Enterobacter hormaechei for degrading nicosulfuron and repairing microbial agent containing enterobacter hormaechei

Abstract

The invention relates to enterobacter hormaechei for degrading nicosulfuron and a repairing fungicide containing the enterobacter hormaechei, and belongs to the technical field of microorganisms. The strain is preserved in the China Center for Type Culture Collection, the preservation date is December 1, 2023, the preservation number is CCTCC NO: M 20232422, the classification name is Enterobacter hormaechei ES1, the strain is gram negative, methyl red and catalase grow at 37 DEG C, V-P reaction is positive, and starch cannot be hydrolyzed. The remediation microbial inoculum prepared by inoculating the microbial inoculum to agricultural wastes (30% of wood chips, 30% of straws and 20% of biochar) and mushroom bran compost (20%) has a degradation rate of 95.50% on nicosulfuron-polluted soil. Compared with an existing soil remediation method, the method has the advantages of being efficient, low in toxicity, free of environment pollution, not prone to generating drug resistance and the like, and has wide application prospects and good application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Enterobacter hormaechei ES1 (Enterobacter hormaechei ES1) capable of degrading nicosulfuron and a repairing bacterial agent containing the strain. Background Art

[0002] Since its development in the 1970s, sulfonylurea herbicides have been widely used for weed control in corn, wheat, oat and other fields because they have the characteristics of inhibiting the activity of acetolactate synthase in plants to prevent the growth of sensitive plants. Nicosulfuron, as a sulfonylurea herbicide, is widely used in corn and other fields because of its high efficiency and low toxicity. Due to its poor photodegradation and low volatility, it remains in corn and soil, affecting the environment and human health. Nicosulfuron, a long-residue nicosulfuron, has a DT50 value of about 75 days and a slow degradation rate in water and soil. In North America and Europe, nicosulfuron is often detected in surface water, wetland sediments and soil, and even trace amounts of nicosulfuron have been detected in animals and humans. With the increase in the use of nicosulfuron, the residue problem has become increasingly prominent, and environmental problems are threatened.

[0003] Nicosulfuron can be degraded in many ways, such as photodegradation under natural light conditions under the action of photocatalysts. Chemical hydrolysis breaks the urea bridge by acting on the carboxyl carbon in the urea bridge, decomposing Nicosulfuron. Adsorption can also degrade Nicosulfuron. Biodegradation includes plant, animal and microbial degradation. Microbial degradation is becoming the main trend in pollutant degradation due to its economic applicability and environmental friendliness.

[0004] In recent years, microbial technology has become a hot technology that can effectively reduce the use of chemical products. Agricultural waste such as mushroom husks and corn stalk biochar not only has a biological remediation effect on the environment, but can also be used as a fixed carrier material for processing strains into microbial agents. Summary of the invention

[0005] The invention aims to provide an Enterobacter hormaechei ES1 strain capable of degrading nicosulfuron and a repairing bacterial agent containing the strain.

[0006] The Enterobacter hormaechei ES1 (Enterobacter hormaechei ES1) for degrading nicosulfuron described in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on December 1, 2023, with the address being Wuhan University, Wuhan, China (430072), with the preservation number being CCTCC NO: M 20232422, and the classification name being: Enterobacter hormaechei ES1 (Enterobacter hormaechei ES1).

[0007] The strain of the present invention is identified as Enterobacter hormaechei through physiological and biochemical characteristics and 16S rDNA sequence analysis. The strain grows very fast on LB solid plate culture medium. The physiological and biochemical characteristics are measured to show that the strain is Gram-negative, methyl red, catalase, 37°C growth and VP reaction are positive, and the strain cannot hydrolyze starch.

[0008] The Enterobacter hormaechei ES1 of the present invention can effectively degrade nicosulfuron residues, protect the ecological environment and human health, and is easy to use and low in cost. The repair bacterial agent prepared by using the Enterobacter hormaechei ES1 has the advantages of low production cost, easy use, good removal effect, etc., is suitable for repairing soil systems, achieves sustainable development, and has very important theoretical and application values. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 : Scanning electron micrograph of Enterobacter hormaechei ES1 (LB solid medium, 30°C, 10h);

[0010] Figure 2 : Schematic diagram of the phylogenetic tree of Enterobacter hormaechei ES1;

[0011] Figure 3 : Schematic diagram of the degradation effect of Nicosulfuron by Enterobacter hormaechei ES1 at different temperatures (the horizontal axis represents temperature (℃), the left vertical axis represents the degradation rate of Nicosulfuron (%), and the right vertical axis represents OD 600 );

