World Journal
of Microbiology
& Biotechnology
Biodegradation Pseudomonas W. Chobchuenchom,”
1.7. 607-614
of 3xhlorobenzoate putida 10.2
by
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 hazardous wastes which are usually introduced into the environment through two major routes, namely, as active ingredients in commonly used herbicides (Horvath 1971; Pfister 1973) and as metabolic products of polychlorinated biphenyls (PCBs). Biodegradation of PCBs by soil microorganisms was found to give rise to the accumulation of chlorobenzoate in the environment (Ahmed & Focht 1973; Furukawa et al. 1979; Sylvestre & Fauteux 1982; Masse et al. 1984). There have been reports that 3-chlorobenzoate (3CBa) could be utilized as the sole carbon source by several microorganisms such as Pseudomonas sp. Bl3 (Weisshaar et al. 1987) and Alcaligenes eufrophus JMP134 (Don et al. 1995). The biodegradation of 3CBa in aerobic bacteria commonly proceeds via 3-chlorocatechol (3CC). The ring cleavage through a mechanism known as modified ortho cleavage precedes the spontaneous chlorine removal (Ghosal et al. 1985; Weisshaar et al. 1987; Chaudhry & Chapalamadugu 1991). In some aerobic bacteria, degradation of 3CBa and other W. Chobchuenchom and of Biotechnology, Faculty Bangkok, 10400 Thailand; Laboratory of Biotechnology, Rangsit Highway, Bangkok, @ 1996 Rapid Science
A. Bhumiratana are with the Department of Science, Mahidol University, RAMA VI. fax: (662) 2463026. S. Mongkolsuk is with the Chulaborn Research Institute. Vipavadee10210 Thailand. *Corresponding author.
chlorinated aromatic compounds is incomplete, and this leads to the appearance of a black colour resulting from the polymerization of 3CC. 3CC is a common intermediate in the biodegradation of various halogenated aromatic compounds, including 4-chlorobenzoic acid (Chatterjee & Chakrabarty 1982). In contrast to aerobic bacteria, the anaerobic microbe Desulfomonile fiedjei DCB-1 converts 3CBa to benzoate by a mechanism of reductive dehalogenation (Mohn & Tiedje 1992). Reductive dehalogenation is important in numerous biodegradations, including those of organochlorine pesticides, alkyl solvents and aryl halides. Although reductive dehalogenations are normally seen in anaerobic conditions, they are also found in aerobic conditions, for example, the reductive dechlorination of dichlorobenzoate by Alculigenes denifr$cans (van den Tweel et al. 1987) and reductive dechlorination of pentachlorophenol by Fluvobacferium sp. (Steiert & Crawford 1986), Rhodococcxs chlorophenolicus (Apajalahti & Salkinoja-Salonen 1987) and Rhodococms sp. (Haggblom et al. 1989). This report describes the characteristics of PseuAomonus pufida 10.2, a newly isolated bacterial strain capable of degrading 3CBa. The characteristics of 3CBa degradation and chloride elimination by resting cell suspensions and growing cells were studied.
Publishers World ]oumal of Mmobtology & Biotechnology, Vol 12, 1996
607
W. Chobchuenchom et al.
Materials
and Methods
Media and Growth Conditions The two media used in this study were M9 mineral salt medium (42 mM Na,PO,.IZH,O, 22 mM KH,PO,, 10 mM NaCl, 18 mM NH,CI, 2 mu MgSO,, 0.5 mM CaCI,) and modified M9 medium (MM9) which was composed of 0.02% (w/v) yeast extract in M9 medium. Where indicated, medium was supplemented with 3CBa at a final concentration of 1.0 mM from a stock solution of I M 3CBa in absolute ethanol. When chloride analysis was required NaCI, CaCl,, and NH,CI were omitted from M9 medium, and I ml of trace element stock solution [containing (g/l): 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 2% KOH] was added per liter. For solid media, 1.5% Bacto agar was added to the liquid media. Media and trace element stock solutions were sterilized by autoclaving at 15 lb inch-2 for 20 min. Bacterial cultivation in liquid media was carried out at 30°C with shaking at 150 rev min-‘. Cell growth was determined by measuring optical density at 600 nm using a Milton Roy spectronic 1001 plus. Chemicals The chemicals 3CBa, catechol (CAT) and cis,cis-muconic acid (MUC) were from Sigma Co., St. Louis, USA and 3CC was obtained from Tokyo Kasei Chemical, Japan. Chromatographic grade acetonitrile and methanol were from Merck, Darmstadt, Germany. Chloroform was from Fluka, Buchs, Switzerland. M9 basal salt medium was from Gibco BRL, Paisley, Scotland. Bacto agar and biochemical test media were from Difco, Detroit, MI, USA. All chemicals used in this study were of analytical grade. Preparation of Cell Suspensions and Cell Erfracfsfrom Pseudomonas putida 10.2 Unless specified otherwise, P. pufida 10.2 was precultured in MM9 medium. When preparing cell suspensions, cultures were harvested from the late exponential growth phase (18 h after inoculation), centrifuged for 15 min at 5000 x g, washed once with 50 mM phosphate buffer pH 7.0 and resuspended in the same buffer to obtain the indicated cell concentrations. For preparation of cell extracts, cultures were harvested and washed, and the cell pellets were used fresh or kept at - 20°C until used. Frozen cell pellets were thawed and suspended in 50 mM phosphate buffer pH 7.0 to give a concentration of 0.5 to 1.0 mg ml-‘. Using ahquots of 10 ml, the cell suspensions were disrupted by sonication (Soniprep MSE, UK) for 15 min. To avoid overheating of the preparation, sonication was carried out for 10 s with 10 s stop intervals and all operations were carried out on ice. Unbroken cells and debris were removed by centrifugation at 14,000 x g for 30 min, at 4°C. The supernatant obtained was designated crude extract.
Enzyme Assays Assay for KC-Degrading Enzyme. Assays were performed in test 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 terminated by the addition of 20 ~1 of 1 M trichloroacetic acid. The precipitate was removed by centrifugation at 10,000 x g for 10 min and the catechol content of the supematant determined by HPLC.
