Klebsiella oxytoca PL1 and application thereof in degradation of pretilachlor
By developing Klebsiella acid-producing PL1 strain to prepare biorepair bacteria, the residual pollution problem of primrose in soil and water bodies was solved, efficient and low-cost pollution repair effect was achieved, and the ecological environment and crop growth were protected.
Patent Information
- Application Number
- CN202510582550.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art is difficult to effectively remove residual pollution of puramyl in soil and water, especially in low temperature and low microbial activity, and its degradation time may be extended, resulting in potential harm to the subsequent crops.
A PL1 strain of Klebsiella acid-producing bacteria was developed. By preparing biorepair bacterial agents, it applied to soil or water contaminated by ceramide, and used the degradation ability of the strain to convert ceramide into harmless products. The composition of the biorepair bacterial agents includes fermentation broth, seedling soil, activated carbon, soybean meal, potassium humate and PVAL binder.
In a short period of time, the degradation rate of proxolamine was significantly improved, the ecological environment and human health were protected, and the degradation rate reached 78.57%. At the same time, it had no toxic side effects on crop growth and reduced costs.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and particularly relates to Klebsiella oxytoca PL1 and its application in degrading pretilachlor. Background Art
[0002] Pretilachlor, with the chemical name of 2-chloro-2',6'-diethyl-N-(2-propoxyethyl)acetanilide, is a widely used selective herbicide mainly used to control gramineous weeds and is widely applied to the control of field weeds in crops such as rice, corn, and soybeans. The mechanism of action of pretilachlor is to inhibit the synthesis of fatty acids in weeds, especially during the formation of plant cell membranes. By interfering with the biosynthesis of fatty acids, it further affects the growth and development of plants. Pretilachlor is absorbed quickly in plants, especially showing strong activity at the seedling stage, and can effectively control the germination and growth of weeds. However, the long-term use of pretilachlor will lead to its residue in the soil. Especially in a soil environment with strong water solubility, it is easy to migrate with water, causing soil pollution and potential harm to subsequent crops.
[0003] With the increasing requirements for environmental safety, the pollution problems of pretilachlor in soil and water have attracted increasing attention. The half-life range of pretilachlor is relatively wide, and depending on the environment and soil type, its half-life ranges from 10 days to several months. For example, a study pointed out that pretilachlor degrades relatively quickly in humid tropical soils with high microbial activity, and its half-life is about 15 to 30 days (Babu et al. , 2015). However, in soils with low temperature and low microbial activity, its degradation time will be prolonged, possibly reaching 60 days or even longer (Yu et al. , 2007). Therefore, how to effectively remove pretilachlor residues and repair polluted soil has become an urgent issue to be solved in current agricultural environmental management.
[0004] Bioremediation is a technology that uses microorganisms (such as bacteria, fungi) or plants to decompose or transform harmful compounds in soil and water. Research shows that pretilachlor can be degraded through the metabolic actions of various microorganisms. In particular, certain bacteria, fungi, and algae can decompose pretilachlor through hydrolysis, redox reactions, dealkylation, etc., and transform it into non-toxic or low-toxic products. For example, Bacillus sp. , Sphingomonas sp. and Pseudomonas sp. and other bacteria have been found to be able to effectively degrade pretilachlor and show strong degradation ability in the repair of polluted soil (Kumar et al. , 2011; Choi et al. , 2005). During the microbial degradation process, the chlorine atoms of pretilachlor are removed, and through chemical reactions such as hydrolysis and dealkylation, it is transformed into non-toxic intermediate products. For example, Rhodococcus sp. Pretilachlor is degraded through hydrolysis and redox reactions and ultimately transformed into harmless products (Goh et al. , 2009). These studies indicate that microorganisms reduce the residual pollution of pretilachlor in the soil by degrading it.
[0005] Therefore, it is of great significance to explore pretilachlor removal solutions based on microbial degradation technology, especially to develop biodegradable strains that can efficiently degrade pretilachlor. Summary of the Invention
[0006] The purpose of the present invention is to provide a biodegradable strain that can efficiently degrade the amide herbicide pretilachlor, and develop it into a bioremediation agent for the remediation of pretilachlor pollution.
