Vol. 53, No. 4APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1987, p. 810-8150099-2240/87/040810-06$02.00/0Copyright © 1987, American Society for Microbiology

Reductive Dechlorination of 2,4-Dichlorobenzoate to4-Chlorobenzoate and Hydrolytic Dehalogenation of 4-Chloro-,

4-Bromo-, and 4-Iodobenzoate by Alcaligenes denitrificans NTB-1WILL J. J. VAN DEN TWEEL,* JAN BART KOK, AND JAN A. M. DE BONT

Department of Microbiology, Agricultural University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, TheNetherlands

Received 30 October 1986/Accepted 16 January 1987

Akcaligenes denitrificans NTB-1, previously isolated on 4-chlorobenzoate, also utilized 4-bromo-, 4-iodo-, and2,4-dichlorobenzoate but not 4-fluorobenzoate as a sole carbon and energy source. During growth,stoichiometric amounts of halide were released. Experiments with whole cells and cell extracts revealed that4-bromo- and 4-iodobenzoate were metabolized like 4-chlorobenzoate, involving an initial hydrolyticdehalogenation yielding 4-hydroxybenzoate, which in turn was hydroxylated to 3,4-dihydroxybenzoate. Theinitial step in the metabolism of 2,4-dichlorobenzoate was catalyzed by a novel type of reaction for aerobicorganisms, involving inducible reductive dechlorination to 4-chlorobenzoate. Under conditions of low andcontrolled oxygen concentrations, A. denitrificans NTB-1 converted all 4-halobenzoates and 2,4-dichlorobenzoate almost quantitatively to 4-hydroxybenzoate.

Halogenated compounds have been produced syntheti-cally on a large scale during the last few decades, and theyhave entered the biosphere either by accidental spillage orby deliberate release. In many cases these chemicals may betransformed or fully eliminated from the environment bymicrobial degradation.

Aerobically, the majority of haloaromatic compounds aredegraded by microorganisms that leave the carbon-halogenbond intact until chlorocatechols are formed and the ring iscleaved by a dioxygenase (4, 5, 7, 8, 25, 28, 35, 36).However, aerobic catabolism of haloaromatic compoundsmay also proceed along an initial oxidative or hydrolyticdehalogenation reaction. For instance, 4-fluorophenyl-alanine was oxidized by a monooxygenase to tyrosine (12).In a similar way 2-fluorobenzoate (6) and 4-chlorophenylace-tate (20) were oxidized by a dioxygenase, yielding catecholand 3,4-dihydroxyphenylacetate, respectively. An analo-gous reaction is probably involved in the metabolism of2-chlorobenzoate (2-CBA) in Pseudomonas cepacia INMI-KZ-2 (38). Hydrolytic dehalogenation reactions have beenreported for the metabolism of 3- and 4-chlorobenzoates(3-CBA and 4-CBA) (11, 13, 14, 18, 26, 37). Aerobic metab-olism of halogenated aliphatic compounds also may involveeither oxidative or hydrolytic dehalogenation reactions.Methylchloride was oxidized by a methanotroph via themethane monooxygenase activity present, although methylchloride did not serve as a carbon source for growth (29).Dichloromethane dehalogenation in a Hyphomicrobiumstrain was catalyzed by a reduced glutathione-dependentdehalogenase, with formaldehyde as a product (16). Gluta-thione-independent hydrolytic dehalogenation of haloge-nated aliphatic compounds has also been observed (10, 21).To our knowledge, however, there are no reports on theaerobic metabolism of halogenated aliphatic or aromaticcompounds involving reductive dehalogenation.Under anaerobic conditions, haloaromatic compounds are

degraded by reductive dechlorination before ring cleavage,as shown for chlorinated phenols (3) and chlorinated benzo-

* Corresponding author.

