Curr Microbiol DOI 10.1007/s00284-015-0852-4
Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated Pseudomonas aeruginosa sp. Strain JQ-41 Haihai Song1 • Zhiren Zhou1 • Yuanxiu Liu1 • Si Deng1 • Heng Xu1
Received: 22 January 2015 / Accepted: 28 April 2015 Springer Science+Business Media New York 2015
Abstract A soil bacterium designated strain JQ-41, capable of growth on fenpropathrin as the sole carbon source and energy source, was isolated from a long-term pyrethroid insecticide-treated orchard. Based on the morphology, physio-biochemical characteristics, and 16S rDNA gene analysis, as well as the G?C content of the genomic DNA, the strain JQ-41 was identified as Pseudomonas aeruginosa. Up to 92.3 % of 50 mg l-1 fenpropathrin was degraded by P. aeruginosa strain at 30 C and pH 7 within 7 days. The kinetic parameters qmax, Ks, and Ki were established to be 1.14 day-1, 38.41 mg l-1, and 137.67 mg l-1, respectively, and the critical inhibitor concentration was determined to be 72.72 mg l-1. Cell surface hydrophobicity of P. aeruginosa strain was enhanced during growth on fenpropathrin. Three metabolites from fenpropathrin degradation were identified by gas chromatography mass spectrometry, and then a possible degradation pathway was proposed. In addition, this isolate was also able to degrade a wide range of synthetic pyrethroid insecticides including cypermethrin, deltamethrin, bifenthrin, and cyhalothrin with the degradation process following the first-order kinetic model. Taken together, our results provide insights into the kinetics and mechanism of fenpropathrin degradation by P. aeruginosa strain and also Haihai Song and Zhiren Zhou have equally contributed to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s00284-015-0852-4) contains supplementary material, which is available to authorized users. & Heng Xu
[email protected] 1
Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, China
highlight its promising potential in bioremediation of pyrethroid-contaminated environment.
Introduction Over the last 40 years, the pyrethroid insecticides, a class of the pyrethrin analogues extracted from the flowers of Chrysanthemum species [26], have been widely used in agricultural, home and garden insecticides for their high efficiency, broad-spectrum, relatively low toxicity [11]. Fenpropathrin (a-cyano-3-phenoxybenzyl-2,2,3,3-tetramethyl cyclopropanecarboxylate) is one of the most popular pyrethroid insecticides, used for the prevention and control of agricultural pests and diseases on fruits, tea, vegetables and other crops [8]. However, continuous use of fenpropathrin has caused widespread environmental contamination problems. Recent reports showed that residues of fenpropathrin have been detected in nearly all the tested samples of agricultural zones and urban area [11, 13]. This situation also increased the potential of human exposure to the fenpropathrin which may damage to the reproductive system, nervous system, respiratory system, and immune system of human beings with developmental exposure [19, 29]. Therefore, the need for effective measures to remove fenpropathrin from environment is urgent. Microbial bioremediation is considered to be the most efficient and promising strategy compared to the conventional physicochemical methods including ozonation, photodecomposition, chemical washing and photodegradation, which are known for comparatively high cost and low efficiency [10, 25]. To date, a few fenpropathrin-degrading microorganisms such as Sphingobium cloacae [4], Clostridium celerecrescens [34], Ochrobactrum tritici [31], and
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H. Song et al.: Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated…
Candida peliculas [3] have been isolated. However, these isolates commonly degraded only single or a couple of pyrethroid insecticides, which may limit their use in bioremediation for pyrethroid combined pollution. In addition, to our knowledge, the assessments of the bacterial cell surface hydrophobicity (CSH) still have not been reported for any fenpropathrin-degrading bacteria by now. The CSH is one of the most important factors that govern bacterial adhesion, uptake and degradation of hydrophobic organic compounds [7]; the study of CSH could thus lay a theoretical basis for the bioremediation of organic pollution such as fenpropathrin in the environment. Furthermore, there was still little information available on biodegradation pathway and degradation kinetics of fenpropathrin at a wide range of different concentrations. The objectives of the present study were (1) to isolate and identify a promising degradation bacterium for the treatment of pesticide-contaminated environment; (2) to investigate the effects of abiotic factors (temperature and pH) on biodegradation of fenpropathrin; (3) to determine the kinetic parameters for the biodegradation of fenpropathrin and other pyrethroids; and (4) to elucidate the biodegradation mechanism of fenpropathrin by the strain.
