Vol. 55, No. 10

Evidence for a New Pathway in the BacterialDegradation of 4-Fluorobenzoate

RUDIGER H. OLTMANNS,lt RUDOLF MULLER,l* MANFRED K. OTTO,2 AND FRANZ LINGENS'

Institut fur Mikrobiologie der Universitat Hohenheim, Garbenstrasse 30, D-7000 Stuttgart 70,1and Fraunhofer Institut fur Grenzflachen- und Bioverfahrenstechnik, D-7000 Stuttgart 80,2 Federal Republic of Germany

Received 30 June 1989/Accepted 11 July 1989

Six bacterial strains able to use 4-fluorobenzoic acid as their sole source of carbon and energy were isolatedby selective enrichment from various water and soil samples from the Stuttgart area. According to theirresponses in biochemical and morphological tests, the organisms were assigned to the genera Alcaligenes,Pseudomonas, and Aureobacterium. To elucidate the degradation pathway of 4-fluorobenzoate, metabolicintermediates were identified. Five gram-negative isolates degraded this substrate via 4-fluorocatechol, as

described in previous studies. In growth experiments, these strains excreted 50 to 90% of the fluoride fromfluorobenzoate. Alkaligenes sp. strains RHO21 and RHO22 used all three isomers of monofluorobenzoate.Alcaligenes sp. strain RHO22 also grew on 4-chlorobenzoate. Aureobacterium sp. strain RHO25 transientlyexcreted 4-hydroxybenzoate into the culture medium during growth on 4-fluorobenzoate, and stoichiometricamounts of fluoride were released. In cell extracts from this strain, the enzymes for the conversion of4-fluorobenzoate, 4-hydroxybenzoate, and 3,4-dihydroxybenzoate could be detected. All these enzymes were

inducible by 4-fluorobenzoate. These data suggest a new pathway for the degradation of 4-fluorobenzoate byAureobacterium sp. strain RHO25 via 4-hydroxybenzoate and 3,4-dihydroxybenzoate.

Fluorinated hydrocarbons are widely used as herbicides,fungicides, and pharmaceuticals (41, 46). Unlike organochlo-rine compounds, whose biodegradation has been investi-gated intensively, fluorinated hydrocarbons were rarely theobjects of microbial-degradation studies. The exceptionalbiological activities of fluorinated aromatic compounds canbe explained by the dichotomic chemical behavior of thefluorine substituent with its +M and -I effects together withits hydrogen-resembling size, reflected by the van der Waalsradii of 120 pm (for H) and 135 pm (for F) (36). Thisrelationship shows that monofluorobenzoates undergo diox-ygenation, forming fluorocatechols, or become subject toanaerobic degradation by benzoate-degrading bacteria (40).On the other hand, the exchange of a hydrogen atom by afluorine atom can create analogs that proceed along normalmetabolic pathways until the fluorine substituent produces aspecific enzyme inhibitor. Such a metabolic transformationis termed lethal synthesis (37). This effect was found withfluoroacetate and fluorocitrate as dead-end products (1, 13).The critical step in the degradation of fluorohydrocarbons isthe fission of the carbon-fluorine bond. For the cleavage ofthis bond in fluoroacetic acid, a rather specific enzyme hasbeen found (16, 45). In monofluorobenzoates, fluoride re-lease can occur after oxidative decarboxylation and ringfission of fluorocatechol (17, 18, 41). Alternatively, thefluorine is replaced in a dioxygenase-catalyzed reaction, asin the degradation of 2-fluorobenzoate, in which unsubsti-tuted catechol and 3-fluorocatechol are the products ofmetabolic transformation (6, 32, 46). The ability of microor-ganisms isolated on hydrocarbons without fluorine substitu-ents to grow on fluorinated compounds was demonstratedseveral times (12, 22, 23). In contrast, very few bacterialstrains with fluorinated hydrocarbons as the only growthsubstrates have been isolated directly (19, 24, 25, 46). In this

* Corresponding author.t Present address: Institut fur Industrielle Genetik, Universitat

Stuttgart, D-7000 Stuttgart 1, Federal Republic of Germany.

paper, we report the isolation and characterization of bacte-ria growing on 4-fluorobenzoate as the sole carbon source aswell as the identification of the first intermediates in thedegradation pathways.