ates (9, 27). Reductive dehalogenation was also responsiblefor the degradation of halogenated aliphatic hydrocarbonsunder methanogenic conditions (1, 34) and under denitrifyingconditions (2).Recently we reported on the bioformation of 4-

hydroxybenzoate (4-HBA) from 4-chlorobenzoate (4-CBA)by Alcaligenes denitrificans NTB-1 (32). The results de-scribed in this paper demonstrate that this species also growson 2,4-dichlorobenzoate (2,4-DCBA), 4-bromobenzoate(4-BBA), and 4-iodobenzoate (4-IBA). 4-CBA, 4-BBA, and4-IBA were all hydrolytically dehalogenated to 4-HBA, andevidence will now be presented for a reductive dehalogena-tion reaction under aerobic conditions. This novel type ofreaction is involved in the catabolism of 2,4-DCBA via4-CBA to 4-HBA.

MATERIALS AND METHODSMedia and culture conditions. A. denitrificans NTB-1 was

routinely grown in a chemostat (volume, 1.0 liter; dilutionrate, 0.03 h-1) under carbon-limited conditions. The contin-uous culture (30°C, pH 7) was supplied with a mineral saltsmedium (32) to which 2,4-DCBA, 4-CBA, or 4-BBA wasadded at 1 g/liter and 4-IBA was added at 0.5 g/liter.Experiments with whole cells. Simultaneous adaptation

experiments and incubation experiments at controlled oxy-gen concentrations were performed as described previously(32).Enzyme assays. Cell extracts were prepared by ultrasonic

treatment as described previously (31). All assays wereperformed at 30'C. Attempts to assay for 2,4-DCBAdehalogenase activity were done by measuring 2,4-DCBAconcentrations in various incubation mixtures in the pres-ence of cell extract and NADPH. 4-CBA dehalogenase wasassayed by measuring the conversion of 4-CBA in 4-HBA.4-HBA hydroxylase was determined by means of 4-HBA-dependent NADPH oxidation (18). Protocatechuatedioxygenase was measured by means of a polarographicoxygen probe (18).

Analytical methods. Chloride, bromide, and iodide concen-trations were determined with a Micro-Chlor-o-Counter

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METABOLISM OF HALOBENZOATES 811

TABLE 1. Rates of oxygen uptake by washed-cell suspensions ofA. denitrificans NTB-1 grown on various carbon sources

Oxygen uptakeb (nmol of 02 consumed/min per mgSubstratea of protein) with indicated carbon source

4-CBA 4-BBA 4-IBA 2,4-DCBA 4-HBA Succinate

4-FBA <5 10 <5 5 5 <54-CBA 100 170 45 45 20 <54-BBA 75 140 60 50 25 <54-IBA 100 100 50 35 15 <52,4-DCBA 50 80 30 30 5 <54-HBA 90 220 55 40 20 <5Protocatechuate 20 60 5 10 5 <5Succinate <5 <5 <5 <5 <5 55

a No activity was observed with the following substrates: 2-chloro-4-hydroxybenzoate, 3,5-dichlorocatechol, gentisate, benzoate, 2- or 3-HBA, 2-or 3-CBA, 2-FBA, 3-BBA, 2,6-DCBA, 2,5-DCBA, 3,4-DCBA, 3,5-DCBA,catechol, or 2,3-dihydroxybenzoate.

