Presence of esterase and laccase in Bacillus subtilis facilitates biodegradation and detoxification of cypermethrin

Ubiquitous presence of cypermethrin as a contaminant in surface stream and soil necessitates to develop potential bioremediation methods to degrade and eliminate this pollutant from the environment. A cypermethrin utilizing bacterial strain (MIC, 450  ppm) was isolated from the soil of pesticide contaminated agriculture field and characterized by using polyphasic approach. On molecular basis bacterial isolate showed 98% homology with Bacillus subtilis strain 1D. Under optimized growth conditions, bacteria showed 95% degradation of cypermethrin after 15 days and the end products of cypermethrin biodegradation under aerobic conditions were cyclododecylamine, phenol, 3-(2,2-dichloroethenyl 2,2-dimethyl cyclopropane carboxylate,1-decanol,chloroacetic acid, acetic acid, cyclopentan palmitoleic acid, and decanoic acid. Amplification of esterase (700 bp) and laccase (1200 bp) genes was confirmed by PCR which showed a possible role of these enzymes in biodegradation of cypermethrin. In the presence of cypermethrin Km value(s) of both the enzymes was low than the control. A nobel cypermethrin degradation pathway followed by B. subtilis was proposed on the basis of characterization of biodegraded products of cypermethrin using GC-MS. Cypermethrin biodegradation ability of Bacillus subtilis strain 1D without producing any toxic end product reveals the potential of this organism in cleaning of pesticide contaminated soil and water.

Use of pesticide-degrading microbial systems for the removal of pollutants from the contaminated systems requires the understanding of ecological, physiological, and biochemical mechanisms of the degrading organisms. Objective of the present study was to isolate and characterize cypermethrin degrading bacterial isolates from of an agricultural field soil. This study may also be exploited in bioremediation and cleaning practices of pesticide contaminated soil, water using the best isolates or gene/enzymes. The present work is very significant in order to degrade the xenobiotic or toxic chemicals into nontoxic end product within a short period of time via ecofriendly and economically.

Conclusion
Bacillus subtilis strain 1D isolated in the present study almost completely metabolized cypermethrin in15 days under laboratory conditions. This is the first report to show the involvement of laccase enzyme in the cypermethrin biodegradation. Metabolism of cypermethrin a bacterial same strain is of vital importance because cypermethrin possess antimicrobial activities hence it prevents the beneficial microflora of the soil. The bacterial isolate harbours the metabolic pathway for the detoxification of the cypermethrin and it completely degrades cypermethrin without leaving any persistent or toxic metabolite. The strain utilizes cypermethrin as a sole source of carbon for growth, which suggests adaptation of B. subtilis to oligotrophic environment. The ability of the organism to survive at higher concentration of cypermethrin with enhanced degradation potential makes this isolate an ideal candidate for its application in cypermethrin biodegradation.

Results
Isolation and characterization of cypermethrin degrading bacterial isolates. After five rounds of transfer, a sum of 10 bacterial isolates able to grow on cypermethrin as a sole carbon source were recovered from the pesticide contaminated soil of an agriculture field using enrichment culture technique. Recovered bacterial isolates were grown with cypermethrin upto 500 ppm to check their maximum tolerance level for cypermethrin. Out of 10 isolates only one bacterial strain (1D) was able to grow at 450 ppm of cypermethrin. On the basis of above results bacterial strain 1D was selected for further study. Bacterial isolate 1D was aerobic, gram positive, pink in color and had rod shaped cells. The isolate was characterized on the basis of biochemical, physiological and molecular chaacters. Phylogenetic analysis of the 16 s rDNA gene sequences revealed that strain 1D could be grouped among Bacillus species as it showed 99% homology with Bacillus subtilis (Fig. 1).
Response surface methodology for cypermethrin degradation by strain 1D. Box-behnken design, based on central composite rotatable design (CCRD) was employed to investigate interactive effect of significant variables like (inoculum size (A), cypermethrin concentration (B), and RPM (C)) on cypermethrin degradation by strain 1D. The experimental design and the response of dependent variables for cypermethrin are described in methods section. Data was processed by response surface regression procedure of Design Expert version-11, software, and results were obtained by fitting with the quadratic model equation (Table 1). where (Y) is predicted as % biodegradation of cypermethrin by strain 1D; A, B, and C are the coded values for RPM, concentration and inoculum size respectively. R1 = %Biodegradation of cypermethrin (response optimized by RSM). Figure (S5) shows interaction of one factor with others individually. The factors are speed, pesticide concentration, and inoculum size. From the Fig. S5 it is clear that among the three combinations, optimum percent degradation was 95%. Centre point lies in the parallel to their optimum which, indicates as optimum range of cypermethrin degradation, that is 10 ml(inoculum), 120(rpm) and160 ppm (concentration).

