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FEMS Microbiology Ecology 37 (2001) 251^258
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Microbial hydrolysis of methyl aromatic esters by
Burkholderia cepacia isolated from soil
Geraldine Philippe, Danielle Vega *, Jean Bastide
Centre de Phytopharmacie, UMR CNRS 5054, Universitë de Perpignan, 52 Avenue de Villeneuve, 66860 Perpignan Cedex, France
Received 26 April 2001; received in revised form 27 July 2001; accepted 30 July 2001
First published online 24 September 2001
Abstract
A bacterial strain that could utilise methyl benzoate as the sole source of carbon and energy was isolated from soil. This strain was
identified as a Burkholderia cepacia. In minimum mineral medium, the strain hydrolysed the methyl ester to form benzoic acid, which is then
also degraded. This strain was also able to hydrolyse the ester bond of substituted chlorobenzoic esters and methyl thiophene-2-carboxylate,
but did not metabolise their reaction products. A crude enzymatic extract obtained from this strain was relatively stable, and hydrolysed
more compounds than the microorganism itself: for instance, methyl 3-(N,N-diethylsulfamoyl)thiophene-2-carboxylate was hydrolysed by
the enzyme but not by the microorganism. The bacterial strain was unable to hydrolyse the ester bond of two sulfonylurea herbicides,
thifensulfuron methyl and metsulfuron methyl, in solution or after reintroduction in sterile soil. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Soil degradation; Methyl ester; Methyl benzoate degradation; Esterase; Burkholderia cepacia isolation
1. Introduction
For pesticides carrying a methyl ester group, degradation pathways in soil can involve hydrolysis of the methyl
ester bond, and lead to formation of the corresponding
acids [1^4].
Microorganisms can carry out this degradation, but the
number of strains so far isolated is very small. The microorganisms that have been isolated can hydrolyse aliphatic
esters such as fenoxaprop-ethyl [5^7], diclofop-methyl [8]
and £uazifop-butyl [9,10], but to our knowledge, no microorganism able to hydrolyse the methyl ester group of
aromatic pesticides has yet been reported. Grant [11] described the hydrolysis of ethyl and phenyl benzoate by a
Corynebacterium strain. From the environment, several
microorganisms that have the ability to metabolise dimethylphthalate have been isolated. In some cases the initial step
in the degradation of phthalate esters was a de-ester-
* Corresponding author. Tel. : +33 4 68 66 22 56;
Fax: +33 4 68 66 22 23.
E-mail address : [email protected] (D. Vega).
i¢cation reaction [12,13]. Some of the microorganisms in
soil degrade the methyl ester functions of sulfonylurea
herbicides, such as thifensulfuron methyl [14]; however,
previous attempts to isolate microorganisms from soil
able to perform this degradation were unsuccessful
[14,15]. In this paper, we describe the isolation of a microorganism degrading methyl benzoate, and its ability to
degrade di¡erent methyl aromatic esters.
2. Materials and methods
2.1. Chemicals
Methyl benzoate (1), benzoic acid (1a), methyl 4-chlorobenzoate (2), 4-chlorobenzoic acid (2a), methyl 2,5-dichlorobenzoate (3), 2,5-dichlorobenzoic acid (3a) and 2thiophenecarboxylic acid (4a), were purchased from Aldrich Chemical. Thifensulfuron methyl (methyl 3-[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate) (7) was a gift of Procida
(Marseille, France). Metsulfuron methyl (methyl 2-(4-methoxy-6-methyl-1,3,5-triazin-2-ylcarbamoylsulfamoyl)benzoate) (9) was a gift of Dupont de Nemours. Synthesis of
methyl thiophene-2-carboxylate (4), methyl 3-(N,N-dieth-
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 6 0 - X
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G. Philippe et al. / FEMS Microbiology Ecology 37 (2001) 251^258
ylsulfamoyl)thiophene-2-carboxylate (5), 3-(N,N-diethylsulfamoyl)thiophene-2-carboxylic acid (5a), methyl 3-sulfamoylthiophene-2-carboxylate (6), 3-sulfamoylthiophene2-carboxylic acid (6a), thifensulfuron (3-[[(4-methoxy-6-
methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]2-thiophenecarboxylic acid) (7a), and methyl 2-(N-N-diethylsulfamoyl) benzoate (8) and methyl 2-(N,N-diethylsulfamoyl) benzoate (8a) were performed according to
Table 1
Hydrolysis of compounds to their corresponding acids by B. cepacia and by crude cell extract
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the methods of Bastide et al. [16]. All chemicals were of
analytical grade. Structures of all the compounds and corresponding acids are presented in Table 1.