608
Worki]oumal
ofA4icrobmlogy & Biotechnology, Vof 12, 1996
Cafechol 1.2 Dioxygenase Assay. Catechol 1,2-dioxygenase (EC 1.13.11.1) assays were based on HPLC detection of MUC rather than an optical density at 260 nm as previously reported (Dom & Knackmuss 1978). Reactions were carried out in 2-ml Eppendorf tubes containing 1.5 ml of either non-treated or boiled enzyme preparations and 0.1 mM CAT, 1.28 mM MgSO, and 0.5 mM NADPH. Each reaction tube was incubated at 30°C in a water bath for 3 h. At intervals of 0, 0.5, 1, 2, and 3 h, aliquots of reactions and controls were taken and the reaction stopped by the addition of 20 ~1 1 M trichloroacetic acid. After removal of precipitate by centrifugation for 10 min at 10,000 x g, the concentration of MUC in the supematants was determined by HPLC. Protein Deferminafion Protein concentrations ford protein detection bovine serum albumin at 280 nm.
were determined either by using the Bradkit (Bio-Rad Laboratories, CA, USA) with as standard or by measuring optical density
Analytical Procedures High Pressure Liquid Chromatography. HPLC was performed with a Shimadzu model LC 6A HPLC machine with a reverse phase Shimpack clc/ODS column (6 x I50 mm). The column temperature was set at 40°C and the peaks were detected by using a u.v.vis spectrometric detector (SPD-6AV). For determination of 3CBA, orthophosphoric acid (10 rnr.4) containing 50% acetonitrile was used as the mobile phase with a flow rate of 1 ml min-’ and the peaks were detected at 278 nm. For analysis of 3CC in culture broth, 2 1 of culture broth was extracted with ethyl acetate, dried over MgSO, and then evaporated with a rotary evaporator. Extracted preparations were resuspended in chloroform and analysed for 3CC using HPLC as described above with a mobile phase of 50% methanol containing 40 mM acetic acid and a detection wavelength of 284 nm. For the analysis of CAT, the supematant from resting cell suspensions was analysed for appearance of CAT by HPLC. The retention times of metabohte and standard peaks were compared in eight mobile phase systems by both separate and co-inject methods. For determination of CAT, peak areas of samples were converted to concentration by reading from the CAT standard curve prepared as for the detection of 3CBa using a detection wavelength of 230 nm and a flow rate of 0.5 ml min-‘. Liquid Chromatography/Mass Spectrometry (K/MS). For analysis of catechol appearance in reactions containing 3CC and crude enzyme extract, the supematants of each reaction and authentic catechol were studied using LC/MS. HPLC was performed using a Waters TM model 600s. Supematant from the reaction was directly injected and separated on a reverse phase Shimpack clc/ ODS column (6 x 150) with a 0.5 ml min-’ flow rate and 50% acetonitrile in water as the mobile phase. Interphase was performed by using atmospheric pressure chemical ionization (APCI) with a 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 supematant at indicated time intervals was monitored by silver nitrate titration with potassium chromate as indicator.
Results Isolation P. pufidu agricultural
and Idenf$cafion 10.2
was
of P. putida isolated
field in Chiang
from Mai,
10.2 a soil
Thailand
sample by using
from
an
a batch
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 hcubation
15 time
20
25
30
(II)
Figure 1. Growth and degradation of 3-chlorobenzoate by Pseudomonas putida 10.2 in MM9 supplemented with 3CBa. Optical density at 600 nm (a); amount of 3CBa (m) and chloride ion (A).
0 0.0
0.2
0.4
0.6
Cell concentration Figure
culture enrichment technique; approximately 10 g of each soil sample was resuspended in 25 ml of distilled water and filtered once through Whatman filter paper No 4 and twice through Whatman filter paper No I. Aliquots of 500 ,~l were then inoculated into 125-ml Erlenmeyer flasks containing 50 ml of M9 supplemented with I mM 3CBa and incubated with shaking at 30°C. All turbid cultures were subcultured 3 to 5 times by transferring 100 ~1 of turbid culture broth to 50 ml of fresh medium. Finally, broth from each turbid culture was streaked on mineral salt agar supplemented with 3CBa to obtain pure cultures. Growth of purified isolates was then compared in M9 medium with and without 3CBa supplementation. Ten isolates which showed higher growth yield in the presence of 3CBa were studied further for their ability to degrade 3CBa. Based on growth rate, growth yield and the rate of 3CBa degradation by resting cell suspensions, it was found that isolate 10.2 was the best 3CBa degrader. For identification of isolate 10.2, microscopic examinations and biochemical tests were performed (Table 1). Based on the identification scheme in Bergey’s manual of determinative bacteriology (Holt et al. 1994, strain 10.2 was identified as Pseudomonas putida. Growth and 3CBa Degradation by P. putida 10.2 As illustrated in Figure I, the growth of P. putida 10.2 in MM9 supplemented with 2 mM 3CBa resulted in the decrease of 3CBa at a rate of 25 nmol ml-’ h- ’ and an increase in chloride concentration of 16.5 nmol ml - 1 h - *. The effects of presence of other carbon sources in MM9 medium on 3CBa degradation by P. putida 10.2 were studied. A decrease of 3CBa utilization by P. ptrtida 10.2 was observed when media were supplemented with other easily degraded carbon sources such as glucose and pyruvate (Table 2). At low concentrations of glucose (O.l%),
putida treated
0.8
1.0
(mg ml ‘)
2. The effect of resting cell suspensions of Pseudomonas 10.2 on the rate of 3-chlorobenzoate degradation. Noncell suspension (a) and heat treated cell suspension
(rn).
3CBa degradation was reduced from 17.6 to 4.5% and at higher levels (1.0%) inhibition was complete. Similarly, lactose supplementation at 0.1% caused a slight reduction in 3CBa utilization (17.6% to 11.5%) and a marked inhibition at 1%, despite the fact that the biochemical tests showed that this organism could not assimilate lactose. This pattern of inhibition was also found with succinate supplementation. Degradation of 3CBa by Resting Cell Suspensions The rate of 3CBa degradation was studied using cell suspensions at the concentrations shown in Figure 2. The rate of 3CBa degradation was approximately proportional to the cell mass. The rate of 3CBa degradation was defined as the amount of 3CBa lost from the medium, and the values were calculated when the loss was directly proportional to the time of incubation. Presence of 3CC in Spent Culture Broth Containing 3CBa To obtain information concerning the biodegradation pathway of 3CBa by P. ptrtida 10.2, culture media were extracted with ethyl acetate and the extracts analysed by HPLC. Peaks with retention times equivalent to authentic 3CC were observed. A sample comparison of HPLC chromatograms of authentic 3CC and a metabolic extract is illustrated in Figure 3. Presence of CAT in Resting Cell Suspension of P. putida 10.2 which contained 3CBa Attempts to identify CAT as a metabolite in the spent 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.