Materials and Methods Isolation and Identification of Strain JQ-41 The strain JQ-41 was isolated from a long-term pesticidetreated orchard in Chengdu, China. 1 g of soil was inoculated on the Luria–Bertani (LB) medium and incubated at 30 C for 2 days. The well-isolated colonies were transferred into mineral salt medium (MSM) containing (NH4)2SO4 (1 g l-1), MgSO47H2O (0.5 g l-1), CaCl2 2H2O (0.05 g l-1), FeSO47H2O (0.005 g l-1), Na2HPO4 2H2O (1.25 g l-1), and KH2PO4 (1 g l-1), supplemented with 50 mg l-1 fenpropathrin at 30 C and 1809g in 250-ml conical flask for 7 days. The transfer was conducted five times successively until the fenpropathrin concentration gradually increased to 800 mg l-1. The eugenic colonies were picked out as the potential strains and their degrading abilities of fenpropathrin were monitored by high-performance liquid chromatography (HPLC). The strain JQ-41 showed highest degrading efficiency of fenpropathrin. Identification of strain JQ-41 was done according to Bergey’s Manual of Determinative Bacteriology [6]. The 16S rDNA gene was amplified by PCR using 27F and 1492R as primer set [17]. This sequence was then used as query for BLAST homology searches. Homology analysis was performed using Clustal X and Mega 4.1, a phylogenetic tree was constructed by the Neighbor-Joining method, and the
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dataset was bootstrapped 1000 times. The G?C content of the genomic DNA was determined by thermal denaturation using E. coli K-12 DNA as Ref. [16]. Degradation Experiment of Fenpropathrin The isolated strain was pre-cultured in 100 ml Luria–Bertani (LB) medium for 24 h, harvested by centrifugation (50009g for 5 min at 4 C) and washed twice with 0.9 % (w/v) sterile saline at pH 7. Colony-forming unit (cfu) of this suspension was quantified by the dilution plate count technique. Unless otherwise specified, 1 ml of this suspension (*5 9 107 cfu ml-1) was used as inoculums for all subsequent experiments. Triplicate cultures (100 ml) were grown in MSM supplemented with 50 mg l-1 fenpropathrin at 30 C and 1809g in 250-ml conical flask. Non-inoculated cultures served as controls. Samples were collected at 24 h interval for 7 days. The accumulation of bacterial cell cultures was evaluated by counting the colony-forming unit per liter of serial dilutions, and the amount of residual fenpropathrin was determined by HPLC. The effect of initial conditions on the degradation of fenpropathrin by strain JQ-41 was studied in batch cultures. Single factor test was designed in this study under different conditions of temperature (20, 25, 30, 35, 40 C), pH (5, 6, 7, 8, 9) and initial concentrations of fenpropathrin (50, 100, 200, 400, 600, 800 mg l-1). Residual fenpropathrin concentration was determined at 24 h interval for 7 days. Tests were run in triplicate. Biodegradation Kinetics of Various Pyrethroid Insecticides The strain was inoculated into 100 ml sterile MSM in 250-ml conical flask supplemented with 50 mg l-1 cypermethrin, deltamethrin, bifenthrin and cyhalothrin, respectively. Triplicate cultures were grown in MSM at 30 C and pH 7 for 7 days. Identification of Fenpropathrin Metabolites and Analysis of Biodegradation Pathway The cell-free extract of the cultures grown in MSM containing 50 mg l-1 fenpropathrin were collected at 24 h interval for 7 days. Then the cultures were acidified to pH 2 with 2 M HCl and extracted with ethyl acetate. The organic layer was dehydrated over anhydrous sodium sulfate, dried under nitrogen, and dissolved in the same volume of methanol [18]. After filtration with a 0.45 lm membrane, the samples were subjected to gas chromatography mass spectrometry (GC/MS) for detection and analysis of the metabolites.
H. Song et al.: Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated…
Evaluation of Bacterial Cell Surface Hydrophobicity
where C0 is the initial concentration of substrate (mg l-1), Ct is the concentration of remaining substrate at time t (mg l-1), and k and t are degradation rate constant (day-1) and the degradation period in days, respectively. The half-life (t1/2) of substrate was calculated by Eq. (4) [27].
CSH was determined based on the bacterial adherence to hydrocarbons (BATH) test [21]. The n-hexadecane was used for CSH determination of strain JQ-41. The concrete operation process of BATH had been described with some modifications [30]. The CSH value was calculated using Eq. (1).
t1=2 ¼
CSH ¼ ðA0 A1 Þ=A0 ;
where k is the degradation rate constant (day-1).