[0007] To achieve the above purpose, the present invention adopts the following technical solutions: The present invention isolates a new Klebsiella oxytoca strain from soil that has been chronically polluted by pretilachlor ( Klebsiella oxytoca ). Its biological characteristics are as follows: The colonies formed on LB medium are milky white and have a neat and smooth edge; the strain morphology is short rod-shaped or oval; the strain is a Gram-negative bacterium, has coagulase activity, can decompose carbohydrates, etc., and is easy to grow on ordinary inorganic salt medium. The 16S rDNA sequence of this strain is shown in SEQ ID NO.1. Through sequence alignment and construction of a phylogenetic tree, it is identified that this strain belongs to the kingdom Bacteria ( Bacteria kingdom ), phylum Proteobacteria ( Proteobacteria ), class Gammaproteobacteria (γ- Proteobacteria ), family Enterobacteriaceae ( Enterobacterales ), genus Klebsiella oxytoca ( Klebsiella oxytoca ). Therefore, it is classified and named Klebsiella oxytoca ( Klebsiella oxytoca ) PL1.
[0008] The strain Klebsiella oxytoca PL1 was deposited at the China Center for Type Culture Collection (CCTCC) on December 30, 2024. The deposit address: Wuhan, China, Wuhan University; the deposit number: CCTCC NO: M 20242959.
[0009] Furthermore, the culture medium of Klebsiella oxytoca PL1 is LB medium, and the culture temperature is 28 - 30 °C.
[0010] The present invention's research discovers that the strain Klebsiella oxytoca PL1 can effectively degrade pretilachlor residues in a short time.
[0011] Therefore, the present invention provides the application of Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 in degrading pretilachlor.
[0012] Furthermore, the application is the bioremediation of pretilachlor-polluted soil or water body. The strain Klebsiella oxytoca PL1 reduces pollution residues by degrading and converting pretilachlor in the polluted soil or water body into harmless products to achieve the purpose of remediation.
[0013] Furthermore, the application is to prepare the strain Klebsiella oxytoca PL1 into a biological bacterial fertilizer, and the biological bacterial fertilizer is applied to the pretilachlor-polluted soil or water body to play the role of degrading pretilachlor.
[0014] Another object of the present invention is to provide a bioremediation agent for degrading pretilachlor, and the active ingredient of the bioremediation agent includes the Klebsiella oxytoca ( Klebsiella oxytoca ) PL1.
[0015] Preferably, by mass percentage, the composition of the bioremediation agent includes: 10-20% of the fermentation broth containing Klebsiella oxytoca ( Klebsiella oxytoca ) PL1, 50-70% of nursery soil, 5-10% of activated carbon, 5-10% of soybean meal, 1-2% of potassium humate, 1-2% of plant-derived amino acids, 2-3% of PVAL binder, and the balance is water.
[0016] More preferably, by mass percentage, the composition of the bioremediation agent is: 10% of fermentation broth, 70% of nursery soil, 5% of activated carbon, 5% of soybean meal, 1% of potassium humate, 1% of plant-derived amino acids, 3% of PVAL binder, and the balance is water.
[0017] In the bioremediation agent provided by the present invention, the nursery soil is the main filler; the activated carbon is used as the strain carrier; the soybean meal is used as a nutrient to provide an N source; the potassium humate and plant-derived amino acids provide organic matter to increase soil fertility; the PVAL binder is a granulation binder.
[0018] The fermentation broth is obtained by inoculating Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 into a fermentation medium and performing biological fermentation. Preferably, the composition of the fermentation medium by mass percentage includes: 0.5-1.5% of NH4NO3, 0.4-0.6% of KH2PO4, 1.0-2.0% of K2HPO4, 0.4-0.6% of NaCl, 1-3% of MgSO4•7H2O, the balance is water, and the pH is 7.0±0.2.
[0019] More preferably, the composition of the fermentation medium in terms of mass percentage is: NH4NO3 1%, KH2PO4 0.5%, K2HPO4 1.5%, NaCl 0.5%, MgSO4•7H2O 2%, the balance being water, and the pH is adjusted to 7.0.
[0020] Preferably, the method for preparing the fermentation broth includes: inoculating the activated Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 into a seed medium, and culturing it with shaking at 25 - 30 °C until the logarithmic growth phase to obtain a seed solution; inoculating 15 - 20 mL of the seed solution into every 1000 g of the fermentation medium, and simultaneously adding 15 - 20 mL of a nutrient solution, and performing bioconversion at 25 - 30 °C for 24 - 72 h to obtain the fermentation broth; The composition of the seed medium in terms of mass percentage includes: sucrose 0.3 - 0.5%, MgSO4•7H2O 0.04 - 0.08%, K2HPO4 0.15 - 0.2%, NaCl 0.08 - 0.1%, KH2PO4 0.05 - 0.07%, yeast extract 0.15 - 0.2%, the balance being water, and the pH is 7.0 ± 0.2.
