World Journal of Microbiology & Biotechnology 1.7.

607-614

Biodegradation of 3xhlorobenzoate by
Pseudomonas putida 10.2

W. Chobchuenchom,” S. Mongkolsuk and A. Bhumiratana

Pseudomonas putidu 10.2, a 3-chlorobenzoate (3CBa)-degrading bacterium, was isolated from a soil sample
obtained from an agricultural area in Chiang Mai, Thailand. This bacterium could degrade 2 mu 3CBa very rapidly
with the concomitant formation of chloride ion when grown in mineral salt-yeast extract medium. The presence of
glucose, lactose and pyruvate in the medium reduced the capability of this bacterium to degrade 3CBa.
Metabolites such as 3-chlorocatechol (3CC), catechol and cis,ris-muconic acid (muconate) could be detected in the
growth medium or in cell suspensions when 3CBa was used as the substrate. Furthermore, when crude enzyme
extract prepared from 3CBa-grown P. putida 10.2 was incubated with 3CC, catechol and muconate could be
detected in the reaction mixtures. Thus, the biodegradation pathway of 3CBa by P. putidu 10.2 was proposed to
involve transformation of 3CBa to 3CC. The dehalogenation step is believed to involve removal of chloride from
3CC to form catechol, which is subsequently converted to muconate.

Key words: Biodegradation, 3-chlorobenzoate, reductive dechlorination.

Chlorobenzoates are environmentally contaminating hazard- chlorinated aromatic compounds is incomplete, and this
ous wastes which are usually introduced into the environ- leads to the appearance of a black colour resulting from the
ment through two major routes, namely, as active ingredi- polymerization of 3CC. 3CC is a common intermediate
ents in commonly used herbicides (Horvath 1971; Pfister in the biodegradation of various halogenated aromatic
1973) and as metabolic products of polychlorinated biphenyls compounds, including 4-chlorobenzoic acid (Chatterjee &
(PCBs). Biodegradation of PCBs by soil microorganisms was Chakrabarty 1982).
found to give rise to the accumulation of chlorobenzoate in In contrast to aerobic bacteria, the anaerobic microbe
the environment (Ahmed & Focht 1973; Furukawa et al. Desulfomonile fiedjei DCB-1 converts 3CBa to benzoate by
1979; Sylvestre & Fauteux 1982; Masse et al. 1984). a mechanism of reductive dehalogenation (Mohn & Tiedje
There have been reports that 3-chlorobenzoate (3CBa) 1992). Reductive dehalogenation is important in numerous
could be utilized as the sole carbon source by several biodegradations, including those of organochlorine pesti-
microorganisms such as Pseudomonas sp. Bl3 (Weisshaar cides, alkyl solvents and aryl halides. Although reductive
et al. 1987) and Alcaligenes eufrophus JMP134 (Don et al. dehalogenations are normally seen in anaerobic conditions,
1995). The biodegradation of 3CBa in aerobic bacteria they are also found in aerobic conditions, for example, the
commonly proceeds via 3-chlorocatechol (3CC). The ring reductive dechlorination of dichlorobenzoate by Alculigenes
cleavage through a mechanism known as modified ortho denifr$cans (van den Tweel et al. 1987) and reductive
cleavage precedes the spontaneous chlorine removal dechlorination of pentachlorophenol by Fluvobacferium sp.
(Ghosal et al. 1985; Weisshaar et al. 1987; Chaudhry & (Steiert & Crawford 1986), Rhodococcxs chlorophenolicus
Chapalamadugu 1991). (Apajalahti & Salkinoja-Salonen 1987) and Rhodococms sp.
In some aerobic bacteria, degradation of 3CBa and other (Haggblom et al. 1989).
This report describes the characteristics of PseuAomonus
pufida 10.2, a newly isolated bacterial strain capable of
W. Chobchuenchom and A. Bhumiratana are with the Department
of Biotechnology, Faculty of Science, Mahidol University, RAMA VI. degrading 3CBa. The characteristics of 3CBa degradation
Bangkok, 10400 Thailand; fax: (662) 2463026. S. Mongkolsuk is with the
and chloride elimination by resting cell suspensions and
Laboratory of Biotechnology, Chulaborn Research Institute. Vipavadee-
Rangsit Highway, Bangkok, 10210 Thailand. *Corresponding author. growing cells were studied.

