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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
BIODEGRADATION OF THE ORGANOPHOSPHATE PESTICIDE TETRACHLORVINPHOS BY
BACTERIA ISOLATED FROM AGRICULTURAL SOILS IN MÉXICO
Ma. Laura ORTIZ-HERNÁNDEZ and Enrique SÁNCHEZ-SALINAS
Laboratorio de Investigaciones Ambientales, Centro de Investigación en Biotecnología, Universidad Autónoma
del Estado de Morelos. Av. Universidad 1001, Col. Chamilpa; C.P. 62210, Cuernavaca, Mor. México.Tel. +52
777 329 7057, Fax. +52 777 329 7030; E-mail ortizhl@uaem.mx
(Recibido agosto 2008, aceptado junio 2009)
Key words: bacteria, biodegradation, pesticides, tetrachlorvinphos
ABSTRACT
A bacterial consortium which degrades tetrachlorvinphos (phosphoric acid, 2-chloro-
1-(2,4,5-trichlorophenyl) ethenyl dimethyl ester) was isolated from agricultural soil.
This consortium was composed of six pure strains which were characterized based on
their morphological and biochemical characteristics. The strains were presumptively
identi±ed as
Stenotrophomonas malthophilia,
Proteus vulgaris,
Vibrio metschinkouii,
Serratia fcaria,
Serratia
spp. and
Yersinia enterocolitica.
The consortium and the six
bacteria were assessed in order to discover their ability to degrade tetrachlorvinphos
(TCV) in mineral medium and in rich medium. Growth curve experiments showed
that the bacterial consortium was able to grow in mineral medium containing TCV
as the only carbon source. However, only one pure strain was able to remove TCV in
mineral medium, while all of them removed it in rich medium. Hydrolysis products
were detected and identi±ed by gas chromatography-mass spectrometry. These data
indicate that the isolated strains can be used for waste biodegradation or bioremediation
of TCV-contaminated soil or water.
Palabras clave: plaguicidas, agricultura, residuos, biodegradación
RESUMEN
Se aisló un consorcio bacteriano de suelos agrícolas, capaz de degradar tetraclor-
vinfos (
ácido FosFórico, 2-cloro-1-(2,4,5-tricloroFenil) etenil dimetil ester
). Este
consorcio estuvo formado por seis cepas puras que fueron caracterizadas con base en
sus características bioquímicas y morfológicas. Las cepas fueron presumiblemente
identi±cadas como
Stenotrophomonas malthophilia,
Proteus vulgaris,
Vibrio mets-
chinkouii,
Serratia fcaria,
Serratia
spp. and
Yersinia enterocolitica.
El consorcio y
las seis cepas puras fueron cultivados en medio mineral y en medio rico, para evaluar
su capacidad para degradar TCV. Los resultados de las curvas de crecimiento mos-
traron que el consorcio es capaz de crecer en presencia de TCV como única fuente
de carbono. Sin embargo, sólo una cepa pura removió el TCV del medio de cultivo,
pero todas las cepas removieron este plaguicida en medio rico. Los productos de la
hidrólisis fueron detectados por cromatografía de gases acoplada a espectrometría
Rev. Int. Contam. Ambient. 26 (1) 27-38, 2010
M.L. Ortiz-Hernández and E. Sánchez-Salinas
28
de masas. Estos datos indican que las cepas aisladas pueden ser utilizadas para la
biodegradación de residuos o para la biorremediación de suelos o aguas contaminadas
con este plaguicida.
INTRODUCTION
Pesticides are organic compounds manufactured
and used for pest control. When pesticides are dis-
persed in the environment, they become pollutants,
with ecological effects that require remediation.
Environmental pollution is caused by both excessive
and continuous use of pesticides, and begins when
these compounds enter the environment by various
means (accidental spills, direct application, residues
from cleaning of containers, state of equipment used
and methods used to apply the products). The qual-
ity of soils, ground water, inland and coastal waters,
and air are all affected by pesticide contamination
(Chapalamadugu and Chaudry 1992).
Organophosphate pesticides (OP) constitute a
group of widely used, very heterogeneous com-
pounds that share a phosphoric acid derivative
chemical structure. There are currently 140 OP
compounds being used as pesticides and as plant
growth regulators around the world. These com-
pounds are components of more than 100 types of
commercially available pesticides (such as Para-
oxon, Parathion, Coumaphos and Diazinon), and it
has been estimated that over 1500 different OP have
been synthesized during the past century (Kang
et
al.
2006). In the United States of America alone,
60,000 tons/year of these types of compounds are
produced (Chapalamadugu and Chaudry 1992).