[0021] More preferably, the composition of the seed medium in terms of mass percentage is: sucrose 0.5%, MgSO4•7H2O 0.04%, K2HPO4 0.2%, NaCl 0.1%, KH2PO4 0.05%, yeast extract 0.2%, the balance being water, and the pH is adjusted to 7.0.
[0022] The composition of the nutrient solution in terms of mass percentage includes: sucrose 0.5 - 1%, beef extract 1 - 5%, KH2PO4 0.1 - 0.5%, MgSO4•7H2O 0.1 - 0.5%, K2HPO4 0.2 - 0.5%, NaCl 0.1 - 0.5%, yeast powder 1.0 - 10%, the balance being water, and the pH is 7.0 ± 0.2.
[0023] More preferably, the composition of the nutrient solution in terms of mass percentage is: sucrose 0.5%, beef extract 1%, KH2PO4 0.1%, MgSO4•7H2O 0.1%, K2HPO4 0.2%, NaCl 0.1%, yeast powder 1.0%, the balance being water, and the pH is adjusted to 7.0.
[0024] The present invention also provides the application of the biological remediation agent in remediating pretilachlor - contaminated soil, and the application includes: mixing the biological remediation agent into the pretilachlor - contaminated soil to be remediated.
[0025] Preferably, the bioremediation bacterial agent is applied to the pretilachlor-contaminated soil to be repaired at a mass percentage of 0.5%-10%. Research shows that, compared with the control group without using the bacterial agent, the degradation rate of pretilachlor in the contaminated soil after using the bioremediation bacterial agent is significantly increased, and the degradation rate of pretilachlor reaches 78.57% 60 days after adding the bacterial agent to the soil.
[0026] Preferably, the pretilachlor-contaminated soil is the soil for growing cucumbers or rice. Research shows that the bioremediation bacterial agent can not only effectively degrade the pretilachlor contamination in the soil, but also has no toxic or side effects on crop growth. Since the pretilachlor in the soil is degraded, its inhibitory effect on crop growth is effectively reduced, and the germination vitality of sensitive crops such as cucumber and rice seeds in the soil is significantly increased.
[0027] The beneficial effects of the present invention are as follows: (1) The present invention provides a new Klebsiella oxytoca ( Klebsiella oxytoca ) strain PL1, which has the biological activity of efficiently degrading pretilachlor. Applying this strain to the pretilachlor-contaminated soil or water body can effectively degrade the pretilachlor residue in a short time, protect the ecological environment and human health, and is convenient to use and low in cost.
[0028] (2) The bioremediation bacterial agent prepared by using Klebsiella oxytoca PL1 has the advantages of low production cost, convenient use, good removal effect, etc., and is suitable for treating the pollution caused by pretilachlor in the environment such as soil, and has very important theoretical and application values. Brief Description of the Drawings
[0029] 图1 It is a schematic diagram of the morphology of Klebsiella oxytoca strain PL1. Among them, the left figure is a picture of the colonies growing on the plate, and the right figure is a picture under the microscope after Gram staining.
[0030] 图2 It is a scanning electron micrograph of Klebsiella oxytoca strain PL1.
[0031] 图3 It is a phylogenetic tree of Klebsiella oxytoca strain PL1 constructed based on the 16S rDNA sequence.
[0032] 图4 It is the degradation curve of pretilachlor by PL1 under different inoculation rates.
[0033] 图5 It is the degradation curve of pretilachlor by PL1 under different pH conditions.
[0034] 图6 It is the degradation curve of pretilachlor by PL1 under different pretilachlor concentration conditions.
[0035] 图7 It is the apparent diagram of the soil bioremediation microbial agent.
[0036] 图8 It is the degradation dynamics of pretilachlor in soil by PL1 microbial agent. Specific implementation manners
[0037] To better explain the present invention, the main content of the present invention is further clarified below in conjunction with specific embodiments. However, the content of the present invention is not limited to the following embodiments only. The technical solutions involved in the embodiments of the present invention are all conventional solutions in the art unless otherwise specified; the reagents or materials are all from commercial channels unless otherwise specified.
[0038] Example 1: Isolation and identification of pretilachlor-degrading bacteria I. Isolation and screening of strains (1) Preparation of culture media The isolation and purification liquid culture medium is an inorganic salt liquid culture medium (MSM): 1.00 g of NaCl, 1.50 g of K2HPO4, 0.50 g of KH2PO4, 0.20 g of MgSO4•7H2O, 1.0 g of sucrose, pretilachlor (100 mg / L - 500 mg / L), 1000 mL of distilled water, pH 7.0, 115 °C, sterilized for 15 min.