2.2. Analytical methods
Concentrations of all the products were determined by
HPLC. HPLC analyses were performed on a system consisting of a Beckman pump and a Shimadzu SPD 2A UV
detector. The operating parameters were as follows : column Kromasil C8 Hypersil KR 235^5 Wm; mobile phase,
acetonitrile/water/acetic acid (50:50:0.5) (v/v/v) delivered
at a £ow rate of 1 ml min31 . The detecting wavelength
was 235 nm for 1, 1a, 2, 2a, 3, 3a, 8, 8a, 9 and 9a and 254
nm for 4, 4a, 5, 5a, 6, 6a, 7 and 7a. All compounds studied
were quanti¢ed using external standards.
2.3. Methyl benzoate degradation in microbially active and
sterile soils
A soil sample was collected from a depth of 0^20 cm at
Bolquere, in the south of France, air dried, sieved (2 mm)
and stored in plastic bags at 5³C. Soil properties were:
sand 41.3%, silt 39.7%, clay 19.0%, organic carbon
2.95% and pH 6.2. Soil was sterilised by autoclaving at
121³C for 20 min three times at 24 h intervals. Flasks
containing soil equivalent to 20 g oven-dried weight sterile
or non-sterile soil were treated with a ¢ltered (0.2 Wm)
aqueous solution of methyl benzoate (870 mg l31 ) to obtain a ¢nal concentration of 20 mg kg31 of dry soil. Water
was added to give a moisture content of 25% (w/w of dry
weight of soil). Flasks containing the soil^herbicide mixture were capped and kept in an incubator at 30 þ 1³C.
Duplicate samples were periodically removed and frozen
(320³C) until extraction by shaking for 1 h with 25 ml of
methanol/water/acetic acid (45:5:0.5, v/v/v). After decanting and centrifuging for 5 min, the supernatant was HPLC
analysed as described before.
2.4. Culture conditions
Liquid cultures were carried out in a mineral medium
(MM) containing in 1 l of distilled water: 3.5 g K2 HPO4 ,
1.5 g KH2 PO4 , 0.27 g MgSO4 , 1 g NH4 Cl, 0.03 g
Fe2 (SO4 )3 W9H2 O, and 0.03 g CaCl2 . The mineral medium
was adjusted to pH 6.8 and sterilised by autoclaving for 20
min at 121³C before storage. Iron sulfate and magnesium
sulfate were ¢lter-sterilised and added to the medium after
autoclaving to prevent the formation of precipitates.
Aqueous solution (0.2 Wm sterilised) of each tested product was added as carbon source in the liquid cultures. A
culture started with a single colony from the solid nutrient
medium (Bacto nutrient agar, Difco) was used as inoculum. All containers were capped to avoid evaporation.
Cultures were incubated in a rotary shaker at 200 rpm
and at 30³C. Growth rate was monitored by optical den-
253
sity measurements at 550 nm with a spectrophotometer
Beckman (DU 520).
2.5. Isolation, identi¢cation and characterisation of
degrading strains
When total methyl benzoate degradation occurred in a
sample of soil treated with methyl benzoate (20 mg kg31
of dry soil), a subsample (1.5 g) was added to MM (50 ml)
supplemented with methyl benzoate (1.2 mmoles l31 ) in a
100-ml Erlenmeyer £ask, and incubated at 30³C in a rotary shaker at 200 rpm. Samples of the suspension (0.5 ml)
were periodically diluted with acetonitrile (0.5 ml) and
after centrifugation to remove solid matter, analysed by
HPLC.
When the total amount of methyl benzoate was degraded, 1 ml of this soil suspension was subcultured in
50 ml of fresh MM supplemented with methyl benzoate.
This operation was performed twice. Immediately after
total methyl benzoate degradation, a 10-fold dilution series was prepared and 0.1 ml of each dilution was spread
on plates of MM plus agar (15 g l31 ) supplemented with
methyl benzoate and incubated at 30³C for 1 week. Single
colonies growing on these dilution plates were assayed for
their methyl benzoate degradation abilities.
The isolate was maintained by monthly transfers onto
plates containing MM supplemented with methyl benzoate
or onto nutrient agar plates, and stored at 4³C. For longterm storage, the strain was stored in nutrient broth (Difco) containing 15% glycerol (v/v) at 380³C.
Colony morphology was determined on nutrient agar
(Difco). Gram reaction and cell morphology were determined by observing stained cells (Gram stain kit, BioMerieux) with a light microscope. Oxidase and catalase activities were determined with BioMerieux reagents. The
isolate was identi¢ed with API 20NE kit test (BioMerieux).