Although they have very useful properties, their
intensive and indiscriminate use has caused short
and long term environmental hazards and health
problems (Ortiz-Hernández
et al
. 1997).
Most synthetic OP compounds are highly toxic
and are powerful inhibitors of acetylcholinesterase,
a vital enzyme involved in neurotransmission, in
the form of acetylcholine substitutes (Donarski
et al
. 1989, Sultatos 1994, Grimsley
et al
. 1998,
Bakry
et al
. 2006). Organophosphates may also
cause delayed neurotoxic effects which are not due
to acetylcholinesterase inhibition. The function of
other esterases found in animal organisms is not well
understood. In the presence of OP, these enzymes
are phosphorylated and inactivated. Once 80 % of
the enzyme is inactivated, usually within four days
of exposure, potentially lethal symptoms can be
observed, including neck muscle weakness, diarrhea
and respiratory depression (Grimsley
et al.
1998).
Serious contamination issues arise at waste
disposal sites close to agricultural Felds and at OP
production facilities, due to inappropriate handling
and improper storage (Ortiz-Hernández
et al
. 1997).
Another problem is buildup of wastes. In undevel-
oped countries (Africa, Latin America, Asia and
East Europe), there are about a hundred thousand
metric tons of obsolete pesticides that are no longer
usable. These pesticides have simply expired; their
storage conditions are very poor with inadequate
safety measures, resulting in improper containment,
leaks, Fltration into soil and water bodies, and ac-
cidental spills. Environmental hazards and health
risks caused by obsolete pesticides could therefore
potentially affect many countries (Martínez 2004).
Another current problem is the 400,000 liters of
pesticide waste on the México-United States bor-
der, which contains approximately 1,500 mg/L OP
that has been used for parasite control in livestock
(Mulbry
et al.
1996).
Pesticides in soil and water can be degraded by
biotic and abiotic pathways, however biodegrada-
tion by microorganisms is the primary mechanism
of pesticide breakdown and detoxiFcation in many
soils. Thus microbes may have a major effect on
the persistence of most pesticides in soil (Surekha
et al.
2008).
Isolation of indigenous bacteria capable of me-
tabolizing OP compounds has received considerable
attention because these bacteria provide an environ-
mentally friendly method of
in situ
detoxiFcation
(Richins
et al
. 1997, Mulchaldani
et al
. 1999). In
some contaminated environments, autochthonous
microbial populations have evolved over time to
adapt to these contaminants (Pahm and Alexander
1993). These sites are therefore the most appropriate
ecological niches to Fnd and isolate strains capable
of degrading OP compounds (Ramos and Rojo
1990, Oshiro
et al
. 1996, Ortiz-Hernández
et al
.
2001, Horne
et al
. 2002). One of the most important
enzymes is phosphotriesterase (PTE), Frst found in
Pseudomonas diminuta
MG and
Flavobacterium
sp.
ATCC 27551, which is able to hydrolyze a consider-
able number of synthesized OP (Mulbry
et al.
1986,
Serdar
et al.
1989, Mulbry 2000). This enzyme is
called organophosphorus hydrolase (OPH) (Mansee
BIODEGRADATION OF TCV BY BACTERIA ISOLATED FROM AGRICULTURAL SOILS
29
et al.
2005) and exhibits high catalytic activity, hy-
drolyzing a broad range of organophosphates through
cleavage of P-O and P-S bonds in these OP (Ang
et
al.
2005).
The most signi±cant step in OP compound de-
toxi±cation is hydrolysis, since it makes compounds
more vulnerable to further biodegradation (Kumar
et al
. 1996). The mechanism of hydrolysis and
its kinetic characteristics are well known (Brown
1980, Lewis
et al
. 1988, Mulbry and Karns 1989,
Dumas
et al
. 1989, Dumas and Rauschel 1990,
Ortiz-Hernández
et al.
2003). PTE has potential
for use for cleaning up OP-contaminated environ-
ments. However, in previous laboratory trials us-
ing cultures in aqueous medium, it was observed
that this enzyme does not have any effect on some
OP, which suggests a speci±city in its activity that
depends on the type of phosphoric acid substitutes
used to form the OP (Ortiz-Hernández
et al.
2001,
2002 and 2003).
Biodegradation is a common method for the re-
moval of organic pollutants because of its low cost
and low collateral destruction of indigenous animal
and plant organisms (Liu
et al
. 2007). Studies of
microbial biodegradation are useful in the develop-
ment of strategies for detoxi±cation of pesticides by
microorganisms (Qiu
et al.
2006).
In previous studies, ten OP pesticides were tested
with PTE from
Flavobacterium
sp. ATCC 27551.
