Aeromonas lated from Field Crop

Sciknow Publications Ltd.
FBLS 2014, 2(4):67-70
DOI: 10.12966/fbls.12.01.2014
Frontiers of Biological and Life Sciences
©Attribution 3.0 Unported (CC BY 3.0)
Biodegradation of Cladinafop Propargyl by Aeromonas sp. Isolated from Field Crop
Avneesh Kumar, Harmanjit Kaur, Simranbir Kaur, Kashmir Singh, and Baljinder Singh*
Department of Biotechnology, Panjab University, Chandigarh, India
*Corresponding author (Email: gilljwms2@gmail.com)
Abstract - In this study, a highly effective clodinafop propargyl (CF) degrading bacteria strain, B1, was isolated from herbicides
contaminated soil sample. This strain, identified as Aeromonas sp., utilises CF as the sole source of carbon and energy for growth.
81.3% CF was degraded out of initial provided 80 mg/L CF. Degradation of CF was accompanied by release of chloride ion. The
major metabolite [4-(4-chloro-2-fluorophenoxy) phenol] was identified by GC-MS. A metabolic pathway for the degradation of
CF by B1 has been proposed.
Keywords - Aeromonas sp., Clodinafop Propargyl, Biodegradation, GC-MS
1. Introduction
CF
(prop-2-ynyl(R)-2-[4-(5-chloro-3-fluoro-2
pyridyloxy)phenoxy]propionate), is a recently introduced aryloxyphenoxypropionate herbicide and is used for post emergence
control of annual grasses in cereals (Singh, 2013). CF is absorbed by the leaves and interferes with the production of
fatty acids needed for plant growth in susceptible grassy
weeds (Hammami et al., 2011).
India is an agriculture based country. About 60-70% of its
population is dependent on agriculture (Singh et al., 2011) and
today, high-yielding agriculture heavily depends on chemical
weed control (Baghestani et al., 2007). The Government of
India has given provisional registration to cladinafop along
with other herbicides (Dhaliwal et al., 1998; Brar et al., 1999).
The widespread use of CF has resulted in the discharge of
large amounts of the compound into the environment, which
eventually reach the biosphere (Gherekhloo et al., 2010; Vazan et al., 2011). Several studies have demonstrated that CF
and its derivatives are toxic and carcinogenic to humans and
other living organisms (U.S.E.P.A., 2004; Kashanian et al.,
2008; Gui et al., 2011). Therefore, the degradation of CF in
the environment is of great concern.
Due to low CF persistence; the half-life in soil was reported to be 5 d, dependent on the soil type, pH, and microbial
population (Hou et al., 2011). Hou et al., (2011) describe for
the first time a microbial strain Rhodococcus sp. T1, able to
use CF. They had reported 97.9% CF-degradation without
identifying its metabolites. Singh, (2013) recently isolated a
Pseudomonas sp. that could use CF as the sole carbon, nitrogen and energy source.
For isolation of bacterial species from soil, CF contaminated moist soil sample was collected from two different field
crop area. Enrichment and sub-culturing of two samples
yielded two different genera of bacteria Pseudomonas and
Aeromonas capable of degrading CF. Although CF degradation pathway is same in both bacteria(s) but degradation kinetic is different in both. Compared to the Pseudomonas sp.
strain B1 described previously, there are important differences are as follow:
We have isolated different strain Aeromonas sp. strain B2
that mineralized the CF as sole carbon source up to 80 mg L-1.
87.14 % CF was degraded by Pseudomonas sp. strain B1 out
of initial provided 80 mg/L CF after 9 h incubation whereas
Aeromonas sp. strain B2 was capable of degrading 81.3% CF
after 12 h of incubation. Therefore fast degradation rate was
observed in Pseudomonas sp. strain B1. Importantly, this is
the first report of degradation of CF by genus Aeromonas.
2. Materials and Methods
Soil samples were collected from crop field area with a previous history of CF application, located in the city of Chandigarh, Punjab, India. CF (99.4% purity) was purchased from
Sigma Aldrich (PESTANAL, Fluka analytical). All other
chemicals used in this study were analytical grade or higher
purity.
Due to low CF solubility in water (4 mg/ L), a stock solution of CF was prepared by dissolving it in methanol at concentration of 1 mg/mL and further added to medium to get
final concentration. A selective minimal salt (MS) medium
was prepared containing 40 mg/L CF as a sole source of
carbon in addition to 4 g Na2HPO4*2H2O, 2 g KH2PO4
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Frontiers of Biological and Life Sciences (2014) 67-70
(0.025%), MgSO4*7H2O (0.05%), and 1 mL of trace element
solution (0.1 g of ZnSO4*7H2O, 0.03 g of MnCl2*7H2O, 0.3 g
of H3BO3, 0.2 g of CoCl2*6H2O, 0.01 g of CuCl2*2H2O, 0.02
g of NiCl2*6H2O, in 1 L of the solution). Five grams of soil
sample were inoculated into Erlenmeyer flask (250 mL)
containing 100 ml autoclaved water. Soil (0.5 ml in autoclaved water) was spread on MS media plates and incubated at
30 ºC until bacterial colonies appeared. Single colonies were
subcultured on fresh plates to purity with the CF concentration being increased from 40 to 120 mg/L. The final axenic
strain was reinoculated into MS medium to check for retention of growth. The isolated strain B1 was classified by Gram
staining and 16S rRNA analysis. Genomic DNA extraction
from strain B1 was performed using the method described by
Sambrook et al. (1989). Partial fragment of 16S rRNA gene of
strain B1 was amplified by PCR with set of universal primers
27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R
(5’-TACGGYTACCTTGTTACGACTT-3’) following the
PCR parameters as described by (Singh et al., 2011).
