FEMSMicrobiologyLetters 96 (1992)253-258 © 1992Federationof EuropeanMicrobiologicalSocieties0378-1097/92/$05.00 Publishedby Elsevier
253
FEMSLE05.%1
Metabolism of 3-methyldiphenyl ether by Sphingomonas sp. SS31 Stefan Schmidt ~, Rolf-Michael Wittich a, Peter Fortnagel ~, Dirk E r d m a n n b and Wittko Francke t, lnstitut fiir AUgemeineBotanik. Abteihmgfiir Mikrobiologie, Unicersi,iitHat::bnrg, Hamburg, FRG. and h institut fiir OrganischeChemie. Unh'ersitlitHamburg, tlamburg. FRG
Received 14 May 1992 Revisionreceived6 July 1992 Accepted 7 July 1992 Key words: 3-Methyldiphenyl ether; Biodegradation; Dioxygenolytic ether cleavage; Ortho ring fission; Unproductive side-chain oxidation; Sphingomonas sp. SS31
1. SUMMARY The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methyleatechol, and 4-methyicatechol, respectively. Phenol and 3methylphenol were oxidized to the corresponding catechois which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.
Correspondence to: R.-M.Winich,UniversitiitHamburg,Insti-
tut fiir AIIgemeineBotauik, AbteilungMikrobiologie,OhnhorststraBe 18, D-2000Hamburg52, FRG.
Minor amounts of 3-(hydroxymethyl)-diphenyl ether detected in the supernatant of the culture broth gave evidence for an unproductive oxidation of the side-chain, finally leading to the nondegradable product 3-carboxydiphenyl ether.
2. INTRODUCTION Diphenyl ether (DE) derivatives have been, and in part are still, industrially produced in large amounts as heat transfer liquids, perfume additives, and fire retardants. Extensive utilization of diphenyl ether herbicides now represents a serious problem of environmental concern [1-3]. The fungus Omninghamella echimdata was shown recently to oxidize DE to the dead-end products 4-hydroxydiphenyl ether and 4,4'-dihydroxydiphenyl ether [4]. Bacterial biodegradation of DE by Pseudomonas species has been shown to proceed by 2,3-dioxygenation; the unproductive ortho cleavage of the respective diol yielded 2-phenoxymuconic acid [5]. Meta-cicavage of 2,3dihydroxydiphenyl ether and subsequent in-
254 tramolecular transesteriflcation of 2-hydroxy-6phenoxymuconic acid semialdehyde yielded the dead-end product 2-pyron-6-carboxylic acid [6]. In both cases, solely phenol was utilized for growth. Here, we report on the complete biodegradation of an alkyl-substituted DE.
3. MATERIALS AND METHODS
3.1. Organism The
chromatography (GC), and mass spectrometry (MS) coupled to GC were performed as previously described [7].
3. 4. Chemicals 3-Methylphenol, catechol and both methylcatechols, 3MDE (m-phenoxytoluene), 3-(hydroxymethyl)-diphenyl ether, 3-(hydroxymethyl)-phenol (3-hydroxybenzyl alcohol), and 3-carboxydiphenyl ether were from Aldrich-Chemic, Steinheim, FRG. All other chemicals were of the highest purity available.
diphenyl ether-degrading bacterium
Sphingomonas sp. SS3 (DSM 6432), originally isolated with 4-fluorodiphenyl ether as the target carbon source, was used for adaptation experiments to achieve complete utilization of the new carbon source 3-methyldiphenyl ether (3MDE). For this purpose, the strain was incubated in the presence of both DE and 3MDE, by stepwise replacing 3MDE for DE. The derivative of strain SS3 capable to utilize also 3MDE, was termed strain SS31 and used throughout our investigations.
3.2. Growth For growth of the organisms a previously described mineral salts medium was used [7]. The carbon source, 3MDE, which is water-soluble to a limited extent only (37/zM, corresponding to 7 mg/I at 28°C), was fed over the gas phase as described by Sander et al. [7] or was added to the freshly autoclaved mineral salts medium in case of growth of mass cultures. Substrate amounts corresponded to a concentration of 5 mM. Measurement of growth parameters was done as previously reported [71.