ð1Þ
lnð2Þ ; k
ð4Þ
where A0 represents the initial optical densities at 400 nm of the bacterial suspension, A1 is the final optical densities at 400 nm of the aqueous phase.
Nucleotide Sequence Accession Number
Chemical Analyses
The 16S rDNA gene sequence of P. aeruginosa strain was deposited at the GenBank, with the accession number registered as KM948588.
Pyrethroid quantification was analyzed by HPLC (Shimadzu LC-06A, Japan), using a Shimadzu SPDM10AVP photodiode detector and an Intersil ODS-SP C18 reversedphase column (5 lm 9 4.6 mm 9 150 mm). Elution was done with acetonitrile/water (90:10, v/v) at 1.0 ml min-1. Detection was carried out at 150–400 nm (total scan) based on retention time and peak area of the pure standard. The metabolites of fenpropathrin were identified on a GC/MS system (Shimadzu QP2010 Plus, Japan) equipped with an HP-5MS capillary column (30 m 9 250 lm 9 0.25 lm). The operating conditions were as follows: the column was held initially at a temperature of 80 C for 2 min, then at 5 C min-1 to 150 C for 1 min, at 10 C min-1 to 180 C for 4 min, and finally at 20 C min-1 to 260 C for 10 min. The temperatures corresponding to the transfer line and the ion trap were 280 and 230 C, respectively. The injection volume was 1 ll with splitless sampling at 250 C. Ultra-high purity helium was used as a carrier gas at a flow rate of 1.5 ml min-1. Kinectics Analyses Haldane-Andrews model (Eq. 2) was used to describe the specific degradation rate (q) at different initial fenpropathrin concentrations [24]. q¼
qmax S ; S þ Ks þ ðS2 =Ki Þ
ð2Þ
where qmax is the maximum specific fenpropathrin degradation rate (day-1), Ki is the substrate inhibition constant (mg l-1), KS is the half-saturation constant (mg l-1), and S is the inhibitor concentration (mg l-1). The first-order kinetic model (Eq. 3) was applied to study the biodegradation kinetics of fenpropathrin and other pyrethroids in MSM [12]. Ct ¼ C0 ekt ;
ð3Þ
Results and Discussion Identification and Characterization of Strain JQ-41 The strain JQ-41 was a Gram-negative, obligately aerobic, nonspore forming, and rod-shaped bacterium with dimensions of 1.5–4.0 lm in length and 0.7–1.0 lm in width. Colonies grown on LB agar plate were big, greenish yellow, roundish, smooth, and semitransparent with irregular margin. The physiological-biochemical properties of strain JQ-41 were summarized in Supplementary Table 1. PCR amplification of 16S rDNA gene from strain JQ-41 was obtained and completely sequenced (1414 bp, Supplementary Fig. 1). Analysis of the 16S rDNA gene sequences demonstrated that strain JQ-41 belonged to Pseudomonas species (Supplementary Fig. 2). The G?C content of strain JQ-41 was 65.8 mol%, which fell within the expected range for the genus Pseudomonas (58–70 mol%) [20] and was very similar to reported P. aeruginosa strains’ data [22, 32]. In conclusion, based on the morphology, physiobiochemical characteristics, and 16S rDNA gene analysis, as well as the G?C content of the genomic DNA, strain JQ-41 was identified as Pseudomonas aeruginosa. However, to our knowledge, there have been no reports of fenpropathrin biodegradation by P. aeruginosa strain. Utilization of Fenpropathrin for Growth by Strain JQ-41 The kinetics of fenpropathrin degradation and the growth of the strain on 50 mg l-1 fenpropathrin were investigated simultaneously in MSM. The strain degraded fenpropathrin rapidly with 74.5 % of the initial dose during the first 4 days. After incubation for 7 days, 92 % of the added
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H. Song et al.: Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated…
fenpropathrin was degraded by strain JQ-41 (Fig. 1). In contrast, no significant change was observed in cultures that were not inoculated. Fenpropathrin degradation was concerned with a concomitant increase of bacterial biomass. The isolate grew rapidly and reached its maximum within 3 day, and then it was obvious decreased as shown in Fig. 1. Additionally, the strain grew well on MSM with fenpropathrin without any other carbon source, showing its excellent environmental adaption. This is an important feature of a pesticide-degrading microorganism to be used for bioremediation of variable contaminated environments.