Table 1. Microscopic monas pufida 10.2.
and biochemical
Mlcroscoplc
characteristics
Pseudomonas pufida 10.2
Pseudomonas pufida as described in Bergey’s manual
rod negative not found
rod negative not found
characteristics
Morphology Gram’s strain Spore formation Biochemical
characteristics
+ + + -
Catalase Catechol ortho-cleavage Citrate production Denitrification Gelatin dissemination Growth at 41°C H,S production lndole Methyl red Motility Nitrate reduction Oxidase O/F glucose* O/F maltose O/F lactose O/F arabinose O/F sucrose O/F galactose O/F mannose Phenylalanine deaminase Starch hydrolysis TSlt Urease test VP test$ Yellow orange cellular pigment
carbon
MM9 MM9 MM9 MM9 MM9 MM9 MM9 MM9 MM9 NG -
610
3CBa 3CBA 3CBA 3CBA 3CBA 3CBa 3CBa 3CBa 3CBa
no growth;
+ + + + + + + +
1.0% 0.1% 1.0% 0.1% 1.0% 0.1% 1.0% 0.1% ND -
K/N
sources
composition
+ + + + + + + + +
+
-
Table
of varlous
+
K/N
utilization:
2. Effects
Presence of KC Degrading Enzyme and Catechol 1,~ dioxygenase in Crude Exfracfs of P. putida 10.2 The identification of 3CC and CAT in culture broths and resting cell suspensions suggested that P. putida 10.2 could degrade 3CBa via these compounds. If this is were true, 3CC-degrading enzyme and catechol 1,2-dioxygenase should be found in crude extracts of P. pufida 10.2. When 3CC was incubated with such extracts from P. pufida 10.2 grown in MM9 medium, the formation of CAT was observed, and this activity was dependent on the presence of NADPH (Table 4). Assays of catechol l,&dioxygenase in crude enzyme extracts of P. putida 10.2 were carried out at intervals of 0, 0.5, I, 2 and 3 h, by determining the concentration of MUC by HPLC, and this activity was destroyed by boiling (Table 4). Furthermore, no enzyme activity could be observed when the appropriate substrates were not added to the cell free extracts (data not shown). Examination of the crude enzyme reaction mixture containing 3CC as substrate by HPLC showed two metabolite peaks which corresponded to authentic CAT and MUC. The presence of CAT in 3CC reactions were further confirmed by LC/MS. As shown in Figure 4, the metabolite mass spectrum corresponded to the mass spectrum of authentic catechol. Peaks corresponding to MUC could not be separated from buffer peaks in various mobile phase systems used in
+ f +
+ + +/-l-/-/-/-/-/-
‘-xidative and fermentative t-triple sugar iron agar; $-Voges-Proskauer.
Medium
nature of the compound. Therefore, the appearance of CAT in resting cell suspensions of P. pufidu 10.2 was analysed by HPLC after 44 h incubation (Table 3). Metabolite and authentic CAT peaks were superimposed in all solvent systems used in this study. The result was confirmed by observation of higher peak areas when samples were coinjected with authentic CAT (data not shown). The concentrations of 3CC and CAT produced in the supematant at 44 h were approximately 0.034 and 0.007 mM, respectively; as expected these were very low levels.
of Pseudo-
glucose glucose pyruvate pyruvate lactose lactose succinate succinate not determined;
on growth
and degradatlon
of S-chlorobenzoate
by Pseudomonas
Growth rate hr-l
Growth yield mg ml-’
Initial concentration of 3CBa (mM)
Final concentration of 3CBa (mr4)
3CBa used (mM)
0.13 0.19 0.16 0.16 0.15 0.13 0.14 NG 0.18
0.088 0.275 0.275 0.154 0.149 0.105 0.110 NG 0.231
1.0 1.0 1.0 1.0 1.0 1.0 1.0 ND 1.0
0.824 1.0 0.955 1.0 0.987 0.978 0.885 ND 0.897
0.176 0.00 0.045 0.00 0.013 0.022 0.115 ND 0.103
l
-
at 24 h of incubation.
World Journal of Microbiology 6 Biofechnology, Vol 12, 1996
pufida
10.2. Percentage 3CBa utilization’ WJ) 17.6 0.0 4.5 0.0 1.3 2.2 11.5 ND 10.3
Biodegradation of 3-chlorobenzoafe
6.51
optical density of authentic MUC. It was found that the metabolite peaks showed the same absorption spectra as authentic MUC, with maximum optical density at 265 nm.
I
A. I\ I I
Discussion
6.50
B.
6.50 I
I
C. \
I
Retention time (min) Figure 3. HPLC chromatograms and retention times showing the possible presence of 3-chlorocatechol in the culture broth of Pseudomonas putida 10.2 grown in 3CBa-containing medium. (A) authentic 3CC; (B) co-injection of extract and authentic 3CC; (C) extract only.
HPLC. For further identification of MUC, peaks which showed retention times corresponding to MUC were scanned for maximum optical density at wavelengths between 230 and 290 nm and compared to the maximum
Table Solvent
3. The presence
of catechol
in resting
cell suspension
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 concentrations of 3CBa are required if it is to serve as a carbon source for this organism. Dom et al. (1974) also reported 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, because cells can use these compounds as alternative carbon sources. Goulding et al. (1988) reported that 12 microorganisms 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 slightly decreased rate when compared to the rate in media without glucose. Since, P. pfidu 10.2 cannot utilize lactose, lactose should not interfere with its degradation of 3CBa. However, for some unknown reason, high concentrations of lactose (1%) did show inhibitory effects. The presence of 1% of succinate was found to be toxic to the cells, but low concentrations (0.1%) had no effect on cell growth and 3CBa degradation. The formation of CAT in the reaction containing 3CC and crude extract was shown to require NADPH. This supports the results obtained by Mohn & Tiedje (1992) that reductive dehalogenation reactions require an electron donor. This constitutes evidence of a reductive dechlorina-
of Pseudomonas
putida
system
mM rnM mM rnM rnM mM mM mM
phosphoric phosphoric phosphoric acetic acid acetic acid acetic acid acetic acid acetic acid
with Jchlorobenzoate.