[0039] The isolation and purification solid culture medium is an inorganic salt solid culture medium (AMSM): 1.00 g of NaCl, 1.50 g of K2HPO4, 0.50 g of KH2PO4, 0.20 g of MgSO4•7H2O, 1.00 g of sucrose, pretilachlor (100 mg / L - 500 mg / L), 20.00 g of agar, 1000 mL of distilled water, pH 7.0, 115 °C, sterilized for 15 min.
[0040] Preparation of sterile water: The sterile water is obtained by autoclaving distilled water at 121 °C and 15 psi for 20 min.
[0041] The pH of the above culture media is adjusted with 1 mol / L NaOH solution and 1 mol / L HCl solution.
[0042] (2) Isolation and purification of strains Weigh 10.00 g of soil that has been contaminated by pretilachlor for a long time (from Hangzhou, Zhejiang Province) into a 250 mL conical flask, add 100 mL of inorganic salt medium, and place it on a shaker (30 °C, 150 rpm) for shaking culture for 7 days; let it stand at room temperature for 30 min, take 5 mL of the bacterial supernatant and inoculate it into 100 mL of separation medium (containing 100 mg / L of pretilachlor), and place it on a shaker (30 °C, 150 rpm) for shaking culture for 7 days; take 10 mL of the bacterial liquid obtained from the above separation medium and inoculate it into 100 mL of separation medium (containing 200 mg / L of pretilachlor), and place it on a shaker (30 °C, 150 rpm) for shaking culture for 7 days; and so on. The inoculation amount of the bacterial liquid each time is 10%, and the concentration of pretilachlor in the separation medium is increased by 100 mg / L each time, until the concentration of pretilachlor in the enrichment liquid reaches 500 mg / L.
[0043] Gradually dilute the above bacterial supernatant with sterile water to suspensions with gradient concentrations of 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 . Pipette 100 μL of the suspension of each concentration gradient and drop it onto the center of the separation and purification solid medium, spread it evenly with a spreader, repeat each gradient 3 times, and invert the plate and culture it in an incubator at 30 °C for 2 days. Repeat the above operation until single colonies with the same morphology appear on the separation and purification medium, and inoculate the obtained strains on the slant of the solid separation and purification medium for storage and standby.
[0044] Centrifuge the domesticated degrading bacteria at 4 °C and 5000 r / min to discard the supernatant and collect the bacterial cells. Use an inoculation loop to pick up the bacterial strain and spread it on an LB solid plate according to the quartering method for culturing for 12 h. Use an inoculation loop to pick 3 loops of single colonies on the plate and put them into 100 mL of LB liquid medium for activation culture. Wait until OD 600 = 1.0, then centrifuge at 4 °C and 5000 r / min to discard the supernatant and collect the bacterial cells, add an equal volume of sterile water and mix evenly to prepare a seed solution for standby.
[0045] Finally, a degrading strain that can grow on the separation medium containing 100 mg / L of pretilachlor was isolated, and its degradation effect was verified by high performance liquid chromatography (HPLC).
[0046] HPLC determination conditions: Agilent 1100 high performance liquid chromatograph (Agilent, USA); detector: Agilent 1100 ultraviolet detector (Agilent, USA); chromatographic column: C18 reverse phase column (Waters, 150 mm × 4.60 mm, 5 μm); mobile phase: acetonitrile∶acidified water (0.1% acetic acid) = 7:3, V / V), flow rate 1.0 mL / min; column temperature 30 °C; detection wavelength: 230 nm; injection volume: 10 μL.
[0047] The calculation method of degradation rate is as follows: Degradation rate (%) = (residual amount of control sample - residual amount of treated sample) / residual amount of control sample × 100%.
[0048] The strain with the best degradation effect was named PL1. This strain could degrade 85.52% of pretilachlor with an initial concentration of 50.00 mg / L in 7 d under the environmental conditions of inorganic salt medium.
[0049] II. Identification of degradation bacterium PL1 (1) Colony characteristics The obtained strain PL1 was inoculated on LB medium and cultured in a constant temperature incubator at 30 °C for 16 h. As 图1 shown, the colony edge was neat and smooth, the colony was opaque milky white, the surface was rough, and a large, round bulge was formed.
[0050] (2) Observation results by scanning electron microscope As 图2 shown, the strain was observed by electron microscope to be rod-shaped, with a diameter of 0.5 - 0.8 μm and a length of 0.9 - 2.0 μm. Both ends were smooth and round, showing short rod-shaped or oval bacteria.