The results showed that the enzyme does not have
activity towards some chemical structures of OP. In
particular, PTE does not have activity towards TCV
(Ortiz-Hernández
et al.
2003). In this paper, we
describe the isolation and characterization of a TCV-
degrading bacterial consortium from agricultural
soils with potential use in bioremediation. Metabolite
analysis was also carried out.
MATERIALS AND METHODS
Pesticide description
TCV is an organophosphate pesticide used in
México for external parasites of livestock and poul-
try; for pests of fruit, vegetable, ornamental and forest
plants; and in recreational areas and on agricultural
equipment. It is also added to soil before crop culti-
vation and is useful for control of ²ies in livestock
manure (CICOPLAFEST 2004). It is moderately
toxic, affecting the human respiratory system, and
is quickly incorporated into the body through the
skin. Exposure to this OP causes acetylcolinesterase
inhibition, and it is also responsible for carcinogenic
problems, liver damage (cancer), and damage to
thyroid cells. The chemical structure of TCV and its
general characteristics are shown in
table I
.
Reagents and materials
Analytical grade tetrachlorvinphos (97.0 % pure)
was purchased from Ultra Scienti±c (Analytical
Standards). Solvents for gas chromatography (GC)
and gas chromatography-mass spectra (GC-MS)
were purchased from Mallinckrodt ChromAR
®
HPLC. Tripticasein soy agar (TS) was obtained from
Bioxon; potassium monobasic phosphate, potassium
dibasic phosphate, ammonium sulphate, magnesium
sulphate, sodium chloride, calcium chloride, iron
sulphate, sodium molybdate and manganese sulphate
and Tris buffer were purchased from J. T. Baker.
Isolation of bacteria that degrade TCV
The soil used for isolation of microorganisms
was collected from a commercial corn±eld in central
México which had been treated with organophos-
phate pesticides twice a year for the previous ±fteen
years. The soil was a vertisol type and was collected
TABLE I.
CHEMICAL STRUCTURE AND TCV CHARACTERISTICS (EXTOXNET 1998)
Chemical structure
General characteristics
CH
3
CH
3
-O
O
-
O
-
P
-
O
-
C
-
Cl
Cl
Cl
CHCl
Phosphoric acid, 2-chloro-1-
(2,4,5-trichlorophenyl) ethenyl
dimethyl ester
Physical state: solid
- Color: tan to brown
- Odor: mild chemical
- Molecular weight and formula:
366.0; C
10
H
9
Cl
4
O
4
P
- Boiling point: 97-98 ºC
- Vapor pressure: 4.2x10
-
8
mm de Hg
- Solubility in various solvents at 0 ºC:
40 mg/L in chloroform,
40 mg/L in dichloromethane,
20 mg/L in acetone,
8 mg/L in xylene,
15 mg/L in water at 24 ºC
- LD
50
in rats: 50 mg/kg
M.L. Ortiz-Hernández and E. Sánchez-Salinas
30
at 10 cm below the soil surface at the selected sites.
The characteristics of the soil were 35 % moisture;
pH 7.52; electric conductivity 730 mS/cm; 2.88 %
organic matter; 0.12 % total nitrogen; 35.49 mg/kg
available phosphorus; and cation exchange capacity
18.85 cmol/kg. The soil analysis was performed ac-
cording to Ortiz-Hernández
et al
. (1993a and 1993b).
Sterile Petri dishes were used to store and trans-
port samples at 4 ºC until isolation of the bacteria
(Van-Elsas and Smalla 1997).
One gram of soil was
suspended in 5 mL of sterile mineral medium (MM)
and this suspension was considered the inoculum.
The MM had the following composition (per liter):
0.2 g KH
2
PO
4
; 0.5 g K
2
HPO
4
(sterilized separately
at 125 ºC for 25 min to prevent precipitation and
later aseptically added to the rest of the salts); 1 g
(NH
4
)
2
SO
4
; 0.2 g MgSO
4
•7H
2
O; 0.2 g NaCl; 0.05
g CaCl
2
•2H
2
O; 0.025 g FeSO
4
•7H
2
O; 0.005 g
Na
2
MoO
4
; 0.0005 g MnSO
4
(pH 7.0 ± 0.3).
Flasks (125 mL) were supplemented with TCV as
the only carbon source, which was added as a ²lter-
sterilized methanol solution (Millipore membrane,
pore size 0.25 mm). The methanol was evaporated
to dryness before the addition of MM, and 20 mL of
MM and 0.2 mL of the inoculum (about 2 × 10
7
CFU/
mL of bacteria) were then transferred to the ³asks and
thoroughly homogenized. The ²nal concentrations of
TCV were 10, 15 and 25 mg/L. In this acclimation
period, bacteria that had grown in the presence of 10
mg/L of TCV were sequentially cultured ²rst in 15
mg/L and then in 25 mg/L of TCV. The culture was
incubated at room temperature (about 25 ºC), without
contact with the ambient atmosphere, and shaken con-
tinuously (120 rpm, Lab Line shaker) for seven days.
