Sphingomonas inoculant compound and application thereof in promoting degradation of chlorpyrifos

Abstract

The invention discloses a sphingomonas compound and application thereof in promoting degradation of chlorpyrifos, and relates to the technical field of biology. The compound is composed of a sterile fermentation broth of acinetobacter and a sphingomonas solution, and the acinetobacter fermentation broth obtained through screening can effectively promote growth and colonization activity of chlorpyrifos degradation bacteria sphingomonas, so that the degradation efficiency of chlorpyrifos is improved; and an efficient novel method is provided for bioremediation of pesticide pollution.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for improving the degradation efficiency of chlorpyrifos by using a composite of a sphingomonas agent and a bacterial fermentation liquid. Technical Background

[0002] Chlorpyrifos, as an organophosphorus pesticide, is widely used due to its high insecticidal properties. However, its residues in soil and water bodies pose serious hazards to the environment and human health. Currently, biodegradation is considered an effective method for pesticide pollution control. Since the 1970s, many strains of degrading bacteria that use chlorpyrifos as a carbon source and energy source have been isolated and identified. Chlorpyrifos-degrading bacteria from multiple genera such as Pseudomonas, Bacillus, Arthrobacter, Stenotrophomonas, and Sphingomonas have shown good degradation capabilities for chlorpyrifos under pure culture conditions.

[0003] However, in practical applications, there are often problems such as low survival rate and weak degradation activity of the introduced degradation bacteria. Therefore, it is necessary to find a method that can promote the growth of chlorpyrifos-degrading bacteria, promote their colonization in the soil, and enhance their ability to degrade chlorpyrifos. Summary of the invention

[0004] In view of the above problems, the present application provides a Sphingomonas agent compound and its application in promoting the degradation of chlorpyrifos.

[0005] Specifically, the above objectives are achieved through the following solutions:

[0006] First, the present application provides a sphingomonas agent compound, which is composed of bacterial fermentation broth and sphingomonas liquid; the above-mentioned bacterial fermentation broth is prepared by the following method: culturing rice Acinetobacter oryzae in LB liquid culture medium for 48 hours, then centrifuging the culture solution at 10000rpm for 10 minutes, taking the supernatant and filtering it with a 0.22μm filter membrane to filter out the bacteria in the culture solution, and the filtrate is the bacterial (sterile) fermentation broth.

[0007] The above-mentioned sphingomonas liquid was prepared by the following method: the activated sphingomonas was inoculated into LB liquid culture medium, cultured overnight in a shaker at 180 rpm and 30°C, centrifuged at 6000 rpm for 3 min, the supernatant was discarded, the bacterial precipitate was taken and sterile water was added to adjust the absorbance value (OD) of the bacterial liquid at a wavelength of 600 nm. 600 )=1, the bacterial content is 10 8 CFU / mL of Sphingomonas liquid.

[0008] Furthermore, in the above-mentioned sphingomonas agent compound, the OD of the sphingomonas liquid is 600=1; the volume ratio of the bacterial fermentation broth to the sphingomonas broth is preferably 20:1.

[0009] Preferably, the above-mentioned Sphingomonas is a strain with a deposit number of CGMCC No.11031.

[0010] Secondly, the present application provides the use of the above-mentioned sphingomonas agent compound in promoting the degradation of chlorpyrifos, that is, applying the sphingomonas agent compound to soil (such as soil in the rhizosphere of rice) to degrade the chlorpyrifos remaining in the soil.

[0011] Compared with the existing chlorpyrifos degradation methods, the present application discovered for the first time that the sterile fermentation broth of Acinetobacter oryzae can significantly improve the degradation efficiency of chlorpyrifos-degrading bacteria, thereby providing a new efficient and environmentally friendly method for the bioremediation of pesticide pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The statistical results of the aseptic fermentation broth of strain B10 in Example 1 promoting the growth of strain HJY are shown in FIG.

[0013] Figure 2 This is the test result of Example 2 for improving the degradation efficiency of chlorpyrifos by using a composite of Sphingomonas liquid and the sterile fermentation liquid of strain B10.

[0014] Figure 3 This is a photo of the culture dish in Example 3.

[0015] Figure 4 The statistical results of the number of HJY in the soil of Example 3 are shown.

[0016] Figure 5 The results of the chlorpyrifos concentration test in rice root tissue and root soil in Example 4 are shown. DETAILED DESCRIPTION

[0017] 1. Strains used in the examples:

[0018] The chlorpyrifos-degrading bacteria is Sphingomonas sp., which was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on July 2, 2015. The address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101; the deposit number is CGMCC No. 11031. The strain has been disclosed by Chinese patent CN105274031A, and the applicant named it the degradation bacteria HJY.

[0019] Strain B10 is Acinetobacter oryzae, which the applicant named B10. It was provided by Jiangsu Academy of Agricultural Sciences. The strain is a conventional strain in the field, as disclosed in the document "Genetic Diversity of Endophytic Diazotrophs of the Wild Rice, Oryza alta and Identification of the New Diazotroph, Acinetobacter oryzae sp. nov.", and can also be purchased through commercial channels (such as CGMCC1.10689).