MATERIALS AND METHODS

Bacterial strains. The strains isolated and investigated inthis work are listed in Table 1.Media and culture conditions. The media and culture

conditions were those previously described for the isolationof chlorobenzoate-degrading bacteria (10).Measurement of growth. Growth of the cultures was mon-

itored turbidimetrically at 546 nm with a Lambda 15 spec-trophotometer (The Perkin-Elmer Corp., Norwalk, Conn.)or an Eppendorf photometer (ilOiM; Netheler und Hinz,Hamburg, Federal Republic of Germany) equipped with a

400- to 600-nm filter (10 Klett units correspond to an opticaldensity at 546 nm of 0.063).Enrichment and isolation of 4-flporobenzoate-degrading

bacteria. Bacterial strains were isolated by selective enrich-ment from various water and soil samples from the Stuttgartarea. A soil sample (100 g) was added to 300 ml of mineralsalts medium (10) containing 2.5 mM 4-fluorobenzoate (0.35g/liter) and 2.5 mM succinic acid (0.3 g/liter) in closedErlenmeyer flasks. Alternatively, 1 volume of a water sam-

ple was mixed with 1 volume of medium containing 5.0 mM4-fluorobenzoate and 5.0 mM succinic acid. After beingshaken for 4 days at 30°C, the substrate concentrationdecreased; subcultures were transferred every second dayfor five passages into fresh mineral salts medium containing2.5 mM 4-fluorobenzoate. The cultures were plated on agarplates containing mineral salts medium with or without 2.5mM 4-fluorobenzoate. After 7 days, colorless or slightlybrown colonies approximately 1 mm in diameter appearedon the plates with 4-fluorobenzoate, whereas no colonieswere observed on plates without the substrate. After severaltransfers on solid medium with 4-fluorobenzoate as the only

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TABLE 1. Bacterial strains used in this work

Strain Source of isolate Doublingtime (h)

Alcaligenes sp. strain RHO21 Water from Neckar 6.8River

Alcaligenes sp. strain RHO22 Water from Korsch 6.4Brook

Pseudomonas sp. strain RHO23 Soil from Hohenheim 6.3Pseudomonas sp. strain RHO24 Soil from Hohenheim 5.6Aureobacterium sp. strain Soil from Hohenheim 7.0RHO25

Pseudomonas sp. strain RHO26 Soil from Hohenheim 5.0

growth substrate, the six strains designated RHO21 toRHO26 were isolated.Taxonomic classification of the bacteria. For the taxonomic

classification of the isolated bacteria, the following methodswere applied: Gram staining by the methods of Drews (11)and Suslov et al. (43), flagella staining by the method ofMayfield and Inniss (31), spore staining as described in Conn(7), and cyst staining according to Bergey's Manual ofSystematic Bacteriology (29). The G+C molar ratios weredetermined by the method of Flossdorf (14). Physiologicaltests were carried out by using the API 20E system (Appa-reils et Procddds d'Identification-System S.A., La Balme lesGrottes, France) and by the methods of Drews (11), Conn(7), Blazevic and Ederer (2), King et al. (26), Cerny (4), andHugh and Leifson (20). The classification of the strains wasdone according to Bergey's Manual (29, 42).

Analytical methods. Substrates and metabolites in culturemedia were analyzed by high-performance liquid chromatog-raphy. The conditions were the same as those described byOltmanns et al. (34). Thin-layer chromatography was per-formed with analytical silica gel layers (0.25 mm; SIL G-25UV254; Macherey-Nagel, Duren, Federal Republic of Ger-many) with toluene-ethylacetate-acetic acid (60:30:5, vol/vol/vol) as solvent. Fluorine ion concentrations were mea-sured with a halide flow injection sensor containing afluoride-selective lanthanum fluoride electrode as previouslydescribed (35). Gas-liquid chromatography (GLC) was car-ried out with a Carlo-Erba GC Mega 5300 equipped with aPS086 column and a flame ionization detector. Metaboliteswere identified by CI(C4H1O)-GLC mass spectrometry with aCarlo-Erba Fractovap 2151 (MILAN) interfaced directly to aFinnigan 4023 mass spectrometer with an INCOS 2300 datasystem (Finnigan MAT, Bremen, Federal Republic of Ger-many).