All cultures were made in triplicate. The consortium
bacteria isolated at 25 mg/L of TCV was selected for
colony puri²cation and for further evaluation of the
capacity of isolated bacteria to hydrolyze TCV
Several different colonies were chosen from the
isolated consortium. They were streaked separately
on TS agar plates containing 25 mg/L of the pesticide.
Those colonies considered as visually different were
plated on TS agar with pesticide and incubated at
37 ºC. This procedure was repeated several times to
ensure the purity of the isolated colonies.
In order to characterize and identify the isolated
bacteria, a BBL crystal system (Becton Dickinson)
was used. Additional biochemical tests were also
performed. The shape and morphology of bacterial
cells were determined by light microscopy (Granados
and Villaverde 1996, 1997). Identi²cation was made
following
Bergey’s Manual of Systematic Bacteriol-
ogy
(Krieg and Holt 1984).
Evaluation of the capacity of isolated bacteria to
hydrolyze TCV in MM
We carried out the growth and degradation experi-
ments with the consortia and pure colonies isolated
in 25 mg/L TCV. The cells were seeded in MM and
maintained in agitation as described above. One mil-
liliter sub-samples for TCV analysis were taken every
12 h from each ³ask and were placed in glass tubes.
These portions of the cultures were extracted twice
with equal volumes of ethyl acetate as the extracting
reagent. The mixture was centrifuged at 3000 rpm for
ten minutes. The ethyl acetate with residual TCV was
²ltered and dried with anhydrous sodium sulfate fol-
lowed by ²ltration through glass-²ber paper (What-
man GF/B). This operation was conducted sequen-
tially and the ²ltrates were mixed. The ²ltrate was
evaporated to dryness and resolved in 50 µL HPLC
grade dichloromethane for analysis. The amount
of pesticide was quanti²ed on a Hewlett-Packard
6890 gas chromatograph equipped with a nitrogen-
phosphorus detector (NPD) on a cross-linked 5 %
phenylmethylsilicone capillary column (30 m by 0.2
mm inner diameter). The operating conditions were:
injector temperature 240 ºC; detector temperature 280
ºC; oven temperature 100 to 250 ºC at 12 ºC/min; and
carrier ³ow 2 ml/min. Calibration curves from 0 to 25
mg/L were made for the TCV. The experiments were
set for 72 h in triplicate. Systems of mineral medium
and TCV without bacteria, and mineral medium with
inoculum without TCV were run as controls.
Evaluation of the capacity of isolated bacteria to
hydrolyze TCV in a rich medium
In order to measure bacterial growth and TCV
degradation in a rich culture, an experiment was per-
formed using tripticasein soy broth (Bioxon, Becton
Dickinson de México). Nutrient broth was prepared
according to the manufacturer’s instructions. The
experiment was made under the same experimental
conditions as those described above.
Bacterial growth
Cell growth was measured spectrophotometrically
by measuring OD
600
in a Spectronic 601 Milton
Roy spectrophotometer ²tted with deuterium and
tungsten lamps.
Metabolite analysis
Each isolated strain was cultured under the condi-
tions described above. We extracted the culture using
ethyl acetate as described above. Each of these extrac-
tions was later analyzed by gas chromatography (Trace
GC) coupled to a mass spectrometer (Thermo Finnigan
BIODEGRADATION OF TCV BY BACTERIA ISOLATED FROM AGRICULTURAL SOILS
31
Polaris Q) under the following conditions: Equity-5
column 30m × 0.25 mm ID, 0.25 µm, oven at 120 ºC (3
min) and at 270 ºC at 5 ºC/min, 250 ºC injector, MSD
detector, scan range 45-450 amu, 325 ºC transfer line,
30cm/sec at 120 ºC helium ±ow, 1.0 µL, splitless (0.3
min) injection, splitless liner, double taper.
RESULTS
Isolation of bacteria using TCV as a carbon source
Six bacterial strains capable of utilizing TCV as
the sole carbon source for growth were isolated from
the agricultural soil sample in the presence of TCV.
They were presumptively identi²ed according to
their physiological and biochemical characteristics.
The YS strain was identi²ed as
Stenotrophomonas
malthophilia;
A1 as
Proteus vulgaris;
A5 as
Vib-
rio metschinkouii
; A3 and C2 as
Serratia fcaria
and
Serratia
spp. respectively and A2 as
Yersinia
enterocolitica.