(Marius, Utrecht, The Netherlands). Protein contents ofwhole cells and cell extracts were determined by the methodof Lowry et al. (17) with crystalline bovine serum albumin asthe standard. Concentrations of 4-fluorobenzoate (4-FBA),4-CBA, 4-BBA, 4-IBA, 2,4-DCBA, and 4-HBA were quan-titatively analyzed by reverse-phase high-pressure liquidchromatography (HPLC) with a C-18 column (200 by 3 mm;Chrompack, Middelburg, The Netherlands). The mobilephase was methanol-water-acetic acid (60:40:1), the flowrate was 0.4 ml/min, and detection was by UV absorbance at254 nm. Under these conditions the following retention times(in minutes) were observed: 4-FBA, 4. 1; 4-CBA, 8.5;4-BBA, 11.4; 4-IBA, 10.6; 2,4-DCBA, 9.8; and 4-HBA, 2.9.Accumulating metabolites were initially identified by com-parison of retention times with those of authentic samplesand by in situ scanning of the UV spectra after the flow hadbeen stopped. Ether extraction of the accumulating productswas done as described by Marks et al. (19). The resultingether extracts were methylated with diazomethane (33) andanalyzed by gas-liquid chromatography-mass spectrometry(GC-MS). Samples were analyzed on a capillary column(SIL19CB; 26 m by 0.22 mm inner diameter; Chrompack).The temperature was programmed to rise from 120°C (2 min)to 260°C at a rate of 4°C/min. The methyl esters of 2,4-DCBA, 4-CBA, and 4-HBA under these conditions hadretention times of 6.95, 4.43, and 12.52 min, respectively.Mass spectra of these compounds were obtained on a VGMM7070F mass spectrometer at 70 eV electron impact. Theoxygen concentration in the gas phase was measured asdescribed previously (32).

Chemicals. All biochemicals were from Boehringer, Mann-heim, Federal Republic of Germany. 2-Chloro-4-hydroxy-benzoate and 2,3-dichlorobenzoate (2,3-DCBA) were fromICN Biomedicals Inc., Plainview, N.Y. 4-CBA, 4-FBA, and2,4-DCBA were purchased from Janssen Chimica, Beerse,Belgium. 4-BBA was from Fluka, Buchs, Switzerland. Allother chemicals were of commercially available analyticalgrade and were used without further purification.

RESULTS

Growth of A. denitrificans NTB-1 on various substrates. A.denitrificans NTB-1 was able to use the following com-pounds as sole carbon and energy sources: 4-CBA, 2,4-DCBA, 4-BBA, 4-IBA, 4-HBA, 3-hydroxybenzoate(3-HBA), benzoate, phenylacetate, glucose, succinate, py-ruvate, acetate, and ethanol. No growth was observed with

2-CBA, 3-CBA, 2-HBA, 3,5-DCBA, 3,4-DCBA, 2,5-DCBA,2,6-DCBA, 4-FBA, 4-chlorQ- or 4-fluorophenylacetate, or2-chloro-4-hydroxybenzoate as substrates (substrate con-centration, 2 mM). Previous results have shown that boththe lag phase and the growth rate of A. denitrificans NTB-1during growth on 4-CBA were dependent on the concentra-tion of 4-CBA (32). Similar results were obtained duringgrowth on 4-BBA and 2,4-DCBA. At a 2 mM concentrationof these two compounds, doubling times of 12 and 13 h,respectively, were observed, while with 4 mM 4-BBA or2,4-DCBA, strain NTB-1 doubled in 68 and 26 h, respec-tively. No growth of NTB-1 on 4-IBA (2 mM) was observedin batch culture. However, by growing NTB-1 in a chemo-stat under carbon-limited conditions (dilution rate, 0.03 h-'),good growth was obtained. During all growth experimentsthe halide was quantitatively released, as determined bymeasuring halide accumulation.

Simultaneous adaptation experiments. To investigate themetabolism of 4-CBA, 4-BBA, 4-IBA, and 2,4-DCBA, cellswere grown on several substrates and incubated with possi-ble intermediates, and rates of oxygen uptake were re-corded. Cells grown on halobenzoates oxidized 4-CBA,4-BBA, 4-IBA, 2,4-DCBA, 4-HBA, and 3,4-dihydroxybenz-oate but not 4-FBA (Table 1). Succinate-grown cells did notoxidize 4-CBA, 4-BBA, 4-IBA, or 2,4-DCBA, but surpris-ingly, 4-HBA-grown cells oxidized these halogenated com-pounds. 2-Chloro-4-hydroxybenzoate, an intermediate of2,4-DCE3A metabolism in initial hydrolytic dehalogenation atthe 4-position, was not oxidized by 2,4-DCBA-grown cells,nor was 3,5-dichlorocatechol, an intermediate in initialdioxygenation, oxidized by washed cells grown on 2,4-DCBA (Table 1).