Isolation of metabolites. For the isolation of metabolicintermediates, cells were removed from the culture fluid bycentrifugation at 4,500 x g. The supernatant was acidified topH 2 with H3PO4 and extracted with diethyl ether at 0°C.The organic layer was dried over anhydrous Na2SO4 andevaporated. Metabolites were methylated with diazometh-ane and identified by GLC-mass spectrometry.

Chemicals. 4-Fluorocatechol was a generous gift fromK.-H. Engesser, Universitat Stuttgart, Stuttgart, FederalRepublic of Germany. All other chemicals were from com-mercial sources and of analytical grade.Oxygen measurements. Oxygen uptake studies with whole

cells of Aureobacterium sp. strain RHO25 were performedwith a Clarkelectrode (YSI 4004; Yellow Springs Instru-ments Co., Yellow Springs, Ohio) connected to a polarizingunit (Rank Brothers, Cambridge, United Kingdom). Thereaction chamber (335 ,ul) was stirred with a magnetic stirrer.The electrode was calibrated with oxygen-saturated water

TABLE 2. Utilization of carbon sources by newlyisolated microorganisms

Substrate Reaction of strain':(2.5 mM) RHO21 RH022 RH023 RH024 RH025 RH026

4-Fluorobenzoate +b + b + + + +3-Fluorobenzoate +b +b _ _ _b2-Fluorobenzoate + b + b4-Chlorobenzoate - + - - - -Phenylacetic acid + + + +4-Hydroxybenzoate + + + + +Protocatechuate + + + + +Benzoate + + + + + +Succinate + + + + + +Acetate + + + + + +Toluene + + - - +Benzene + + - - +Fluorobenzene + + - - +

a +, Growth observed; -, no growth observed.b Brownish coloring of culture fluid was observed.

and with dithionite solution. Cells were suspended in mineralsalts medium without substrate, and the optical density at546 nm was adjusted to 1.5. Oxygen consumption rates weremeasured before and after the addition of the substrates(final concentration, 0.5 mM).

Preparation of cell extracts. Cells (10 g) were suspended in10 ml of 50 mM Tris hydrochloride buffer (pH 7.9). Cellswere disrupted by sonification (10 times, 30 s each time) witha sonifier (model J17A; Branson Sonic Power Co., Danbury,Conn.). Cell debris was removed by centrifugation for 20min at 37,000 x g. The supernatant was used as crudeextract in the enzyme assays.Enzyme assays. The standard enzyme assay contained, in

1 ml, 700 pl of crude extract, 0.05 ,umol of (NH4)2Fe(SO4)2,and 1.5 p,mol of NADH. The reaction was started by adding1 ,umol of substrate. The disappearance of the substrates wasmonitored by high-performance liquid chromatography andthin-layer chromatography. Fluoride release was detectedwith the fluoride sensor as described above, and oxygenconsumption was measured with the oxygen electrode asdescribed above.

RESULTS

Isolation of bacteria growing on 4-fluorobenzoate. Six bac-terial strains which could grow on 4-fluorobenzoate as theirsole source of carbon and energy were isolated by using aselective enrichment technique. The enrichment procedurefor the strains isolated independently from various water andsoil samples is described in detail in Materials and Methods.

Classification of the newly isolated strains. For the taxo-nomic classification of the isolated strains, the morphologi-cal properties of the strains were investigated and biochem-ical tests were carried out. On the basis of Bergey's Manualof Systematic Bacteriology (29, 42), the strains were classi-fied as shown in Table 1.

Catabolic activities of the isolated strains. A number oforganic compounds were tested as growth substrates. 4-Fluorobenzoate-grown bacteria were inoculated into mineralsalts medium containing aliphatic and aromatic substrates(Table 2). It is interesting that Alcaligenes sp. strains RHO21and RHO22 used all three isomers of monofluorobenzoate.Alcaligenes sp. strain RHO22 also grew on 4-chloroben-zoate. Furthermore, all strains grew with benzoic acid astheir sole carbon source. All strains did not grow with

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A100

_ _

3%IA._ 50

- _'oUo

o In

B50

-

to

C _

_

&_'o 10O.-0

10 20 30

Time (h)

I,oa

SovMEn

C _

e

o. o_

0

2L

a a

2,0 c

_,l

-2I~

10 20 30

Time (h)FIG. 1. Growth of Aureobacterium sp. strain RH025 (A) and

Pseudomonas sp. strain RHO26 (B) on 2 mM 4-fluorobenzoate.Optical density (0) and substrate (-), and fluoride (A) concentra-tions are indicated.

