CN114908007A

CN114908007A - Rhodococcus pyridinivorans capable of degrading pyrethroid insecticides and application thereof

Info

Publication number: CN114908007A
Application number: CN202210468820.8A
Authority: CN
Inventors: 陈少华; 黄耀华; 吴小珍; 李嘉仪; 雷棋棋; 张文平; 余虹晓
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-08-16
Anticipated expiration: 2042-04-29
Also published as: CN114908007B

Abstract

The invention discloses Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) capable of degrading pyrethroid insecticides and application thereof. The strain is preserved in Guangdong province microorganism culture collection center at 21.7.2021, with the preservation number being GDMCC NO: 61812. the strain has remarkable biodegradability of pyrethroid insecticides, can efficiently degrade 9 different pyrethroid pesticides such as allethrin, beta-cypermethrin and the like, can completely degrade 50mg/L of D-cyphenothrin within 30 hours, and has the optimal temperature and pH of 31.76 ℃ and 7.85 respectively. After the strain is used for treating the contaminated soil containing 50mg/kg of D-cyphenothrin for 12 days, the biodegradation rate reaches 76.25%, and the degradation half-life period is 6.14 days, which shows that the strain also has efficient bioremediation effect on the contaminated soil. The invention enriches the germplasm resource library of pesticide degrading bacteria for solving the problem of residual pollution of the pyrethroid insecticides in agricultural production, and has important practical application value for producing nontoxic and pollution-free green agricultural products.

Description

Rhodococcus pyridinivorans capable of degrading pyrethroid insecticides and application thereof

Technical Field

The invention belongs to the technical field of microbial degradation. More particularly, relates to Rhodococcus pyridinivorans capable of degrading pyrethroid insecticides and application thereof.

Background

The pyrethroid is an analogue of natural pyrethrin (Pyrethrum) extracted from Chrysanthemum cinerariae aefolia folium, is a broad-spectrum pesticide, and has good killing effect on diptera and hymenoptera pests. At the same time, pyrethroids are a nerve poison, whose mechanism of action is mainly to cause repeated nerve discharges, affecting voltage-gated sodium and calcium channels. In addition, it can inhibit the function of gamma-aminobutyric acid (GABA) -gated chloride channels, thereby preventing the transmission of neurotransmitters between cells. In addition to neurotoxicity, pyrethroids also have reproductive and endocrine toxicity. Studies have shown that exposure of humans to pyrethroids is mainly via non-dietary routes (61%), but also ingestion (26%), inhalation (8%) and exposure (5%). Pyrethroids are highly hydrophobic and rapidly adsorb to the sediment after entering the water, some of which are then utilized by local microbes, and another part enters the aquatic food net and eventually accumulates in the body, possibly through bio-amplification.

Numerous results demonstrate that pyrethroids are highly toxic to aquatic organisms and it has been reported that mitochondrial/microsomal cytochrome P450 s in humans and insects are involved in the metabolism of pyrethroids in vivo. 3-phenoxybenzoic acid (3-PBA) is a representative intermediate metabolite of pyrethroids and has been widely detected in milk and human body fluids. A series of ecological problems due to the unreasonable use of pyrethroids has attracted public attention over the last decade.

The large-scale application of pyrethroids has caused ecological pollution of soil, sediments and surface water, and also caused severe indoor residues. After applying pyrethroids in the environment, as long as a small fraction of the active ingredient reaches the target organism, the majority remains on the plant and soil surface. The frequent use of pyrethroids has led to their parent and intermediary metabolites being widely detected worldwide, especially in bodies of water in urban areas. Cyfluthrin and cypermethrin are frequently detected in northern vietnam on fish and vegetable samples and exceed the daily allowable intake (ADI). To monitor pesticide contamination levels, the state of california, usa developed a city monitoring program. The results show that the pesticide content of the surface water of california is extremely high, with bifenthrin being detected the most frequently. In addition, freshwater crustaceans in major watersheds, california, were also investigated from which the emergence of pyrethroid resistance alleles was discovered at high frequency, suggesting the prevalence of drug resistance.

The development of pesticide degrading microorganisms for 30 years due to the unique advantages thereof has become an effective strategy for scientific research hotspot and controlling pesticide residue. Microorganisms can catabolize heterologous contaminants remaining in the environment into non-toxic or low-toxic inorganic small molecule compounds by various means.

Related technical contents for degrading pyrethroid by using pesticide degrading microorganisms are disclosed in the prior art, for example, Chinese patent ' a Microbacterium estericum and application thereof for efficiently degrading fenvalerate ' discloses a Microbacterium estericum capable of degrading fenvalerate ' and Chinese patent ' a Rhodococcus rhodochrous for efficiently degrading cyhalothrin and application thereof ' discloses a Rhodococcus rhodochrous capable of degrading cyhalothrin. However, the above-mentioned Microbacterium estericum and Rhodococcus rhodochrous have been disclosed to have a degrading effect on only one pyrethroid (fenvalerate or cyhalothrin), and the degrading effect is limited; and the degradation effect difference is large for fenvalerate or cyhalothrin with different concentrations.