Analysis of intermediates of cypermethrin biodegradation after 15 th day of incubation. GCMS
study of biodegraded cypermethrin was conducted at JNU, Delhi, India. Peaks of different compounds were identified on the basis of their mass spectra and library identification program. Peak at retention time of 16.620 min corresponded to cypermethrin standard (control) (Fig. 1). This peak disappeared concomitantly with the formation of another peak with a change in retention time (Fig. 4), which reveals that some new compounds were formed subsequently ( Table 2). These compounds were non toxic, as confirmed by literature and library.     Enzyme kinetics. The enzyme kinetics of esterase and laccase was analysed in strain 1D by using Lineweaver   Amplification of laccase and esterase genes indicates that these gene are present in the bacterial genome and their expression is activated in response to the pesticide.

Discussion
Pyrethroid pesticides are excessively used in agricultural fields to control different pests. Cypermethrin, a member of pyrethroid group of pesticides is invariably used in different crops to get quality yield. Bioremediation using microbial cultures provides a cheap and environmentally friendly way to remove toxic pollutants from the environment. In the present study, a cypermethrin degrading bacterial strain was recovered from pesticide contaminated soil of an agricultural field using enrichment technique and characterized on the basis of biochemical, morphological and molecular (16S rDNA sequencing) characters. The organism showed 99% homology with Bacillus subtilis (Fig. 1).
Bacillus subtilis strain 1D was able to tolerate 450 ppm of cypermethrin and could degrade 240 ppm of the same in minimal medium. This feature gives the advantage of pesticide biodegradation in variable environments, because the organism survived and utilized the toxic compounds when exposed to high concentrations. In a rapid degradation of cypermethrin by Pseudomonas spp.and Bacillus spp. 4,22 was observed at 125 ppm with degradation percent of 83%. Bacillus subtilis strain 1D could degrade cypermethrin at higher concentration. Possibility of continuous expression of key enzyme(s) responsible for cypermethrin degradation at higher concentration of the contaminant by 1D cannot be ignored. Bacterial isolates engaged in efficient degradation of cypermethrin may be able to grow on the hydrolyzed products of cypermethrin. In present study we presume that metabolic activity of strain 1D was not subjected to complete catabolite repression by high cypermethrin concentration. High cypermethrin tolerance and degradation capability of Bacillus subtilis strain 1D, makes this strain suitable for decontamination and remediation of contaminated sites.
Optimum culture conditions for cypermethrin biodegradation by Bacillus subtilis Strain 1D were also determined by using response surface methodology (RSM). RSM is an empirical modelling system that has been successfully applied to improve and optimize complex processes, including fermentation for variety of microorganisms [23][24][25] . Previous studies have shown that application of statistical experimental design in biodegradation processes can result in improved yields of biodegradation with rapid and economical determination of optimum culture conditions using minimal resources 14,26 . In the present study, RSM has been employed to optimize culture conditions which favour cypermethrin degradation. The optimized parameters for cypermethrin biodegradation include speed (rpm), concentration ( ppm) and inoculums size (mL). The results of the experiments were statistically analyzed and the significance and effect of each factor on responses were evaluated (Fig. 2).
In vitro percent degradation of cypermethrin using Bacillus subtilis in minimal medium was maximum for trans α-(95%) and cis β-(93.7%) after 15 days (Fig. 3). Degradation of trans form of cypermethrin was maximum because this form is less stable than cis. Bacillus spp. degraded cypermethrin up to 81.6% within 15 days under standard growth conditions (temperature 32 °C pH 7 and shaking at 116 rpm) in minimal medium 4 . To implement effective biodegradation strategy, it was necessary to analyse the nature of intermediate compounds biodegraded by the bacterial strain. Degradation of pyrethroid insecticides produces more toxic intermediates in biodegradation processes 27 . Our results showed that the strain 1D not only efficiently degraded cypermethrin but also transformed cypermethrin into non toxic forms.
It is presumed that break down of the ester bond of a compound indicates the expression of enzyme carboxylesterases. This enzyme acts as a regulatory enzyme for pyrethroid biodegradation and results in acid and alcohol production 11,21 . Metabolites of cypermethrin have been arranged sequentially to propose a biodegradation pathway in Bacillus subtilis (Fig. 5). A new hypothetical degradation pathway of cypermethrin using strain 1D has been proposed because some new intermediates were found during cypermethrin biodegradation (Table 3).