Table II
shows the various pheno-
typical characteristics of the selected TCV-degrading
bacteria strains. Additionally, the pattern resulting
from the 30 reactions in the BBL crystal system was
considered, which converted into a ten digit pro²le
number was used as the basis for identi²cation.
TCV degradation by isolated bacteria in MM
Figure 1
shows growth and TCV degradation by
the isolated consortium. This consortium was able to
decrease TCV concentration from 25 mg/L to 10.70
mg/L (57 %) in MM within the ²rst 36 hours. No
degradation of TCV was observed in un-inoculated
controls.
TCV degradation with the pure strain separated
from the isolated consortium was analyzed.
Figure 2
shows the results obtained from measuring the opti-
cal density of the bacterial culture medium (MM)
of each type of bacteria from the consortium, in the
presence of TCV, during a 72-h period. It can be
seen that the A3 strain showed the most growth in
comparison to the other strains. YS, A1, A5, C2,
A2 strains showed some growth without signi²cant
differences between them.
TABLE II.
STRAIN ISOLATED FROM AGRICULTURAL SOIL CAPABLE OF UTILIZING TCV AS SOLE CARBON SOURCE
FOR GROWTH AND OVERVIEW OF THE PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF
BACTERIAL ISOLATES
Strain
MT
CI
IN
MR
OX
GR
FC
Specie
BBL Cristal System
(% con²ability)
YS
+
+
+
+
+
-
Alc-Ac.
Stenotrophomonas malthophilia
98.88
A1
+
-
+
+
-
-
Ac-Ac
Proteus vulgaris
99.75
A5
-
-
+
-
-
-
Alc-not changes
Vibrio metschinkouii
99.73
A3
+
-
-
+
-
-
Ac-Ac
Serratia fcaria
93.34
C2
+
-
+
+
+
-
Alc- not changes
Serratia spp.
92.60
A2
+
+
+
+
-
-
Alc-Ac
Yersinia enterocolitica
99.29
MT = Motility; CI =Citrate; IN = Indol; RM = Methyl red; OX = Oxidase; GR = Gram tintion; CF = Carbohydrates fermentation;
Alc = Alcalin; Ac = Acid
+ = positive reaction;
-
= negative reaction
25
20
15
mg/L
10
5
0
0
Concentration (mg/L)
Control concentration (mg/L)
O.D
O.D control
12
24
36
Time (hours)
48
60
72
2.10
1.80
1.50
1.20
Growth (O.D)
0.90
0.60
0.30
0.00
30
Fig. 1.
Growth of bacterial consortium in MM containing
TCV and changes in the concentration of TCV as time
function. O.D = Optical density (600 nm).
1.80
1.60
1.40
1.20
Growth (O.D)
1.00
0.80
0.60
0.40
0.20
0.00
0
12
24
36
Time (hours)
A3
YS
A5
C2
A2
A1
48
60
72
Fig. 2.
Growth rate comparison of different bacteria in presence
of 25 mg/L of TCV in MM, during 72 hours of culture.
O.D = Optical density (600 nm).
M.L. Ortiz-Hernández and E. Sánchez-Salinas
32
The fnal TCV concentration and the percentage
oF TCV remaining in solution are displayed in
table
III.
Growth oF the A3 strain
increased during 72 h
oF culture, and thereFore was the most eFfcient strain
For removing TCV (48.48 %).
Figure 3
shows TCV
concentration as a Function oF time.
Results of pesticide concentration in TS broth
The ability oF the consortium to degrade TCV in
TS broth was measured. The results indicate that 100
% oF the pesticide was removed From the rich me-
dium. Additionally,
±gure 4
shows growth and TCV
degradation in the TS-enriched medium. It can be
observed that the concentration oF pesticide decreased
by almost 100 % in the culture oF all isolated strains.
The strain that showed the lowest removal rate was
C2 (81.20 %). These results suggest a co-metabolic
process, since they diFFer greatly From those obtained
in mineral medium.
In this study, the A3 strain showed a 49 % TCV
decrease in MM; this measurement was obtained
without the addition oF an additional carbon source.
When the bacteria were cultured in presence oF a rich
medium, including a carbon source, the percentage oF
TCV removed From the medium increased consider-
ably (98 %). This suggests a co-metabolism process.
±or the other bacteria isolated From the sample, the
TCV removal percentage was lower in the mineral
medium, but when they were cultured in a medium
rich in carbon and nutrients, TCV removal capacity
equaled that oF the A3 strain. Under these conditions,
any oF the strains isolated could be eFFective For treat-
ing wastes or restoring contaminated environments.