2.6. Degradation of the methyl esters by an isolated pure
culture of bacteria
The degradation of the di¡erent products was studied in
liquid cultures (25 ml) inoculated with 2 ml of a starter
culture containing 107 bacteria ml31 of the strain into 125
ml capped £asks. Samples of culture (0.5 ml) were removed at regular intervals, mixed with acetonitrile (0.5
ml), and centrifuged brie£y to remove solid matter before
injection into the HPLC. The concentration of products
and their metabolites was monitored by HPLC. A control
experiment with non-inoculated mineral medium was also
carried out and samples were periodically analysed as described above.
2.7. Crude cells lysates
From a large culture (2 l) the newly isolated strain
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(Burkholderia cepacia) was incubated with methyl benzoate, cells were harvested by centrifugation (10 000Ug,
10 min, 4³C), washed with 100 ml of 1/15 M phosphate
bu¡er, pH 7.0 (58.7 ml Na2 HPO4 1/15 M and 41.3 ml
KH2 PO4 1/15 M) and suspended in this bu¡er at 4³C
(6 ml g31 of pellet). The cell suspension was disrupted
with a 375 W ultrasonic oscillator (Heat System-Ultrasonic) in ice for a total of 6 min with two intervals of
3 min each. The unbroken cells and cellular debris were
removed by centrifugation at 36 000Ug for 20 min at 4³C.
The supernatant £uid was precipitated with ammonium
sulfate 60% (w/v). The precipitate was collected by centrifugation at 9000Ug for 15 min, dissolved in 15 ml 0.1 M
Tris^HCl bu¡er, pH 7.2, then ¢ltered through a 0.22-Wm
membrane (Millipore). This extract was stored at 320³C
until testing.
2.10. Enzyme addition to sterile soil
2.8. Enzyme assay and protein determination
In non-sterile soil, methyl benzoate was rapidly transformed, and total hydrolysis was observed in less than 1 h
(Fig. 1). This hydrolysis led to benzoic acid (1a) which was
also quickly degraded. In sterilised soil, the methyl benzoate concentration decreased slowly, with 75% remaining
after 100 h of incubation, but no benzoic acid was detected (see Fig. 4).
The esterase activity was assayed by measuring the formation of the corresponding acids from the di¡erent tested
aromatic esters. Crude extracts (50 Wl) were incubated with
the di¡erent compounds in 0.1 M Tris^HCl bu¡er, pH 7.2
in a 0.5 ml ¢nal volume. The reactions were performed at
30³C. At time intervals aliquot (50 Wl) was taken and the
reaction was stopped by the addition of 50 Wl acetonitrile,
after centrifuging, HPLC analysis was carried out. Controls samples containing boiled extract were treated and
analysed in an identical way.
Protein concentrations were determined using the Bradford assay [17] with crystalline serum albumin (Sigma) as
the protein standard. In each case, 1 U of enzyme activity
was de¢ned as that amount of enzyme producing 1 Wmole
of product in 1 min at 30³C. For the temperature stability
study, crude extract was incubated at 30³C and the esterase activity was determined after 24, 72 and 168 h incubation as described above.
Sterile soil (1 g) was mixed with crude cell extract (100
Wl). Phosphate bu¡er (1/15 M) pH 7.0 was added to give a
moisture content of 25% (w/w of dry weight of soil). After
addition of a sterile solution (50 Wl) of methyl benzoate
(870 mg l31 ) or thifensulfuron methyl (500 mg l31 ) the
tubes were capped and incubated at 30 þ 1³C. Samples
were extracted (by 1 ml of the extracting mixture) and
analysed after 6, 24, 72, and 120 h incubation as previously described.
3. Results
3.1. Methyl benzoate degradation in soils
3.2. Isolation of bacterial strains from soils
After the enrichment procedure with methyl benzoate,
we obtained 12 bacterial colonies able to grow on MM
plates supplemented with methyl benzoate, as the sole
source of carbon and energy. When tested in liquid MM
supplemented with methyl benzoate, one of these strains
grew well and was able to degrade methyl benzoate totally
in less than 1 week. The isolated strain was a Gram-negative mobile rod and displayed the characteristics of the
genus Pseudomonas. On nutrient agar or on MM agar
supplemented with methyl benzoate, it formed smooth,
2.9. B. cepacia addition to sterile soil
The bacterial strain was grown on 50 ml nutrient broth
(Difco) for 48 h; cells were harvested by centrifugation
(10 min, 6000Ug, 10³C). The cells were washed three
times in 1/15 M phosphate bu¡er pH 7.0 and resuspended
in 10 ml mineral medium ; the ¢nal optical density was
5.1 at 550 nm. Cell suspension (2.5 ml) was added to
a £ask containing sterile soil equivalent to 20 g of
oven-dried soil, and sterile water was added to give a
moisture content of 25% (w/w of dry weight of soil). After
mixing, the £ask was incubated for 4 days at 30³C. An
aqueous sterile solution of methyl benzoate was added to
obtain a ¢nal concentration of 20 mg kg31 of dry soil. The
£ask containing the soil^herbicide mixture was kept in an
incubator at 30 þ 1³C. Aliquots (2.5 g) were removed at
various times and extracted and analysed as previously
described.