GC-MS analysis of biodegraded cypermethrin showed the presence of non toxic metabolites which indicates
Bacillus subtilis is an effective and suitable strain for the degradation of pesticide belonging to pyrethroid group. To confirm the nature of cypermethrin metabolites, bacterial strain was grown in 15 days old filter sterilized broth containing intermediate compounds of cypermethrin. Surprisingly bacterial strain was able to grow in this broth, which shows the absence of toxic metabolites even after complete degradation of cypermethrin (Fig. 4). However more specific studies are required to test the toxicity of the intermediates of the pesticide.
Because of the specificity, esterase and laccase play important role in the degradation of wide variety of pollutants in the environment. Activities of esterase and laccase were elucidated qualitatively and quantitatively for their possible role in cypermethrin biodegradation. Increase in esterase and laccase activity was observed in the presence of cypermethrin as compared to the control. Results indicate induction of esterase and laccase activities in 1D bacterial strain under cypermethrin stressed condition to overcome the stress. Cypermethrin biodegradation was brought about by esterase enzyme which is present in B. subtilis strain 1D. Role of bacterial esterase has been elucidated for biodegradation of carbamate, organophosphate and cypermethrin. Different forms of pyrethroid hydrolases and their expression have been reported in Ochrobacterium, Bacillus and Sphingobium spp. Esterase belongs to hydrolase group of enzymes and found capable of hydrolyzing a large number of ester bond and ester bond containing compounds [28][29][30] .
Presence of bacterial laccase is reported in Azospirillum lipoferum 31 , Pseudomonas syringae 32 , B. subtilis 33 . Role of fungal laccase is reported in the degradation of chlorpyrifos, liluron and metribuzin 34 . Bacterial laccase is also involved in dye decoloration in Bacillus subtilis 35,36 Bacillus vallismortis 37 , Bacillus subtilis X1 38 . Very few studies have been reported on biodegradation of pesticide using laccase. Most of the studies were conducted on fungal laccase. Role of laccase in the degradation of cypermethrin was observed in strain 1D. So the presence of these two enzymes in the bacteria makes the organism more prominent candidate for the degradation of wide range of the pollutants.
Minimal medium supplemented with cypermethrin showed low km values which confirms that cypermethrin induces production of esterase and laccase while under normal conditions, km values were high when 1/Vmax is constant. This indicates production of enzyme is under competitive inhibition. This may be because some chemical constituents of the medium may bind to enzyme and act as competitive inhibitors. In the presence of the cypermethrin, laccase and esterase may undergo conformational changes and finally enzymes are free to work which leads to decrease in km value (Fig. 6). The role of esterase in the degradation of indoxacarb has been described which helped in the detoxification of the xenobiotic compounds 39,40 . Thermophillic bacterium Alicyclobacillus tengchongensis completely degraded malathion by producing esterase 41 . Presence of laccase gene was also confirmed by gene amplification. The same set of primers used and found amplicons of 600-1500 bp size for laccase 42 . Our results are also relevant to the findings of 42 , as amplicon size of 1200 bp in B. subtilis was reported. Amplification of esterase gene was observed in strains of Bacillus spp. And product was of approximately 550 bp whereas in SA2 it was at approximately 700 bp 3 .  Laccase and esterase genes are major regulatory genes which are responsible for cypermethrin degradation. Esterase is responsible for the formation of alcohol and acid when it reacts with water. Laccase uses molecular oxygen as a co-substrate and converts it into water. The results of EST and laccase amplification were also supported by the result of GCMS where acid and alcohols are formed. Various authors have used fungi, bacteria, plant, animal and microbial enzyme to study the biodegradation of Carbon nanotubes (CNTs), graphene (GRA), and their derivatives along with experimental and molecular simulation methods [43][44][45][46] showed that single-walled carbon nanotube (SWCNT) release would significantly affect the microbial enzyme-catalyzed processes of organic pollutants and lignin model compounds (LMCs) in nature. They found that microbial degradation appears to be the most promising practical application as compared with enzymatic degradation because enzymatic degradation strictly requires a suitable temperature and pH. If environmental conditions are not appropriate, the enzyme activity could be inhibited or disappear. The limitations for microbial degradation are relatively lower because microorganisms can grow under a variety of conditions 47,48 . Composting or addition of compost can simultaneously increase soil organic matter content and soil fertility besides bioremediation (pesticides, hydrocarbon and phenol), and thus it is believed to be one of the most cost-effective methods for soil remediation 47,49 explored laccase on molecular level for lignin degradation by using molecular docking and molecular dynamics (MD) simulations which provide detailed information about interaction mechanism between laccase and lignin. This is useful to develop new laccases with high lignin-degrading ability in the field of environmental protection and industrial applications.