4-iodo-, 4-bromo-, and 4-methylbenzoate; 4-fluoro- and 4-chlorophenylacetic acid; chlorobenzene; the three isomericdifluorobenzenes; fluoroacetate; or 2,4-, 2,6-, and 3,4-diflu-orobenzoate.Growth characteristics. The effects of various 4-fluoroben-

zoate concentrations on the growth of the organisms and onthe release of fluoride were determined in liquid culture (Fig.1). The maximum optical density reached was not propor-tional to the amount of 4-fluorobenzoate added to the culturemedium. Turbidity increased at concentrations ofup to 5 and4 mM 4-fluorobenzoate for Aureobacterium sp. strainRH025 and Alcaligenes sp. strain RH022, respectively; theother strains showed optimum growth at a substrate concen-tration of 3 mM (Fig. 2). Higher concentrations of 4-fluo-robenzoate inhibited growth of the cultures. Except in thecases of Pseudomonas sp. strains RH023 and RH026,fluoride recovery was nearly stoichiometric at lower sub-strate concentrations, while the release of fluoride decreasedrapidly at higher concentrations of 4-fluorobenzoate. It isremarkable that in the culture medium of Aureobacteriumsp. strain RH025, the organically bound fluorine was re-leased in stoichiometric quantities up to a concentration of 4mM 4-fluorobenzoate (Fig. 2). While being grown on 4-fluorobenzoate, Alcaligenes sp. strains RHO21 and RHO22caused a brownish coloration of the medium. The sameeffect could be observed with strains RHO23 and RHO24when succinic acid was added to the growth medium. OnlyAureobacterium sp. strain RHO25 caused no coloring. Thechange in color of the culture fluid was due to the accumu-lation of a compound we have identified as 4-fluorocatechol(see below).

0,1

0,011 2 3 4 5

0

ben

a00L..0

0

4- Fluorobenzoate (mM)FIG. 2. Influence of initial 4-fluorobenzoate concentration in the

medium for Aureobacterium sp. strain RHO25 (circles) and Pseu-domonas sp. strain RHO26 (triangles) on optical density (closedsymbols) and fluoride release (open symbols).

In the growth medium for Aureobacterium sp. strainRH025, 4-hydroxybenzoic acid was detectable (see below).This metabolite appeared transiently at the beginning of logphase in the culture fluid and disappeared from the mediumwithin 3 to 4 h. This indicated that 4-hydroxybenzoate wasan intermediate in the degradation of 4-fluorobenzoate bythis strain.

Identification of metabolites. The initial steps in the meta-bolic pathways for 4-fluorobenzoate degradation utilized bythe isolated bacteria were established from the structures ofthe intermediate metabolites formed during growth on 4-fluorobenzoate. The structures were based on the identitiesof relative GLC retention times of methylated derivativesand confirmed by comparison of their mass spectra withthose of authentic compounds. Figure 3A shows the GLCprofile of the methylated compounds obtained by extractionof the culture fluid of Alcaligenes sp. strain RH022. Besides4-fluorobenzoic acid methylester (peak a, mle = 155 [M +1]+), four more peaks were observed. These peaks corre-sponded to 4-fluoro-2-methoxyphenol and 5-fluoro-2-meth-oxyphenol (peaks b and c, mle = 143 [M + 1]+) and to4-fluorocatechol (peak e, mle = 129 [M + 1]+). Figure 3Bshows the mass spectrum of the double-methylated 4-fluo-rocatechol (peak d, mle = 157 [M + 1]+) isolated from theculture medium of Alcaligenes sp. strain RH022. Figure 4Ashows the GLC profile of the compounds obtained byextraction and methylation of the growth medium of Aureo-bacterium sp. strain RH025. In addition to the unconvertedsubstrate (peak a), two new peaks (b and c) were observedand identified as 4-methoxybenzoic acid methylester (mle =167 [M + 1]+) and 4-hydroxybenzoic acid methylester (mle= 153 [M + 1]+) by comparison with the authentic sub-stances.

