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: [email protected]) 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 68 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. 5. References Baghestani, M. A., Zand, E., Soufizadeh, S., Mirvakili, M., Jaafarzadeh, N. (2007). Response of winter wheat (Triticum aestivum L.) and weeds to tank mixtures of 2,4-D plus MCPA with clodinafop propargyl. Weed Biol. Manage., 7, 209-218. Brar, L. S., Walia, U. S., Dhaliwal, B. K. (1999). Efficacy of new herbicides for control of resistant Phalaris minor in wheat. Pestic. Res. J., 11, 177-180. 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