@ 1996 Rapid Science Publishers

World ]oumal of Mmobtology & Biotechnology, Vol 12, 1996 607

 W. Chobchuenchom et al.

Materials and Methods Cafechol 1.2 Dioxygenase Assay. Catechol 1,2-dioxygenase (EC
1.13.11.1) assays were based on HPLC detection of MUC rather
Media and Growth Conditions than an optical density at 260 nm as previously reported (Dom &
The two media used in this study were M9 mineral salt medium Knackmuss 1978). Reactions were carried out in 2-ml Eppendorf
(42 mM Na,PO,.IZH,O, 22 mM KH,PO,, 10 mM NaCl, 18 mM tubes containing 1.5 ml of either non-treated or boiled enzyme
NH,CI, 2 mu MgSO,, 0.5 mM CaCI,) and modified M9 medium preparations and 0.1 mM CAT, 1.28 mM MgSO, and 0.5 mM
(MM9) which was composed of 0.02% (w/v) yeast extract in M9 NADPH. Each reaction tube was incubated at 30°C in a water
medium. Where indicated, medium was supplemented with 3CBa bath for 3 h. At intervals of 0, 0.5, 1, 2, and 3 h, aliquots of
at a final concentration of 1.0 mM from a stock solution of I M reactions and controls were taken and the reaction stopped by the
3CBa in absolute ethanol. When chloride analysis was required addition of 20 ~1 1 M trichloroacetic acid. After removal of precipi-
NaCI, CaCl,, and NH,CI were omitted from M9 medium, tate by centrifugation for 10 min at 10,000 x g, the concentration
and I ml of trace element stock solution [containing (g/l): of MUC in the supematants was determined by HPLC.
H,BO,, 11.4; ZnS0,.7H,O, 2.2; MnC1.4H,O, 0.5; CoCl,, 0.16;
CuSO,.SH,O, 0.16; (NH,)Mo,0,,.4H,O, 1.61; NaEDTA, 50 and Protein Deferminafion
2% KOH] was added per liter. For solid media, 1.5% Bacto agar Protein concentrations were determined either by using the Brad-
was added to the liquid media. Media and trace element stock ford protein detection kit (Bio-Rad Laboratories, CA, USA) with
solutions were sterilized by autoclaving at 15 lb inch-2 for 20 bovine serum albumin as standard or by measuring optical density
min. Bacterial cultivation in liquid media was carried out at 30°C at 280 nm.
with shaking at 150 rev min-‘. Cell growth was determined by
measuring optical density at 600 nm using a Milton Roy spec-
tronic 1001 plus. Analytical Procedures
High Pressure Liquid Chromatography. HPLC was performed with a
Shimadzu model LC 6A HPLC machine with a reverse phase
Chemicals
Shimpack clc/ODS column (6 x I50 mm). The column tempera-
The chemicals 3CBa, catechol (CAT) and cis,cis-muconic acid
ture was set at 40°C and the peaks were detected by using a u.v.-
(MUC) were from Sigma Co., St. Louis, USA and 3CC was vis spectrometric detector (SPD-6AV). For determination of 3CBA,
obtained from Tokyo Kasei Chemical, Japan. Chromatographic orthophosphoric acid (10 rnr.4) containing 50% acetonitrile was
grade acetonitrile and methanol were from Merck, Darmstadt, used as the mobile phase with a flow rate of 1 ml min-’ and the
Germany. Chloroform was from Fluka, Buchs, Switzerland.
peaks were detected at 278 nm. For analysis of 3CC in culture
M9 basal salt medium was from Gibco BRL, Paisley, Scotland. broth, 2 1 of culture broth was extracted with ethyl acetate, dried
Bacto agar and biochemical test media were from Difco, Detroit, over MgSO, and then evaporated with a rotary evaporator.
MI, USA. All chemicals used in this study were of analytical