Figure 4B shows the mass spectrum obtained from peak b.These results suggest that 4-hydroxybenzoic acid is the firstmetabolic intermediate in 4-fluorobenzoate degradation byAureobacterium sp. strain RH025.Oxygen uptake studies. Additional evidence for the new

degradation pathway was obtained when we checked theoxygen uptake rates of 4-fluorobenzoate-, benzoate-, andsuccinate-grown cells of Aureobacterium sp. strain RH025.The enzymes for the conversion of 4-fluorobenzoate and itspresumed degradation products, 4-hydroxybenzoate and3,4-dihydroxybenzoate, were induced by 4-fluorobenzoateor unsubstituted benzoate (Table 3).

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100-

50

A a

C1;~~~d L-.L-O 10 2o 30

Time (min)

B

ItIW,L.A 4-80 ^X3 150 200

100-

0

._i

Id*1

m/0FIG. 3. GLC and mass spectrometry analyses of the culture fluid

of Alcaligenes sp. strain RHO22. (A) GLC profile of diazomethane-derivatized extracts of the culture fluid from Alcaligenes sp. strainRHO22. Peaks: a, 4-fluorobenzoic acid methylester; b and c, the twofluoromethoxyphenols; d, 1,2-dimethoxy-4-fluorobenzene; e, 4-flu-orocatechol. (B) Mass spectrum of 1,2-dimethoxy-4-fluorobenzene(peak d).

Enzymatic studies. When we incubated crude extractsfrom Aureobacterium sp. strain RH025 with 4-fluoroben-zoate in the presence of NADH and Fe2 , oxygen wasconsumed, the substrate disappeared, and fluoride was re-leased concomitantly. However, only trace amounts of thepresumed reaction product 4-hydroxybenzoate and traces of3,4-dihydroxybenzoate were detectable. This is not surpris-ing, since under the assay conditions used, the enzymes forthe conversion of 4-hydroxybenzoate as well as 3,4-dihy-droxybenzoate were also active. Activities for 4-fluoroben-zoate dehalogenase, 4-hydroxybenzoate-3-hydroxylase, and3,4-dihydroxybenzoate-3,4-dioxygenase were 0.50, 16.10,and 0.45 mU/mg of protein, respectively. In extracts ofsuccinate-grown cells, none of these enzymes was detected.

DISCUSSION

Generally, benzoate derivatives are degraded via intro-duction of two hydroxyl groups either ortho or para to eachother. The oxidative cleavage of the resulting diols yieldscis,cis-muconic acids or muconate semialdehyde derivativesas intermediates (9, 15). The general route of 4-fluoroben-zoate degradation by bacteria appears to involve a 1,2-dioxygenase attack followed by decarboxylation, yielding4-fluorocatechol (3, 6, 18, 21, 22, 41). The data presentedabove indicate that the five new gram-negative isolatesdescribed here also degraded 4-fluorobenzoate via 4-fluoro-

50-

A 0l C

b

O 10 20Time (min)

B

,jpIp I n,11A L. A A. A-,ALI I-

80 100 150 200mfVe

FIG. 4. GLC and mass spectrometry analyses of the culture fluidofAureobacterium sp. strain RHO25. (A) GLC profile of diazometh-ane-derivatized extract of the culture medium of Aureobacteriumsp. strain RHO25. Peaks: a, 4-fluorobenzoic acid methylester; b,4-methoxybenzoic acid methylester; c, 4-hydroxybenzoic acid me-thylester. (B) Mass spectrum of 4-methoxybenzoic acid methylester(peak b).

catechol. As strains RHO21 and RH022 grew, this interme-diate accumulated and inhibited further growth. The accu-mulation of this metabolite in the culture fluid suggests thatthe ring cleavage enzyme in these strains may have a lowspecific activity with regard to this substrate. The five strainsseem to remove the fluorine substituent after ring cleavage.The conversion of the 4-fluorocatechol may proceed viaoxidative ring cleavage to the corresponding fluoromu-conate, lactonization, and elimination of fluoride, accordingto the pathway described in previous studies (18, 41) forbiodegradation of 4-fluorobenzoate.The results obtained with Aureobacterium strain RH025

suggest a new pathway for 4-fluorobenzoate metabolism,one in which the fluorine substituent is replaced in the initial

TABLE 3. Oxygen consumption of induced cells ofAureobacterium sp. strain RHO25a

Oxygen consumption (nmol/min)Substrate of cells induced by:

Benzoate 4-Fluorobenzoate

4-Fluorobenzoate 0.71 0.894-Hydroxybenzoate 0.29 0.353,4-Dihydroxybenzoate 0.21 0.21Benzoate 1.17 0.82

aUninduced cells consumed no oxygen.