Screening of strain B1 as nitrogen fixing bacteria was
done using nitrogen free malate media (Smith-Grenier et al.,
1996) containing bromothymol blue as an indicator and incubated at 37ºC and 50 ºC up to 24 h. Nitrogen fixer bacteria
will produce blue coloured zone in the solid culture conditions.
Strain B1 was pre-cultured in 5 mL fresh MS medium
containing 50 mg/L of CF at 30 ºC with shaking at 100 rpm
for 12 h. This cell culture ( OD600 = 1, 0.2 mL) were inoculated in 50 ml of MS medium containing 80 mg/ L CF for 24 h
incubated at 30 ºC, under pH 7 to study the degradation of CF
by strain B1 in liquid culture. To determine the effect of initial
concentration of CF on degradation, the MS medium was
fortified with CF at concentrations of 40, 80 and 120 mg/L.
Heat-killed strain B1 was used as control. Each treatment was
performed in three replicates. At regular intervals, 5-mL
samples were collected from each flask. Biomass was monitored by measuring the OD600 with a spectrophotometer
(Shimadzu UV-1650 PC, Japan). The CF concentration and
its metabolites produced by biodegradation were determined
by HPLC and GC-MS as described Singh, (2013). The HPLC
analysis was performed using system (Dionex UltiMate®
3000) consisting of P680 HPLC pump, a C18 reversed-phase
analytical column (Acclaim 120, 4.6 mm X 250 mm, dp= 5
μm) with suitable guard column and a D170U UV-detector
using acetonitrile:water (50:50) as mobile phase at flow rate
1.0 ml min-1 and an injection volume 20 µl. For GC-MS
analysis, GCMS-QP2010 Plus (Shimadzu Corporation, Kyoto,
Japan) analysis was used. Capillary column used in the GC
was Rtx-1MS (30 m x 0.25mm ID x 0.25μm df) supplied by
Restek U.S. (Bellefonte, PA, U.S.A.). GC column oven
temperature was programmed for an initial hold of 1 min at
100 ºC; then temperature was increased at 10 ºC min−1 to 200
ºC; then upto 260 ºC at the rate of 15 ºC min−1; followed upto
300 ºC at the rate of 3 ºC min−1 and then hold at 300 ºC for 2
min. The gas flow rate was 1 mL min−1 in splitless mode with
injection temperature of 270 ºC.
The chloride ion concentration was determined using
Mohr method (Singh, 2013). Two hundred microliters of a
sample was diluted so that the chloride concentration was up
to 0.1 mM was added to 50 ml of 0.25 M potassium chromate.
The reaction mixture was titrated with 0.1 M silver nitrate
solution. Chloride ion concentrations were calculated by
using volumetrically analysis.
3. Results and Discussion
Three pure isolates that could grow by using CF as the sole
source of carbon were obtained from the soil samples. The
ability of these strains to degrade CF was confirmed in liquid
MS media supplemented with CF. One isolate, designated as
strain B1, showed the highest CF-degrading ability and was
selected for subsequent experiments. When grown on LB agar,
cells of this strain are non-spore-forming, gram negative,
motile, and globular- or globular-rod-shaped. The nucleotide
sequence of the 16S rRNA of strain B1 (1145 bp) was deposited into the GenBank database under the accession number
KC844266. BLASTN analysis of 16S rRNA gene sequence
revealed that strain B1 belonged to Aeromonas sp (99 %
similarity). Aeromonas spp. are known as nitrogen-fixing
bacteria but isolated strain B1 does not fix atmospheric nitrogen. Samples collected from the growth media were subjected to HPLC analysis. HPLC chromatograms of control
and test reactions are recorded. HPLC analysis showed a
substantial reduction in the levels of CF. Limits of detection
(LODs) were calculated using a peak-to peak height signal to
noise ratio of 3:1, at the lowest calibration concentration of
analyte. LOD for CF was 2 ng/L.CF and its major metabolite
peaks were observed after retention time 2.779 min and 1.874
min respectively. Strain B1 could degrade 81.3% of initial
provided 80 mg/L CF within 12 h. Interestingly, the organism
showed maximum growth (biomass 0.35 g/l after 12 h of
incubation) with 80 mg/L concentration of CF whereas with
40 mg/L CF lesser growth could be observed (Fig 1). The
degradation of CF by strain B1 could be affected by substrate
concentration (Fig. 1). After incubation for 12 h, 9.2 mg/L and
53.6 mg CF remained in culture with initially added concentration 40, and 120 mg/L, respectively to the MS medium.