Catabolism of halobenzoates and excretion of intermediaryproducts from these halobenzoates by washed cell suspensions.To study the metabolism of the halobenzoates more directly,the disappearance of various halobenzoates from incubationmixtures was studied by HPLC. 4-CBA-grown cells readilycatabolized 4-CBA, 4-BBA, 4-IBA, and 2,4-DCBA but not4-FBA (Table 2). Similar results were obtained for ceUsgrown on 4-BBA, 4-IBA, 2,4-DCBA, and 4-HBA (Table 2).Although the activities varied depending on the carbonsource used for growth, no significant variation was ob-served in the relative rates of consumption of 4-CBA,4-BBA, and 4-IBA (Table 2). Furthermore, the ratio of4-halobenzoate to 2,4-DCBA consumption for cells grownon various haloaromatics was in all cases about 0.65. Aninteresting observation was made with 4-HBA-grown cells.These cells, when incubated with 2,4-DCBA, transientlyaccumulated a product (Fig. 1) which, when analyzed byHPLC, had the same retention time and UV characteristicsas authentic 4-CBA. After methylation of the reaction mix-ture sampled at 60 min (Fig. 1), two compounds were

TABLE 2. Dehalogenation rates of various substrates byA. denitrificans NTB-1 cells grown on various carbon sources

Dehalogenation rate (nmol of substrate consumed/minSubstrate per mg of protein) with indicated carbon source

4-CBA 4-BBA 4-IBA 2,4-DCBA 4-HBA Succinate

4-FBA <1 <1 <1 <1 <1 <14-CBA 18 11 12 28 8 <14-BBA 15 11 12 30 8 NDa4-IBA 18 9 10 28 7 ND2,4-DCBA 11 7 6 20 5 <1

a ND, Not determined.

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1.5-

C:0

.

0.5/a, 1.-

0 40 80 120

time (min)FIG. 1. Formation of 4-CBA (0) and chloride ([) from 2,4-

D)CBA (0) by 4-HBA-grown A. denitrificans NTB-1 cells. Totalvolume of the reaction mixture was 10 ml, containing 17.5 mg ofprotein.

detected by GC-MS analysis. One compound showed themass spectrum of methylated 2,4-DCBA, and the othercompound showed a mass spectrum (Fig. 2A) identical to themass spectrum of authentic 4-chlorobenzoic acidmethylester. This accumulation of 4-CBA from 2,4-DCBAwas not expected since the rate of 4-CBA consumption bywhole cells was greater than the rate of2,4-DCBA consump-

tion (Table 2). However, when 4-CBA and 2,4-DCBA were

100 c+ 139 A

75 170[r EH

.~0100 150 2600

C

100- 121

Ir?l I- [OOCH

m/eFIG. 2. MS of (A) methylated 4-CBA produced by 4-HBA-grown

cells from 2,4-DCBA and (B) methylated 4-HBA formed from2,4-DCBA at 1.2% oxygen in the gas phase.

added simultaneously, it was observed that the 4-CBAconcentration remained constant until 2,4-DCBA was me-tabolized to leave a concentration of approximately 0.25 mM(Fig. 3).

Previous results for the metabolism of 4-CBA by A.denitrificans NTB-1 had shown that 4-CBA was almoststoichiometrically converted to 4-HBA under conditions oflow oxygen concentration (32). Similar experiments havenow been done with ceils grown on 4-BBA, 4-IBA, and2,4-DCBA with the same substrates (Fig. 4). In addition, theproduct obtained from the 2,4-DCBA incubation mixturewas methylated, and GC-MS analysis showed that thismethylated product was indeed 4-hydroxybenzoic acidmethylester (Fig. 2B). The effect of the oxygen concentra-tion in the gas phase on the rate of 2,4-DCBA consumptionand the rate of4-HBA formation was identical to the effect ofoxygen on 4-CBA consumption and 4-HBA formation, asshown previously (32), in that conversion was maximal atabout 1.2% oxygen in the gas phase, while under anaerobicconditions almost no 2,4-DCBA consumption ahd hence no4-HBA formation was obtained.No decrease in concentration as measured by HPLC and

no halide release was observed when 2,4-DCBA-grownNTB-1 cells were incubated under normal atmospheric con-ditions with 2-chloro-4-hydroxybenzoate, 4-chlorophenyl-acetate, 2-CBA, 3-CBA, 4-FBA, 2,6-DCBA, 2,5-DCBA,3,4-DCBA, or 3,5-DCBA.