Enrichment and isolation of cypermethrin-degrading Bacteria. Pesticide contaminated soil samples
were collected from the agricultural fields of Udham Singh Nagar, Uttarakhand, India. Enrichment and isolation of pesticide degrading bacterial strains was carried out in MSM (minimal Salt Medium) by using enrichment culture technique 17,18,50 . Bacterial colonies with different morphologies appeared on the plates were picked, purified and preserved on nutrient agar in refrigerator. One bacterial isolate showing maximum tolerance for cypermethrin was selected for further study.
Identification and Characterization of strain 1D. Cypermethrin-degrading bacterial isolate 1D was grown on Nutrient agar at 33 °C for 24 h and identified on the basis of morphological, biochemical, and molecular characters. Genomic DNA of the bacterial strain was extracted 51 . 16S rDNA gene was amplified using universal primers (27 f;5′AGAGTTTGATCMTGGCTCAG3′ and1492r:5′TACGGYTACCTTGTTACGACTT-3′). Amplified PCR product was run on agarose gel and sequenced by Biotech Centre, South Campus,Delhi University. Resulting 16S rDNA gene sequences were compared using BLAST program and phylogeny of the organism was deduced by MEGA 7.0 software 52 .

Inoculum preparation.
To test cypermethrin biodegradation under laboratory condition bacterial inoculum was prepared by growing the isolate in 50 mL Nutrient broth for 24 h at 33 °C under shaking condition at 120 rpm. After incubation, bacterial culture (2 mL) was transferred to MSM (50 mL) containing 20 ppm cypermethrin under aseptic conditions and allowed to grow for 20 days at 33 °C with shaking at 120 rpm. Samples were withdrawn after 10th and 15th day of incubation and residual pesticide was quantified by GC after extraction 17 .

