Hydroxylation of the Herbicide Isoproturon by Fungi Isolated from Agricultural Soil

Several asco-, basidio-, and zygomycetes isolated from an agricultural field were shown to be able to hydroxylate the phenylurea herbicide isoproturon [N-(4-isopropylphenyl)-N′,N′-dimethylurea] to N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea and N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Bacterial metabolism of isoproturon has previously been shown to proceed by an initial demethylation to N-(4-isopropylphenyl)-N′-methylurea. In soils, however, hydroxylated metabolites have also been detected. In this study we identified fungi as organisms that potentially play a major role in the formation of these hydroxylated metabolites in soils treated with isoproturon. Isolates of Mortierella sp. strain Gr4, Phoma cf. eupyrena Gr61, and Alternaria sp. strain Gr174 hydroxylated isoproturon at the first position of the isopropyl side chain, yielding N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea, while Mucor sp. strain Gr22 hydroxylated the molecule at the second position, yielding N-(4-(1-hydroxy-1-methylethyl)phenyl)-N′,N′-dimethylurea. Hydroxylation was the dominant mode of isoproturon transformation in these fungi, although some cultures also produced traces of the N-demethylated metabolite N-(4-isopropylphenyl)-N′-methylurea. A basidiomycete isolate produced a mixture of the two hydroxylated and N-demethylated metabolites at low concentrations. Clonostachys sp. strain Gr141 and putative Tetracladium sp. strain Gr57 did not hydroxylate isoproturon but N demethylated the compound to a minor extent. Mortierella sp. strain Gr4 also produced N-(4-(2-hydroxy-1-methylethyl)phenyl)-N′-methylurea, which is the product resulting from combined N demethylation and hydroxylation.     

Growth in coculture stimulates metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the beta-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of (14)C-labeled isoproturon to (14)CO(2) and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Growth in Coculture Stimulates Metabolism of the Phenylurea Herbicide Isoproturon by Sphingomonas sp. Strain SRS2

Metabolism of the phenylurea herbicide isoproturon by Sphingomonas sp. strain SRS2 was significantly enhanced when the strain was grown in coculture with a soil bacterium (designated strain SRS1). Both members of this consortium were isolated from a highly enriched isoproturon-degrading culture derived from an agricultural soil previously treated regularly with the herbicide. Based on analysis of the 16S rRNA gene, strain SRS1 was assigned to the β-subdivision of the proteobacteria and probably represents a new genus. Strain SRS1 was unable to degrade either isoproturon or its known metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, or 4-isopropyl-aniline. Pure culture studies indicate that Sphingomonas sp. SRS2 is auxotrophic and requires components supplied by association with other soil bacteria. A specific mixture of amino acids appeared to meet these requirements, and it was shown that methionine was essential for Sphingomonas sp. SRS2. This suggests that strain SRS1 supplies amino acids to Sphingomonas sp. SRS2, thereby leading to rapid metabolism of ¹⁴C-labeled isoproturon to ¹⁴CO₂ and corresponding growth of strain SRS2. Proliferation of strain SRS1 suggests that isoproturon metabolism by Sphingomonas sp. SRS2 provides unknown metabolites or cell debris that supports growth of strain SRS1. The role of strain SRS1 in the consortium was not ubiquitous among soil bacteria; however, the indigenous soil microflora and some strains from culture collections also stimulate isoproturon metabolism by Sphingomonas sp. strain SRS2 to a similar extent.     

Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon.

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the alpha-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Isolation from Agricultural Soil and Characterization of a Sphingomonas sp. Able To Mineralize the Phenylurea Herbicide Isoproturon

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the α-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.     