[0051] (3) Analysis of physiological and biochemical characteristics Refer to "Microbiology Experiments" (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments (Third Edition). Beijing: Higher Education Press, 1999.) and "Handbook for Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. Handbook for Systematic Identification of Common Bacteria. Beijing: Science Press, 2011.) to determine the physiological and biochemical characteristics of degradation bacterium PL1.
[0052] The determination results showed that this strain was a Gram-negative bacterium, with rennet activity, could decompose carbohydrates, etc., and was easy to grow on ordinary inorganic salt medium.
[0053] (4) Homology analysis of 16S rDNA of degradation bacterium The genomic DNA of the degrading bacterium PL1 was extracted using an extraction kit (Genetically Modified Biotechnology Co., Ltd., Beijing, China), and the 16S rDNA gene was amplified by polymerase chain reaction (PCR) using universal primers. The primers were 27F (5’-AGAGTTGATCCTGCTCAG-3’) and 1492R (5’-GTTACCTTTACGACTT-3’).
[0054] 50 µL amplification system: 25 µL of Premix taq, 500 ng of template (genomic DNA of the degrading bacterium PL1), 10 µL each of 1492R and 27F, and made up to 50 µL with sterile water.
[0055] The PCR cycling parameters were: preheating at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1.5 min, and final extension at 72°C for 10 min.
[0056] The PCR products were purified by 1% agarose gel electrophoresis and EasyPure gel. The PCR products were purified and recovered for the 16S rDNA fragment using the V-gene nucleic acid purification kit (TIANGEN), and then sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). The obtained 16S rDNA sequence of PL1 is shown as SEQ ID NO.1.
[0057] The sequencing results were compared for homology with the 16S rDNA sequences in the Genbank using the Blast software, and it was found that the sequence had 99% homology with the gene sequences of strains such as Klebsiella oxytoca TL2. A phylogenetic tree of the biocontrol bacteria was constructed as shown in 图3 and it was determined that PL1 belongs to the Bacteria kingdom ( Bacteria kingdom ), Proteobacteria phylum ( Proteobacteria ), Gammaproteobacteria class (γ- Proteobacteria ), Enterobacteriaceae family ( Enterobacterales ), Klebsiella oxytoca genus ( Klebsiella oxytoca ), and thus it was named Klebsiella oxytoca ( Klebsiella oxytoca ) PL1.
[0058] Strain Klebsiella oxytoca PL1 was deposited at the China Center for Type Culture Collection (CCTCC) on December 30, 2024. Deposit address: Wuhan University, Wuhan, China; Deposit number: CCTCC NO: M 20242959; It was detected as viable on January 6, 2025.
[0059] Example 2: Analysis of the degradation characteristics of strain Klebsiella oxytoca PL1 I. Influence of Different Inoculum Sizes of PL1 on the Degradation Rate of Pretilachlor PL1 was cultured on an LB plate with an initial concentration of 100 mg / L of pretilachlor for 12 h. The culture condition was constant temperature culture at 30 °C. After observing the growth of single colonies, they were transferred to an LB liquid medium and incubated for 12 h. When OD 600 = 1.0, the supernatant was discarded by centrifugation at 5000 r / min at 4 °C, and the bacterial cells were collected. An equal volume of sterile water was added and mixed evenly to prepare a seed solution, which was stored at 4 °C for later use. It was inoculated into 100 mL of an inorganic salt liquid medium with a pH of 7 (containing 50 mg / L of pretilachlor) at an inoculum size of 2%, and cultured at 150 rpm at 1%, 2%, 3%, 4%, 5%, 6%, and 7%. Samples were taken at 0 d, 1 d, 3 d, and 7 d to measure the growth of the pretilachlor-degrading bacterium PL1 and the content of pretilachlor. The growth of PL1 was measured using an ultraviolet spectrophotometer for OD 600 measurement; the content of pretilachlor was measured using HPLC. Each treatment was repeated three times, and the uninoculated one was used as a control to culture, observe, and record the growth of the strain.
[0060] The results are as 图4 shown. The optimal inoculum size of the degrading bacterium PL1 is 2%.
[0061] II. Influence of Different pH Conditions on the Degradation Rate of Pretilachlor by PL1 The PL1 bacterial suspension was inoculated into 100 mL of an inorganic salt liquid medium with pH values of 5, 7, and 9 (containing 50 mg / L of pretilachlor) at an inoculum size of 2%, and cultured at 30 °C and 150 rpm. Samples were taken at 0 d, 1 d, 3 d, and 7 d to measure the growth of the pretilachlor-degrading bacterium PL1 and the content of pretilachlor. Each treatment was repeated three times, and the uninoculated one was used as a control to culture, observe, and record the growth of the strain.
