Bacillus siamensis TWS79 and application thereof
By screening the strain of Bacillus Siam with strong cellulase-producing ability, the existing heavy metal repair microorganisms have poor colonization ability, and the effect of effectively repairing cadmium-contaminated soil in mildly heavy metal-contaminated farmlands was achieved.
Patent Information
- Application Number
- CN202411977994.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-05-02
AI Technical Summary
Existing heavy metal repair microorganisms have poor colonization ability in soil, resulting in poor repair results.
By screening the strain of Bacillus Siam TWS79 with strong cellulase production ability, this strain has good colonization ability in soil rich in straw resources and can effectively adsorb cadmium ions.
The strain of Bacillus Siam TWS79 can effectively repair and control cadmium-contaminated soil in mildly heavy metal-contaminated farmlands, reduce the cadmium content in soil and rice, and has high application prospects and economic value.
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Figure CN119913064A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of agricultural microorganisms, and in particular to a siam bacillus TWS79 and an application thereof. Background Art
[0002] According to statistics, more than 10% of the cultivated land area in the country is currently polluted by heavy metals. About half of the cultivated land in some cities in South China is polluted by toxic heavy metals such as cadmium, arsenic, mercury and petroleum organic matter. In some cities in the Yangtze River Delta, contiguous farmland is polluted by multiple heavy metals. Commonly used soil heavy metal remediation methods include physical remediation, chemical remediation, phytoremediation and microbial remediation. Among them, microbial remediation uses microorganisms to passivate or convert heavy metals into non-toxic forms in the form of adsorption, reduction, detoxification, stabilization, etc., to prevent plant absorption. Compared with physical, chemical remediation and phytoremediation technologies, microbial remediation is a green, low-cost, sustainable remediation technology that does not affect normal agricultural production.
[0003] During the process of microbial remediation of soil heavy metal pollution, proteins, polysaccharides and other substances in the microorganisms can form complexes with heavy metal ions, thereby adsorbing them on the surface of the microorganisms and reducing their pollution to the soil. However, whether the exogenously added microorganisms can stably reproduce in the soil is the key to the success of microbial remediation. Conventional screening of heavy metal remediation microorganisms only focuses on the tolerance of microorganisms to heavy metals and their remediation performance, and their colonization ability is often overlooked. In fact, the content of heavy metals in cultivated soil is relatively low, and the requirements for the heavy metal tolerance of microorganisms are not high. Only microorganisms that can maintain stable reproduction can exert a sustained remediation effect. The present invention uses the production of cellulase as the first screening factor to screen out an extracellular polysaccharide-producing strain that is more suitable for colonization in cultivated soil rich in straw resources, and its Cd 2+ Its tolerance capacity reaches 20mg / L, it can effectively adsorb cadmium ions, and can effectively repair and treat cadmium-contaminated cultivated soil, with high application prospects and economic value. Summary of the invention
[0004] The purpose of the present invention is to provide a siam bacillus TWS79 and an application thereof, so as to solve the deficiencies of the prior art.
[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is:
[0006] A strain of Bacillus siamensis TWS79, the Bacillus siamensis TWS79 (Bacillus siamensis) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32347.
[0007] Bacillus siamensis TWS79 cells prepared by the Bacillus siamensis TWS79.
[0008] The preparation method of Siamese Bacillus TWS79 bacterial cells is as follows: Siamese Bacillus TWS79 (Bacillussiamensis) is inoculated into LB liquid culture medium, and the liquid fermentation conditions are as follows: fermentation temperature is 28-30°C, rotation speed is 170-190r / min, and fermentation time is 24-48h. After the fermentation is completed, Siamese Bacillus TWS79 bacterial cells are obtained by centrifugation.
[0009] Furthermore, the siam bacillus TWS79 cells are resuspended in sterile water to prepare a bacterial suspension, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / ml.
[0010] Furthermore, the microbial agent is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by adsorption, mixing and solid fermentation, that is, the microbial fertilizer.