Disclosure of Invention

The invention provides a new excellent strain capable of degrading pyrethroid insecticides, which has excellent degradation effect on various pyrethroid insecticides, the degradation rate of the D-cyphenothrin with the concentration of 50mg/L is 100% within 30 hours, the degradation rate of up to 7 pyrethroid insecticides is higher than 95% within 72 hours, and the degradation effect is stable. The excellent strain provided by the invention has good application value in the pollution and environmental protection of pyrethroid pesticides.

The first purpose of the invention is to provide a Rhodococcus pyridinivorans strain Y6 capable of degrading pyrethroid insecticides with high efficiency.

The second purpose of the invention is to provide the application of the strain Y6.

The third purpose of the invention is to provide a microbial inoculum for degrading pyrethroid insecticides.

A fourth object of the present invention is to provide a biological metabolic pathway for degrading D-cypermethrin by Rhodococcus pyridinovorans strain Y6.

The above purpose of the invention is realized by the following technical scheme:

rhodococcus pyridinivorans strain Y6 capable of degrading pyrethroid insecticides, which is deposited at 21.7.2021 in the microbial culture collection center of Guangdong province and has the deposit number of GDMCC No: 61812.

the strain is separated from activated sludge collected near a chemical plant in Guangdong province, and is identified as Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) through morphological characteristics, 16SrDNA phylogenetic analysis and whole genome analysis. An ultra-high resolution field emission scanning electron microscope (U-R SEM) shows that the strain Y6 is long rod-shaped at the early stage, is split into short rods or is approximately spherical at the middle and later stages, and has the length of about 0.5-2 mu m. After the strain Y6 is cultured on an LB agar plate for 48 hours, the bacterial colony is round and convex, has a smooth surface, is opaque and has neat edges. The physiological and biochemical test results are as follows: the reaction is tested to be positive by catalase, nitrate reduction test, urease test, esculin hydrolysis, lipase test, hydrogen sulfide test, fructose fermentation, glycerol fermentation, mannitol fermentation, saligenin fermentation and ribose fermentation; oxidase, starch hydrolysis, V-P determination, methyl red test, glucose fermentation and maltose fermentation test show negative reaction.

Further, the strain Y6 has the ability to significantly degrade various pyrethroid insecticides. Therefore, the application of the strain Y6 or the bacterial suspension thereof in degrading pyrethroid insecticides is within the protection scope of the invention.

Further, the application of the strain Y6 or its bacterial suspension in preparing products for degrading pyrethroid insecticides is also within the protection scope of the present invention. The product may be a bacterial preparation or other form of biological product involving the degradation of pyrethroid insecticides.

Further, the invention also provides application of the strain Y6 in repairing natural environment or artificial environment polluted by pyrethroid insecticides.

Preferably, the natural environment comprises a natural body of water and/or soil; the artificial environment includes one or more of an agricultural production area, an industrial production area, an urban greening area, and a residential area.

Further, the pyrethroid insecticide comprises one or more of d-cyphenothrin, tetramethrin, fenvalerate, permethrin, allethrin, permethrin, beta cypermethrin, prallethrin and deltamethrin.

Preferably, the pyrethroid insecticide comprises one or more of d-cyphenothrin, allethrin, beta-cypermethrin, fenvalerate, prallethrin, tetramethrin and permethrin.

Further preferably, the pyrethroid insecticide comprises one or more of d-cyphenothrin, allethrin, fenvalerate, prallethrin and tetramethrin.

Most preferably, the pyrethroid insecticide is d-cypermethrin.

An agent for degrading pyrethroid insecticides, which comprises the strain Y6, and is also within the protection scope of the invention.

Preferably, the bacterial number OD of the strain Y6 ₆₀₀ The value is not less than 1.04.

Based on the above, the method for degrading pyrethroid insecticides or treating and restoring the polluted environment by using the above fungicide also belongs to the protection scope of the invention.

In order to control better and more stable effect, the degradation or treatment conditions using the microbial inoculum are preferably controlled as follows: the temperature is 30-35 deg.C, and the pH is preferably 7-8.

Further, when the pyrethroid insecticide is degraded or the natural environment or the artificial environment polluted by the pyrethroid insecticide is repaired, the inoculation temperature of the strain Y6 is 30-35 ℃, the pH value is 7-8, and the thallus number OD of the strain Y6 ₆₀₀ The value is not less than 1.04.

Further preferably, the inoculation temperature of the strain Y6 is 30-32 ℃, the pH is 7.5-8, and the cell number OD of the strain Y6 ₆₀₀ The value is 1.04-1.1.

Most preferably, the inoculation temperature of the strain Y6 is 31.76 ℃, the pH value is 7.85, and the thallus number OD of the strain Y6 ₆₀₀ The value was 1.04.

Under the condition of a laboratory, Rhodococcus pyridinivorans Y6 is directly added into MSM liquid culture medium, and the degradation rate of 50mg/L of D-cyphenothrin can reach 100% within 30 hours.

The invention has the following beneficial effects:

(1) the invention provides a strain capable of efficiently degrading pyrethroid insecticides, namely Rhodococcus pyridinivorans strain Y6. The strain Y6 can grow by using pyrethroid insecticides such as D-cyphenothrin and the like as a unique carbon source, and can effectively degrade pyrethroid insecticides in a short time. The degradation rate of the fenpropathrin to the MSM liquid culture medium with the concentration of 50mg/L is 100 percent within 30 hours, the degradation rate of the allethrin, the beta-cypermethrin and the fenvalerate within 72 hours reaches 100 percent, the degradation rate of the prallethrin is 98.62 percent, the degradation rate of the tetramethrin is 97.94 percent, and the degradation rate of the permethrin is 93.07 percent. The strain is shown to have excellent pyrethroid insecticide degradation capability and good pyrethroid bioremediation capability.