Experiments with cell extracts. The feasibility of a degra-dation route of 2,4-DCBA involving 4-CBA and subsequentmetabolism of 4-CBA was further investigated, as was themetabolism of 4-BBA and 4-IBA, by measuring specificenzyme activities in cell extracts. NTB-1 cells grown oneither 4-HBA, 4-CBA, 4-BBA, 4-IBA, or 2,4-DCBA had anNADPH-dependent 4-hydroxybenzoate hydroxylase and

2.5 -

20 X

E-1:150

C-

1.0-

0.5

0 T_

0 40 80 120 160time (min)

FIG. 3. Simultaneous consumption of 4-CBA (0) and 2,4-DCBA(0) by 4-HBA-grown NTB-1 cells; [1, chloride release. Totalvolume of the reaction mixture was 10 ml, containing 17.5 mg ofprotein.

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METABOLISM OF HALOBENZOATES 813

E 1.5 60 360c0

4"10 1.0 20-

0

0.5 0.5- 0

0 00 20 40 60 80 100 0 20 40 60 80 100 0 10 20 30

time (min)

FIG. 4. Bioconversion of 4-BBA (A), 4-IBA (B), and 2,4-DCBA (C) to 4-HBA by 4-BBA-, 4-IBA-, and 2,4-DCBA-grown cells,respectively, at 1.2% oxygen in the gas phase. Symbols: 0, 4-BBA; A, 4-IBA; A, 2,4-DCBA; 0, 4-HBA; O, halide. All incubation mixtures(total volume, 10 ml) contained 29.5 mg of protein.

also a 3,4-dihydroxybenzoate dioxygenase, whereas suc-cinate-grown cells did not contain these enzymes (Table 3).Several attempts were made to show the presence of areductive 2,4-DCBA dehalogenase and a hydrolytic 4-CBAdehalogenase in extracts of 2,4-DCBA-grown cells, but noactivities were detected.

DISCUSSIONAerobic microorganisms isolated on 4-CBA hydrolytically

dehalogenate this substrate to 4-HBA (13, 14, 18, 26, 37), incontrast to the dioxygenase reactions used for the initial stepin the catabolism of various other halobenzoates in severalorganisms (5, 7, 36). Microorganisms which degrade 4-CBAvia 4-chlorocatechol so far have not been isolated by classi-cal enrichment techniques, probably because the benzoate1,2-dioxygenase of ordinary benzoate degraders is not activeon 4-CBA (15, 23). Some years ago Hartmann et al. (8)isolated a Pseudomonas sp. by continuous enrichmentwhich degraded 3-CBA, 4-CBA, and 3.5-DCBA via thecorresponding chlorocatechols. They assumed that thisstrain was naturally evolved from two defined strains, Pseu-domonas sp. strain B13 and Pseudomonas sp. strain mt-2,which were used as coinocula. Later on, Reineke andKnackmuss (24) confirmed this hypothesis by constructingin vitro a 4-CBA degrader by combining the genes of the3-CBA pathway of Pseudomonas sp. strain B13 and somegenes of the TOL plasmid of Pseudomonas sp. strain mt-2.A. denitrificans NTB-1 is no exception to the general rule

for 4-CBA metabolism (32), and apart from 4-CBA it also

TABLE 3. Specific activities of the 4-HBA hydroxylase (NADPHdependent) and the 3,4-dihydroxybenzoate dioxygenase ofA. denitrificans NTB-1 after growth on various substrates

Sp act (nmol/lmin per mg of protein)Growthsubstrate 4-HBA 3,4-Dihydroxybenzoate

hydroxylase dioxygenase

4-CBA 20 1754-BBA 30 1804-IBA 15 1752,4-DCBA 15 2104-HBA 15 190Succinate <1 <1

metabolized 4-BBA and 4-IBA to 4-HBA. 4-FBA, however,did not serve as a growth substrate, nor was it defluorinatedby cells grown on 4-CBA. In contrast, Arthrobacter sp.strain TM-1 grown on 4-CBA was able to defluorinate 4-FBA(18).