This limited growth at higher concentrations of CF could
again be attributed to toxicity at higher concentrations of CF.
Similarly, Singh (2013) observed that the growth of Pseudomonas sp. strain B2 was also dependent on initial CF concentration added to the medium. The growth of strain B1 on
CF and its ability to degrade CF is shown in Fig. 2. With CF as
the carbon, nitrogen and energy source, strain B1 produced a
typical sigmoidal growth curve consisting of a relatively very
short lag phase and an exponential phase of approximately 12
h, followed by abrupt transition to the stationary phase (Fig.
2). The GC-MS spectrum pattern of standard (without inoculum) and its metabolites were recorded. The major metabolites,
clodinafop
acid
and
4-(4-Chloro-2-fluoro-phenoxy)-phenol peaks were observed
at 4.519 min and 1.874 min respectively. No change in CF
Frontiers of Biological and Life Sciences (2014) 67-70
concentration was observed in culture that was inoculated
with heat-killed strain B1. Singh, (2013) reported that higher
intracellular CF concentration would result in slower degradation rate and this is in consistent with our observation.
Standard exhibited molecular ion peak (M+) at 349 m/z and
characteristic fragment ions at 323 m/z, 266 m/z, and 238 m/z.
4-(4-Chloro-2-fluoro-phenoxy)-phenol displayed a molecular
ion at m/z 240 (M+) and characteristic fragment ions at 183
69
m/z, 165 m/z and 100 m/z. Only trace amounts of
4-(4-Chloro-2-fluoro-phenoxy)-phenol were detected during
the early stages of growth (1-2 h), high concentrations of this
metabolite in the growth medium during the log and stationary
phases
(15-30
h)
suggested
that
4-(4-Chloro-2-fluoro-phenoxy)-phenol was the major degradation product. Other possible breakdown product, including
clodinafop acid was also observed.
CF 40 ppm
CF 80 ppm
CF 120 ppm
120
CF conc. (ppm)
100
80
60
40
20
0
-2
0
2
4
6
8
10
12
14
16
Time (h)
Fig. 1. Effect of concentration on CF degradation. Data is presented as mean and standard error of three independent observations. Some error bars are not present because they are smaller than the diameter of the symbol.
Degradation of CF
Uninoculated medium
Growth on MS medium
90
80
0.32
0.24
60
50
0.16
40
0.08
30
Biomass (g/l)
CF conc. (ppm)
70
20
0.00
10
-2
0
2
4
6
8
10
12
14
16
Time (h)
Fig. 2. Degradation of CF by strain B1. A time course study of CF degradation in MS medium supplemented with 80 mg/L CF.
These metabolites were in accordance with previous study
(Singh, 2013). During the reaction amounts of chloride ion
(1.8±0.4 mg/L) were released from initial provided 80 mg/L
CF within12 h. Therefore, it is possible that the chloride ion
70
Frontiers of Biological and Life Sciences (2014) 67-70
release leads to catabolism of the pyridyl moiety in CF (Singh,
2013). The increase in chloride concentration was accompanied with decrease of 4-(4-chloro-2-fluoro-phenoxy)-phenol
concentration and support further degradation of
4-(4-chloro-2-fluoro-phenoxy)-phenol metabolite. However,
no any other metabolites were observed by adopted methods
of GC-MS detection. Strain B1grew in MS medium containing CF with 4-(4-Chloro-2-fluoro-phenoxy)-phenol as metabolite was observed during growth and was in agreement
with previous observations (Smith-Grenier and Adkins, 1996;
Singh, 2013). Smith-Grenier and Adkins, (1996) reported the
degradation of diclofop-methyl by Chryseomonas luteola and
Sphingomonas
paucimobilis
and
formation
4-(2,4-dichlorophenoxy)phenol as metabolite. The formation
of phenol as metabolite during growth of strain B1 in MS
medium provided an indication that it might be due to esterase
activity as reported previously (Hou et al., 2011; Singh, 2013).
The structures of the metabolites revealed that the initial
degradation of the compound to take place via cleavage of the
C-O
bond.
The
presence
of
metabolite,
[4-(4-chloro-2-fluorophenoxy) phenol], supported this suggestion. In summary, the results indicate that strain B1 is
capable of rapidly hydrolyzing the ester bond of CF to produce clodinafop acid, which in turn may either be directly
hydrolyzed to form 4-(4-Chloro-2-fluoro-phenoxy)-phenol.
4. Conclusion
In this report, a CF-degrading strain, B1, was isolated from
crop field area. The degradation of CF by this strain was
simple, rapid and highly effective. Furthermore, a possible
metabolite of CF was identified for the first time. This strain
could be a potential candidate to remove CF from contamination sites due to its high degradation efficiency.
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