(2) The strain Y6 can be used for repairing natural environments such as water bodies, soil and the like polluted by pyrethroid insecticides, and has efficient bioremediation effect on the polluted soil. Can be used for treating the problems of excessive pyrethroid insecticide residue and environmental pollution in agricultural production, thereby producing green and nuisanceless healthy agricultural products and protecting the ecological environment and human health.

(3) The invention provides a new idea for solving the problem of residual pollution of the pyrethroid insecticides in agricultural production, enriches the germplasm resource library of pesticide degrading bacteria, and has important practical application value for producing nontoxic and nuisanceless green agricultural products.

Drawings

FIG. 1 is a 16S rDNA phylogenetic analysis of strain Y6.

FIG. 2 is a colony morphology and scanning electron micrograph of strain Y6; wherein a is a scanning electron micrograph (2.5 multiplied by 10K); b is the form of strain Y6 on LB plates.

FIG. 3 is a design of a response surface for degrading D-cyphenothrin by strain Y6; wherein a is designed for a temperature response curved surface, and b is designed for a pH response curved surface; c is the inoculum response surface design.

FIG. 4 is a graph of the effect of temperature and pH interaction on the degradation of D-cypermethrin; wherein a is a response surface graph and b is an isotherm graph.

FIG. 5 shows the growth and degradation dynamics of strain Y6 during the degradation of D-cyphenothrin.

FIG. 6 shows the degradation ability of strain Y6 to other pyrethroid insecticides at other concentrations of 50 mg/L.

FIG. 7 shows the degradation ability of strain Y6 to other pyrethroid insecticides at a concentration of 200 mg/L.

FIG. 8 shows the degradability of strain Y6 for D-cyphenothrin at different concentrations.

FIG. 9 is an inhibition curve during the degradation of D-cyphenothrin at different concentrations by strain Y6.

FIG. 10 shows the ability of strain Y6 to degrade D-cyphenothrin in soil environment.

FIG. 11 is the biological metabolic pathway of strain Y6 to metabolize D-cyphenothrin.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The media formulations described in the examples below are as follows:

basal salt Medium (MSM, g/L): (NH) ₄ ) ₂ SO ₄ 2.0g；CaCl ₂ ·2H ₂ O 0.01g；FeSO ₄ ·7H ₂ O 0.001g；Na ₂ HPO ₄ ·12H ₂ O 1.5g；MgSO ₄ ·7H ₂ O 0.2g；KH ₂ PO ₄ 1.5g。

Luria-Bertani medium (LB, g/L): 5.0g of yeast extract; peptone 10.0 g; 10.0g of sodium chloride.

The above culture medium is prepared with distilled water, pH is 7.2, and sterilized in autoclave at 121 deg.C for 20 min.

Solid medium: 15g of agar powder was added per 1L of the medium.

Example 1 isolation and characterization of the strains

1. Isolation and screening of strains

Sample preparation: activated sludge collected from a chemical plant in Guangdong province.

The separation and screening method adopts an enrichment culture method, and comprises the following specific steps:

10g of an activated sludge sample is added into a 50mL basal medium, and meanwhile, a D-cyphenothrin mother solution (acetone is used as a solvent) is added to ensure that the final mass concentration of the D-cyphenothrin is 50mg/L, and the tolerant bacteria enrichment culture is carried out under a shaking table at 30 ℃ and 200 r/min. After 7 days of culture, the cells were inoculated into a second batch of MSM medium containing 100mg/L D-cyphenothrin at an inoculum size of 10%. After culturing for 7 days under the same conditions, transferring the strain into MSM culture medium containing the D-cyphenothrin with the concentration of 200mg/L according to the inoculation amount of 10 percent, and continuing culturing for 7 days. By analogy, the mass concentration of the d-cyphenothrin is continuously increased by times, and the enrichment culture is continuously carried out to 800 mg/L. Gradually diluting the enriched culture solution into diluents with different concentrations, uniformly coating 20 mu L of diluents to an MSM solid LB plate containing 50mg/L of d-cyphenothrin, culturing at 30 ℃ until a single colony grows out, repeatedly streaking the separated tolerant bacteria on an LB solid culture medium for 3 times to purify the strain, and finally preserving the strain by using 30% of glycerol at-80 ℃. The sample was then subjected to extraction treatment and its degradation effect was verified using High Performance Liquid Chromatography (HPLC).

The method is adopted to successfully separate and obtain the high-efficiency D-cyphenothrin degrading strain with the serial number of Y6 from the environmental sample, the strain can grow by using the D-cyphenothrin as a unique carbon source, and the degradation rate of the D-cyphenothrin with the concentration of 50mg/L in 30 hours reaches 100%.

2. Identification of strains

After culturing the strain Y6 obtained by separation on an LB solid plate at 30 ℃ for 48 hours, the colony is round and convex, and has smooth surface, non-transparent and neat edge. A super-high resolution field emission scanning electron microscope (U-R SEM) shows that the strain Y6 is long rod-shaped in the early stage, is split into short rods or approximate spheres in the middle and later stages, is about 0.5-2 mu m in length and is split in a propagation mode.

