG chain has lost its identity and is further based on triethylene glycol degradation studies employing a degraded as a constituent of the polyglycol structure (Fig. 4). Pseudomonas flluorescens strain. Characterization of [14C]acetic acid as a prominent low- In our study, the molecular mechanism of PEG depolymer- molecular-weight metabolite, even of the EO-labeled model ization has not been investigated in as much detail as by surfactant (Table 4), could indicate its direct origin from a some others (11, 22, 33). However, one conclusion drawn cleaved EO-unit. Acetate may be the oxidation product of from the experimental results is that there are strong differ- acetaldehyde formed either directly by PEG depolymeriza- ences between the degradation products of neutral and tion (22) or after the dehydration of ethylene glycol (38). carboxylated [14C]PEG model metabolites (Table 5): The Acetic acid and ethylene glycol and its oxidation products, nEO-intermediates resulting from primary surfactant degra- glycolic, glyoxylic, and oxalic acids, represent simple me- dation are not degraded further by only one depolymeriza- tabolites that are subject to ultimate biodegradation. Apart tion mechanism. In the case of the carboxylated PEGs, the from the central metabolic role of acetic acid, the final chain shortening proceeds oxidatively, i.e., leaving acidic degradation of the other 14C-labeled C2 compounds can be nEO oligomers. On the other hand, microbial depolymeriza- outlined as well. From various studies on ethylene glycol tion of neutral PEGs seems to be affected by hydrolytic as degradation by individual bacterial strains (2, 4, 7), it is 536 STEBER AND WIERICH APPL. ENVIRON. MICROBIOL.

'NdN.,6No,\,o6N ,O\,c e neutral /carboxylated which in an isolate from sewage sludge, grown on triethylene IN CO2H PEG chain glycol, has been indicated by studies by Schoberl (25). Final proof of this proposed additional catabolic sequence that non - 1/leavage of [C2 units\\xidative may involve several groups of microorganisms requires further investigation. Thus, results of this degradation study give evidence for HOCH2- CH20H o---daton HOCH2-C02H glycolic acid the simultaneous action of different mechanisms for each ethylene glycol catabolic step in the course of the biodegradation of alcohol ethoxylate, indicating that the complete biodegradation proc- - 2 [H] ess of the surfactant molecule is affected by several distinct bacterial groups. Therefore, degradation of FAEs by microbial biocenoses of sewage treatment plants offers a charac- OHC - CO2H glyoxylic acid teristic example for complex, but very effective mechanisms for the biodegradation of xenobiotic compounds, in the - 2 [H] environment. LITERATURE CITED -2 HCO2H -- HO2C- CO2H oxalic acid 1. Anthony, H. D. J., and R. S. Tobin. 1977. Immiscible solvent formic acid extraction scheme for biodegradation testing of polyethoxylate FIG. 5. for the oxidation of Additionally proposed pathway 2. nonionicChild, J.,surfactants.and A. Willets.Anal. 1978.Chem.Microbial49:398-401.metabolism of ali- PEG-derived C2 units by microbial biocenoses. phatic glycols. Bacterial metabolism of ethylene glycol. Bio- chim. Biophys. Acta 538:316-327. assumed that glyoxylic acid, as the oxidation product of 3. Cook, K. A. 1979. Degradation of the non-ionic surfactant glycolic acid and ethylene glycol, is channel ed into the Dobanol 45-7 by activated sludge. Water Res. 13:259-266. 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