3.3. Enzyme assays and analytical methods Preparation of cell-free extracts, determination of protein, estimations of enzyme activities and oxygen demands, extraction of metabolites from the culture medium and their purification by preparative high-performance liquid chromatography (HPLC), identifications and quantitations of metabolites and co-oxidation products by HPLC, thin-layer chromatography (TLC), gas
4. RESULTS
4.1. Organisms; adaptation for, and utilization of, 3MDE The bacterial strain SS3 was formerly identified as a Pseudomonas sp. isolated from an enrichment culture with 4-fluorodiphenyl ether as the only carbon and energy source [8]. The strain utilized also benzoate and 4-hydroxybenzoate, but no other hydroxybenzoates, dihydroxybenzoates, nor any of the three isomeric methylbenzoates. Strain SS31, obtained as a derivative of the DEdegrading strain SS3 after an about 4-month adaptation period, now additionally utilized 3MDE, 3-methylphenol, 3-methyl- and 4-methylcatechol as new carbon sources; however, methylbenzoates were not utilized for growth, Further investigations performed by Deutsche Sammlung von Mikroorganismen (DSM; Braunschweig, FRG) upon the organisms fatty acids, lipids, and ubiquinones patterns (Elke Lang, DSM, personal communication) allowed the assignment of strain SS3 to the new genus Sphingomonas sp, recently proposed by Yabuuchi et al. [9]. The capability of Sphingomonas sp. SS31 to utilize 3MDE for growth is demonstrated in Fig. 1. The doubling times during the exponential phase of growth strongly depended on the bioavailability of the carbon source (if fed over the gas phase; use of flasks with baffles, on the other hand, killed the cells due to the too rapid dissolution kinetics) and was estimated to be 8.2 h in the course of the early growth phase. Control experiments performed in the absence of 3MDE
255 Table i Specific oxygen uptake rates by resting cells of strain
gomonas sp. SS3i Substrate assayed
[ ._~
0.1
e=
o.ot
- ~ - ~ , A - ~ " ~ 50
100
150
"~*'~''A-* 200
20
250
Time [hi
Sphin-
Specific oxygen demand (nmol 0 2 min- : mg protein- i) after growth with 3MDE
Acetate
126 108 90 18 145 36 72 45 27 27
il 9 7
253
FEMSLE05.%1
Metabolism of 3-methyldiphenyl ether by Sphingomonas sp. SS31 Stefan Schmidt ~, Rolf-Michael Wittich a, Peter Fortnagel ~, Dirk E r d m a n n b and Wittko Francke t, lnstitut fiir AUgemeineBotanik. Abteihmgfiir Mikrobiologie, Unicersi,iitHat::bnrg, Hamburg, FRG. and h institut fiir OrganischeChemie. Unh'ersitlitHamburg, tlamburg. FRG
Received 14 May 1992 Revisionreceived6 July 1992 Accepted 7 July 1992 Key words: 3-Methyldiphenyl ether; Biodegradation; Dioxygenolytic ether cleavage; Ortho ring fission; Unproductive side-chain oxidation; Sphingomonas sp. SS31
1. SUMMARY The bacterium Sphingomonas sp. SS31, which was obtained from the diphenyl ether-degrading strain Sphingomonas sp. SS3 by an adaptation process, utilized 3-methyldiphenyl ether for growth in addition to diphenyl ether. The initial enzymatic attack onto this compound proceeded by a regioselective, but non-specific dioxygenation at the carbon carrying the ether bridge and the adjacent carbon of the unsubstituted as well as the methyl-substituted aromatic nucleus. Upon spontaneous decomposition, the resulting unstable hemiacetal structure yielded 3-methylphenol and catechol, or phenol, 3-methyleatechol, and 4-methyicatechol, respectively. Phenol and 3methylphenol were oxidized to the corresponding catechois which, after subsequent ortho-cleavage, were channeled into the oxoadipate pathway.
Correspondence to: R.-M.Winich,UniversitiitHamburg,Insti-
tut fiir AIIgemeineBotauik, AbteilungMikrobiologie,OhnhorststraBe 18, D-2000Hamburg52, FRG.
Minor amounts of 3-(hydroxymethyl)-diphenyl ether detected in the supernatant of the culture broth gave evidence for an unproductive oxidation of the side-chain, finally leading to the nondegradable product 3-carboxydiphenyl ether.