Fig. 1. Methyl benzoate (1,
b, benzoic acid (1a).
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E)
degradation in non-sterile soil at 30³C.
G. Philippe et al. / FEMS Microbiology Ecology 37 (2001) 251^258
255
light beige, convex circular colonies, opaque and with a
regular border. The strain was catalase negative, oxidase
positive, strictly aerobic and reduced nitrate to nitrite. Results with the API 20 NE kit test indicated 98.4% similarity with B. (Pseudomonas) cepacia.
3.3. Degradation of methyl benzoate by the B. cepacia
strain
In a liquid culture inoculated with the B. cepacia strain,
methyl benzoate was rapidly degraded. Total degradation
took 30 h (Fig. 2A). Only small amounts of benzoic acid
were detected during this degradation, suggesting that it
was probably further metabolised. Fig. 2B con¢rms that
B. cepacia rapidly degrades benzoic acid in a liquid MM
supplemented with benzoic acid alone. An increase in
OD550 , suggesting bacterial growth, was observed during
these experiments. In sterile medium alone, methyl benzoate concentration decreased slightly over time, but without the formation of benzoic acid. B. cepacia retained its
ability to degrade methyl benzoate despite sub-culturing
Fig. 2. Degradation of methyl benzoate (1, E) (A) and benzoic acid (1a,
b) (B) by B. cepacia cultivated in MM pH 6.8 at 30³C. Growth (a) of
B. cepacia was measured at 550 nm. Insert shows the formation of benzoic acid (1a) during methyl benzoate degradation.
Fig. 3. Degradation of methyl esters by B. cepacia cultivated in MM
and in non-inoculated MM (- - -); pH 6.8 at 30³C. A: Methyl 4-chlorobenzoate (2) (102 Wmoles l31 ); B: methyl 2,5-dichlorobenzoate (3) (38
Wmoles l31 ); C: methyl thiophene-2-carboxylate (4) (70 Wmoles l31 ). The
corresponding acids are: 2a, 3a and 4a. Refer to Table 1 for structures
of compounds.
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Table 2
Activities of the esterase in cell extracts of B. cepacia grown on methyl esters of aromatic acids
Methyl esters
Concentration (mM)
Activity (U/mg of protein) in the cell extract
Methyl benzoate (1)
Methyl 4-chlorobenzoate (2)
Methyl 2,5-dichlorobenzoate (3)
Methyl thiophene-2-carboxylate (4)
Methyl 3-(N,N-diethylsulfamoyl)thiophene-2-carboxylate (5)
Methyl 3-sulfamoylthiophene-2-carboxylate (6)
Thifensulfuron methyl (7)
Methyl 2-(N-N-diethylsulfamoyl) benzoate (8)
0.72
0.44
0.16
0.73
0.14
0.40
0.24
0.15
0.12
0.20
0.03
0.05
0.005
0
0
0
four times in MM supplemented with glucose (1 g l31 ) but
with no methyl benzoate present (result not shown).
3.3.1. Degradation of other methyl esters of aromatic acids
When methyl esters 2, 3 and 4 were used as the sole
source of carbon, they were rapidly degraded, with halflives respectively of 5, 25 and 6 h (Fig. 3), and with the
appearance of large quantities of the corresponding acid.
In sterile medium alone, the concentration of the methyl
esters decreased slightly, but the corresponding acid was
not formed. Under similar conditions, B. cepacia was not
able to degrade methyl esters 5, 6, 7, 8 or 9 (Table 1).