[0011] Furthermore, the microbial agent is used to reduce the effective cadmium content in soil and the cadmium content in rice.
[0012] Furthermore, any one or more of the Bacillus siamensis TWS79 (Bacillus siamensis) and / or the Bacillus siamensis TWS79 cells and / or the microbial agent and / or the microbial fertilizer are used for straw degradation, cellulase production and exopolysaccharide production.
[0013] Furthermore, the application method of Bacillus siamensis TWS79 (Bacillus siamensis) includes root irrigation and / or soil spraying and / or flushing the Bacillus siamensis TWS79 (Bacillus siamensis) and / or the Bacillus siamensis TWS79 cells and / or the microbial agent and / or the microbial fertilizer.
[0014] Furthermore, the application scenario is farmland with mild heavy metal pollution.
[0015] Beneficial effects of the present invention: In view of the problem that conventional heavy metal remediation microorganisms have poor colonization ability in the soil and thus cause poor remediation effect, the present invention uses cellulase production as the first screening factor to screen out a siam Bacillus TWS79 with the functions of degrading straw, producing extracellular polysaccharides, and adsorbing heavy metals. The strain can utilize straw to form good colonization ability in the soil where straw is returned to the field, has better environmental adaptability, can effectively repair and treat cadmium-contaminated cultivated soil, and has high application prospects and economic value. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The colony morphology of strain TWS79 on LB medium;
[0017] Figure 2 This is a diagram of the cellulose decomposition effect of strain TWS79;
[0018] Figure 3 This is the filter paper disintegration effect diagram of strain TWS79;
[0019] Figure 4 is the 16S rRNA phylogenetic tree of strain TWS79;
[0020] Figure 5 is the effective cadmium content in soils with different treatments;
[0021] Figure 6 Cadmium content in rice with different treatments.
[0022] TWS79, whose classification name is Bacillus siamensis, is deposited in the General Microbiology Center of China Culture Collection Administration, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is October 25, 2024, and the deposit number is CGMCC NO.32347. DETAILED DESCRIPTION
[0023] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.
[0024] It should be noted that the details of the present invention are known to those skilled in the art. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer, and the experimental steps not described in detail refer to pathophysiological experiments, online databases, etc.
[0025] Unless otherwise specified, the raw materials used in the following examples are as follows:
[0026] LB liquid medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, dilute to 1 L with deionized water, and sterilize at 121 °C for 30 min.
[0027] LB solid medium (1 L): 10 g of tryptone, 5 g of yeast powder, 10 g of NaCl, 15 g of agar, dilute to 1 L with deionized water, and sterilize at 121 °C for 30 min.
[0028] CMC-Na medium (1 L): CMC-Na 15 g, NH4NO3 1 g, yeast extract 1 g, MgSO4·7H2O 0.5 g, KH2PO4 1 g, agar 15 g, dilute to 1 L with deionized water, sterilize at 121°C for 30 min.
[0029] Filter paper culture medium: 5 filter paper strips, NH4NO3 1 g, yeast extract 1 g, MgSO4·7H2O 0.5 g, KH2PO4 1 g, dilute to 1 L with deionized water, sterilize at 121℃ for 30 min.
[0030] Example 1
[0031] A strain of Bacillus siamensis TWS79, Bacillus siamensis TWS79 (Bacillus siamensis), was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32347.
[0032] Isolation, screening and identification of Bacillus siamensis TWS79:
[0033] (1) Isolation of strains
[0034] Soil samples were collected from a vegetable garden in Yiyang City. 10 g of soil sample was added to a triangular flask containing 100 ml of sterile saline. After standing for 20 min, the sample was shaken at 30 °C and 180 rpm for 30 min. 1 ml of the sample was added to 9 ml of sterile saline and then diluted 10 2 , 10 3 , 10 4 times, take 100 μl of the above soil suspension and evenly spread it on the CMC-Na medium plate, culture it in a 30℃ incubator for 48 h, pick up a single bacterial colony with a sterile toothpick, streak it, purify it, and save it for later use.