16S rDNA molecular biology identification:

extracting the genome DNA of the strain Y6, performing PCR amplification by using a 16S rDNA bacteria universal primer (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1429R: 5'-GGTTACCTTGTTACGACTT-3') by using the genome DNA as a template, and trusting a PCR product to perform sequencing by Jinwei Zhi (Guangzhou) Biotechnology limited company. And comparing and analyzing the 16S rDNA sequence measured by the strain in a GenBank database by using BLAST, selecting related sequences with higher homology, and constructing a phylogenetic tree and analyzing the evolutionary relationship by using MAGE software.

As shown in FIG. 1, the phylogenetic tree of 16S rDNA … shows that the similarity between the strain obtained by separation and purification of the invention and Rhodococcus pyridinivorans (PDB 9) strain can reach more than 99%. The colony morphology of strain Y6 and its scanning electron micrograph are shown in FIG. 2. And storing in Guangdong province microorganism culture collection center at 21/7/2021, wherein the preservation number is GDMCC No. 61812, and the preservation address is: guangzhou city, first furious Zhonglu No. 100 large yard No. 59 building No. 5.

Physiological and biochemical characteristics:

on the basis of the results of rDNA sequencing analysis of strain Y616S, the biochemical test contents related to Rhodococcus in Berger's Manual of identification of bacteria (eighth edition) in 1984 were referred to, and the following items were selected for physiological and biochemical tests, and the specific results are shown in Table 1. The strain Y6 is a gram-positive bacterium, aerobic, positive in the reactions of catalase, nitrate reduction test, urease test, esculin hydrolysis, lipase test, hydrogen sulfide test, fructose fermentation, glycerol fermentation, mannitol fermentation, saligenin fermentation and ribose fermentation test; the test shows negative reaction in oxidase, starch hydrolysis, V-P measurement, methyl red test, glucose fermentation and maltose fermentation test. According to the results of physiological and biochemical tests, the results were compared with Berger's Manual of identification of bacteria (eighth edition). Strain Y6 was identified as Rhodococcus pyridinivorans (Rhodococcus pyridinivorans) based on morphological, 16S rDNA and physiological and biochemical characterization.

TABLE 1 physiological and biochemical tests of Strain Y6

Physiological and biochemical test	Results	Physiological and biochemical experiment	Results
				Anaerobic growth	-	Methyl Red test	-
Contact enzyme	+	Lipase test	+
				Oxidases	-	Fructose fermentation	+
V-P assay	-	Glycerol fermentation	+
				Starch hydrolysis	-	Maltose utilization	-
Nitrate reduction	+	Fermentation of mannitol	+
				Citrate utilization	+	Fermentation of saligenin	+
Hydrolysis of esculin	+	Fermentation of glucose	-
				Urease	+	Ribose fermentation	+

Example 2 optimization of the optimal degradation conditions for Strain Y6

1. Experimental method

The Box-Behnken design based on the response surface technology (RSM) was used to optimize the d-cyphenothrin degradation conditions of strain Y6. Based on the preliminary results of the single-factor experiment, three independent variables were selected, respectively temperature (X) ₁ )，pH(X ₂ ) And inoculum size (X) ₃ ) To predict the optimal degradation parameters of strain Y6 for D-cyphenothrin (Table 2). D-cyphenothrin (Y) ₁ ) The residual amounts were obtained by HPLC analysis and represent the effect of three variables at different levels on strain Y6.

TABLE 2 three independent variables based on Box-Behnken design and their coding and uncoding levels

2. Results

Table 3 lists the experimental design variables corresponding to the degradation of d-cyphenothrin. In Design Expert software, polynomial regression analysis is carried out on relevant degradation data through a response curve, and the experimental value of the degradation of the D-cyphenothrin is fitted to obtain the following quadratic polynomial regression equation:

Y ₁ ＝100.00+1.49X ₁ +3.36X ₂ –0.21X ₃ –1.86X ₁ X ₂ +0.55X ₁ X ₃ –0.16X ₂ X ₃ –3.05X ₁ ² –9.53X ₂ ² –2.6X ₃ ²

wherein: x ₁ As the correlation factor temperature, X ₂ Is the pH value, X ₃ For inoculation amount, Y ₁ Is the response value.

Table 3 Box-Behnken experiment design matrix and corresponding degradation rate of d-cyphenothrin

Note: y is ₁ The degradation rate (%) of the D-cyphenothrin is shown; x ₁ 、X ₂ And X ₃ Respectively, the encoding values of temperature, pH and inoculum size; y is ₁ Values are mean ± s.e., letters after the value indicate that there is no significant difference at the 5% level between having the same letter sequence (DMRT method).