[0062] The results are as 图5 shown. The optimal growth pH of the degrading bacterium PL1 is 7.
[0063] III. Influence of Different Concentrations of Pretilachlor on the Degradation Rate of PL1 The PL1 bacterial suspension was inoculated into 100 mL of an inorganic salt liquid medium with a pH of 7 and pretilachlor (PC) concentrations of 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, and 200 mg / L at an inoculum size of 2%, and cultured at 30 °C and 150 rpm. Samples were taken at 0 d, 1 d, 3 d, 7 d, 10 d, 12 d, and 15 d to measure the growth of the pretilachlor-degrading bacterium PL1 and the content of pretilachlor (PC). Each treatment was repeated three times, and the uninoculated one was used as a control to culture, observe, and record the growth of the strain.
[0064] The results are as 图6 shown. The optimal initial degradation concentration of pretilachlor by the degrading bacterium PL1 is 50 mg / L.
[0065] IV. Kinetic equation for the degradation of pretilachlor by strain PL1 The formula for the first-order kinetic equation is: C = C 0 exp(- k1t ); In the above formula, C is the final concentration of the reaction; t is the reaction time; K is the first-order kinetic reaction rate constant; C0 is the initial concentration of the chemical.
[0066] The results of the effect of the initial concentration of pretilachlor on the degradation of pretilachlor by strain PL1 are shown in Table 1.
[0067] Table 1: Data of the first-order kinetic equation for the degradation of pretilachlor by strain PL1
[0068] The experimental results show that when the initial concentration of pretilachlor is 50 - 200 mg / L, the degradation of pretilachlor conforms to the pesticide degradation kinetic equation. It can be seen from Table 1 that when the concentration of pretilachlor is 50 mg / L, the degradation rate constant of pretilachlor by strain PL1 is 0.99, the half-life is 7.86 days, the half-life of 100 mg / L pretilachlor is 6.06 days, and the half-life of 200 mg / L pretilachlor is 17.96 days. For untreated pretilachlor in the medium, the half-life gradually shortens with the increase in concentration, which are 137.6, 58.8, and 36.1 days respectively. In the case of high-concentration pesticides, photolysis and hydrolysis are also the reasons for the increase in the degradation rate of pretilachlor. Therefore, with the increase in concentration, its degradation half-life shortens.
[0069] In summary, the optimal degradation conditions of the PL1 bacterial agent in the MSM medium: when the initial concentration of pretilachlor is 50 mg / L, the inoculation amount is 2%, the pH is 7, at 30 °C, and it can degrade 85.1% after culturing for 7 days. The optimal degradation conditions of the PL1 bacterial agent in the medium: when the initial concentration of pretilachlor is 100 mg / L, the inoculation amount is 2%, the pH is 7, and it is cultured at a constant temperature of 30 °C. The half-life of pretilachlor degradation is 6.06 days.
[0070] Example 3: Preparation of the PL1 soil bioremediation bacterial agent The soil bioremediation bacterial agent containing strain Klebsiella oxytoca PL1 is prepared by using Klebsiella oxytoca strain PL1 as the living component, and the steps are as follows: (1) The strain Klebsiella oxytoca PL1 was inoculated into LB liquid medium and cultured with shaking at 30 °C and 150 rpm until the logarithmic growth phase. The cells were washed three times with 50 mmol phosphate buffer saline (PBS) at pH 7 to obtain the bacterial strain; (2) The bacterial strain obtained in step (1) was inoculated into the seed flask medium at an inoculation amount of 10% by volume and cultured with shaking at 30 °C and 150 rpm until the logarithmic growth phase to obtain the seed liquid; (3) 20 mL of the seed liquid prepared in step (2) (OD 600 nm = 1) was added to 1000 g of the fermentation medium. At the same time, 20 mL of the nutrient solution was added to the above 1000 g of the fermentation medium, and biological fermentation was carried out at 28 °C for 72 h to obtain the fermentation broth. Then, according to the mass percentage, 10% of the fermentation broth, 70% of the seedling-growing soil, 5% of activated carbon, 5% of soybean meal, 1% of potassium humate, 1% of plant-derived amino acids, 3% of PVAL binder, and 5% of distilled water were mixed and granulated. Ventilation drying was carried out at 25 °C for 12 h, and after air drying, it was hermetically packaged to obtain the soil bioremediation agent of the degrading bacterium PL1 strain of the present invention, as 图7 shown.