[0012] Figure 4: Schematic diagram of the degradation effect of Enterobacter hormaechei ES1 on nicosulfuron at different pH (the horizontal axis represents pH, the left vertical axis represents the degradation rate of nicosulfuron (%), and the right vertical axis represents OD 600 );

[0013] Figure 5 : Schematic diagram of the degradation effect of Enterobacter hormaechei ES1 at different inoculation amounts (the horizontal axis represents the inoculation amount of strain ES1 (%), the left vertical axis represents the degradation rate of nicosulfuron (%), and the right vertical axis represents the OD 600 );

[0014] Figure 6 Schematic diagram of degradation of nicosulfuron by Enterobacter hormaechei ES1 at different initial concentrations (the horizontal axis represents the concentration of nicosulfuron substrate (mg L -1 ), the left ordinate represents the degradation rate of nicosulfuron (%), and the right ordinate represents the OD 600 );

[0015] Figure 7 Schematic diagram of the restoration of Nicosulfuron-contaminated soil by restoration bacteria (the horizontal axis represents time (d), the vertical axis represents Nicosulfuron concentration (mg kg -1 ). DETAILED DESCRIPTION

[0016] Example 1: Isolation, Identification and Growth of Enterobacter hormaechei ES1 1. Isolation and Screening of Enterobacter hormaechei ES1

[0017] 1) Soil samples that had been long-term treated with nicosulfuron were collected from the teaching and research base of Jilin Agricultural University, and 5.0 g of the soil samples were taken and placed in 95.0 mL of inorganic salt liquid medium (MSM);

[0018] 2) Add nicosulfuron to the system of step 1) to a final concentration of 50 mg L -1 , cultured at 30°C and 150rpm for 7 days to obtain inorganic salt culture solution 1;

[0019] 3) Take 1 mL of the inorganic salt culture solution 1 obtained in step 2) and add nicosulfuron to a final concentration of 100 mg L -1 , cultured at 30°C and 150rpm for 7 days to obtain inorganic salt culture solution 2;

[0020] 4) Take 1 mL of the inorganic salt culture solution 2 obtained in step 3) and add nicosulfuron to a final concentration of 200 mg L -1 , cultured at 30°C and 150rpm for 7 days to obtain inorganic salt culture solution 3;

[0021] 5) The inorganic salt culture solution 3 obtained in step 4) was diluted continuously with sterile water (1 mL of inorganic salt culture solution 3 was added to 9 mL of sterile water, which was considered as a 1-fold dilution. The dilution was repeated 6 times to a concentration of 200 mg L -1 *10 -6 ), and 50 μL was applied to a nicosulfuron solution with a concentration of 200 mg L -1 The culture medium was plated on an inorganic salt solid medium at 30° C. and 150 rpm for 3 days, and strains with inconsistent colors, shapes, sizes, etc. were picked up and streaked onto LB solid medium respectively. The operation was repeated until strains with consistent colors, shapes, sizes, etc. appeared, and the separation of the strains was completed to obtain Enterobacter hormaechei ES1. The degradation rate of nicosulfuron by Enterobacter hormaechei ES1 was determined by HPLC.

[0022] HPLC conditions: Agilent 1260 high performance liquid chromatograph (Agilent, USA); detector: Agilent 1260 UV detector (Agilent, USA); chromatographic column: C 18 Reversed phase column (Waters, 150 mm×4.60 mm, 5 μm); mobile phase: acetonitrile: 0.1% acetic acid water 70:30, V / V), flow rate 1.0 mL / min; column temperature 25°C; detection wavelength: 240 nm; injection volume: 10 μL.

[0023] The degradation rate is calculated as follows:

[0024]

[0025] Residue in control sample: the residual concentration of Nicosulfuron when only Nicosulfuron was added; Residue in treated sample: the residual concentration of Nicosulfuron when Nicosulfuron and Enterobacter hormaechei ES1 were added.

[0026] The composition of the inorganic salt liquid medium (MSM) is: NaCl 1.00g, K2HPO4 1.00g, KH2PO41.00g, MgSO4·7H2O 0.20g, glucose 5.0g, NH4Cl 1.80g, distilled water 1000mL, pH 7.0, sterilized at 115℃ for 15min;

[0027] The composition of the inorganic salt solid medium is: NaCl 1.00g, K2HPO4 1.00g, KH2PO4 1.00g, MgSO4·7H2O 0.20g, glucose 5.0g, NH4Cl 1.80g, agar powder 20.00g, distilled water 1000mL, pH 7.0, sterilized at 115℃ for 15min;