Table 4 gives the analysis of variance (ANOVA) of the quadratic polynomial model. Note that the F value of the model term is 36.75 and the P value is<0.0001, indicating that the equation fits very significantly to the degradation process of d-cyphenothrin. The statistical analysis result shows that the coefficient of determination R is ² 0.9793, indicating that the actual values of this experiment are highly fitted to the predicted values of the model. In addition, the correction decision coefficient R of the model ² 0.9526, and a lower coefficient of variation (c.v. ═ 1.45%) also indicate that the model is accurate and reliable. The regression analysis results show that the temperature (X) ₁ ) And pH (X) ₂ ) The single factor term and the square term of (2), namely X ₁ 、X ₂ 、X ₁ X ₂ 、X ₁ ² And X ₂ ² Has significant effect on degrading the D-cyphenothrin by the strain Y6 (P)<0.05) (fig. 3). Wherein, the square term X ₂ ² Has extremely significant influence level on the degradation of the D-cyphenothrin by the strain Y6 (P)<0.0001). And the size of the inoculum size (X) ₃ ) And amount of inoculation and pH (X) ₂ ) The interaction term (a) had no significant effect on the metabolic activity of strain Y6 (P)>0.05)。

TABLE 4 Dexpanthemum cyphenothrin degradation Process fitting quadratic polynomial model and analysis of variance (ANOVA)

Note: p <0.0001 indicates that this factor has a very significant effect on the strain.

In order to present the effect of various factors and their interactions on the metabolism of D-cyphenothrin by strain Y6 in a more intuitive multidimensional visual model, the inoculum size (X) ₃ ) The code value is fixed at 0, i.e. OD ₆₀₀ The value is 1.0, and a three-dimensional response surface plot and a contour plot are plotted, as shown in fig. 4. The maximum theoretical value of d-cyphenothrin degradation in the response surface plot was 100.23%, which corresponds to the actual value of strain Y6. And three variables X ₁ ，X ₂ And X ₃ The optimal levels of (a) were 0.176, 0.282 and 0.085 at the encoded level, i.e. the optimal temperature and pH at the unencoded level were 31.76 ℃ and 7.85, respectively, and the OD600 value of the inoculum was 1.04.

EXAMPLE 3 Effect of Strain Y6 on the degradation of pyrethroid insecticides

1. Experimental methods

(1) Preparation of inoculated bacteria: the strain Y6 purified in example 1 was inoculated into 10mL of LB liquid medium and activated overnight until logarithmic phase (24-48 hours), OD was adjusted to 1.0, 1mL of the bacterial solution was centrifuged at 6000rpm for 3 minutes, and then the cells were washed twice with sterile physiological saline (0.9% NaCl) to obtain an inoculum.

(2) And (3) determining the degradation performance: and respectively transferring the resuspended bacterial liquid into 50mL of MSM culture medium containing 50mg/L of D-cyphenothrin, tetramethrin, fenvalerate, permethrin, allethrin, permethrin, beta-cypermethrin, prallethrin, deltamethrin, flumethrin and cyhalothrin, continuously culturing at 30 ℃ and 200rpm, repeating the experiment for 3 times, taking the MSM culture medium which is not inoculated as a blank control, and measuring the residual quantity of each pyrethroid by HPLC.

An ultraviolet/visible spectrophotometer was used to monitor the growth of strain Y6 in MSM medium containing d-cyphenothrin at 600nm, and the degradation of d-cyphenothrin by strain Y6 was analyzed by a primary kinetic model:

C _t ＝C ₀ ×e ^-kt

in the formula, C ₀ Is the initial concentration (mg/L) of D-cyphenothrin, C _t Is the amount of D-cyphenothrin at time t, k is the degradation constant (h) ^-1 ) And t is the degradation time (h).

The following formula was used to calculate the theoretical half-life (t) of D-cyphenothrin _1/2 )：

t _1/2 ＝ln(2)/k

Where ln (2) is the natural logarithm of 2 and k is the degradation constant.

(3) Chromatographic conditions are as follows: with a device equipped with Phenomenex C ₁₈ The various classes of pyrethroids were quantified on a reverse phase chromatography column (250 nm. times.4.60 mm, 5 μm) and a Waters 2690HPLC system with UV detector. The mobile phase consisted of acetonitrile and deionized water (75:25) at a flow rate of 1.0mL min ^-1 . The sample injection amount and the detection wavelength were 10. mu.L and 253nm, respectively. The retention times of the D-cyphenothrin, the tetramethrin, the fenvalerate, the permethrin, the allethrin, the permethrin, the beta cypermethrin, the prallethrin, the deltamethrin, the flumethrin and the Meperfluthrin are 13.229, 6.801, 13.579, 14.302, 7.304, 11.127, 11.473, 5.559, 12.785, 21.393 and 9.706min respectively.

The degradation rate of each type of pyrethroid was calculated according to the following formula: percent degradation (%) - (1-A) ₁ /A ₀ )×100，

Wherein: a. the ₁ The amount of pyrethroid remaining after treatment with strain Y6, A ₀ As a control for the amount of pyrethroid remaining after treatment.

Quality control: and correcting the standard substance by adopting an external standard method to prepare a standard curve.