3.4. A cell-free extract from B. cepacia
A crude cell extract was obtained by sonication and
ammonium sulfate precipitation (60%). The extract, total
volume 15 ml, contained 0.48 mg l31 protein, with a speci¢c activity of 0.12 units mg31 with methyl benzoate as
substrate. The extract was able to break down the ester
bond in methyl esters 1, 2, 3, 4 and 5, with the concomitant production of the corresponding acids, but methyl
esters 6, 7, and 8 were not degraded (Table 1). Quantitative analysis by HPLC revealed that the corresponding
Fig. 4. Methyl benzoate (1, E) degradation in sterile soil inoculated with
a suspension of B. cepacia and in non-inoculated sterile soil (- - -) at
30³C. b, benzoic acid (1a).
acids were not metabolised further, during the experiment
time. Table 2 shows the activities of methyl esterase in
crude extract on di¡erent substrates. No hydrolysis was
observed when the crude extract was boiled for 5 min
before incubation. The enzymatic activity in the extract
appeared stable : after 168 h of incubation at 30³C, more
than 25% of the original activity remained.
3.5. Degradation of methyl benzoate in sterile soil,
following addition of a suspension of B. cepacia
When a suspension of B. cepacia was added to sterile
soil, methyl benzoate was rapidly degraded, with a half-life
of about 30 h (Fig. 4). Simultaneously, benzoic acid appeared, with a maximum concentration at 24 h, reducing
to zero by 98 h.
3.6. Degradation of methyl benzoate in soil, following
addition of crude cell extract
When 100 Wl of crude cell extract were added to sterile
soil, methyl benzoate was degraded to benzoic acid, with a
half-life of about 40 h (Fig. 5). However, under the same
conditions, the extract has no activity on the herbicide
thifensulfuron methyl.
Fig. 5. Methyl benzoate (1, E) degradation in sterile soil (1 g) mixed
with 100 Wl of crude cell extract. b, benzoic acid (1a).
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4. Discussion
A strain of B. cepacia, isolated from soil, is able to
hydrolyse the ester bond of methylbenzoic ester, and to
degrade the resulting benzoic acid. A comparison of B.
cepacia growth on either methyl benzoate or benzoic
acid (Fig. 2) shows that benzoic acid from methyl benzoate hydrolysis can be used as carbon source. B. cepacia
is also able to hydrolyse the ester bond of compounds 2, 3,
4, but it could not degrade their corresponding acids, respectively 4-chlorobenzoic acid (2a), 2,5-dichlorobenzoic
(3a) acid or thiophene carboxylic acid (4a). In these conditions the growth rate is very low (data not shown),
although the degradation rates of compounds 2, 3, and 4
are very close to that observed for methyl benzoate.
Numerous bacterial isolates have been reported that
are capable to degrade both benzoic acid and chlorobenzoic acids [18]. B. cepacia was not able to hydrolyse
the substituted thiophenes 5, 6, 7, the compound 8 and
the herbicide metsulfuron methyl 9 (Table 1). This di¡erence of reactivity may be related to the selectivity
of microorganism activity. Actually, microorganisms are
generally very speci¢c in their ability to hydrolyse ester
bonds, and this speci¢city is su¤cient for the synthesis
of optically pure substances. For instance, an esterase
was used in the resolution of ethyl chrysanthemate
derivatives during the synthesis of pyrethrin insecticides
[19].
The crude enzymatic extract obtained from B. cepacia
keeps its ability to hydrolyse the ester bond in compounds
1, 2, 3, 4 and 5, but does not degrade the resulting acids.
Interestingly, the enzyme's spectrum of activity was broader than that of the microorganism itself (Table 1). Compound 5 is hydrolysed slowly by the enzyme but not by the
microorganism ; however, the other compounds, 6, 7 and
8, are not degraded by the microorganism and not hydrolysed by crude enzymatic extract. This di¡erence between the whole microorganism and the enzymatic extract
could be related to problems of penetration of the product into B. cepacia. The enzymatic extract is relatively
stable: it retains more than 50% of its activity after 72 h
at 30³C.
The addition of degrading microorganisms to soil can
increase pesticide transformation rates [20,21]. B. cepacia
introduced into sterile soil quickly degrades methyl benzoate to benzoic acid, which itself is also degraded. Under
similar conditions, the crude enzymatic extract keeps this
activity when added to sterile soil. This enzymatic activity
persists for a long time, the degradation rate being still
signi¢cant after 72 h. However, this esterase activity of
soil does not hydrolyse the herbicide thifensulfuron methyl
to thifensulfuron, the ¢rst step of this compound degradation in soil [14].
The results show that hydrolysis of methyl aromatic
ester derivatives is possible by a strain of B. cepacia.
Although only the ester bond is involved in the reaction,
257
the strain is not able to hydrolyse all molecules carrying
this functional group. This selectivity does not seem to
be related to the capacity of the strain to degrade the
aromatic ring, because with chlorinated derivatives, a
fast hydrolysis of the ester bond without degradation of
the ring is observed. These di¡erences may be related to
questions of reactivity, as previously shown for chemical
reactions [16].
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