Retention Authentic catechol
10 10 10 40 40 40 40 40
10.2 incubated
acid containing acid containing acid containing containing 25% containing 20% containing 30% containing 35% containing 50%
25% acetonitrile and 10% methanol 20% acetonitrile 30% acetonitrile methanol and 10% acetonitrile methanol methanol methanol methanol
5.098 7.408 5.057 5.737 9.600 6.760 6.04 4.260
time
Supernatant of resting cell suspension 5.083 7.365 5.043 5.683 9.817 6.683 5.94 4.200
(min)
of
Supernatant of resting cell suspenslon + authentic catechol 5.087 7.312 5.047 5.732 9.625 6.758 6.01 4.268
WorldJoumal of Microbiology 6 Biotechnology, Vol 12. 1996
611
W. Chobchuenchom et al. Reductive KC dechlorination activity should be important for the biodegradation of many chlorinated aromatic compounds; previous reports have suggested that 3CC is a
tion biodegradative pathway in P. ptlfidu 10.2, an aerobic bacterium. Reductive dehalogenation reactions of XC by aerobic microorganisms have not been previously reported.
6!
i0, ,
56 5Q +4%&LL
' Dale 65
-z-
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 Table 4. Specific in crude extracts Enzyme
activities of 3chlorocatechol-degrading of Pseudomonas pufida 10.2.
enzyme
Specific
preparation SCC-degrading (nmol hr-’
Non-treated crude with all required without NADPH Boiled with l
enzyme cofactors
treated crude enzyme’ all required cofactors
-Crude extract was placed ND - Not determined.
in a boiling
water
common intermediate in the biodegradation of such compounds (Sondossi et al. 1992) and that the problem in degrading halosubstituted aromatic compounds is the inefficiency of ring-cleavage enzymes in halocatechol transformation (Knackmuss 1983). To further clarify whether CAT was an intermediate produced in reactions containing 3CC as substrate, supernatants were directly filtered and analysed using LC/MS. It was found that the mass spectrum of the metabolite peak corresponded to that of authentic catechol. The result indicated that catechol in the culture broth could have resulted from the activity of a 3CC-degrading enzyme. In addition to the formation of CAT in reactions containing XC and crude extract, the accumulation of MUC was also observed. The presence of MUC could have resulted from the activity of catechol l&dioxygenase (Table 4).
Acknowledgement This work was supported by National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency and UNDP. We are grateful for the help of Dr Timothy W. Flegel in reading this manuscript.
References Ahmed, M. & Focht, D.D. 1973 Degradation of polychlorinated biphenyl by two species of Achromobucter. Canadian ]oaurnal of Microbiology 19, 47-52. Apajalahti, J.H.A. & Salkinoja-Salonen, M.S. 1987 Complete dechlorination of tetrachlorohydroquinone by cell extracts of pentachlorophenol-induced Rhodococcus chlorophenolicus. ]ownal of Bacteriology 169, 5125-5130. Chatterjee, D.K. & Chakrabarty, A.M. 1982 Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids. Mo\ecu~ur and General Genetics 188, 279-285. Chaudhry, G.R. & Chapalamadugu, S. 1991 Biodegradation of halogenated organic compounds. Microbiologic& Reviews 55, 59-79. Don, R.H. Weightman, A.J., Knackmuss, H.-J., Timmis, K.N. 1985
and catechol
activity
enzyme mg-‘)
1,2-dioxygenase
of enzyme Catecholl (nmol
,bdioxygenase mln-I mg-‘)
1.9 0
0.7 ND
0
0
bath for 5 min before
of 3-chlorobenzoafe
enzyme
assay;
Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcufigenes etttrophus JMP134(pJP4). Journal of Bacteriology 161, 85-90. Dom, E., Hellwig, M., Reineke, W., Knackmuss, H.-J. 1974 Isolation and characterization of a &chlorobenzoate degrading Pseudomonad. Archives of Microbiology 99, 61-70. Dom, E. & Knackmuss H.-J. 1978 Chemical structure and biodegradability of halogenated aromatic compounds, substituent effects on 1,2-dioxygenation of catechol. Biochemical ]oumul 174,85-95. Furukawa, K., Tomizuka, N. & Kamibayashi, A. 1979 Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Applied and Environmental Microbiology 38, 301-310. Ghosal, D., You, I.-S., Chatterjee, D.K. & Chakrabarty, A.M. 1985 Microbial degradation of halogenated compounds. Science 228, 135-142. 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 Hydroxylation and dechlorination of tetrachlorohydroquinone by Rhodococcw sp. strain CP-2 cell extracts. Applied and Environmental Microbiology 55, 51&519. Holt, J.G. 1994 Bergey’s Mum& of Determinative Bacteriology. 9th edition. Baltimore: Williams & Wilkins. Horvath, R.S. 1971 Cometabolism of the herbicide 2,3,6-trichlorobenzoate. ]ournul of Agricultwul and Food Chemisfry 19, 291293. Knackmuss, H.J. 1983 Xenobiotic degradation in industrial sewage: haloaromatics as target substrates. Biochemical Society Symposium 48,173-190. Masse, R., Messier, F., Peloquin, L., Ayotle, C. & Sylvestre, M. 1984 Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Applied and Environmental Microbiology. 47, 947-95 1. Mohn, W.W. & Tiedje, J.M. 1992 Microbial reductive dehalogenation. Microbiological Reviews 56, 482-507. Pfister, R.M. 1973 Interactions of halogenated pesticides and microorganisms: a review. In CRC Handbook of Microbiology, ed La&n, A.I. & Lechevalier, H. pp. l-33. Cleveland: CRC Press. Sondossi, M., Sylvestre, M. & Ahmad, D. 1992 Effects of chlorobenzoate transformation on the Pseudomonas fesfosferoni biphenyl and chlorobiphenyl degradation pathway. Applied and Environmental Microbiology 58, 458-495.
World Joumi of Microbroiogy & Btohchnofogy. ‘701 12. 1996
613
W. Chobchuenchom
et al.