Extracted preparations were resuspended in chloroform and
grade. analysed for 3CC using HPLC as described above with a mobile
phase of 50% methanol containing 40 mM acetic acid and a
Preparation of Cell Suspensions and Cell Erfracfsfrom Pseudomonas detection wavelength of 284 nm. For the analysis of CAT, the
putida 10.2 supematant from resting cell suspensions was analysed for appear-
Unless specified otherwise, P. pufida 10.2 was precultured in ance of CAT by HPLC. The retention times of metabohte and
MM9 medium. When preparing cell suspensions, cultures were standard peaks were compared in eight mobile phase systems by
harvested from the late exponential growth phase (18 h after both separate and co-inject methods. For determination of CAT,
inoculation), centrifuged for 15 min at 5000 x g, washed once peak areas of samples were converted to concentration by reading
with 50 mM phosphate buffer pH 7.0 and resuspended in the same from the CAT standard curve prepared as for the detection of
buffer to obtain the indicated cell concentrations. 3CBa using a detection wavelength of 230 nm and a flow rate of
For preparation of cell extracts, cultures were harvested and 0.5 ml min-‘.
washed, and the cell pellets were used fresh or kept at - 20°C Liquid Chromatography/Mass Spectrometry (K/MS). For analysis
until used. Frozen cell pellets were thawed and suspended in of catechol appearance in reactions containing 3CC and crude
50 mM phosphate buffer pH 7.0 to give a concentration of 0.5 to enzyme extract, the supematants of each reaction and authentic
1.0 mg ml-‘. Using ahquots of 10 ml, the cell suspensions were catechol were studied using LC/MS. HPLC was performed using
disrupted by sonication (Soniprep MSE, UK) for 15 min. To avoid a Waters TM model 600s. Supematant from the reaction was
overheating of the preparation, sonication was carried out for 10 s directly injected and separated on a reverse phase Shimpack clc/
with 10 s stop intervals and all operations were carried out on ice. ODS column (6 x 150) with a 0.5 ml min-’ flow rate and 50%
Unbroken cells and debris were removed by centrifugation at acetonitrile in water as the mobile phase. Interphase was performed
14,000 x g for 30 min, at 4°C. The supernatant obtained was by using atmospheric pressure chemical ionization (APCI) with a
designated crude extract. cone voltage of 40. The CAT peak was detected by mass
spectrometry using a Waters model VG trio 2000.
Chloride Release Assay. Determination of chloride released in culture
Enzyme Assays supematant at indicated time intervals was monitored by silver
Assay for KC-Degrading Enzyme. Assays were performed in test nitrate titration with potassium chromate as indicator.
tubes in a total volume of 2.5 ml containing 0.1 mM 3CC, 1.28 mM
MgSO,, 0.5 mM NADPH and diluted enzyme preparations. The
reactions were incubated in a water bath at 37°C. At the indicated
time intervals, 200 ~1 aliquots were taken and the reactions Results
terminated by the addition of 20 ~1 of 1 M trichloroacetic acid.
Isolation and Idenf$cafion of P. putida 10.2
The precipitate was removed by centrifugation at 10,000 x g for
10 min and the catechol content of the supematant determined by P. pufidu 10.2 was isolated from a soil sample from an
HPLC. agricultural field in Chiang Mai, Thailand by using a batch