[0071] The LB medium was prepared with distilled water, and the weight percentages of each component were: 10% NaCl, 5% yeast extract powder, 10% tryptone, the pH was adjusted to 7.0, and it was sterilized at 121 °C for 20 min.
[0072] The seed flask medium was prepared with distilled water, and the mass percentages of each component were: 0.5% sucrose, 0.04% MgSO4•7H2O, 0.2% K2HPO4, 0.1% NaCl, 0.05% KH2PO4, 0.2% yeast extract, the pH was adjusted to 7.0, and it was sterilized at 115 °C for 15 min.
[0073] The fermentation medium was prepared with distilled water, and the weight percentages of each component were: 1% NH4NO3, 0.5% KH2PO4, 1.5% K2HPO4, 0.5% NaCl, 2% MgSO4•7H2O, the pH was adjusted to 7.0, and it was sterilized at 115 °C for 15 min.
[0074] The nutrient solution was prepared with distilled water, and the weight percentages of each component were: 0.5% sucrose, 1% beef extract, 0.1% KH2PO4, 0.1% MgSO4•7H2O, 0.2% K2HPO4, 0.1% NaCl, 1.0% yeast powder, the pH was adjusted to 7.0, and it was sterilized at 115 °C for 15 min.
[0075] The above-mentioned culture medium and nutrient solution were adjusted to the appropriate pH with 1 mol / L NaOH solution and 1 mol / L HCl solution.
[0076] The seedling-growing soil was purchased from Jiangsu Peilei Substrate Technology Development Co., Ltd., the activated carbon was purchased from Jiangsu Pengzhan Activated Carbon Co., Ltd., the soybean meal was purchased from Shandong Hongbo Biotechnology Co., Ltd., the potassium humate was purchased from Shandong Tenghong Biotechnology Co., Ltd., the plant-derived amino acid was purchased from Shandong Tenghong Biotechnology Co., Ltd., and the PVAL binder was purchased from Jiangsu Langsheng New Materials Technology Co., Ltd.
[0077] Example 4: The effect of PL1 bacterial agent on soil remediation First, 1.00 kg of garden soil was weighed and 100 mL of 1000 mg / L pretilachlor solution was added to make the concentration of pretilachlor in the soil 100 mg / kg (i.e., the soil contaminated with pretilachlor was prepared). The soil bioremediation bacterial agent containing PL1 strain obtained in Example 3 was applied to the soil at a content percentage of 0.56%, thoroughly mixed, and placed in a constant-temperature incubator at 30 °C for incubation in the dark. Samples were taken at 60 d of incubation in the dark incubator to determine the residue of pretilachlor in the soil.
[0078] The calculation method of the degradation rate is as follows: Degradation rate (%) = (residue in the control sample - residue in the treated sample) / residue in the control sample × 100%.
[0079] The experimental results are as 图8 shown. When using the soil bioremediation bacterial agent containing PL1 strain, the degradation rate of pretilachlor increased significantly. The degradation rate of pretilachlor reached 78.57% at 60 d after adding the PL1 bacterial agent to the soil, while the degradation rate of pretilachlor was only 49.1% at 60 d without adding the bacterial agent.
[0080] Example 5: The effect of the soil remediation by PL1 bacterial agent on the remediation of sensitive crops Cucumber and rice were used as the test crops, and the seeds with consistent germination after soaking in water and germination acceleration were selected for standby; the pretilachlor solution was prepared for standby.
[0081] Treatment 1: 1.00 kg of air-dried soil (sieved through a 20-mesh sieve) was taken and an appropriate amount of water was added to make the water content 20%.
[0082] Treatment 2: 1.00 kg of air-dried soil (sieved through a 20-mesh sieve) was taken respectively, and 50, 10, 8, 5, 2, 1 mL of 1000 mg / L pretilachlor stock solution was added. After thorough mixing, the pretilachlor concentration in the soil was 50, 10, 8, 5, 2, 1 mg / kg. Then, a bioremediation bacterial agent (prepared in Example 3) based on the above PL1 strain with a weight percentage of 10% was added and mixed evenly.
[0083] Treatment 3: Take 1.00 kg of air-dried soil (passed through a 20-mesh sieve), add 50, 10, 8, 5, 2, 1 mL of pretilachlor 1000 mg / L solution, and make the medicated soil with pretilachlor concentrations of 50, 10, 8, 5, 2, 1 mg / kg after thorough mixing.