Steiert, J.G. & Crawford, R.L. 1986 Catabolism of pentachlorophenol by a Flavobacterium sp. Biochemical and Biophysical Research Communications 141, 825-830. Sylvestre, M. & Fauteux, J. 1982 A new facultative anaerobe capable of growth on chlorobiphenyls. ]ournal of General and Applied Microbiology 28, 61-72. Van den Tweel, W.J.H., ter Burk, N., Kok, J.B. & DeBont, J.M.M. 1987 Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro, 4-bromo, and a-iodobenzoate by Alcaligenes denitrijcans
NTB-I. Applied and Environmental Microbiology 53, 810815. Weisshaar, M.-P., Franklin, F.C.H. & Reineke, W. 1987 Molecular doning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain BI3. Journal of Bacteriology
169,394-402. (Received in revised form 28 March 1996)
1996; accepted 2 April
of Microbiology
& Biotechnology
Biodegradation Pseudomonas W. Chobchuenchom,”
1.7. 607-614
of 3xhlorobenzoate putida 10.2
by
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 hazardous wastes which are usually introduced into the environment through two major routes, namely, as active ingredients in commonly used herbicides (Horvath 1971; Pfister 1973) and as metabolic products of polychlorinated biphenyls (PCBs). Biodegradation of PCBs by soil microorganisms was found to give rise to the accumulation of chlorobenzoate in the environment (Ahmed & Focht 1973; Furukawa et al. 1979; Sylvestre & Fauteux 1982; Masse et al. 1984). There have been reports that 3-chlorobenzoate (3CBa) could be utilized as the sole carbon source by several microorganisms such as Pseudomonas sp. Bl3 (Weisshaar et al. 1987) and Alcaligenes eufrophus JMP134 (Don et al. 1995). The biodegradation of 3CBa in aerobic bacteria commonly proceeds via 3-chlorocatechol (3CC). The ring cleavage through a mechanism known as modified ortho cleavage precedes the spontaneous chlorine removal (Ghosal et al. 1985; Weisshaar et al. 1987; Chaudhry & Chapalamadugu 1991). In some aerobic bacteria, degradation of 3CBa and other W. Chobchuenchom and of Biotechnology, Faculty Bangkok, 10400 Thailand; Laboratory of Biotechnology, Rangsit Highway, Bangkok, @ 1996 Rapid Science
A. Bhumiratana are with the Department of Science, Mahidol University, RAMA VI. fax: (662) 2463026. S. Mongkolsuk is with the Chulaborn Research Institute. Vipavadee10210 Thailand. *Corresponding author.
chlorinated aromatic compounds is incomplete, and this leads to the appearance of a black colour resulting from the polymerization of 3CC. 3CC is a common intermediate in the biodegradation of various halogenated aromatic compounds, including 4-chlorobenzoic acid (Chatterjee & Chakrabarty 1982). In contrast to aerobic bacteria, the anaerobic microbe Desulfomonile fiedjei DCB-1 converts 3CBa to benzoate by a mechanism of reductive dehalogenation (Mohn & Tiedje 1992). Reductive dehalogenation is important in numerous biodegradations, including those of organochlorine pesticides, alkyl solvents and aryl halides. Although reductive dehalogenations are normally seen in anaerobic conditions, they are also found in aerobic conditions, for example, the reductive dechlorination of dichlorobenzoate by Alculigenes denifr$cans (van den Tweel et al. 1987) and reductive dechlorination of pentachlorophenol by Fluvobacferium sp. (Steiert & Crawford 1986), Rhodococcxs chlorophenolicus (Apajalahti & Salkinoja-Salonen 1987) and Rhodococms sp. (Haggblom et al. 1989). This report describes the characteristics of PseuAomonus pufida 10.2, a newly isolated bacterial strain capable of degrading 3CBa. The characteristics of 3CBa degradation and chloride elimination by resting cell suspensions and growing cells were studied.
Publishers World ]oumal of Mmobtology & Biotechnology, Vol 12, 1996
607
W. Chobchuenchom et al.
Materials
and Methods
Media and Growth Conditions The two media used in this study were M9 mineral salt medium (42 mM Na,PO,.IZH,O, 22 mM KH,PO,, 10 mM NaCl, 18 mM NH,CI, 2 mu MgSO,, 0.5 mM CaCI,) and modified M9 medium (MM9) which was composed of 0.02% (w/v) yeast extract in M9 medium. Where indicated, medium was supplemented with 3CBa at a final concentration of 1.0 mM from a stock solution of I M 3CBa in absolute ethanol. When chloride analysis was required NaCI, CaCl,, and NH,CI were omitted from M9 medium, and I ml of trace element stock solution [containing (g/l): 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 2% KOH] was added per liter. For solid media, 1.5% Bacto agar was added to the liquid media. Media and trace element stock solutions were sterilized by autoclaving at 15 lb inch-2 for 20 min. Bacterial cultivation in liquid media was carried out at 30°C with shaking at 150 rev min-‘. Cell growth was determined by measuring optical density at 600 nm using a Milton Roy spectronic 1001 plus. Chemicals The chemicals 3CBa, catechol (CAT) and cis,cis-muconic acid (MUC) were from Sigma Co., St. Louis, USA and 3CC was obtained from Tokyo Kasei Chemical, Japan. Chromatographic grade acetonitrile and methanol were from Merck, Darmstadt, Germany. Chloroform was from Fluka, Buchs, Switzerland. M9 basal salt medium was from Gibco BRL, Paisley, Scotland. Bacto agar and biochemical test media were from Difco, Detroit, MI, USA. All chemicals used in this study were of analytical grade. Preparation of Cell Suspensions and Cell Erfracfsfrom Pseudomonas putida 10.2 Unless specified otherwise, P. pufida 10.2 was precultured in MM9 medium. When preparing cell suspensions, cultures were harvested from the late exponential growth phase (18 h after inoculation), centrifuged for 15 min at 5000 x g, washed once with 50 mM phosphate buffer pH 7.0 and resuspended in the same buffer to obtain the indicated cell concentrations. For preparation of cell extracts, cultures were harvested and washed, and the cell pellets were used fresh or kept at - 20°C until used. Frozen cell pellets were thawed and suspended in 50 mM phosphate buffer pH 7.0 to give a concentration of 0.5 to 1.0 mg ml-‘. Using ahquots of 10 ml, the cell suspensions were disrupted by sonication (Soniprep MSE, UK) for 15 min. To avoid overheating of the preparation, sonication was carried out for 10 s with 10 s stop intervals and all operations were carried out on ice. Unbroken cells and debris were removed by centrifugation at 14,000 x g for 30 min, at 4°C. The supernatant obtained was designated crude extract.
Enzyme Assays Assay for KC-Degrading Enzyme. Assays were performed in test 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 terminated by the addition of 20 ~1 of 1 M trichloroacetic acid. The precipitate was removed by centrifugation at 10,000 x g for 10 min and the catechol content of the supematant determined by HPLC.