608 Worki]oumal ofA4icrobmlogy & Biotechnology, Vof 12, 1996

 Biodegradation of 3-chlorobenzoate

1.7

T 0.80

g
a 0.75
g
5 0.70
z
s
E 0.65
5
e&I 0.60

0.55

0.50
0 5 10 15 20 25 30

hcubation time (II)

Figure 1. Growth and degradation of 3-chlorobenzoate by

Pseudomonas putida 10.2 in MM9 supplemented with 3CBa. 0
Optical density at 600 nm (a); amount of 3CBa (m) and chloride
0.0 0.2 0.4 0.6 0.8 1.0
ion (A).
Cell concentration (mg ml ‘)

Figure 2. The effect of resting cell suspensions of Pseudomonas

culture enrichment technique; approximately 10 g of each putida 10.2 on the rate of 3-chlorobenzoate degradation. Non-
soil sample was resuspended in 25 ml of distilled water and treated cell suspension (a) and heat treated cell suspension
filtered once through Whatman filter paper No 4 and twice (rn).
through Whatman filter paper No I. Aliquots of 500 ,~l
were then inoculated into 125-ml Erlenmeyer flasks contain- 3CBa degradation was reduced from 17.6 to 4.5% and at
ing 50 ml of M9 supplemented with I mM 3CBa and higher levels (1.0%) inhibition was complete. Similarly,
incubated with shaking at 30°C. All turbid cultures were lactose supplementation at 0.1% caused a slight reduction
subcultured 3 to 5 times by transferring 100 ~1 of turbid in 3CBa utilization (17.6% to 11.5%) and a marked inhibition
culture broth to 50 ml of fresh medium. Finally, broth from at 1%, despite the fact that the biochemical tests showed
each turbid culture was streaked on mineral salt agar that this organism could not assimilate lactose. This pat-
supplemented with 3CBa to obtain pure cultures. Growth tern of inhibition was also found with succinate
of purified isolates was then compared in M9 medium with supplementation.
and without 3CBa supplementation. Ten isolates which
showed higher growth yield in the presence of 3CBa were Degradation of 3CBa by Resting Cell Suspensions
studied further for their ability to degrade 3CBa. Based on The rate of 3CBa degradation was studied using cell suspen-
growth rate, growth yield and the rate of 3CBa degradation sions at the concentrations shown in Figure 2. The rate of
by resting cell suspensions, it was found that isolate 10.2 3CBa degradation was approximately proportional to the
was the best 3CBa degrader. For identification of isolate cell mass. The rate of 3CBa degradation was defined as the
10.2, microscopic examinations and biochemical tests were amount of 3CBa lost from the medium, and the values
performed (Table 1). Based on the identification scheme in were calculated when the loss was directly proportional to
Bergey’s manual of determinative bacteriology (Holt et al. the time of incubation.
1994, strain 10.2 was identified as Pseudomonas putida.
Presence of 3CC in Spent Culture Broth Containing 3CBa
Growth and 3CBa Degradation by P. putida 10.2 To obtain information concerning the biodegradation path-
As illustrated in Figure I, the growth of P. putida 10.2 way of 3CBa by P. ptrtida 10.2, culture media were extracted
in MM9 supplemented with 2 mM 3CBa resulted in the with ethyl acetate and the extracts analysed by HPLC. Peaks
decrease of 3CBa at a rate of 25 nmol ml-’ h- ’ and an with retention times equivalent to authentic 3CC were ob-
increase in chloride concentration of 16.5 nmol ml - 1 h - *. served. A sample comparison of HPLC chromatograms of
The effects of presence of other carbon sources in MM9 authentic 3CC and a metabolic extract is illustrated in Figure 3.
medium on 3CBa degradation by P. putida 10.2 were
studied. A decrease of 3CBa utilization by P. ptrtida 10.2 Presence of CAT in Resting Cell Suspension of P. putida 10.2
was observed when media were supplemented with other which contained 3CBa
easily degraded carbon sources such as glucose and pyru- Attempts to identify CAT as a metabolite in the spent
vate (Table 2). At low concentrations of glucose (O.l%), medium were not successful, which may due to the utilizable