Biodegradation of the Phenylurea Herbicide Isoproturon and its Metabolites in Agricultural Soils

Degradation of the phenylurea herbicide isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) and several phenylurea and aniline metabolites was studied in agricultural soils previously exposed to isoproturon. The potential for degradation of the demethylated metabolite 3-(4-isopropylphenyl)-1-methylurea in the soils was much higher compared to isoproturon. In the most active soil only 6% of added ¹⁴C-labelled isoproturon was mineralised to ¹⁴C₂ within 20 days while in the same period 45% of added ¹⁴C-labelled 3-(4-isopropylphenyl)-1-methylurea was mineralized. This indicates that the initial N-demethylation may be a limiting step in the complete mineralization of isoproturon. Repeated addition of 3-(4-isopropylphenyl)-1-methylurea to the soil and further subculturing in mineral medium led to a highly enriched mixed bacterial culture with the ability to mineralize 3-(4-isopropylphenyl)-1-methylurea.The culture did not degrade either isoproturon or the didemethylatedmetabolite 3-(4-isopropylphenyl)-urea when provided as sole source of carbon and energy. The metabolite 4-isopropyl-aniline was also degraded and utilised for growth, thus indicating that 3-(4-isopropylphenyl)-1-methylurea is degraded byan initial cleavage of the methylurea-group followed by mineralizationof the phenyl-moiety. Several attempts were made to isolate pure bacterial cultures degrading 3-(4-isopropylphenyl)-1-methylurea or 4-isopropyl-aniline,but they were not successful.     

Residues effects of isoproturon in mature earthworm (Aporrectodea caliginosa) under laboratory conditions

This study was conducted to investigate the residues of isoproturon and its metabolites, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl) urea, and 4-isopropylanilin in soil and mature earthworms under laboratory conditions. Mature earthworms (Aporrectodea caliginosa) were exposed for various durations (7, 15, 30, and 60 days) to soils contaminated with isoproturon concentrations (2, 4, 6, 8, and 10 mg.kg(-1) soil). The decrease in isoproturon concentration in the soil depended on initial concentration it was slower at higher concentrations. The isoproturon and its metabolites accumulated in earthworms it increased during the first 15 days and decreased thereafter. Acute toxicity of isoproturon was determined together with total soluble protein content and glycogen of worms. These parameters were related to isoproturon concentration in soil and earthworms. No lethal effect of isoproturon was observed even at the concentration 1000 mg.kg(-1) soil after 60 days of exposure. A reduction of total soluble protein was observed in all treated worms (maximum 59.54%). This study is suggesting the use of the total soluble protein content and glycogen of earthworms as biomarker of exposure to isoproturon.     

Inducible hydroxylation and demethylation of the herbicide isoproturon by Cunninghamella elegans

A screening of 27 fungal strains for degradation of the phenylurea herbicide isoproturon was performed and yielded 15 strains capable of converting the herbicide to polar metabolites. The zygomycete fungus Cunninghamella elegans strain JS/2 isolated from an agricultural soil converted isoproturon to several known hydroxylated metabolites. In addition, unknown metabolites were produced in minor amounts. Inducible degradation was indicated by comparing resting cells pregrown with or without isoproturon. This shows that strain JS/2 is capable of partially degrading isoproturon and that one or more of the enzymes involved are inducible upon isoproturon exposure.     

Isolation of isoproturon-degrading bacteria from treated soil via three different routes

Three different isolation routes (flask enrichment/flask degradation assay, flask enrichment/microplate degradation assay, MPN assay/microplate degradation assay) were used to obtain pure cultures of bacteria which degraded isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) as sole carbon and nitrogen source in a mineral salts medium from a field soil treated with isoproturon in the laboratory. All three isolation routes were successful, but the microplate assay of degradation was more successful than the flask assay. Characterization of 36 isolates indicated that they formed 16 distinct phenotypes (10 Gram-positive phenotypes, six Gram-negative phenotypes) which are likely to represent distinct species. Low concentrations of the degradation product 3-(4-isopropylphenyl)-1-methylurea (IPPMU) were occasionally found in the culture solutions. When provided as the sole source of carbon and nitrogen, the monomethyl degradation product was itself rapidly degraded by several of the isolates. Some isolates were also able to use the demethylated degradation product 3-(4-isopropylphenyl)-urea (IPPU) as sole source of carbon and nitrogen, although there was occasionally an extended lag-phase before rapid degradation commenced. One isolate was particularly active and degraded isoproturon, the monomethyl and demethylated degradation products of isoproturon, and demethylated the related phenylureas diuron and linuron.     