[0020] 2. Culture medium and reagents involved in the examples:

[0021] LB liquid medium: Weigh 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl, dissolve them in deionized water, make up to 1000 mL, and adjust the pH to 7.0. Autoclave at 121°C for 20 min.

[0022] 10% LB liquid medium: obtained by mixing deionized water and LB liquid medium in a volume ratio of 9:1. Autoclave at 121°C for 20 min.

[0023] LB solid medium: Add agar to a final concentration of 2% to LB liquid medium, i.e., add 20 g agar to 1000 mL of liquid LB medium. Autoclave at 121°C for 20 min.

[0024] Chlorpyrifos / 10% LB liquid medium: Chlorpyrifos was added to 10% LB liquid medium at a final concentration of 5 mg / L.

[0025] Chlorpyrifos standard (99.9%) was purchased from Dr. Ehrenstorfer GmbH, Germany; acetonitrile (chromatographic grade) was purchased from Merck, Germany.

[0026] The 0.22 μm filter membrane was purchased from Merck-Millipore, specification: 33 mm, 0.22 μm.

[0027] Example 1 Screening of specific bacteria that promote the growth of chlorpyrifos-degrading bacteria HJY by fermentation broth culture method 1. Collection of sterile bacterial fermentation broth

[0028] This example tests the effects of sterile fermentation broths of 10 different bacteria (Table 1) screened from soil in the laboratory in the early stage on the growth of chlorpyrifos-degrading bacteria HJY, and screens bacterial (sterile) fermentation broths that promote the growth of HJY.

[0029]

[0030] First, the 10 activated bacterial strains were cultured in LB liquid medium for 48 h, and then the culture solution was centrifuged at 10000 r / min. -1 Centrifuge for 10 minutes, filter the supernatant after centrifugation with a filter membrane with a pore size of 0.22 μm to sterilize, and obtain the sterile fermentation broth of each bacterial strain for later use.

[0031] 2. Screening of specific bacteria that promote the growth of chlorpyrifos-degrading bacteria HJY

[0032] The activated strain HJY was inoculated into LB liquid medium and heated at 180 r·min. -1 , incubate overnight in a shaker at 30°C, 6000 r·min -1 Centrifuge for 3 minutes, discard the supernatant, take the bacterial precipitate and add sterile water to adjust the absorbance value of the bacterial solution at a wavelength of 600nm (OD 600 )=1, the bacterial content is about 10 8 CFU / mL of HJY bacterial solution for later use.

[0033] 20 μL of sterile fermentation liquid of each of the 10 bacterial strains and 180 μL of chlorpyrifos / 10% LB liquid medium were added to a 96-well plate, and 1 μL of HJY bacterial solution was added to each treatment; the positive control was set to add 200 μL of chlorpyrifos / 10% LB liquid medium + 1 μL of HJY bacterial solution; the negative control was 200 μL of chlorpyrifos / 10% LB liquid medium; each treatment was repeated 3 times. The 96-well plate was placed at 180 r·min -1 , shaker at 30°C, and measure OD after 36h of culture 600 The value characterizes the growth of HJY. The bacteria whose fermentation broth has a significant promoting effect on the growth of HJY were screened out. Figure 1 As shown, among the 10 bacterial fermentation broths in this experiment, the sterile fermentation broth of B10 had the most significant effect in promoting the growth of HJY.

[0034] Example 2 Indoor effect of aseptic fermentation broth of strain B10 on improving the efficiency of HJY in degrading chlorpyrifos

[0035] The effects of the aseptic fermentation broth of strain B10 on the degradation of chlorpyrifos by HJY were analyzed as follows:

[0036] CK treatment: 3 mL chlorpyrifos / 10% LB liquid medium;

[0037] HJY treatment: 2985 μL chlorpyrifos / 10% LB liquid medium + 15 μL HJY bacterial solution (preparation method is the same as Example 1);

[0038] HJY+B10 sterile fermentation liquid treatment: 2685 μL of chlorpyrifos / 10% LB liquid culture medium + 15 μL of HJY bacterial liquid (prepared in the same manner as in Example 1) + 300 μL of B10 sterile fermentation liquid (prepared in the same manner as in Example 1).

[0039] The above treatments were added into sterile 10 mL glass centrifuge tubes and placed in a shaker at 30 °C and 180 r / min. -1 Culture in dark. Take samples at 0, 24, 48, 72, 96, and 120 hours respectively; set 4 replicates for each sampling time point, extract chlorpyrifos from the culture solution by the full extraction method, and detect the concentration of chlorpyrifos (the sampling and detection methods of this embodiment refer to the method disclosed in the literature "Zhan Honglin, Ge Jing, Cheng Jiangfeng, et al. Effect of endophytic bacteria colonization of rice on the degradation of chlorpyrifos in paddy soil [J]. Jiangsu Agricultural Science, 2020, 48(14): 278-282."). The experimental results are as follows Figure 2 As shown, it can be seen that the sterile fermentation broth of strain B10 significantly improved the efficiency of HJY in degrading chlorpyrifos under laboratory culture conditions.