30 C. Nevertheless, the strain degraded 76.4 and 71.7 % of the fenpropathrin at 20 and 40 C, which still kept relatively high degradation rate. Figure 2b shows the most rapid removal of fenpropathrin by strain occurred at pH 7, followed by pH 6 and pH 8, with removal rates of 91.8, 83.3, and 78.4 %, respectively. However, at pH 5 or 9, degradation rates of fenpropathrin by the strain JQ-41 were both over 40 %. All of those results demonstrated that the strain was capable of rapidly degrading fenpropathrin over a wide range of temperature and pH values, which were significant characteristics of the microorganism to be employed for bioremediation.
Effect of Temperature and pH on Biodegradation Previous studies have two important abiotic biotics [2, 31]. Figure degradation rate (92.3
shown that temperature and pH are factors for biodegradation of xeno2a shows that highest fenpropathrin %) for strain JQ-41 was achieved at
Fig. 1 Utilization of fenpropathrin during growth of strain JQ-41. The strain was grown in mineral salt medium supplemented with 50 mg l-1 fenpropathrin as sole source of carbon. Values are means of three replicates with standard deviation
Fig. 2 Effect of temperature and pH upon degradation of fenpropathrin by strain JQ-41. a Effect of temperature on degradation of 50 mg l-1 fenpropathrin by the strain. b Effect of pH on degradation
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Effect of Different Initial Fenpropathrin Concentrations on Biodegradation Kinetic curves of fenpropathrin degradation at various initial concentrations ranging from 50 to 800 mg l-1 by the strain are shown in Fig. 3a. At low concentration of 50 and 100 mg l-1, the removal rate reached 91.6 and 89.5 % after 7 days of incubation, respectively. Noticeably, the isolate experienced a prolonged slow growth phase for utilizing fenpropathrin at higher initial concentrations (over 100 mg l-1), but was not completely inhibited even at concentration as high as 800 mg l-1. However, studies in the past reported that the fenpropathrin-degrading strains usually transform the pesticides with concentration lower than 200 mg l-1 [4, 34]. This feature gives the isolate a competitive advantage in diverse environments, especially exposed to a high concentration of fenpropathrin pollution. Figure 3b shows the relationship between specific degradation rate (q) and initial fenpropathrin concentration. The kinetic parameters qmax, Ks, and Ki of strain determined by nonlinear regression using matrix laboratory (MATLAB) software package (version 7.0) were 1.14 day-1, 38.41 mg l-1, and 137.67 mg l-1, respectively. The value of R2
of 50 mg l-1 fenpropathrin by the strain. Values are means of three replicates with standard deviation
H. Song et al.: Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated…
Fig. 3 Biodegradation of fenpropathrin by strain JQ-41 in mineral salt medium at different initial concentrations. a Degradation kinetics of fenpropathrin by the strain. Values are means of three replicates
with standard deviation. b Relationship between specific degradation rate and initial fenpropathrin concentration by the strain
was 0.977 indicating that the experimental data were fitted well with Haldane-Andrews model. The critical inhibitor concentration was established to be 72.72 mg l-1 showing that q value was gradually increased when the initial concentrations of fenpropathrin were below 72.72 mg l-1, and the inhibition of fenpropathrin would be remarkable at higher concentrations. These results suggested that the fenpropathrin degradation activity of strain JQ-41 could be partially inhibited at a high concentration of fenpropathrin but may not cause a complete repression.