Hydroxylation of quinocetone and carbadox is mediated by CYP1As in the chicken (Gallus gallus)

Quinoxaline derivatives (quinoxalines) comprise a class of drugs that have been widely used as animal antimicrobial agents and feed additives. Although the metabolism of quinoxaline drugs has been mostly studied using chicken liver microsomes, the biochemical mechanism of biotransformation of these chemicals in the chicken has yet to be characterized. In this study, using bacteria produced enzymes, we demonstrated that both CYP1A4 and CYP1A5 participate in the oxidative metabolism of quinoxalines. For CYP1A5, three hydroxylated metabolites of quinocetone were generated. In addition, CYP1A5 is able to hydroxylate carbadox. For CYP1A4, only one hydroxylated product of quinocetone on the phenyl ring was identified. Neither CYP1A5 nor CYP1A4 showed hydroxylation activity towards mequindox and cyadox. Our results suggest that CYP1A4 and CYP1A5 have different and somewhat overlapping substrate specificity in quinoxaline metabolism, and CYP1A5 represents a crucial enzyme in hydroxylation of both quinocetone and carbadox.     

Isolation and characterization of an isoproturon mineralizing <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. strain SH from a French agricultural soil

The phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was found to be rapidly mineralized in an agricultural soil in France that had been periodically exposed to IPU. Enrichment cultures from samples of this soil isolated a bacterial strain able to mineralize IPU. 16S rRNA sequence analysis showed that this strain belonged to the phylogeny of the genus Sphingomonas (96% similarity with Sphingomonas sp. JEM-14, AB219361) and was designated Sphingomonas sp. strain SH. From this strain, a partial sequence of a 1,2-dioxygenase (catA) gene coding for an enzyme degrading catechol putatively formed during IPU mineralization was amplified. Phylogenetic analysis revealed that the catA sequence was related to Sphingomonas spp. and showed a lack of congruence between the catA and 16S rRNA based phylogenies, implying horizontal gene transfer of the catA gene cluster between soil microbiota. The IPU degrading ability of strain SH was strongly influenced by pH with maximum degradation taking place at pH 7.5. SH was only able to mineralize IPU and its known metabolites including 4-isopropylaniline and it could not degrade other structurally related phenylurea herbicides such as diuron, linuron, monolinuron and chlorotoluron or their aniline derivatives. These observations suggest that the catabolic abilities of the strain SH are highly specific to the metabolism of IPU.     

Isolation and characterization of three <Emphasis Type="Italic">Sphingobium</Emphasis> sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains

Three strains of bacteria (designated as YBL1, YBL2, YBL3 respectively) capable of degrading isoproturon, 3-(4-isopropylphenyl)-1, 1-dimethylurea, were isolated from the soils of two herbicide plants. Based on the comparative analysis of the 16S rRNA gene, and phenotypic and biochemical characterization, these strains were identified as Sphingobium sp. The optimum conditions for isoproturon degradation by these strains were pH 7.0, and temperature 30°C. Mg²⁺ (1 mM) enhanced the isoproturon degradation rate, while Ni²⁺ and Cu²⁺ (1 mmol l⁻¹) inhibited isoproturon degradation significantly. These three strains also showed the ability to remove the residues of other phenylurea herbicides such as chlorotoluron, diuron and fluometuron in mineral salt culture medium. The N-demethylation was the first step of degradation of dimethylurea-substituted herbicides. Strain YBL1 was found capable of degrading both dimethylurea-substituted herbicides and methoxymethylphenyl-urea herbicides i.e. linuron (3-(3,4-dichlorophenyl)-1-methoxy-1-methylurea). Using the PCR method, partial sequences of the catechol 1,2-dioxygenase gene were obtained from these strains.     

Microbial conversion of jasmonates-hydroxylations by Aspergillus niger

Aspergillus niger is able to hydroxylate the pentenyl side chain of (-)-jasmonic acid (JA) leading to (11S)-(-)-hydroxy-JA/(11R)- (-)-hydroxy-JA (2:1) and (-)-11,12-didehydro-JA. Methyl (-)-jasmonate (JA-Me) is converted upon hydrolysis. During prolonged cultivation or at non-optimized isolation procedures, the 11-hydroxy-(9Z)-pentenyl side chain may isomerize to (10E)-9-hydroxy- and (9E)-11-hydroxy-compounds by allylic rearrangement. The fungus hydroxylates (+/-)-9,10-dihydro-JA at position C-11 into 11 xi-hydroxy-9,10- dihydro-JA. As JA-ME, the methyl dihydro-JA is hydroxylated only upon hydrolysis into the free acid.     