[0028] LB liquid medium was prepared by adding 10.00 g NaCl, 5.00 g yeast extract powder and 10.00 g tryptone to 1000 mL distilled water, adjusting the pH to 7.0, and sterilizing at 121°C for 20 min;

[0029] LB solid medium is prepared by adding 10.00g NaCl, 5.00g yeast extract powder, 10.00g tryptone, and 20.00g agar powder to 1000mL distilled water, adjusting the pH to 7.0, and sterilizing at 121℃ for 20min; 2. Identification of Enterobacter hormaechei ES1

[0030] (1) Homology analysis of 16S rDNA of degrading bacteria

[0031] The genomic DNA of Enterobacter hormaechei ES1 was extracted according to the instructions of the DNA extraction kit (Tiangen, Beijing) and sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were compared with the National Center for Biotechnology Information (NCBI) database, and the strain ES1 was identified by MEGA10.0 and the phylogenetic tree was constructed by the neighbor-joining method. The strain ES1 had 95% homology with Enterobacter homaechei 10-17, and the strain ES1 was identified as Enterobacter homaechei (Genbank OQ345654) ( Figure 2 ) and deposited with China Center for Type Culture Collection (accession number: CCTCCM20232422). The 16SrDNA sequence of Enterobacter hormaechei ES1 is shown in SEQ ID NO.1.

[0032] (2) Morphological identification of Enterobacter hormaechei ES1

[0033] Enterobacter hormaechei ES1 was inoculated into LB liquid medium, cultured at 30°C and 160rpm, 1mL of Enterobacter hormaechei ES1 bacterial solution was centrifuged, washed 3 times, added with 2.5% pentanediol, fixed overnight at 4°C, and morphological characteristics were observed using a scanning electron microscope. The Enterobacter hormaechei ES1 isolated and purified above grew very fast on the LB solid plate medium, and was observed under an electron microscope to be a smooth, rod-shaped bacterium ( Figure 1 ).

[0034] (3) Analysis of physiological and biochemical characteristics

[0035] The physiological and biochemical characteristics of the degradation bacteria FH-1 were determined with reference to Microbiology Experiment (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiment (3rd Edition). Beijing: Higher Education Press, 1999.) and Manual of Identification of Common Bacteria System (Dong Xiuzhu, Cai Miaoying. Manual of Identification of Common Bacteria System. Beijing: Science Press, 2011.). The strain was Gram-negative, positive for methyl red, catalase, growth at 37°C and VP reaction, and could not hydrolyze starch, as shown in Table 1.

[0036] Table 1 Physiological and biochemical characteristics of Enterobacter hormaechei ES1 Symbol "+" indicates positive, and "-" indicates negative.

[0037] Physiological and biochemical characteristics ES1 Methyl red reaction + VP reaction + Starch hydrolysis - Gram stain - Catalase + Growth at 37°C +

[0038] Example 2: Degradation characteristics of Nicosulfuron by Enterobacter hormaechei ES1

[0039] 1. Preparation of Bacterial Suspension

[0040] Use an inoculation loop to pick up a small amount of Enterobacter hormaechei ES1 and inoculate it into 100 mL of LB liquid culture medium. Shake and culture in a shaker at 30°C and 150 rpm for 48 h. Take an appropriate amount of the bacterial solution and centrifuge it. Discard the supernatant, wash the precipitate twice with sterile water, and suspend it with an equal amount of sterile water to obtain ES1 bacterial suspension for use.

[0041] 2. Effect of temperature on the degradation of nicosulfuron by Enterobacter hormaechei ES1

[0042] In the temperature experiment, five different temperatures (20°C, 25°C, 30°C, 35°C, and 40°C) were selected. -1 In the inorganic salt liquid medium, ES1 bacterial suspension was added at a volume percentage of 4%, and cultured on a shaker at 150 rpm. Samples were taken at 1 day and 4 days to measure OD 600 and nicosulfuron. Figure 3 Enterobacter hormaechei ES1 was greatly affected by temperature. At 35°C, Enterobacter hormaechei ES1 grew best (OD 600 1.25), the degradation rate of nicosulfuron reached 95.26% at 4 days. At 20℃, the growth and degradation of Enterobacter hormaechei ES1 were inhibited.

[0043] 3. Effect of pH on the degradation of nicosulfuron by Enterobacter hormaechei ES1

[0044] The Enterobacter hormaechei ES1 suspension was inoculated with nicosulfuron at a concentration of 50 mg L -1 The pH of the medium was adjusted to 5, 6, 7, 8, and 9 with hydrochloric acid and sodium hydroxide solution, respectively, and the medium was cultured in a shaking incubator at 35°C and 150 rpm. Samples were taken at 1 day and 4 days to measure the OD 600 and nicosulfuron. Figure 4 , Enterobacter hormaechei ES1 has the optimum pH of 6.0, OD 600 1.24, the degradation rate of nicosulfuron reached 95.12%, which was more suitable for degradation in a weakly acidic environment. At pH 9.0, the growth and degradation of Enterobacter hormaechei ES1 were inhibited, which was not suitable for degradation under alkaline conditions.