[0084] Select cucumber and soybean seeds with consistent germination degrees from the seeds that have been soaked and germinated, sow them in different treated soils, and cultivate them in the greenhouse for 7 days. At the same time, observe and record the germination number, germination rate, germination potential, germination index, and vigor index of different treatments. The results are shown in Table 2 and Table 3.
[0085] Table 2 Effects of bioremediation agent PL1 on cucumber germination indexes
[0086] Table 3 Effects of bioremediation agent PL1 on rice seed germination indexes
[0087] The experimental results show that after applying the microbial agent to the medicated soil, the germination number, germination rate, germination potential, germination index, and vigor index of cucumbers and rice can be significantly increased. When the pretilachlor concentration in the soil is 10 mg / kg, after adding the microbial agent with a mass fraction of 10%, the germination numbers of cucumbers and rice are significantly higher than those of cucumbers and rice cultivated without applying the microbial agent treatment. The germination rates are increased by 26.67% and 25% respectively, the germination potentials are increased by 33.75% and 21.15% respectively, the germination indexes are increased by 20.96 and 22.48 GI respectively, and the vigor indexes are increased by 0.38 and 29.05 VI respectively. This indicates that the PL1 microbial agent has a good degradation effect on pretilachlor, can improve the seed activity of cucumbers and rice, and effectively reduce the growth inhibition effect of pretilachlor on seeds.
Claims
1. Klebsiella oxytoca ( Klebsiella oxytoca ), characterized in that The strain was isolated from soil and classified and named as Klebsiella oxytoca PL1, which is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242959.
2. The Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 as claimed in claim 1, characterized in that The Klebsiella oxytoca culture medium of PL1 is LB medium, and the culture temperature is 28 - 30 °C.
3. Use of Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 in degrading pretilachlor.
4. The application according to claim 3, characterized in that, The application is the bioremediation of pretilachlor-contaminated soil or water body.
5. A bioremediation bactericide for degrading pretilachlor, characterized in that, Comprising Klebsiella oxytoca as claimed in claim 1 as an active ingredient ( Klebsiella oxytoca ), PL1.
6. The bioremediation bacterium agent according to claim 5, wherein By mass percentage, its composition includes: the fermentation broth containing Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 10-20%, seedling soil 50-70%, activated carbon 5-10%, soybean meal 5-10%, potassium humate 1-2%, plant-derived amino acids 1-2%, PVAL binder 2-3%, and the balance is water.
7. The bioremediation bacterial agent according to claim 6, wherein The fermentation broth is obtained by inoculating Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 into a fermentation medium and performing biologic fermentation. The composition of the fermentation medium in mass percentage includes: NH4NO3 0.5 - 1.5%, KH2PO4 0.4 - 0.6%, K2HPO4 1.0 - 2.0%, NaCl 0.4 - 0.6%, MgSO4•7H2O 1 - 3%, with the balance being water and the pH being 7.0 ± 0.
2. Klebsiella oxytoca 8. The bioremediation bacterial agent according to claim 7, characterized in that, The preparation method of the fermentation broth comprises: inoculating the activated Klebsiella oxytoca ( Klebsiella oxytoca ) PL1 into a seed culture medium, and culturing with shaking at 25-30 °C until the logarithmic growth phase to obtain a seed solution; inoculating 15-20 mL of the seed solution into every 1000 g of the fermentation culture medium, and simultaneously adding 15-20 mL of a nutrient solution, and performing biological fermentation at 25-30 °C for 24-72 h to obtain the fermentation broth; Klebsiella oxytoca The composition of the seed culture medium includes, by mass percentage: sucrose 0.3 - 0.5%, MgSO4•7H2O 0.04 - 0.08%, K2HPO4 0.15 - 0.2%, NaCl 0.08 - 0.1%, KH2PO4 0.05 - 0.07%, yeast extract 0.15 - 0.2%, with the balance being water and the pH being 7.0 ± 0.2; The composition of the nutrient solution includes, by mass percentage: sucrose 0.5 - 1%, beef extract 1 - 5%, KH2PO4 0.1 - 0.5%, MgSO4•7H2O 0.1 - 0.5%, K2HPO4 0.2 - 0.5%, NaCl 0.1 - 0.5%, yeast powder 1.0 - 10%, with the balance being water and the pH being 7.0 ± 0.
2.
9. Use of the bioremediation bacterial agent according to any one of claims 5-8 in remediating pretilachlor-contaminated soil, characterized in that, Mix the bioremediation agent with the pretilachlor-contaminated soil to be repaired.
10. The application according to claim 9, characterized in that, The pretilachlor-contaminated soil is the soil for growing cucumbers or rice.