Diprenylated chalcones and other constituents from the twigs of Dorstenia barteri var. subtriangularis

The twigs of Dorstenia barteri var. subtriangularis yielded three diprenylated chalcones: (-)-3-(3,3-dimethylallyl)-5'-(2-hydroxy-3-methylbut-3-enyl)-4,2',4'-trihydroxychalcone, (+)-3-(3,3-dimethylallyl)-4',5'-[2'-(1-hydroxy-1-methylethyl)-dihydrofurano]-4,2'-dihydroxychalcone and 3,4-(6",6"-dimethyldihydropyrano)-4',5'-[2',-(1-hydroxy-1-methylethyl)-dihydrofurano]-2'-hydroxychalcone for which the names bartericins A, B and C, respectively, are proposed. Stipulin, beta-sitosterol and its 3-beta-D-glucopyranosyl derivative were also isolated. The structures of these secondary metabolites were determined on the basis of spectroscopic analysis, especially, NMR spectra in conjunction with 2D experiments, COSY, HMQC and HMBC. The structural relationship of bartericins B and C was further established by the chemical cyclization of one to the other.     

Transformation of a monoterpene ketone, piperitenone, and related terpenoids using Mucor piriformis

Biotransformation of piperitenone (I), 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II), and 2-(1-ethyl-1-propylidene)-5-methylcyclohexanone (III) was studied using a versatile fungal strain, Mucor piriformis. The organism initiates transformation of these compounds by hydroxylation at the allylic positions or at the tertiary carbon. Transformation of piperitenone (I) by this strain yielded 5-hydroxypiperitenone (Ic), 7-hydroxypiperitenone (Id), 7-hydroxypulegone (Ie), 10-hydroxypiperitenone (If), and 4-hydroxypiperitenone (Ig) as metabolites. It was possible to block some of the metabolic activities of the organism through structural modification of piperitenone (I). This was evidenced by the fact that biotransformation of 5,5-dimethyl-2-(1-methylethylidene)-cyclohexanone (II) yielded 5,5-dimethyl-2-(1-hydroxy-1-methylethyl)-2-cyclohexen-1-one (IIb) and 5,5-dimethyl-3-hydroxy-2-(1-methylethylidene)-cyclohexanone (IIa), whereas 2-(1-ethyl-1-propylidene)-5-methylcyclohexanone (III) yielded 6-(1-ethyl-1-propylidene)-5-methyl-2-cyclohexen-1-one (IIIb) and 6-(1-ethyl-1-propylidene)-5-hydroxy-5-methylcyclohexanone (IIIa) as metabolites. Based on the identification of the metabolites, pathways for the biotransformation of I, II, and III have been proposed. The mode of biotransformation of these compounds by M. piriformis also compared to their modes of metabolism in the rat system.     

Metabolic degradation of phenazine-1-carboxylic acid by the strain <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. DP58: the identification of two metabolites

The biotransformation of phenazine-1-carboxylic acid (PCA) by PCA-degrading strain Sphingomonas sp. DP58 yielded small quantities of metabolites and was demonstrated for the first time. The metabolites were isolated by using preparative high-performance liquid chromatography (HPLC). In addition, these were subsequently characterized by gas chromatography (GC)-mass spectrum (MS) after N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization and (1)H-nuclear magnetic resonance (NMR). They were identified as 4-hydroxy-1-(2-carboxyphenyl) azacyclobut-2-ene-2-carbonitrile (HPAEC) and 4-hydroxy-1-(2-carboxyphenyl)-2-azetidinecarbonitrile (HPAC). The two metabolites had transformational relationship between each other.     

Hydroxylated 2-amino-3H-phenoxazin-3-one derivatives as products of 2-hydroxy-1,4-benzoxazin-3-one (HBOA) biotransformation by Chaetosphaeria sp., an endophytic fungus from Aphelandra tetragona

The biotransformation of the phytoanticipin HBOA and its major degradation metabolites 2-hydroxy-N-(2-hydroxyphenyl)acetamide (7) and N-(2-hydroxyphenyl)acetamide (8) by Chaetosphaeria sp., an endophytic fungus isolated from Aphelandra tetragona, was studied. Three new metabolites could be identified as 2-amino-7-hydroxy-3H-phenoxazin-3-one (12), 2-acetylamino-7-hydroxy-3H-phenoxazin-3-one (13) and 7-hydroxy-2-(2-hydroxyacetyl)-amino-3H-phenoxazin-3-one (14). Structure elucidation of 12 and 13 was performed by MS, 1H, 13C NMR and 2D NMR techniques and confirmed by chemical transformation.     