Optimization of growth conditions of strain 1D for cypermethrin biodegradation. Box-behnken
design was explored to optimize the degradation conditions of cypermethrin using bacterial strain 1D. Box-behnken design consisting of 17 experimental runs with three replicates at the centre point was used to optimize the independent variables which significantly influenced cypermethrin biodegradation by Bacillus subtilis strain 1D. Three critical factors and their optimal ranges selected in this experiment for the analysis of cypermethrin biodegradation were; inoculum size (5, 10 and 15 mL), cypermethrin concentration (80,160 and 240 ppm) and shaking speed (80, 120 and 160) rpm. Experiment was conducted in minimal medium for 15 days (Table 3).
Chemical analysis. Extraction of cypermethrin from MSM. Supernatant was collected by centrifugation after 10 th , and 15 th day of bacterial growth in the presence of cypermethrin in minimal salt medium, Un-inoculated medium served as control. Two milliliter of MSM sample was centrifuged at 10,000 rpm for 10 min. Supernatant (1 ml) was transferred to Buchner funnel and mixed with sodium sulphate (1 g) and hexane (1 ml). After formation of two separate layers in separating funnel, bottom layer was discarded and upper layer was collected in a round bottom flask and evaporated completely in an evaporator at room temperature. To the left out of the pesticide, 2 ml hexane was added and mixed properly. After filtration, extracted solution was collected in the eppendorf tube and analyzed by GC.
Degradation products of cypermethrin in MSM containing 20 ppm of cypermethrin were determined by gas chromatography-mass spectrometry (GC-MS) equipped with auto-sampler, an on-column, split/ split less capillary injection system, and HP-5MS capillary column ( 30-500 nm (total scan). The operating conditions were as follows: the column was held at 80 °C for 5 min, ramped at 8 °C.min −1 to 200 °C (first ramp), held at 200 °C for 5 min, ramped at 15 °C.min −1 to 260 °C (second ramp), and then held at 260 °C for 5 min. The temperatures corresponding to transfer line and the ion trap were 280 °C and 230 °C, respectively, and the ionization energy was 70 eV. The injection volume was 1.0 mL with a split ratio of 1:7 at 260 °C. Helium was used as a carrier gas at a flow rate of 1.0 mL min −1 . The metabolic products of cypermethrin were matched with authentic standard compounds on the basis of mass spectrum by using library database.
Estimation of esterase and laccase enzyme in bacterial strain 1D. Laccase. Tryptone yeast extract medium (0.2% yeast extract and 0.2% tryptone, pH, 7.2) inoculated with 1.0% of 12-14 h old bacterial inoculum was incubated at 37 °C, 150 rpm for 120 h. After incubation, bacterial culture was centrifuged at 6,000 × g for 20 min at 4 °C to obtain pellets. Obtained bacterial pellets were washed with phosphate buffer (0.1 M; pH 6.5) containing 10 mM of phenylmethylsulfonyl fluoride (PMSF) to check protease activity in the supernatant before sonication (5 times, 45 s each time with 30 s between each sonication, 20 MHz). The cell extract obtained by centrifugation (14,000 × g) at 4 °C for 20 min was used as a source of crude intracellular laccase enzyme. In principle oxidation of guaiacol by laccase results into reddish brown color which is used to measure enzyme activity at 465 nm. Enzyme assay was performed 53 . A blank with 1 ml buffer instead of enzyme acted as control. One activity enzyme unit (U) was defined as the amount of enzyme that oxidizes 1 μmol of guaiacol per min at 25 °C and the activity is expressed in U/L. Esterase. One mL bacterial culture as grown for laccase was transferred to eppendorf tube and centrifuged at 8000 rpm for 10 min (4 °C). Obtained pellets were suspended in 100 mM potassium phosphate buffer (pH 7.5) and centrifuged at 8000 rpm for 15 min at 4 °C. Esterase activity was determined spectrophotometrically at 450 nm according to 40 . The rate of hydrolysis was expressed as micromoles of αnaphthol produced per minute at room temperature and the specific activity of the enzyme is expressed as micromoles of α-naphthol produced per minute per milligram protein at room temperature.
Kinetics of esterase and laccase by lineweaver burk model. The ability of strain 1D to degrade cypermethrin was investigated in the presence of cypermethrin by Lineweaver Burk quation for esterase and laccase.