[0045] 4. Effect of Enterobacter hormaechei ES1 inoculum dose on degradation of nicosulfuron

[0046] The Enterobacterhormaechei ES1 suspension was inoculated with a nicosulfuron concentration of 50 mg L-1 at a volume percentage of 1%, 2%, 3%, 4%, and 5%.-1 The culture was shaken in an inorganic salt liquid medium at 35°C and 150 rpm, and samples were taken at 1d and 4d to measure OD 600 and nicosulfuron. Figure 5 , the inoculum amount of Enterobacter hormaechei ES1 had little effect on growth and degradation. Among them, when 4% strain ES1 was inoculated, OD 600 The highest value was 1.23, and the degradation rate of nicosulfuron was 95.22% after 4 days.

[0047] 5. Effect of initial concentration of nicosulfuron on degradation of nicosulfuron by Enterobacter hormaechei ES1

[0048] Nicosulfuron was added to the inorganic salt liquid culture medium to a final concentration of 10, 20, 50, 100, and 200 mg / L. -1 The Enterobacter hormaechei ES1 bacterial suspension was inoculated into an inorganic salt medium with different nicosulfuron concentrations, and cultured in a shaking incubator at 35°C and 150 rpm. Samples were taken at 1 day and 4 days to measure the OD 600 and nicosulfuron. Figure 6 , when the initial concentration of nicosulfuron was 20 mg / L -1 When OD 600 1.25, the degradation rate of nicosulfuron was 94.41% at 4 days. With the increase of concentration, the growth and degradation of Enterobacter hormaechei ES1 were inhibited.

[0049] Example 3: Preparation of repair bacterial agent

[0050] The repairing bacterial agent containing Enterobacter hormaechei ES1 is prepared by using the strain of Enterobacter hormaechei ES1 as a living component, and the steps are as follows:

[0051] 1) transferring Enterobacter hormaechei ES1 into LB liquid culture medium, shaking and culturing at 30° C. and 150 rpm until the logarithmic growth phase, and washing with a buffer solution with a pH of 7.00 to obtain a bacterial suspension;

[0052] 2) the bacterial suspension obtained in step 1) was inoculated into LB liquid culture medium at a volume ratio of 4%, and cultured at 30° C. and 150 rpm with shaking until the logarithmic growth phase to obtain Enterobacter hormaechei ES1 seed liquid;

[0053] 3) adding 20 mL of the Enterobacter hormaechei ES1 seed solution obtained in step 2) to 500 g of fermentation medium, wherein each 500 g of the fermentation medium is composed of 80% of agricultural waste (30% of sawdust, 30% of straw and 20% of biochar) and 20% of fungus bran culture material (15% of wheat bran, 3% of soybean meal, 1% of lime and 1% of light calcium carbonate) by weight, and then adding 10 mL of nutrient solution culture medium, and fermenting under ventilation at 30° C. for 4 days, and then packaging to obtain the bioremediation bacterial agent for nicosulfuron-contaminated soil of the present invention. The nutrient solution culture medium is prepared from 1.00 g of NaCl, 1.00 g of K2HPO4, 1.00 g of KH2PO4, 0.20 g of MgSO4·7H2O, 5.0 g of glucose, 1.80 g of NH4Cl, and 1000 mL of distilled water, the pH value is adjusted to 7.0, and sterilized at 115° C. for 15 min.

[0054] The screening and determination process of the fermentation medium components is as follows:

[0055] Orthogonal test analysis was conducted with sawdust (20%, 30%, 40% by mass), straw (20%, 30%, 40% by mass), and biochar (10%, 20%, 30% by mass) in different proportions, with the degradation rate of Nicosulfuron as the main basis. The remaining part of the above three materials was filled with bran culture material (each portion of bran culture material consisted of 75% wheat bran, 15% soybean meal, 5% lime and 5% light calcium carbonate by weight). After the materials were fully mixed in proportion, 4% ES1 seed solution (20mL 500g -1 ), and after being fully fermented in an incubator at 30°C, it was used as a repair agent. Table 2 shows the results of optimizing the composition of the agent by orthogonal test, which shows that when sawdust is 30%, straw is 30%, and biochar is 20%, and 20% of the bran culture material (15% wheat bran, 3% soybean meal, 1% lime and 1% light calcium carbonate) is filled, the degradation rate of nicosulfuron by the repair agent ES1 is 98.34%.