2,4-Dichlorophenol degradation by the soil fungus Mortierella sp

We investigated the degradation pathways and kinetics of 2,4-dichlorophenol (DCP) by an endemic soil fungus, Mortierella sp. (Zygomycetes). Mortierella sp. degraded 32% of added DCP (final concentration, 250 microM) within 1 h. We identified four aromatic metabolites and found two DCP degradation pathways (a hydroxylation pathway and a dechlorination pathway). This is the first report of a dechlorination pathway in Zygomycetes.     

Aryl hydroxylation of isopropyl-3-chlorocarbanilate by soybean plants

Isopropyl-3-chlorocarbanilate-phenyl UL-¹⁴C (CIPC-¹⁴C) is absorbed, translocated and metabolized by soybean plants. Water-soluble metabolites in root and shoot were purified and the root major metabolite characterized. The acetylated aglucones from the β-glucosidase hydrolysis and the esters from the direct acetylation of CIPC-¹⁴C polar metabolites were purified by GLC and analysed by mass spectrometry. The data showed that the phenyl riong of CIPC-¹⁴C was hydroxylated by both root and shoot tissues. Isopropyl-5-chloro-2-hydroxycarbanilate (hydroxy-CIPC) was the predominant aglucone liberated by β-glucosidase from polar metabolites in root and shoot. The o-glucoside of hydroxy-CIPC was shown to be present, by direct acetylation and characterization. In shoot tissue the major metabolites were dechlorinated hydroxy-CIPC and were not hydrolysed by β-glucosidase. These data show that soybean root or shoot tissues hydroxylate the phenyl ring of CIPC-¹⁴C but do not alter either the isopropyl alcohol moiety or the earbamate bond.     

New metabolites in the degradation of fluorene by Arthrobacter sp. strain F101.

Identification of new metabolites and demonstration of key enzyme activities support and extend the pathways previously reported for fluorene metabolism by Arthrobacter sp. strain F101. Washed-cell suspensions of strain F101 with fluorene accumulated 9-fluorenone, 4-hydroxy-9-fluorenone, 3-hydroxy-1-indanone, 1-indanone, 2-indanone, 3-(2-hydroxyphenyl) propionate, and a compound tentatively identified as a formyl indanone. Incubations with 2-indanone produced 3-isochromanone. The growth yield with fluorene as a sole source of carbon and energy corresponded to an assimilation of about 34% of fluorene carbon. About 7.4% was transformed into 9-fluorenol, 9-fluorenone, and 4-hydroxy-9-fluorenone. Crude extracts from fluorene-induced cells showed 3,4-dihydrocoumarin hydrolase and catechol 2,3-dioxygenase activities. These results and biodegradation experiments with the identified metabolites indicate that metabolism of fluorene by Arthrobacter sp. strain F101 proceeds through three independent pathways. Two productive routes are initiated by dioxygenation at positions 1,2 and 3,4, respectively. meta cleavage followed by an aldolase reaction and loss of C-1 yield the detected indanones. Subsequent biological Baeyer-Villiger reactions produce the aromatic lactones 3,4-dihydrocoumarin and 3-isochromanone. Enzymatic hydrolysis of the former gives 3-(2-hydroxyphenyl) propionate, which could be a substrate for a beta oxidation cycle, to give salicylate. Further oxidation of the latter via catechol and 2-hydroxymuconic semialdehyde connects with the central metabolism, allowing the utilization of all fluorene carbons. Identification of 4-hydroxy-9-fluorenone is consistent with an alternative pathway initiated by monooxygenation at C-9 to give 9-fluorenol and then 9-fluorenone. Although dioxygenation at 3,4 positions of the ketone apparently occurs, this reaction fails to furnish a subsequent productive oxidation of this compound.