2. Results of the experiment

The growth and degradation process of strain Y6 in MSM medium with D-Cyanuric ether as growth substrate is shown in FIG. 5. The strain Y6 shows rapid growth in the early stage (0-6 h), which shows that no lag phase exists in the growth process of the strain Y6, and can effectively utilize the D-cyphenothrin and degrade 77.51% of 50mg/L D-cyphenothrin in 6 h. By 24 hours of culture, cell growth reached a peak and then began to decline, indicating that after d-cyphenothrin was mostly metabolized, population numbers of strain Y6 began to decline due to lack of a growth energy source. After 30 hours of treatment, strain Y6 completely degraded the initially added 50mg/L D-cyphenothrin. Notably, the MSM medium contained no additional carbon source other than d-cyphenothrin as the sole source of carbon and energy, indicating that strain Y6 was able to grow with d-cyphenothrin as the sole source of energy, as well as metabolize d-cyphenothrin. The growth log phase of the strain Y6 is 24-48 h, the strain can completely metabolize the D-cyphenothrin within 30h, and the strain Y6 has a large degradation space. As shown in Table 5, the kinetic parameters for the degradation of D-cyphenothrin for strain Y6 indicate that the degradation process of D-cyphenothrin by strain Y6 follows a first order kinetic model. The degradation constants (k) for strain Y6 and the control were 0.1114 and 0.0012, respectively. The theoretical half-life (t) of D-cyphenothrin was calculated for strain Y6 treatment and control, respectively, by the formula described in 2.3.1 _1/2 ). Correlation coefficient (R) between Strain Y6 and control ² ) 0.9643 and 0.7627, respectively, indicate that the degradation data fit well with the first order kinetic model. Theoretical half-life (t) of D-cyphenothrin degradation by strain Y6 _1/2 ) 6.22 hours, significantly shorter than 577.62 hours for the non-inoculated control, i.e., 24 days.

TABLE 5 kinetic parameters of Strain Y6 treatment of D-Cypermethrin in MSM Medium

Note: c _t The degradation rate (mg/L) of the D-cyphenothrin; k is a degradation rate constant; t is t _1/2 Is the treatment time; r is ² To determine the coefficients.

To investigate the degradation potential of strain Y6 in various stress environments, a test of the ability of strain Y6 to degrade different pyrethroid insecticides was tested. After shaking culture for 72h in an incubator protected from light, samples were collected and analyzed. As shown in FIG. 6, the degradation rates of allethrin, beta-cypermethrin and fenvalerate were the highest and reached 100% at a concentration level of 50 mg/L. The second is prallethrin 98.62%, tetramethrin 97.94% and permethrin 93.07%. Then, the degradation rates of permethrin and deltamethrin were 60.93% and 50.10%, respectively. The lowest degradation occurred in both meperfluthrin and flumethrin with a degradation rate of 0 (data not shown in figure 6).

The results are shown in FIG. 7, where the degradation rate of strain Y6 to some pyrethrins decreased significantly after increasing the concentration level to 200mg/L, especially the degradation rate of permethrin and deltamethrin decreased rapidly from 61.78 and 50.13% to 0 and 17.61%, respectively. The inhibition effect of the permethrin on the strain Y6 is obviously enhanced along with the increase of the concentration, and the inhibition effect of the permethrin is second. The degradation rates of permethrin and beta-cypermethrin showed a nearly 18% decrease with increasing concentration, while allethrin, tetramethrin, prallethrin and fenvalerate were not significantly changed. The ability of strain Y6 to degrade different pyrethroid insecticide pesticides indicates that the strain has the potential and advantages of removing pyrethroid residues from complex environments.

Example 4 degradation concentration test of Strain Y6 on D-Cyanuthrin

1. Experimental methods

The strain Y6 purified in example 1 was inoculated into 10mL LB liquid medium overnight for activation culture to logarithmic phase (24-48 hours), and OD was adjusted ₆₀₀ The value is 1.0, 1mL of the bacterial liquid is taken outAfter the heart (6000rpm, 3 minutes), the cells were washed twice with sterile physiological saline (0.9% NaCl). Then, the cells were inoculated into 50mL of MSM medium containing D-cyphenothrin (100, 200, 300, 400, 500, 600, 700 and 800mg/L) at different concentrations, and each set was repeated three times without inoculation as a control. The culture was carried out at 30 ℃ and 200rpm with a constant temperature shaking table for 48 hours, during which samples were taken periodically and HPLC was used to determine the degradation of D-cyphenothrin at different concentrations. Since the pyrethroid insecticide can be used as a growth substrate of the strain Y6 and can also be used as a growth inhibitor, the Andrews equation is adopted to fit the process of degrading the D-cyphenothrin with different concentrations by the strain Y6. The equation is as follows:

wherein: s is the concentration (mg/L) of the d-cyphenothrin; q is the specific degradation rate (h) of D-cyphenothrin ^-1 )；q _max Is the maximum specific degradation rate (h) of d-cyphenothrin ^-1 )；K _s Is a half rate constant (mg/L); k _i Is the inhibition coefficient (mg/L) of the D-cyphenothrin to the strain Y6.

2. Results of the experiment

The initial concentrations of the mother solutions of D-cyphenothrin were 100, 200, 300, 400, 500, 600, 700 and 800mg/L, respectively, in flasks containing 50mL of sterile MSM medium. As shown in FIG. 8, strain Y6 was able to rapidly utilize D-cyphenothrin at concentrations of 100 and 200mg/L, and completely metabolize D-cyphenothrin within 48 hours; when the concentration was increased to 300mg/L to 800mg/L, it was observed that the strain Y6 exhibited a slower degradation of D-cypermethrin, but still maintained a higher degradation rate. In particular, within 12 hours, strain Y6 rapidly metabolized about 50% of the d-cyphenothrin without significant lag phase. After 72 hours of treatment, the degradation rate of the D-cyphenothrin with the concentration of 300mg/L reaches about 97 percent, and the degradation rates of the treatment groups with the concentrations of 400-800 mg/L are respectively 83.92, 83.40, 78.83, 79.74 and 71.53 percent. The strain Y6 can still rapidly degrade the D-cypermethrin at the concentration of 800mg/L and can grow as the only carbon source and energy source. This indicates that strain Y6 can be a valid candidate in the area contaminated with D-cyphenothrin.