WorldJournal of Minobiology 6 Biotechnology. Vol 12, 1996 609

W. Ch&ch~enchom et al.

nature of the compound. Therefore, the appearance of CAT

Table 1. Microscopic and biochemical characteristics of Pseudo-
monas pufida 10.2. in resting cell suspensions of P. pufidu 10.2 was analysed
by HPLC after 44 h incubation (Table 3). Metabolite and
Pseudomonas Pseudomonas authentic CAT peaks were superimposed in all solvent
pufida 10.2 pufida systems used in this study. The result was confirmed by
as described in observation of higher peak areas when samples were co-
Bergey’s manual
injected with authentic CAT (data not shown). The concen-
Mlcroscoplc characteristics
trations of 3CC and CAT produced in the supematant at
Morphology rod rod 44 h were approximately 0.034 and 0.007 mM, respectively;
Gram’s strain negative negative as expected these were very low levels.
Spore formation not found not found
Biochemical characteristics Presence of KC Degrading Enzyme and Catechol 1,~
Catalase + + dioxygenase in Crude Exfracfs of P. putida 10.2
Catechol ortho-cleavage + f The identification of 3CC and CAT in culture broths and
Citrate production + + resting cell suspensions suggested that P. putida 10.2 could
Denitrification -
- degrade 3CBa via these compounds. If this is were true,
Gelatin dissemination
3CC-degrading enzyme and catechol 1,2-dioxygenase
Growth at 41°C
H,S production should be found in crude extracts of P. pufida 10.2. When
lndole 3CC was incubated with such extracts from P. pufida 10.2
Methyl red - grown in MM9 medium, the formation of CAT was ob-
Motility + + served, and this activity was dependent on the presence of
Nitrate reduction -
+ + NADPH (Table 4).
Oxidase
O/F glucose* +/- Assays of catechol l,&dioxygenase in crude enzyme
O/F maltose -l- extracts of P. putida 10.2 were carried out at intervals of 0,
O/F lactose -/- 0.5, I, 2 and 3 h, by determining the concentration of
O/F arabinose -/- MUC by HPLC, and this activity was destroyed by boiling
O/F sucrose -/- (Table 4). Furthermore, no enzyme activity could be ob-
O/F galactose -/-
O/F mannose -/- served when the appropriate substrates were not added to
Phenylalanine deaminase - the cell free extracts (data not shown).
Starch hydrolysis - Examination of the crude enzyme reaction mixture con-
TSlt K/N K/N taining 3CC as substrate by HPLC showed two metabolite
Urease test
peaks which corresponded to authentic CAT and MUC.
VP test$
Yellow orange cellular - The presence of CAT in 3CC reactions were further con-
pigment firmed by LC/MS. As shown in Figure 4, the metabolite
mass spectrum corresponded to the mass spectrum of
‘-xidative and fermentative utilization:
t-triple sugar iron agar; authentic catechol.
$-Voges-Proskauer. Peaks corresponding to MUC could not be separated
from buffer peaks in various mobile phase systems used in

Table 2. Effects of varlous carbon sources on growth and degradatlon of S-chlorobenzoate by Pseudomonas pufida 10.2.

Medium composition Growth Growth Initial Final 3CBa used Percentage

rate yield concentration concentration (mM) 3CBa
hr-l mg ml-’ of 3CBa (mM) of 3CBa (mr4) utilization’
WJ)
MM9 + 3CBa 0.13 0.088 1.0 0.824 0.176 17.6
MM9 + 3CBA + 1.0% glucose 0.19 0.275 1.0 1.0 0.00 0.0
MM9 + 3CBA + 0.1% glucose 0.16 0.275 1.0 0.955 0.045 4.5
MM9 + 3CBA + 1.0% pyruvate 0.16 0.154 1.0 1.0 0.00 0.0
MM9 + 3CBA + 0.1% pyruvate 0.15 0.149 1.0 0.987 0.013 1.3
MM9 + 3CBa + 1.0% lactose 0.13 0.105 1.0 0.978 0.022 2.2
MM9 + 3CBa + 0.1% lactose 0.14 0.110 1.0 0.885 0.115 11.5
MM9 + 3CBa + 1.0% succinate NG NG ND ND ND ND
MM9 + 3CBa + 0.1% succinate 0.18 0.231 1.0 0.897 0.103 10.3

NG - no growth; ND - not determined; l - at 24 h of incubation.

610 World Journal of Microbiology 6 Biofechnology, Vol 12, 1996

 Biodegradation of 3-chlorobenzoafe

6.51 optical density of authentic MUC. It was found that the

metabolite peaks showed the same absorption spectra as
I authentic MUC, with maximum optical density at 265 nm.