0

-4CZ._

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BACTERIAL DEGRADATION OF 4-FLUOROBENZOATE 2503

COOH

Strains F

RHO21-24,26

a,'

OOH

F

ortho cleavagepathway

StrainRH025

COOH

OH

COOH

OOH

OH

ortho cleavagepathway

FIG. 5. Proposed pathways for the degradation of 4-fluoroben-zoate by the newly isolated bacteria.

step to form 4-hydroxybenzoic acid as the first degradationproduct. To date, several microorganisms that degrade theother 4-halobenzoates but not 4-fluorobenzoate via 4-hy-droxybenzoate as the initial metabolic intermediate havebeen found (5, 27, 28, 30, 33, 39, 44). Crude extracts of an

Arthrobacter sp. converted 4-fluorobenzoate to 4-hydroxy-benzoate, but the fluorinated substrate did not support thegrowth of this strain (30). To our knowledge, Aureobacte-rium sp. strain RH025 is the first strain found to degrade4-fluorobenzoate via initial defluorination.

In view of the similarity in size of fluorine and hydrogen,it is not surprising that all organisms investigated here canalso metabolize benzoate. In Aureobacterium sp. strainRHO25, fluorobenzoate induced the enzymes for the con-version of benzoate, 4-hydroxybenzoate, and 3,4-dihydrox-ybenzoate. At the same time, benzoate induced the enzymesfor the conversion of 4-fluorobenzoate. This suggests that inthis strain, 4-fluorobenzoate might be metabolized along aroute normally taken for the conversion of benzoate.The fact that benzoate and 4-fluorobenzoate were used as

growth substrates but 4-chlorobenzoate was not converted,except by Alcaligenes sp. strain RHO22, may be explainedeither by permeability barriers or by the specificity of thefirst degradative enzyme (38). There are indications thatbenzoate enters the cells of Acinetobacter calcoaceticusNCIB 8250 by a facilitated diffusion process (8) whichrequires protein carriers in the cell membranes. It may beassumed that substrate-specific carriers can tolerate fluorinesubstitution but are less likely to be able to deal with analogscontaining the bulkier halogen atoms, i.e., chlorine, bro-mine, and iodine. The ability of Alcaligenes sp. strainsRHO21 and RHO22 to grow on all three isomeric monoflu-orobenzoates may be explained by different enzymes thatconvert the various substrates to catechols or by a singleenzyme with a broad substrate specificity. Normally, 2-

fluorobenzoate is degraded via 3-fluorocatechol or unsubsti-tuted catechol (6, 32, 46), whereas 3-fluorobenzoate is con-verted to 3-fluorocatechol and 4-fluorocatechol (6, 41), and4-fluorobenzoate is metabolized via 4-fluorocatechol.The brownish growth medium of Aureobacterium sp.

strain RH025 in the presence of 3-fluorobenzoic acid sug-gests that this strain produced a catechol from this substrate.

In 4-fluorobenzoate degradation by Aureobacterium sp.strain RH025, however, the predominant metabolite pro-duced is 4-hydroxybenzoic acid. With the initial dehaloge-nation of the aromatic nucleus, this strain circumvents theaccumulation of 4-fluorocatechol and the problems associ-ated with the ring cleavage of this intermediate. The twodifferent pathways for the microbial degradation of 4-fluo-robenzoate are summarized in Fig. 5.The characterization of the 4-fluorobenzoate-dehaloge-

nating enzyme from Aureobacterium sp. strain RH025 isnow in progress in our laboratory.

ACKNOWLEDGMENTS

We thank W. Rude for helpful technical assistance and K.-H.Engesser, Universitat Stuttgart, for a sample of 4-fluorocatechol.We are grateful to H. Hasenfratz-Schreier, Fraunhofer Institut furGrenzflachen- und Bioverfahrenstechnik, Stuttgart, Federal Repub-lic of Germany, and P. Fischer, Universitat Stuttgart, for high-performance liquid chromatography and mass spectral analysis.

This work was supported by the Bundesministerium fur For-schung und Technologie under contract BCT 383.

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