In order to evaluate the eFFect oF pH on TCV hy-
drolysis, 50 mg TCV in 10 mL oF 1:1 ethanol:water
was exposed to pH conditions in a range From 7 to
13 modifed with 1 M NaOH without bacteria For a
period oF 6 h. Chemical hydrolysis occurred at pH>12
only (Ortiz-Hernández
et al
. 2003). This suggests that
TCV removal From the culture medium was due to
bacterial activity.
A3 has not previously been reported as having
a gene that codes For OP pesticide hydrolysis. This
strain is interesting since it has rarely been isolated
From clinical samples, and thereFore its pathogenicity
is low. It would be useFul to test this strain with other
OP pesticides in order to fnd catalytic activity that
might make it a recommendable treatment oF wastes
or polluted environments, with a low potential eFFect
on public health.
Metabolite identi±cation
Figure 5
shows retention times oF the diFFerent
components oF the A3 strain extract cultured For 72 h
in MM. Gas chromatography revealed Four peaks (re-
tention times 11.22, 13.05, 14.19 and 17.54 minutes).
Figure 6
shows mass spectra and the scheme
oF a metabolite that occurs at minute 11.22, identi-
fed as
1-(2,3,4) trichlorophenylethanone
(
Fig. 6a
)
.
This Fragment is equal to
1-(2,4,5)
trichlorophenyl-
ethanone
(
Fig. 6b
), which belongs to a metabolite
resulting From TCV hydrolysis. The peak at 14.19
minutes is the substrate (TCV), a phthalate (retention
time 13.05 min). We Failed to determine the peak at
17.54 min. This procedure was also carried out For
the controls. The pesticide molecule was Found, but
not the metabolite.
In addition, in enzymatic hydrolysis, TCV chem-
istry hydrolysis was assessed using 0.5 M NaOH at
pH 12 in thin layer chromatography with eluent ethyl
acetate:ethanol 60:40. The result oF this reaction was
extracted and analyzed in GC-Mass as described
above. In this chromatogram, we Found the same
metabolite as in enzymatic hydrolysis, but the other
Fraction oF TCV (phosphoric acid) was Found too,
with an ethyl group (
ethyl dimethyl phosphate)
prob-
ably arising From ethanol used in chemical reaction
(
Fig. 7
).
TABLE III.
±INAL CONCENTRATION O± TCV, A±TER 72
HOURS CULTURED IN MM
Bacterial strain
Concentration
(mg/L)
TCV remainder
(%)
Serratia fcaria
12.88 ² 1.35
51.52
Serratia
spp.
18.80 ² 0.81
75.20
Vibrio metschinkouii
17.26 ² 1.90
69.04
Yersinia enterocolitica
18.58 ² 1.32
74.32
Stenotrophomonas malthophilia
18.00 ² 0.98
72.00
Proteus vulgaris
20.90 ² 1.28
83.60
30
mg/L
25
20
15
10
5
0
0
12
24
36
Time (hours)
A3
YS
A5
C2
A2
A1
48
60
Fig. 3.
Concentration oF TCV remainder in culture medium
(MM), From the six isolated bacteria
BIODEGRADATION OF TCV BY BACTERIA ISOLATED FROM AGRICULTURAL SOILS
33
A metabolite of TCV hydrolysis was found in the
A3 and A5 cultures in MM. In the TS broth cultures,
metabolites were obtained in the YS, A2, A5, A1 and
A3 cultures.
From the results of this study, we can see that these
soils hold a great microbiological potential that could
be used to treat stored wastes, pesticide containers
before ±nal disposal and/or contaminated environ-
ments. It is a potential enzyme resource that could be
used in bioremediation processes directly in the ±eld
or in dedicated reactors under controlled conditions.
Further research into the nature of the enzyme, its
optimum activity characteristics and its lifetime in
natural environments are required.
30
2,1
1,8
1,5
1,2
0,9
O.D
0,6
0,3
0
2
1,7
1,4
1,1
0,8
O.D
0,5
0,2
0,1
1,2
0,9
O.D
0,6
0,3
0
1,5
1,2
O.D
0,9
0,6
0
0,3
A3
A5
A2
A1
C2
YS
0
12
Concentration (mg/L)
Growth (O.D)
Concentration (mg/L)
Growth (O.D)
Concentration (mg/L)
Growth (O.D)
Concentration (mg/L)
Growth (O.D)
24
36
Time (hours)
48
60
72
0
12
24
36
Time (hours)
48
60
72
mg/L
mg/L
25
20
15
10
5
0
30
mg/L
25
20
15
10
5
0
30
mg/L
25
20
15
10
5
0
30
20
10
0
0
12
24
36
Time (hours)
48
60
72
0
12
24
36
Time (hours)
48
60
72
Concentration (mg/L)
Growth (O.D)
0
12
24
36
Time (hours)
48
60
72
Concentration (mg/L)
Growth (O.D)
0
12
24
36
Time (hours)
48
60
72
30
mg/L
25
20
15
10
5
0
30
mg/L
25
20
15
10
5
0
2,1
1,8
1,5
1,2
0,9
O.D
0,6
0,3
0
Fig. 4.