Strain NTB-1 was also able to grow on 2,4-DCBA. Bac-teria degrading 2,4-DCBA have also been isolated byVandenbergh et al. (30), but in these strains the catabolicroute of 2,4-DCBA has not been studied. Zaitsev andKarasevich (39) isolated a Corynebacterium sp. which wasable to convert 2,4-DCBA to 4-HBA. They considered threepossible pathways for 2,4-DCBA metabolism: (i) reductivedechlorination to 4-CBA, which in turn would be dechlorin-ated to 4-HBA; (ii) hydrolytic dechlorination, yielding2-chloro-4-hydroxybenzoate, followed by reductive dechlo-rination; and (iii) simultaneous dechlorination of both chlo-rine atoms, yielding 4-HBA. Zaitsev and Karasevich (39)suggested that the third pathway was the most probable,although on the basis of their results the two other pathwayscannot be excluded.

Experiments with whole NTB-1 cells showed that 2-chloro-4-hydroxybenzoate was not an intermediate in themetabolism of 2,4-DCBA in our strain, nor was 2,4-DCBAdegraded via 3,5-dichlorocatechol, a theoretical route resem-bling the aerobic degradation pathway of many haloaromatic

COOH

Br

COOHcL

cl

COOH

Cl

COOH

COOH

OH

COOH

OH ringcleavage

OH

FIG. 5. Proposed catabolic pathway of 2,4-DCBA, 4-CBA,4-BBA, and 4-IBA in A. denitrificans NTB-1.

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814 VAN DEN TWEEL ET AL.

compounds (4, 5, 7, 8, 25, 28, 35, 36). Instead, fromincubation experiments with 4-HBA-grown cells, it is con-cluded that 2,4-DCBA is initially reduced to 4-CBA. To our

knowledge this is the first report on aerobic metabolism ofhalogenated compounds involving reductive dehalogenation.Washed cells grown on 4-HBA had a higher consumptionrate for 4-CBA than for 2,4-DCBA but nevertheless accu-mulated 4-CBA from 2,4-DCBA. Inhibition of the 4-CBAdehalogenase by 2,4-DCBA (Fig. 3) may explain this anom-aly. Surprisingly, however, no 4-CBA accumulated from2,4-DCBA with NTB-1 cells grown on 4-CBA, 4-BBA,4-IBA, or 2,4-DCBA, although the ratio of 2,4-DCBA to4-CBA consumption in these cells was comparable to thisratio in 4-HBA-grown cells (Table 2). We have not yet beenable to measure a 2,4-DCBA reductive dehalogenase in cellextracts, although cell extracts have been prepared andtested in the presence and absence of oxygen with bothNADH and NADPH as reductants.4-HBA was produced from 2,4-DCBA when NTB-1 cells

were incubated under conditions of low oxygen concentra-tions, which is in keeping with 4-CBA dehalogenation as a

hydrolytic process (19, 22). Surprisingly, however, underanaerobic conditions almost no dehalogenation was ob-served, and presently it is being investigated whether thisbehavior is due to energy-dependent active transport of2,4-DCBA.On the basis of the results presented, a degradation route

(Fig. 5) is proposed for halobenzoate metabolism in A.denitrificans NTB-1.

ACKNOWLEDGMENTS

We are grateful to M. A. Posthumus for performing the MSanalyses and to G. Geurtsen and D. A. de Bie for advice andtechnical assistance during the methylation procedure.

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