2. INTRODUCTION Diphenyl ether (DE) derivatives have been, and in part are still, industrially produced in large amounts as heat transfer liquids, perfume additives, and fire retardants. Extensive utilization of diphenyl ether herbicides now represents a serious problem of environmental concern [1-3]. The fungus Omninghamella echimdata was shown recently to oxidize DE to the dead-end products 4-hydroxydiphenyl ether and 4,4'-dihydroxydiphenyl ether [4]. Bacterial biodegradation of DE by Pseudomonas species has been shown to proceed by 2,3-dioxygenation; the unproductive ortho cleavage of the respective diol yielded 2-phenoxymuconic acid [5]. Meta-cicavage of 2,3dihydroxydiphenyl ether and subsequent in-
254 tramolecular transesteriflcation of 2-hydroxy-6phenoxymuconic acid semialdehyde yielded the dead-end product 2-pyron-6-carboxylic acid [6]. In both cases, solely phenol was utilized for growth. Here, we report on the complete biodegradation of an alkyl-substituted DE.
3. MATERIALS AND METHODS
3.1. Organism The
chromatography (GC), and mass spectrometry (MS) coupled to GC were performed as previously described [7].
3. 4. Chemicals 3-Methylphenol, catechol and both methylcatechols, 3MDE (m-phenoxytoluene), 3-(hydroxymethyl)-diphenyl ether, 3-(hydroxymethyl)-phenol (3-hydroxybenzyl alcohol), and 3-carboxydiphenyl ether were from Aldrich-Chemic, Steinheim, FRG. All other chemicals were of the highest purity available.
diphenyl ether-degrading bacterium
Sphingomonas sp. SS3 (DSM 6432), originally isolated with 4-fluorodiphenyl ether as the target carbon source, was used for adaptation experiments to achieve complete utilization of the new carbon source 3-methyldiphenyl ether (3MDE). For this purpose, the strain was incubated in the presence of both DE and 3MDE, by stepwise replacing 3MDE for DE. The derivative of strain SS3 capable to utilize also 3MDE, was termed strain SS31 and used throughout our investigations.
3.2. Growth For growth of the organisms a previously described mineral salts medium was used [7]. The carbon source, 3MDE, which is water-soluble to a limited extent only (37/zM, corresponding to 7 mg/I at 28°C), was fed over the gas phase as described by Sander et al. [7] or was added to the freshly autoclaved mineral salts medium in case of growth of mass cultures. Substrate amounts corresponded to a concentration of 5 mM. Measurement of growth parameters was done as previously reported [71.
3.3. Enzyme assays and analytical methods Preparation of cell-free extracts, determination of protein, estimations of enzyme activities and oxygen demands, extraction of metabolites from the culture medium and their purification by preparative high-performance liquid chromatography (HPLC), identifications and quantitations of metabolites and co-oxidation products by HPLC, thin-layer chromatography (TLC), gas
4. RESULTS
4.1. Organisms; adaptation for, and utilization of, 3MDE The bacterial strain SS3 was formerly identified as a Pseudomonas sp. isolated from an enrichment culture with 4-fluorodiphenyl ether as the only carbon and energy source [8]. The strain utilized also benzoate and 4-hydroxybenzoate, but no other hydroxybenzoates, dihydroxybenzoates, nor any of the three isomeric methylbenzoates. Strain SS31, obtained as a derivative of the DEdegrading strain SS3 after an about 4-month adaptation period, now additionally utilized 3MDE, 3-methylphenol, 3-methyl- and 4-methylcatechol as new carbon sources; however, methylbenzoates were not utilized for growth, Further investigations performed by Deutsche Sammlung von Mikroorganismen (DSM; Braunschweig, FRG) upon the organisms fatty acids, lipids, and ubiquinones patterns (Elke Lang, DSM, personal communication) allowed the assignment of strain SS3 to the new genus Sphingomonas sp, recently proposed by Yabuuchi et al. [9]. The capability of Sphingomonas sp. SS31 to utilize 3MDE for growth is demonstrated in Fig. 1. The doubling times during the exponential phase of growth strongly depended on the bioavailability of the carbon source (if fed over the gas phase; use of flasks with baffles, on the other hand, killed the cells due to the too rapid dissolution kinetics) and was estimated to be 8.2 h in the course of the early growth phase. Control experiments performed in the absence of 3MDE
255 Table i Specific oxygen uptake rates by resting cells of strain
gomonas sp. SS3i Substrate assayed
[ ._~
0.1
e=
o.ot
- ~ - ~ , A - ~ " ~ 50
100
150
"~*'~''A-* 200
20
250
Time [hi
Sphin-
Specific oxygen demand (nmol 0 2 min- : mg protein- i) after growth with 3MDE
Acetate
126 108 90 18 145 36 72 45 27 27
il 9 7