A.
I\
I I Discussion
6.50
Although our new isolate P. ptctidu 10.2 was able to utilize
3CBa as a carbon source, high concentrations of 3CBa (i.e.
KI mM) were found to inhibit growth of the organism (data
not shown). This result demonstrated that suitable concen-
trations of 3CBa are required if it is to serve as a carbon
source for this organism. Dom et al. (1974) also reported
B. that longer incubation periods were required for growth of
Pseudomonas sp. Bl3 in media supplemented with higher
concentrations of 3CBa.
The capability of P. pfida 10.2 to degrade 3CBa was
reduced by the presence of other carbon sources such as
glucose and lactose. Inhibition seen here for glucose and
pyruvate, may have resulted from catabolite repression,
6.50 because cells can use these compounds as alternative carbon
I sources. Goulding et al. (1988) reported that 12 microorgan-
C. isms including five Pseudomonas species, one Klebsiellu, four
Rhodococci and two fungi could degrade 3CBa when the
\ cells were grown in the presence of glucose (0.1%) at a
I I slightly decreased rate when compared to the rate in media
without glucose. Since, P. pfidu 10.2 cannot utilize lactose,
Retention time (min) lactose should not interfere with its degradation of 3CBa.
Figure 3. HPLC chromatograms and retention times showing the However, for some unknown reason, high concentrations
possible presence of 3-chlorocatechol in the culture broth of of lactose (1%) did show inhibitory effects. The presence of
Pseudomonas putida 10.2 grown in 3CBa-containing medium. 1% of succinate was found to be toxic to the cells, but low
(A) authentic 3CC; (B) co-injection of extract and authentic 3CC;
concentrations (0.1%) had no effect on cell growth and
(C) extract only.
3CBa degradation.
The formation of CAT in the reaction containing 3CC
HPLC. For further identification of MUC, peaks which and crude extract was shown to require NADPH. This
showed retention times corresponding to MUC were supports the results obtained by Mohn & Tiedje (1992)
scanned for maximum optical density at wavelengths be- that reductive dehalogenation reactions require an electron
tween 230 and 290 nm and compared to the maximum donor. This constitutes evidence of a reductive dechlorina-

Table 3. The presence of catechol in resting cell suspension of Pseudomonas putida 10.2 incubated with Jchlorobenzoate.

Solvent system Retention time (min) of

Authentic Supernatant of Supernatant of

catechol resting cell resting cell suspenslon
suspension + authentic catechol
10 mM phosphoric acid containing 25% acetonitrile and 10% methanol 5.098 5.083 5.087
10 rnM phosphoric acid containing 20% acetonitrile 7.408 7.365 7.312
10 mM phosphoric acid containing 30% acetonitrile 5.057 5.043 5.047
40 rnM acetic acid containing 25% methanol and 10% acetonitrile 5.737 5.683 5.732
40 rnM acetic acid containing 20% methanol 9.600 9.817 9.625
40 mM acetic acid containing 30% methanol 6.760 6.683 6.758
40 mM acetic acid containing 35% methanol 6.04 5.94 6.01
40 mM acetic acid containing 50% methanol 4.260 4.200 4.268

WorldJoumal of Microbiology 6 Biotechnology, Vol 12. 1996 611

W. Chobchuenchom et al.

tion biodegradative pathway in P. ptlfidu 10.2, an aerobic Reductive KC dechlorination activity should be important
bacterium. Reductive dehalogenation reactions of XC by for the biodegradation of many chlorinated aromatic com-
aerobic microorganisms have not been previously reported. pounds; previous reports have suggested that 3CC is a

i0,
6! ,

56 5Q

+4%&LL ' Dale

-z- 65 70 75 80 85 90 95 100 105 110

Mass/charge

60

Da/e

Mass/charge
Figure 4. Analysis of intermediates produced in crude enzyme reactions with 3-chlorocatechol as substrate by using liquid
chromatography and mass spectrometry. The reaction supernatant was purified by HPLC and the mass spectrum of the assumed
CAT peak was compared to the mass spectrum of authentic catechol. (A) mass spectrum of HPLC peak; (B) mass spectrum of
authentic catechol.