608
Worki]oumal
ofA4icrobmlogy & Biotechnology, Vof 12, 1996
Cafechol 1.2 Dioxygenase Assay. Catechol 1,2-dioxygenase (EC 1.13.11.1) assays were based on HPLC detection of MUC rather than an optical density at 260 nm as previously reported (Dom & Knackmuss 1978). Reactions were carried out in 2-ml Eppendorf tubes containing 1.5 ml of either non-treated or boiled enzyme preparations and 0.1 mM CAT, 1.28 mM MgSO, and 0.5 mM NADPH. Each reaction tube was incubated at 30°C in a water bath for 3 h. At intervals of 0, 0.5, 1, 2, and 3 h, aliquots of reactions and controls were taken and the reaction stopped by the addition of 20 ~1 1 M trichloroacetic acid. After removal of precipitate by centrifugation for 10 min at 10,000 x g, the concentration of MUC in the supematants was determined by HPLC. Protein Deferminafion Protein concentrations ford protein detection bovine serum albumin at 280 nm.
were determined either by using the Bradkit (Bio-Rad Laboratories, CA, USA) with as standard or by measuring optical density
Analytical Procedures High Pressure Liquid Chromatography. HPLC was performed with a Shimadzu model LC 6A HPLC machine with a reverse phase Shimpack clc/ODS column (6 x I50 mm). The column temperature was set at 40°C and the peaks were detected by using a u.v.vis spectrometric detector (SPD-6AV). For determination of 3CBA, orthophosphoric acid (10 rnr.4) containing 50% acetonitrile was used as the mobile phase with a flow rate of 1 ml min-’ and the peaks were detected at 278 nm. For analysis of 3CC in culture broth, 2 1 of culture broth was extracted with ethyl acetate, dried over MgSO, and then evaporated with a rotary evaporator. Extracted preparations were resuspended in chloroform and analysed for 3CC using HPLC as described above with a mobile phase of 50% methanol containing 40 mM acetic acid and a detection wavelength of 284 nm. For the analysis of CAT, the supematant from resting cell suspensions was analysed for appearance of CAT by HPLC. The retention times of metabohte and standard peaks were compared in eight mobile phase systems by both separate and co-inject methods. For determination of CAT, peak areas of samples were converted to concentration by reading from the CAT standard curve prepared as for the detection of 3CBa using a detection wavelength of 230 nm and a flow rate of 0.5 ml min-‘. Liquid Chromatography/Mass Spectrometry (K/MS). For analysis of catechol appearance in reactions containing 3CC and crude enzyme extract, the supematants of each reaction and authentic catechol were studied using LC/MS. HPLC was performed using a Waters TM model 600s. Supematant from the reaction was directly injected and separated on a reverse phase Shimpack clc/ ODS column (6 x 150) with a 0.5 ml min-’ flow rate and 50% acetonitrile in water as the mobile phase. Interphase was performed by using atmospheric pressure chemical ionization (APCI) with a 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 supematant at indicated time intervals was monitored by silver nitrate titration with potassium chromate as indicator.
Results Isolation P. pufidu agricultural
and Idenf$cafion 10.2
was
of P. putida isolated
field in Chiang
from Mai,
10.2 a soil
Thailand
sample by using
from
an
a batch
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 hcubation
15 time
20
25
30
(II)
Figure 1. Growth and degradation of 3-chlorobenzoate by Pseudomonas putida 10.2 in MM9 supplemented with 3CBa. Optical density at 600 nm (a); amount of 3CBa (m) and chloride ion (A).
0 0.0
0.2
0.4
0.6
Cell concentration Figure
culture enrichment technique; approximately 10 g of each soil sample was resuspended in 25 ml of distilled water and filtered once through Whatman filter paper No 4 and twice through Whatman filter paper No I. Aliquots of 500 ,~l were then inoculated into 125-ml Erlenmeyer flasks containing 50 ml of M9 supplemented with I mM 3CBa and incubated with shaking at 30°C. All turbid cultures were subcultured 3 to 5 times by transferring 100 ~1 of turbid culture broth to 50 ml of fresh medium. Finally, broth from each turbid culture was streaked on mineral salt agar supplemented with 3CBa to obtain pure cultures. Growth of purified isolates was then compared in M9 medium with and without 3CBa supplementation. Ten isolates which showed higher growth yield in the presence of 3CBa were studied further for their ability to degrade 3CBa. Based on growth rate, growth yield and the rate of 3CBa degradation by resting cell suspensions, it was found that isolate 10.2 was the best 3CBa degrader. For identification of isolate 10.2, microscopic examinations and biochemical tests were performed (Table 1). Based on the identification scheme in Bergey’s manual of determinative bacteriology (Holt et al. 1994, strain 10.2 was identified as Pseudomonas putida. Growth and 3CBa Degradation by P. putida 10.2 As illustrated in Figure I, the growth of P. putida 10.2 in MM9 supplemented with 2 mM 3CBa resulted in the decrease of 3CBa at a rate of 25 nmol ml-’ h- ’ and an increase in chloride concentration of 16.5 nmol ml - 1 h - *. The effects of presence of other carbon sources in MM9 medium on 3CBa degradation by P. putida 10.2 were studied. A decrease of 3CBa utilization by P. ptrtida 10.2 was observed when media were supplemented with other easily degraded carbon sources such as glucose and pyruvate (Table 2). At low concentrations of glucose (O.l%),
putida treated
0.8
1.0
(mg ml ‘)
2. The effect of resting cell suspensions of Pseudomonas 10.2 on the rate of 3-chlorobenzoate degradation. Noncell suspension (a) and heat treated cell suspension
(rn).
3CBa degradation was reduced from 17.6 to 4.5% and at higher levels (1.0%) inhibition was complete. Similarly, lactose supplementation at 0.1% caused a slight reduction in 3CBa utilization (17.6% to 11.5%) and a marked inhibition at 1%, despite the fact that the biochemical tests showed that this organism could not assimilate lactose. This pattern of inhibition was also found with succinate supplementation. Degradation of 3CBa by Resting Cell Suspensions The rate of 3CBa degradation was studied using cell suspensions at the concentrations shown in Figure 2. The rate of 3CBa degradation was approximately proportional to the cell mass. The rate of 3CBa degradation was defined as the amount of 3CBa lost from the medium, and the values were calculated when the loss was directly proportional to the time of incubation. Presence of 3CC in Spent Culture Broth Containing 3CBa To obtain information concerning the biodegradation pathway of 3CBa by P. ptrtida 10.2, culture media were extracted with ethyl acetate and the extracts analysed by HPLC. Peaks with retention times equivalent to authentic 3CC were observed. A sample comparison of HPLC chromatograms of authentic 3CC and a metabolic extract is illustrated in Figure 3. Presence of CAT in Resting Cell Suspension of P. putida 10.2 which contained 3CBa Attempts to identify CAT as a metabolite in the spent 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.