Growth kinetics and TCV degradation with isolated bacteria cultured during 72 hours in TS broth. O.D = Optical density (600 nm)
M.L. Ortiz-Hernández and E. Sánchez-Salinas
34
DISCUSSION
The bacterial consortium isolated from the sample
was made up of a group of strains whose action was
reFected in signi±cant pesticide depletion. Unfor-
tunately laboratory methods are only capable of
isolating 1 to 10 % of all bacteria growing in soil, so
several of the bacteria interfering in the degradation
processes in natural environments cannot be obtained
in a laboratory. However, isolation of bacteria or
bacterial consortia that might be used in the cleaning
of wastes or polluted environments is very important.
1.5 e+07
1.5 e+07
1.5 e+07
1.5 e+07
1.5 e+07
1.5 e+07
9000000
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
Relative abundance
m/z
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
13.05
11.22
14.19
18.98
17.54
Fig. 5.
Retention times diagram of components from A3 strain extract cultured
in MM during 72 hours. Retention times 11.22, 13.05, 14.19 and 17.54
minutes are
1-(2,3,4) trichloropheniletanone
, a ftalate, TCV and an unk-
nown compound respectively
Relative abundance
m/z
74
109
89
143
51
125
159
179
253
235
222
207
a)
b)
CH
3
O
C
Cl
Cl
Cl
CH
3
O
C
Cl
Cl
Cl
2800000
2600000
2400000
2200000
2000000
1800000
1600000
1400000
1200000
1000000
800000
600000
400000
200000
0
40
60
80
100
120
140
160
180
200
220
240
260
Fig. 6.
Chemical structure and mass spectra of metabolite (a)
1-(2,3,4) trichlorophenyle-
thanone
. (b) Chemical structure of
1-(2,4,5) trichlorophenylethanone.
The culture
conditions of
the
A3 strain
were 72 hours on MM
BIODEGRADATION OF TCV BY BACTERIA ISOLATED FROM AGRICULTURAL SOILS
35
From the results of this study, we cannot be
sure whether this hydrolysis subsequently results
in complete mineralization or if the resulting
metabolites remain in the medium. Other studies
(Chapalamadugu and Chaudry 1992) have shown
that the advantage of an isolated consortium with
pesticide as the sole carbon source is that a bacterial
strain may ±rst carry out the hydrolysis, and other
strains are then able to use the resulting compounds
as phosphorus and carbon sources. Therefore it is
advisable to conduct further studies to evaluate
which bacteria of the consortium carries out the
initial hydrolysis and whether there are others that
produce complete mineralization.
Otherwise it is the case that when bacteria isolated
from soil are in contact with OP pesticides, they gen-
erate new enzymes (such as the phosphotriesterase
of
Flavobacterium
sp. ATCC 27551), resulting in
new metabolic pathways for pesticide degradation.
Environmental conditions, soil pH, agricultural
management and the amount of pesticide added are
important factors for bacterial use of xenobiotic
compounds (such as pesticides) as a growth substrate.
Evolution can occur very quickly, resulting in
microorganisms with new genetic properties which
enable them to degrade compounds. Although further
studies are still necessary, there is evidence to show
that genes for many catabolic functions are located
in plasmids; that an exchange of genes occurs in
the environment; and that the best and most usual
degraders come from environmental samples highly
impacted by pollutant compounds. It is also pos-
sible that the gene that codes for these enzymes was
initially present in a non-expressed form. It has also
been observed that shortly after repeated applications
of a degradable pesticide, the soil becomes richer in
bacterial populations which are capable of degrad-
ing it, which dramatically reduces the effectiveness
of subsequent pesticide applications (LaGrega
et al.
1996, Madigan
et al
. 2004).
Proof of this is the isolation of bacteria from
agricultural soils where repeated applications of OP
pesticides have been reported. Although this isolation
of bacteria was preceded by a stage of adaptation to
the laboratory conditions where they were maintained
with TCV as the only source of carbon, the bacteria
showed different levels of ef±cacy in removing the
pesticide from the culture medium.