[0056] Table 2 Optimization of bacterial composition by orthogonal test method

[0057]

[0058]

[0059] Example 4: Bioremediation of Nicosulfuron-containing contamination by the remediation agent prepared in Example 3

[0060] 500 g of air-dried soil (soil from the experimental field of Jilin Agricultural University that had not been applied with nicosulfuron within 5 years) was added to each treatment group. Four treatment groups were set up: (A) Control: 20 mg kg -1 Nicosulfuron; (B) ES1: add 20mL of Enterobacter hormaechei ES1 suspension and 20mg kg -1 Nicosulfuron; (C) BMF: 20 g of the fermentation medium required for the bacterial agent (30% sawdust, 30% straw, 20% biochar, 20% bacterial bran culture material (15% wheat bran, 3% soybean meal, 1% lime and 1% light calcium carbonate)) was added, but Enterobacter hormaechei ES1 and 20 mg kg -1 (D) BFES1: Add 20 g of the repair bacteria prepared in Example 3 and 20 mg kg -1 Nicosulfuron. Soil samples were taken at 0d, 1d, 3d, 5d, 7d, 14d, and 28d to measure the concentration of Nicosulfuron. For the remediation of Nicosulfuron-contaminated soil, see Figure 7 In the control group, where only nicosulfuron was added, the concentration of nicosulfuron dropped to 17.03 mg kg at 28 days. -1 , which was naturally degraded in the soil with a degradation rate of 15.31%. After adding free bacteria of Enterobacter hormaechei ES1 (ES1 treatment group), the ES1 strain biodegraded Nicosulfuron in the soil, and the concentration of Nicosulfuron dropped to 1.23 mg kg at 28 days. -1 After adding the carrier required by the bacterial agent (BFM treatment group), the nicosulfuron in the soil was adsorbed, and the concentration of nicosulfuron dropped to 15.28 mg kg at 28 days. -1 After adding ES1 bacteria (BFES1 treatment group), agricultural waste and fungus husk culture material adsorbed Nicosulfuron in the soil, and Enterobacter hormaechei ES1 biodegraded Nicosulfuron in the soil. The concentration of Nicosulfuron dropped to 0.92 mg kg at 28 days. -1 , the degradation rate was 95.50%.

Claims

1. An Enterobacterium hallii capable of degrading nicosulfuron, characterized in that: The strain was deposited in the China Center for Type Culture Collection (CCTCC) on December 1, 2023, with the address being Wuhan University, Wuhan, China (430072), with the deposit number being CCTCC NO: M20232422, and the classification name being: Enterobacter hormaechei ES1.

2. A repair bacterial agent containing Enterobacter hormaechei ES1, which is prepared by the following steps: 1) transferring Enterobacter hormaechei ES1 into LB liquid culture medium, shaking and culturing at 30° C. and 150 rpm until the logarithmic growth phase, and washing with a buffer solution with a pH of 7.00 to obtain a bacterial suspension; 2) the bacterial suspension obtained in step 1) was inoculated into LB liquid culture medium at a volume ratio of 4%, and cultured at 30° C. and 150 rpm with shaking until the logarithmic growth phase to obtain Enterobacter hormaechei ES1 seed liquid; 3) 20 mL of the Enterobacter hormaechei ES1 seed solution obtained in step 2) was added to 500 g of the fermentation medium, and then 10 mL of the nutrient solution medium was added. The mixture was fermented with ventilation at 30° C. for 4 days and then packaged to obtain the repair bacterial agent.

3. A repairing bacterial agent containing Enterobacter hormaechei ES1 as claimed in claim 2, characterized in that: The composition of each 500g fermentation medium is composed of 80% agricultural waste and 20% mushroom bran culture medium by weight, the 80% agricultural waste is composed of 30% sawdust, 30% straw and 20% biochar, and the 20% mushroom bran culture medium is composed of 15% wheat bran, 3% soybean meal, 1% lime and 1% light calcium carbonate.

4. A repair bacterial agent containing Enterobacter hormaechei ES1 as claimed in claim 2, characterized in that: The nutrient solution medium was prepared by NaCl 1.00 g, K2HPO4 1.00 g, KH2PO4 1.00 g, MgSO4·7H2O 0.20 g, glucose 5.0 g, NH4Cl 1.80 g, and distilled water 1000 mL, adjusted to pH 7.0, and sterilized at 115°C for 15 min.

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