Table 1. Microscopic monas pufida 10.2.
and biochemical
Mlcroscoplc
characteristics
Pseudomonas pufida 10.2
Pseudomonas pufida as described in Bergey’s manual
rod negative not found
rod negative not found
characteristics
Morphology Gram’s strain Spore formation Biochemical
characteristics
+ + + -
Catalase Catechol ortho-cleavage Citrate production Denitrification Gelatin dissemination Growth at 41°C H,S production lndole Methyl red Motility Nitrate reduction Oxidase O/F glucose* O/F maltose O/F lactose O/F arabinose O/F sucrose O/F galactose O/F mannose Phenylalanine deaminase Starch hydrolysis TSlt Urease test VP test$ Yellow orange cellular pigment
carbon
MM9 MM9 MM9 MM9 MM9 MM9 MM9 MM9 MM9 NG -
610
3CBa 3CBA 3CBA 3CBA 3CBA 3CBa 3CBa 3CBa 3CBa
no growth;
+ + + + + + + +
1.0% 0.1% 1.0% 0.1% 1.0% 0.1% 1.0% 0.1% ND -
K/N
sources
composition
+ + + + + + + + +
+
-
Table
of varlous
+
K/N
utilization:
2. Effects
Presence of KC Degrading Enzyme and Catechol 1,~ dioxygenase in Crude Exfracfs of P. putida 10.2 The identification of 3CC and CAT in culture broths and resting cell suspensions suggested that P. putida 10.2 could degrade 3CBa via these compounds. If this is were true, 3CC-degrading enzyme and catechol 1,2-dioxygenase should be found in crude extracts of P. pufida 10.2. When 3CC was incubated with such extracts from P. pufida 10.2 grown in MM9 medium, the formation of CAT was observed, and this activity was dependent on the presence of NADPH (Table 4). Assays of catechol l,&dioxygenase in crude enzyme extracts of P. putida 10.2 were carried out at intervals of 0, 0.5, I, 2 and 3 h, by determining the concentration of MUC by HPLC, and this activity was destroyed by boiling (Table 4). Furthermore, no enzyme activity could be observed when the appropriate substrates were not added to the cell free extracts (data not shown). Examination of the crude enzyme reaction mixture containing 3CC as substrate by HPLC showed two metabolite peaks which corresponded to authentic CAT and MUC. The presence of CAT in 3CC reactions were further confirmed by LC/MS. As shown in Figure 4, the metabolite mass spectrum corresponded to the mass spectrum of authentic catechol. Peaks corresponding to MUC could not be separated from buffer peaks in various mobile phase systems used in
+ f +
+ + +/-l-/-/-/-/-/-
‘-xidative and fermentative t-triple sugar iron agar; $-Voges-Proskauer.
Medium
nature of the compound. Therefore, the appearance of CAT in resting cell suspensions of P. pufidu 10.2 was analysed by HPLC after 44 h incubation (Table 3). Metabolite and authentic CAT peaks were superimposed in all solvent systems used in this study. The result was confirmed by observation of higher peak areas when samples were coinjected with authentic CAT (data not shown). The concentrations of 3CC and CAT produced in the supematant at 44 h were approximately 0.034 and 0.007 mM, respectively; as expected these were very low levels.
of Pseudo-
glucose glucose pyruvate pyruvate lactose lactose succinate succinate not determined;
on growth
and degradatlon
of S-chlorobenzoate
by Pseudomonas
Growth rate hr-l
Growth yield mg ml-’
Initial concentration of 3CBa (mM)
Final concentration of 3CBa (mr4)
3CBa used (mM)
0.13 0.19 0.16 0.16 0.15 0.13 0.14 NG 0.18
0.088 0.275 0.275 0.154 0.149 0.105 0.110 NG 0.231
1.0 1.0 1.0 1.0 1.0 1.0 1.0 ND 1.0
0.824 1.0 0.955 1.0 0.987 0.978 0.885 ND 0.897
0.176 0.00 0.045 0.00 0.013 0.022 0.115 ND 0.103
l
-
at 24 h of incubation.
World Journal of Microbiology 6 Biofechnology, Vol 12, 1996
pufida
10.2. Percentage 3CBa utilization’ WJ) 17.6 0.0 4.5 0.0 1.3 2.2 11.5 ND 10.3
Biodegradation of 3-chlorobenzoafe
6.51
optical density of authentic MUC. It was found that the metabolite peaks showed the same absorption spectra as authentic MUC, with maximum optical density at 265 nm.
I
A. I\ I I
Discussion
6.50
B.
6.50 I
I
C. \
I
Retention time (min) Figure 3. HPLC chromatograms and retention times showing the possible presence of 3-chlorocatechol in the culture broth of Pseudomonas putida 10.2 grown in 3CBa-containing medium. (A) authentic 3CC; (B) co-injection of extract and authentic 3CC; (C) extract only.
HPLC. For further identification of MUC, peaks which showed retention times corresponding to MUC were scanned for maximum optical density at wavelengths between 230 and 290 nm and compared to the maximum
Table Solvent
3. The presence
of catechol
in resting
cell suspension
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 concentrations of 3CBa are required if it is to serve as a carbon source for this organism. Dom et al. (1974) also reported 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, because cells can use these compounds as alternative carbon sources. Goulding et al. (1988) reported that 12 microorganisms 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 slightly decreased rate when compared to the rate in media without glucose. Since, P. pfidu 10.2 cannot utilize lactose, lactose should not interfere with its degradation of 3CBa. However, for some unknown reason, high concentrations of lactose (1%) did show inhibitory effects. The presence of 1% of succinate was found to be toxic to the cells, but low concentrations (0.1%) had no effect on cell growth and 3CBa degradation. The formation of CAT in the reaction containing 3CC and crude extract was shown to require NADPH. This supports the results obtained by Mohn & Tiedje (1992) that reductive dehalogenation reactions require an electron donor. This constitutes evidence of a reductive dechlorina-
of Pseudomonas
putida
system
mM rnM mM rnM rnM mM mM mM
phosphoric phosphoric phosphoric acetic acid acetic acid acetic acid acetic acid acetic acid
with Jchlorobenzoate.
Retention Authentic catechol
10 10 10 40 40 40 40 40
10.2 incubated
acid containing acid containing acid containing containing 25% containing 20% containing 30% containing 35% containing 50%
25% acetonitrile and 10% methanol 20% acetonitrile 30% acetonitrile methanol and 10% acetonitrile methanol methanol methanol methanol
5.098 7.408 5.057 5.737 9.600 6.760 6.04 4.260
time
Supernatant of resting cell suspension 5.083 7.365 5.043 5.683 9.817 6.683 5.94 4.200
(min)
of
Supernatant of resting cell suspenslon + authentic catechol 5.087 7.312 5.047 5.732 9.625 6.758 6.01 4.268
WorldJoumal of Microbiology 6 Biotechnology, Vol 12. 1996
611
W. Chobchuenchom et al. Reductive KC dechlorination activity should be important for the biodegradation of many chlorinated aromatic compounds; previous reports have suggested that 3CC is a
tion biodegradative pathway in P. ptlfidu 10.2, an aerobic bacterium. Reductive dehalogenation reactions of XC by aerobic microorganisms have not been previously reported.