The physiological base for co-metabolism is not
well known, but the most accepted hypothesis is
related to the speci±city of enzymes. Many of the
enzymes which are present in microbial cells catalyze
reactions on substrates that are different but chemi-
cally related. If the products of any of these activities
are not an adequate substrate for any other of the
activities, this compound will be accumulated, even
if the original enzyme converts its natural substrate
into products that provide energy and a source of
carbon to the active species.
There are several explanations of co-metabolism,
m/z
40
0
200000
400000
79
139
154
109
127
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
60
80
100
120
140
160
180
Relative abundance
CH
3
CH
2
CH
3
CH
3
O
O
O
O
P
Fig. 7.
Mass spectra of
ethyl dimethyl phosphate
M.L. Ortiz-Hernández and E. Sánchez-Salinas
36
which are supported by experimental evidence (Al-
exander 1994):
a) The initial enzymes convert the substrate into an
organic product that is not subsequently converted
by other enzymes in the same microorganism into
metabolic intermediates that would eventually be
used for biosynthesis and energy production.
b) The initial substrate is transformed into products
that inhibit the mineralization activity of later
enzymes or that suppress the growth of organisms.
c) The organism needs a second substrate to carry
out a particular reaction.
According to Alexander (1994), the Frst explana-
tion is the most common one and is based on the fact
that many enzymes act on structurally related sub-
strates. The chemical transformation of a xenobiotic
compound by co-metabolism is interesting from the
environmental point of view for two reasons: ±irstly,
since the size of the microbial population that acts
on an organic compound in the environment is very
low, the compound degrades slowly and the propor-
tion converted does not increase with time. Secondly,
many organic products accumulate as the result of co-
metabolism and these products tend to persist because
one species cannot continue metabolizing the product.
Similar observations have been made in previ-
ous studies, where it has been shown that complete
degradation of methyl parathion takes place more
efFciently in mixed cultures of bacteria than in pure
cultures (Munnecke and Hsiem 1976, Chaudry
et al
.
1988). However, Pahm and Alexander (1993) report
that bacteria cultivated in pure culture are useful for
determining and evaluating the degradation capac-
ity of each species separately. Soil bacteria in their
natural environment have high degradation activity,
however it is difFcult to grow them in the laboratory.
The reason why any particular species cannot use
a xenobiotic compound is because it is possible that
in natural environments, the species takes part in the
metabolism in a very speciFc way. This can be due
to a cooperative process between microorganisms,
which allows the existence of reactions in one meta-
bolic pathway. Many metabolic pathways depend on
enzymes which are codiFed in a plasmid; the ability
of the microorganisms to degrade organic compounds
then depends on the stability of the plasmids or the
microorganism’s genes.
The metabolites found in the A3 culture in both
culture media suggest that this species hydrolyzes the
pesticide in both media, using it as source of carbon
and energy. It also suggests that the mechanism of
TCV hydrolysis is similar to that of PTE hydrolysis
with other OP; namely through a nucleophilic attack
(
Fig.
8
).
After this Frst reaction, with two resulting me-
tabolites, dimethyl phosphate is more soluble in
water and therefore more available for attack by other
microorganisms, being able to use them as a source
of phosphorous (Maloney
et al
. 1988). However, the
other metabolite is a chloride compound which can
become recalcitrant and remain in the environment
for a longer time.
The enzymatic hydrolysis results did not detect
dimethyl phosphate, because this metabolite is more
soluble in water and was not extracted by the solvent
used. Another possible reason is that it was used by
the cultured bacteria. This fraction of the pesticide
was detected in the chemical hydrolysis extracts.
The present study reports isolation of a bacte-
rial consortium which is capable of utilizing TCV
as a source of carbon. Utilization of xenobiotic
compounds by soil microorganisms is a crucial phe-
nomenon by which these compounds are removed
from the environment, thus preventing environmental
pollution. The results of the present study suggest that
the bacteria which were isolated are able to grow in
medium in the presence of added pesticide and may
therefore be used for bioremediation of pesticide-
contaminated soil.
Fig. 8.
±irst TCV hydrolysis reaction according to GC-MS. The
compounds were obtained through pure culture of the
A3 strain with MM and TS broth
Tetrachlorvinphos
Phosphoric acid, 2-chloro-1-(2,4,5-trichlorophenyl) ethenyl dimethyl ester
2,4,5-Trichlorophenacyl chloride
Dimethyl phosphate
+
CH
3
CH
3
CH
3
H
2
O
CH
3
O
P
P
O
O
O
C
CHCl
CHCl
C
Cl
Cl
Cl
Cl
Cl
Cl
O
O
O
HO
OH
BIODEGRADATION OF TCV BY BACTERIA ISOLATED FROM AGRICULTURAL SOILS
37
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