The test shows that the degradation rate of the strain Y6 is relatively reduced along with the increase of the concentration of the D-cyphenothrin, which indicates that the D-cyphenothrin can be used as a substrate for the growth of the strain Y6 in the bioremediation process, and can also play a role in inhibiting the growth of the strain at high concentration. The Andrews equation was therefore used in this test to simulate the kinetic analysis of the biodegradation of d-cyphenothrin at different concentrations. And processing experimental raw data through Matlab software, and performing nonlinear fitting on the raw data according to an Andrews equation. As shown in FIG. 9, the model has a high degree of fitting between the theoretical value and the actual value (R) ² 0.9093) where the maximum specific degradation rate (Q) of d-cyphenothrin _max ) Is 0.8095h ^-1 (ii) a Half rate constant (K) _s ) 568.7754 mg/L; inhibition coefficient (K) _i ) It was 18.7006 mg/L. Q can then be obtained by further derivation of the Andrews equation _max Corresponding concentration of D-cyphenothrin (S) _max ) The concentration is 103.1329mg/L, which is the best predicted concentration value of the strain Y6 for degrading the D-cypermethrin.

Example 5 soil remediation experiment

1. Soil sample for testing

The soil sample of the experiment is collected from surface soil with a depth of 3-20 cm in the vegetable field without pesticide use record within 5 years of southern China agricultural university. The physical and chemical parameters of the soil sample are as follows: the organic content is 10.5 g/kg; nitrate ion content 20 mg/kg; the effective phosphorus content and the effective potassium content are respectively 37.5mg/kg and 105 mg/kg; the conductivity and pH value of the soil are 375 mu S/cm and 6.9 respectively; the soil consisted of 65.0% sandy soil, 28.0% loam and 7.0% clay.

Soil samples were air dried naturally in the room and sieved (2mm) for the next bioremediation simulation study. A portion of the soil was autoclaved at 121 ℃ for 1 hour to completely remove indigenous microbial communities. Then, 250g of sterile and non-sterile soil samples were separately filled into 500mL Erlenmeyer flasks. In thatEach flask was charged with a solution of d-cyphenothrin to a final concentration of 50 mg/kg. And adjusting the water content of the soil to 20-40% by using sterile deionized water. The strain Y6 isolated in example 1 was inoculated into a liquid LB medium and cultured for 24 hours, and OD was adjusted ₆₀₀ To a value of 1.0, 5mL of bacterial suspension was inoculated into sterile and non-sterile soils, respectively, and the experiment was repeated three times with an uninoculated soil sample as a control. After mixing well, the treatment group and the blank group were placed in a climatic chamber and subjected to soil remediation experiments at 30 ℃ in the dark. Subsequently, 10g of soil samples were collected at 2, 4, 6, 8, 10 and 12d for extraction at regular intervals of 1 day.

The soil extraction method comprises the following steps: 10g of sample was added to a 50mL centrifuge tube and 10mL of acetone was added. The tube was vortexed for 10s and then sonicated for 20 min. Then, 30mL of ethyl acetate was added to the centrifuge tube and vortexed for 2 min. The tube was kept at room temperature for about 30min until the aqueous and organic phases separated. The upper organic phase was then removed of water by anhydrous sodium sulfate and transferred to a 250mL flat bottom flask, and the original 50mL centrifuge tube was discarded. Finally, the flat-bottomed flask containing the organic phase was placed in a water pump circulation type vacuum concentrator for concentration. 10mL of chromatographic acetonitrile was added for vortex recovery. The recovered sample was further purified by taking 1mL of the recovered solution using a 1mL syringe and a 0.22 μm filter and stored in a brown bottle at 4 ℃ before HPLC detection.

2. Results of the experiment

As shown in fig. 10, the soil remediation experiment was conducted using sterilized soil and unsterilized soil as two groups, and unsterilized soil as a control. The degradation rates for the sterilized and unsterilized treatments were 76.25 and 67.33%, respectively, after 12 days of continuous treatment with strain Y6, while the natural degradation rate for the sterilized and unsterilized controls was about 30%. The strain Y6 accords with a first-order kinetic model in the soil degradation process, and the degradation rate constants (k) of the sterilized and the non-sterilized treatments are 0.1129 and 0.0900d respectively ^-1 (ii) a Degradation half life (t) _1/2 ) 6.14d and 7.70d, respectively. Specific kinetic parameters are shown in table 6. In addition, no significant lag phase was observed during the experiment. In the case of the non-inoculated control, the degradation rate constants of the sterilized and non-sterilized treatments were given0.0309 and 0.0307d-1, respectively; the degradation half-life is 22.43 d and 22.58d respectively. The strain Y6 is proved to play a biological enhancement role in bioremediation of pyrethroid-polluted sites. Research results show that the strain Y6 can also exert high-efficiency degradation capability in soil, and can be used for restoring application of pyrethroid-polluted soil or water body.