6!
i0, ,
56 5Q +4%&LL
' Dale 65
-z-
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 Table 4. Specific in crude extracts Enzyme
activities of 3chlorocatechol-degrading of Pseudomonas pufida 10.2.
enzyme
Specific
preparation SCC-degrading (nmol hr-’
Non-treated crude with all required without NADPH Boiled with l
enzyme cofactors
treated crude enzyme’ all required cofactors
-Crude extract was placed ND - Not determined.
in a boiling
water
common intermediate in the biodegradation of such compounds (Sondossi et al. 1992) and that the problem in degrading halosubstituted aromatic compounds is the inefficiency of ring-cleavage enzymes in halocatechol transformation (Knackmuss 1983). To further clarify whether CAT was an intermediate produced in reactions containing 3CC as substrate, supernatants were directly filtered and analysed using LC/MS. It was found that the mass spectrum of the metabolite peak corresponded to that of authentic catechol. The result indicated that catechol in the culture broth could have resulted from the activity of a 3CC-degrading enzyme. In addition to the formation of CAT in reactions containing XC and crude extract, the accumulation of MUC was also observed. The presence of MUC could have resulted from the activity of catechol l&dioxygenase (Table 4).
Acknowledgement This work was supported by National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency and UNDP. We are grateful for the help of Dr Timothy W. Flegel in reading this manuscript.
References Ahmed, M. & Focht, D.D. 1973 Degradation of polychlorinated biphenyl by two species of Achromobucter. Canadian ]oaurnal of Microbiology 19, 47-52. Apajalahti, J.H.A. & Salkinoja-Salonen, M.S. 1987 Complete dechlorination of tetrachlorohydroquinone by cell extracts of pentachlorophenol-induced Rhodococcus chlorophenolicus. ]ownal of Bacteriology 169, 5125-5130. Chatterjee, D.K. & Chakrabarty, A.M. 1982 Genetic rearrangements in plasmids specifying total degradation of chlorinated benzoic acids. Mo\ecu~ur and General Genetics 188, 279-285. Chaudhry, G.R. & Chapalamadugu, S. 1991 Biodegradation of halogenated organic compounds. Microbiologic& Reviews 55, 59-79. Don, R.H. Weightman, A.J., Knackmuss, H.-J., Timmis, K.N. 1985
and catechol
activity
enzyme mg-‘)
1,2-dioxygenase
of enzyme Catecholl (nmol
,bdioxygenase mln-I mg-‘)
1.9 0
0.7 ND
0
0
bath for 5 min before
of 3-chlorobenzoafe
enzyme
assay;
Transposon mutagenesis and cloning analysis of the pathways for degradation of 2,4-dichlorophenoxyacetic acid and 3-chlorobenzoate in Alcufigenes etttrophus JMP134(pJP4). Journal of Bacteriology 161, 85-90. Dom, E., Hellwig, M., Reineke, W., Knackmuss, H.-J. 1974 Isolation and characterization of a &chlorobenzoate degrading Pseudomonad. Archives of Microbiology 99, 61-70. Dom, E. & Knackmuss H.-J. 1978 Chemical structure and biodegradability of halogenated aromatic compounds, substituent effects on 1,2-dioxygenation of catechol. Biochemical ]oumul 174,85-95. Furukawa, K., Tomizuka, N. & Kamibayashi, A. 1979 Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Applied and Environmental Microbiology 38, 301-310. Ghosal, D., You, I.-S., Chatterjee, D.K. & Chakrabarty, A.M. 1985 Microbial degradation of halogenated compounds. Science 228, 135-142. 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 Hydroxylation and dechlorination of tetrachlorohydroquinone by Rhodococcw sp. strain CP-2 cell extracts. Applied and Environmental Microbiology 55, 51&519. Holt, J.G. 1994 Bergey’s Mum& of Determinative Bacteriology. 9th edition. Baltimore: Williams & Wilkins. Horvath, R.S. 1971 Cometabolism of the herbicide 2,3,6-trichlorobenzoate. ]ournul of Agricultwul and Food Chemisfry 19, 291293. Knackmuss, H.J. 1983 Xenobiotic degradation in industrial sewage: haloaromatics as target substrates. Biochemical Society Symposium 48,173-190. Masse, R., Messier, F., Peloquin, L., Ayotle, C. & Sylvestre, M. 1984 Microbial biodegradation of 4-chlorobiphenyl, a model compound of chlorinated biphenyls. Applied and Environmental Microbiology. 47, 947-95 1. Mohn, W.W. & Tiedje, J.M. 1992 Microbial reductive dehalogenation. Microbiological Reviews 56, 482-507. Pfister, R.M. 1973 Interactions of halogenated pesticides and microorganisms: a review. In CRC Handbook of Microbiology, ed La&n, A.I. & Lechevalier, H. pp. l-33. Cleveland: CRC Press. Sondossi, M., Sylvestre, M. & Ahmad, D. 1992 Effects of chlorobenzoate transformation on the Pseudomonas fesfosferoni biphenyl and chlorobiphenyl degradation pathway. Applied and Environmental Microbiology 58, 458-495.
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Steiert, J.G. & Crawford, R.L. 1986 Catabolism of pentachlorophenol by a Flavobacterium sp. Biochemical and Biophysical Research Communications 141, 825-830. Sylvestre, M. & Fauteux, J. 1982 A new facultative anaerobe capable of growth on chlorobiphenyls. ]ournal of General and Applied Microbiology 28, 61-72. Van den Tweel, W.J.H., ter Burk, N., Kok, J.B. & DeBont, J.M.M. 1987 Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro, 4-bromo, and a-iodobenzoate by Alcaligenes denitrijcans
NTB-I. Applied and Environmental Microbiology 53, 810815. Weisshaar, M.-P., Franklin, F.C.H. & Reineke, W. 1987 Molecular doning and expression of the 3-chlorobenzoate-degrading genes from Pseudomonas sp. strain BI3. Journal of Bacteriology
169,394-402. (Received in revised form 28 March 1996)
1996; accepted 2 April