[0040] Example 3 Effect of sterile fermentation broth of strain B10 on promoting colonization of HJY in rice rhizosphere soil

[0041] Take rice seeds Nanjing 5055, soak them in 75% alcohol for 1 min, rinse them with sterile water 3-5 times, then soak them in 3% NaClO for 15 min, rinse them with sterile water 7-8 times, plant them on a plate covered with wet filter paper, and place them in a dark incubator at 30°C for 3 days for germination.

[0042] The germinated seeds were transplanted into a 9-well seedling tray filled with sterilized soil containing 10 mg / kg chlorpyrifos, with 6 seeds in each well. When the rice had three leaves and one heart, 4 rice plants with basically the same growth status were retained in each well as experimental treatment objects, and the OD prepared in Example 1 was applied to the roots. 600 =1 HJY bacterial solution and B10 sterile fermentation liquid were directly applied to the rice rhizosphere soil, 2 mL of HJY bacterial solution per well, and 40 mL of B10 sterile fermentation liquid per well; at the same time, a positive control treatment was set up, 2 mL of HJY bacterial solution + 40 mL of sterile water was applied; and 42 mL of sterile water was applied to the negative control treatment. One tray of seedlings was used for each treatment, and 4 seedlings in each well were used as 1 replicate, with 9 replicates per treatment. The rice seedlings were cultured at room temperature of 30°C and a photoperiod of 12 hours of light / 12 hours of darkness.

[0043] Rhizosphere soil samples were collected on the 6th, 12th and 18th day after treatment. 3 g of fresh rhizosphere soil was weighed with a 1% balance and added to a conical flask containing 27 mL of sterile water. The mixture was cultured at 30°C and 170 r / min for 6 h. A 10-fold dilution was obtained. 100 μL of the 10-fold dilution was taken and placed in a centrifuge tube containing 900 μL of sterile water. The mixture was vortexed for 1 min to mix thoroughly. 10 2 times dilution, dilute in this way to get 10 3 , 10 4 , 10 5 , 10 6 Dilution. Pipette 100 μL of 10 4 , 10 5 , 10 6 The diluted solution was evenly spread on the plate containing (final concentration) 25 mg L -1 The LB solid medium containing kanamycin was incubated upside down in a constant temperature incubator at 30°C for 2 days, and the grown HJY colonies were counted (e.g. Figure 3 The amount of HJY in each gram of rice rhizosphere soil was calculated.

[0044] The statistical results of HJY quantity are as follows Figure 4 As shown, compared with the treatment with only chlorpyrifos and its degrading bacteria HJY, the sterile fermentation broth of strain B10 significantly promoted the colonization of HJY in the rice rhizosphere soil.

[0045] Example 4 Effect of the sterile fermentation liquid of strain B10 on promoting HJY to degrade chlorpyrifos in rice root tissue and rhizosphere soil

[0046] The planting and related treatment of rice seedlings were the same as those in Example 3. Rice root tissue samples and rice rhizosphere soil samples were taken on the 6th, 12th and 18th days after treatment. According to the method disclosed in the literature "Zhan Honglin, Ge Jing, Cheng Jiangfeng, et al. Effect of endophytic bacteria colonization of rice on the degradation of chlorpyrifos in paddy soil [J]. Jiangsu Agricultural Science, 2020, 48(14): 278-282.", chlorpyrifos was extracted from rice root tissue samples and rice rhizosphere soil samples at each time point, and the concentration of chlorpyrifos was detected.

[0047] Test results such as Figure 5 As shown in Figure 2, the aseptic fermentation broth of strain B10 significantly promoted the degradation of rice root tissue by chlorpyrifos-degrading bacteria HJY ( Figure 5 (shown in A) and in rhizosphere soil ( Figure 5 The above examples illustrate that the composite prepared by using the sterile fermentation liquid of Acinetobacter oryzae and the liquid of Sphingomonas can significantly promote the growth and colonization of Sphingomonas and improve the degradation efficiency of chlorpyrifos.

Claims

1. A sphingomonas agent compound, characterized in that: The compound is composed of acinetobacter fermentation liquid and sphingomonas liquid.

2. The Sphingomonas agent compound according to claim 1, characterized in that: The Acinetobacter fermentation broth is prepared by the following method: culturing the rice Acinetobacter in LB liquid culture medium for 48 hours, centrifuging the culture broth, filtering the supernatant with a 0.22 µm filter membrane, and the filtrate is the Acinetobacter fermentation broth.

3. The Sphingomonas agent composition according to claim 1, characterized in that: In the compound, the sphingomonas liquid is prepared by the following method: inoculating sphingomonas into LB liquid culture medium, culturing overnight in a shaker at 180 rpm and 30°C, centrifuging, taking the bacterial precipitate and adding sterile water to adjust the bacterial content to 10 8 CFU / mL, that is, the Sphingomonas liquid is obtained.

4. The Sphingomonas agent compound according to claim 1, characterized in that: The volume ratio of the Acinetobacter fermentation broth to the Sphingomonas broth is 20:

1.

5. Use of the Sphingomonas agent compound as claimed in any one of claims 1 to 4 in the degradation of chlorpyrifos.

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