TABLE 6 degradation kinetics parameters of Strain Y6 in D-Cypermethrin contaminated soil

Treatment of	Regression equation	k	R ²	t _1/2
					Sterilized soil + Y6	C _t ＝51.6933e ^-0.1129t	0.1129	0.9234	6.1395
Unsterilized soil + Y6	C _t ＝50.1078e ^-0.0900t	0.0900	0.9716	7.7016
					Sterilized soil	C _t ＝50.3710e ^-0.0309t	0.0309	0.9369	22.4319
Unsterilized soil	C _t ＝50.5159e ^-0.0307t	0.0307	0.8658	22.5781

Note: k represents a degradation constant; r ² Is the decision coefficient; t is t _1/2 Refers to the degradation half-life (h).

Example 6 Biometabolic pathway of Strain Y6 on D-Cypermethrin

1. Experimental methods

To study the metabolic pathway of strain Y6 during d-cyphenothrin degradation, samples were collected from MSM medium at regular intervals at 6, 12, 18, 24 and 30 hours, respectively. The concentrate was finally recovered using chromatographic methanol and passed through a 0.22 μm organic phase filter, and metabolites were detected by gas chromatography-mass spectrometry (GC/MS) (Agilent6890N/5975, USA) and the results were compared to standard compounds in the National Institute of Standards and Technology (NIST) database.

Gas chromatography coupled analysis method: metabolites of D-cyphenothrin were identified in an Agilent6890N/5977B GC/MS system equipped with an autosampler, a split/no-split capillary sampling system and an HP-5MS quartz capillary column (30.0 m.times.250 μm.times.0.25 μm) and an array detector. High-purity helium (purity is more than or equal to 99.99%) is used as carrier gas, and the flow rate is 1.5 mL/min ^-1 . The analysis mode is set as scanning within the range of 30-500 nm. Temperature rising procedure: the column temperature was first maintained at 90 ℃ for 2 minutes at 6 ℃ min ^-1 Rising to 150 deg.C for 1min, and then maintaining at 10 deg.C/min ^-1 Increasing the temperature to 180 DEG CHolding for 4min, and finally heating at 20 deg.C/min ^-1 It was kept at 260 ℃ for 10min (Table 7). MS transmission line temperature: 280 ℃; ion source temperature: 230 ℃; quadrupole temperature: 150 ℃; ionization voltage: 70 eV; sample introduction amount: 1.0. mu.L.

TABLE 7 GC-MS temperature program

Temperature Rate (. degree.C.. min) ^-1 )	Temperature (. degree.C.)	Retention time (min)	Cumulative time (min)
				-	90	2	2
6	150	1	13
				10	180	4	20
20	260	10	34

2. Results of the experiment

Based on the product identification result of the GS-MS on the D-cyphenothrin, the metabolic pathway of the D-cyphenothrin is analyzed. The metabolic pathways are shown in FIG. 11. Strain Y6 was first hydrolyzed by cleaving the carboxylic ester bond to give alpha-hydroxy-3-phenoxyphenylacetonitrile and chrysanthemi nol. The intermediate product alpha-hydroxy-3-phenoxybenzyl cyanide has poor stability in the environment and is quickly converted into 3-phenoxybenzyl alcohol. Subsequently, 3-phenoxybenzyl alcohol is further oxidized to m-phenoxybenzaldehyde. Under the action of the hydrolase of the strain Y6, the m-phenoxybenzaldehyde is further degraded to form phthalic acid and phenol. At the same time, chrysanthemic alcohol is oxidized to chrysanthemic aldehyde, which is then further oxidized to chrysanthemic acid. After 48 hours of degradation, the intermediate product of d-cyphenothrin is eventually degraded into non-toxic small molecules, such as water and carbon dioxide.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. Rhodococcus pyridinivorans strain Y6 capable of degrading pyrethroid insecticides, wherein strain Y6 is deposited at 21.7.2021 in the culture Collection of microorganisms of Guangdong province, and the deposit number is GDMCC No: 61812.

2. the use of strain Y6 or a bacterial suspension thereof according to claim 1 for degrading pyrethroid insecticides.

3. The use of strain Y6 or a bacterial suspension thereof according to claim 1 for the preparation of a product for degrading a pyrethroid insecticide.

4. The use of the strain Y6 or a bacterial suspension thereof according to claim 1 for remediating a pyrethroid insecticide contaminated natural or artificial environment.

5. The use according to claim 4, wherein the natural environment comprises a natural body of water and/or soil; the artificial environment includes one or more of an agricultural production area, an industrial production area, an urban greening area, and a residential area.

6. The use of any one of claims 2 to 5, wherein the pyrethroid insecticide comprises one or more of d-cyphenothrin, tetramethrin, fenvalerate, permethrin, allethrin, permethrin, beta cypermethrin, prallethrin and deltamethrin.

7. A bacterial formulation useful for degrading pyrethroid insecticides comprising the strain Y6 of claim 1.

8. The microbial inoculum according to claim 7, wherein the bacterial count OD of the strain Y6 ₆₀₀ The value is not less than 1.04.

9. A method for degrading pyrethroid insecticides or remediating the environment polluted by the pyrethroid insecticides, characterized by treating with the microbial inoculum of claim 7 or 8.

10. The method of claim 9, wherein the processing conditions are controlled to: the temperature is 30-35 deg.C, and pH is 7-8.