Analysis of Fenpropathrin Biodegradation Metabolites
Characterization of Strain JQ-41 for Cell Surface Hydrophobicity The low water solubility of many hydrocarbons, especially the pyrethroids, has been believed to limit their availability to microbes. Nevertheless, some microorganisms have solved this problem by changing cell surface properties such as hydrophobicity [28]. The CSH of strain JQ-41 grown in 50 mg l-1 fenpropathrin at stationary phase was 68 %, while the CSH of fenpropathrin-free media was 24 %. This suggested that fenpropathrin induced the strain JQ-41 to modify the composition of the cell surface, leading to higher CSH. Fenpropathrin was thus considered to be adsorbed to the cell surface and further degraded. Therefore, the isolate JQ-41 with high hydrophobicity will be potentially useful in bioremediation of the environment polluted by fenpropathrin. Table 1 Chromatographic properties of metabolites of fenpropathrin by strain JQ-41
The fenpropathrin metabolites were extracted and detected by GC/MS. Each product in the light of mass spectrum was matched with authentic standard compound from the National Institute of Standards and Technology (NIST, USA) library database (Table 1). Our results showed three metabolites were present in the degradation process: ahydroxy-3-phenoxybenzeneacetonitrile [B], 3-phenoxybenzaldehyde [C], and 3-phenoxybenzoic acid [D] (Supplementary Fig. 3). Based on analysis of fenpropathrin and the metabolite compounds, the biodegradation pathway of fenpropathrin in P. aeruginosa strain was proposed (Supplementary Fig. 4). Fenpropathrin was originally metabolized by hydrolysis of its ester linkage to yield a-hydroxy3-phenoxybenzeneacetonitrile [B] and 2,2,3,3-tetramethylcyclopropanecarboxylic acid speculated on the basis of compound B, which was not detected in experiment. Then compound B was converted to 3-phenoxybenzaldehyde [C] for its instability in the environment. Subsequent oxidization of compound C produced 3-phenoxybenzoic acid [D]. However, these compounds were transient. At the end of experiment, no metabolite was detected by GC/MS indicating that the metabolite compounds could be further transformed and metabolized. Similar biodegradation pathways have been reported for other pyrethroids such as cypermethrin [14, 28], cyfluthrin [9, 23], and fenvalerate
Code
Retention time (min)
Molecular weight (MW)
Compound
A
24.317
349
Fenpropathrin
B
17.133
225
a-Hydroxy-3-phenoxy-benzeneacetonitrile
C
17.521
198
3-Phenoxybenzaldehyde
D
19.284
213
3-Phenoxybenzoic acid
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H. Song et al.: Kinetics and Mechanism of Fenpropathrin Biodegradation by a Newly Isolated… Table 2 Kinetic parameters of degradation of various pyrethroids by strain JQ-41 Pyrethroids
Regression equation
k (day-1)
t1/2 (days)
R2
Fenpropathrin
Ct = 48.13e-0.535t
0.535
1.30
0.972
Cypermethrin
Ct = 48.41e
-0.486t
0.486
1.43
0.952
Deltamethrin
Ct = 49.74e-0.351t
0.351
1.97
0.989
Bifenthrin
Ct = 45.86e-0.179t
0.179
3.87
0.946
Cyhalothrin
Ct = 48.97 e-0.238t
0.238
2.91
0.981
-1
Ct residual concentration of pyrethroid (mg l ), t degradation period (days), R2 correlation coefficient
Fig. 4 Degradation kinetics of various pyrethroids by strain JQ-41 with the initial concentration of 50 mg l-1. Values are means of three replicates with standard deviation
[15, 33]. It could be inferred that ester hydrolysis by carboxylesterases may be the major biodegradation pathway of pyrethroids in microorganism. Degradation Kinetics of Various Pyrethroid Insecticides The degradation kinetics of various pyrethroid insecticides by the strain are shown in Fig. 4. The strain utilized fenpropathrin, cypermethrin, deltamethrin, bifenthrin, and cyhalothrin as the growth substrates with the degradation rates of 91.7, 87.2, 90.4, 70.1, and 74.8 % within 7 days, respectively. The first-order model was used to represent the biodegradation kinetics of various pyrethroid insecticides by strain. The degradation kinetic parameters of various pyrethroid insecticides are presented in Table 2. The correlation coefficient R2 ranged from 0.946 to 0.989 indicating that the degradation data were well fitted with the model. A broad-spectrum degradation of pyrethroid insecticides by the strain JQ-41 may be attributed to the fact that its degrading enzymes have a relatively wide specificity for substrates. Nevertheless, it was noteworthy that the strain could degrade not only fenpropathrin but also other pyrethroids, which was rarely seen in other microorganisms [1, 5]. In conclusion, an efficient fenpropathrin-degrading strain P. aeruginosa has been isolated and characterized. And for all we know, this is the first report showing biodegradation of fenpropathrin by P. aeruginosa strain. What is more, the strain was capable to degrade a wide range of pyrethroid insecticides including cypermethrin, deltamethrin, bifenthrin, and cyhalothrin. CSH of P. aeruginosa strain was enhanced during growth on fenpropathrin. Three metabolites from fenpropathrin degradation were identified, and a possible degradation pathway
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was deduced. This study highlights the promising potential of the strain in bioremediation of fenpropathrin and other pyrethroid-contaminated environments. Further study on degradation mechanism of P. aeruginosa strain and its application in the filed-scale is underway. Acknowledgments This study was financially supported by the National High Technology Research and Development Program of China (No. 2013AA06A210), the National Natural Science Foundation of China (No. J1103518), and Chengdu Longquanyi District Science and Technology Bureau. The authors wish to thank Professor Guanglei Cheng and Dong Yu from Sichuan University for their technical assistance.
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