612
 Biodegradation of 3-chlorobenzoafe

Table 4. Specific activities of 3chlorocatechol-degrading enzyme and catechol 1,2-dioxygenase

in crude extracts of Pseudomonas pufida 10.2.

Enzyme preparation Specific activity of enzyme

SCC-degrading enzyme Catecholl ,bdioxygenase

(nmol hr-’ mg-‘) (nmol mln-I mg-‘)

Non-treated crude enzyme

1.9 0.7
with all required cofactors
0 ND
without NADPH

Boiled treated crude enzyme’

0 0
with all required cofactors

l -Crude extract was placed in a boiling water bath for 5 min before enzyme assay;
ND - Not determined.

common intermediate in the biodegradation of such com- Transposon mutagenesis and cloning analysis of the pathways
for degradation of 2,4-dichlorophenoxyacetic acid and 3-chloro-
pounds (Sondossi et al. 1992) and that the problem in
benzoate in Alcufigenes etttrophus JMP134(pJP4). Journal of
degrading halosubstituted aromatic compounds is the ineffi- Bacteriology 161, 85-90.
ciency of ring-cleavage enzymes in halocatechol transforma- Dom, E., Hellwig, M., Reineke, W., Knackmuss, H.-J. 1974 Isola-
tion (Knackmuss 1983). tion and characterization of a &chlorobenzoate degrading
To further clarify whether CAT was an intermediate Pseudomonad. Archives of Microbiology 99, 61-70.
Dom, E. & Knackmuss H.-J. 1978 Chemical structure and bio-
produced in reactions containing 3CC as substrate, superna-
degradability of halogenated aromatic compounds, substituent
tants were directly filtered and analysed using LC/MS. It effects on 1,2-dioxygenation of catechol. Biochemical ]oumul
was found that the mass spectrum of the metabolite peak 174,85-95.
corresponded to that of authentic catechol. The result Furukawa, K., Tomizuka, N. & Kamibayashi, A. 1979 Effect of
indicated that catechol in the culture broth could have chlorine substitution on the bacterial metabolism of various
polychlorinated biphenyls. Applied and Environmental Micro-
resulted from the activity of a 3CC-degrading enzyme.
biology 38, 301-310.
In addition to the formation of CAT in reactions contain- Ghosal, D., You, I.-S., Chatterjee, D.K. & Chakrabarty, A.M. 1985
ing XC and crude extract, the accumulation of MUC was Microbial degradation of halogenated compounds. Science 228,
also observed. The presence of MUC could have resulted 135-142.
from the activity of catechol l&dioxygenase (Table 4). Goulding, C., Gillen, C.J. & Bolton, E. 1988 Biodegradation of
substituted benzenes. Jotrrnul of Applied Bacteriology 65, l-5.
Haggblom, M.M., Janke, D. & Salkinoja-Salonen, M.S. 1989
Acknowledgement Hydroxylation and dechlorination of tetrachlorohydroquinone
by Rhodococcw sp. strain CP-2 cell extracts. Applied and Environ-
This work was supported by National Center for Genetic
mental Microbiology 55, 51&519.
Engineering and Biotechnology, National Science and Tech- Holt, J.G. 1994 Bergey’s Mum& of Determinative Bacteriology. 9th
nology Development Agency and UNDP. We are grateful edition. Baltimore: Williams & Wilkins.
for the help of Dr Timothy W. Flegel in reading this Horvath, R.S. 1971 Cometabolism of the herbicide 2,3,6-trichloro-
manuscript. benzoate. ]ournul of Agricultwul and Food Chemisfry 19, 291-
293.
Knackmuss, H.J. 1983 Xenobiotic degradation in industrial sewage:
haloaromatics as target substrates. Biochemical Society Symposium
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4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro, (Received in revised form 28 March 1996; accepted 2 April
4-bromo, and a-iodobenzoate by Alcaligenes denitrijcans 1996)