[0035] (2) Screening of functional strains
[0036] The strain stored at 4°C was inoculated into CMC-Na solid medium, cultured at 30°C for 72h, stained with Congo red solution for 30min, and then rinsed with NaCl solution. The cellulase production capacity of the strain was preliminarily identified based on the ratio of the cellulase decomposition circle to the colony diameter. The strain stored at 4°C was inoculated into LB liquid medium, cultured overnight at 30°C and 160r / min, transferred to filter paper medium, cultured at 30°C and 160r / min, and the disintegration of the filter paper was observed.
[0037] The strain stored at 4°C was inoculated into LB liquid culture medium and cultured at 30°C and 160r / min for 72h to obtain fermentation broth. Extracellular polysaccharides were extracted by alcohol precipitation method. A small amount of distilled water was added to the crude extracellular polysaccharide to dissolve it to obtain a crude polysaccharide test solution. The extracellular polysaccharide content was determined by phenol-sulfuric acid method.
[0038] The strain stored at 4°C was inoculated into LB liquid medium, cultured overnight at 30°C and 160 r / min, and then transferred to medium containing 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L Cd 2+ LB liquid medium, cultured at 30°C, 160r / min for 72h, centrifuged to obtain the supernatant, and the Cd of the centrifuged liquid was determined. 2+ content.
[0039] The strain TWS79 was tested to have the ability to produce cellulase and extracellular polysaccharides ( Figure 2 , Figure 3 ), can tolerate 20mg / LCd 2+ Concentration, at 20mg / L Cd 2+ Under the concentration conditions, the cadmium adsorption rate was 56.87%.
[0040] (3) Strain identification
[0041] After the strain TWS79 was cultured on LB solid medium at 30℃ for 48h, the colonies were round, white, dry and wrinkled, and 1-3mm in diameter. The phylogenetic tree analysis results constructed by 16S rRNA gene sequence showed that the strain TWS79 had the highest homology with Bacillus siamensis ( Figure 4 ), combined with the results of phylogenetic tree comparison analysis based on colony morphology and 16S rRNA gene sequence, strain TWS79 was identified as Bacillus siamensis.
[0042] The invention discloses a Siamese Bacillus TWS79 bacterial cell prepared by Siamese Bacillus TWS79 (Bacillus siamensis), and a preparation method of Siamese Bacillus TWS79 bacterial cell. Siamese Bacillus TWS79 is inoculated into LB liquid culture medium, and the liquid fermentation conditions are as follows: fermentation temperature is 28°C, rotation speed is 170r / min, and fermentation time is 24h. After the fermentation is completed, Siamese Bacillus TWS79 bacterial cell is obtained by centrifugation.
[0043] The Bacillus siamese TWS79 cells were resuspended in sterile water to prepare a bacterial suspension, i.e., a microbial agent, with an effective viable count of ≥ 0.2 × 10 8 cfu / ml.
[0044] Solid microbial agents or bio-organic fertilizers or compound microbial fertilizers prepared by adsorption, mixing and solid fermentation of microbial agents are called microbial fertilizers.
[0045] The application of microbial agents in reducing the effective cadmium content in soil and the cadmium content in rice is in farmland with mild heavy metal pollution. The microbial agents are applied by root irrigation and / or spraying and / or flushing on the soil.
[0046] The application of any one or more of Bacillus siamensis TWS79 and / or Bacillus siamensis TWS79 cells and / or microbial agents and / or microbial fertilizers in straw degradation, cellulase production and extracellular polysaccharide production, and its application scenario is farmland with mild heavy metal pollution.
[0047] Example 2
[0048] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing Bacillus siamensis TWS79 bacterial cells is prepared by using Bacillus siamensis TWS79.
[0049] The invention discloses a Siamese Bacillus TWS79 bacterial cell prepared by Siamese Bacillus TWS79 (Bacillus siamensis), and a preparation method of Siamese Bacillus TWS79 bacterial cell. Siamese Bacillus TWS79 (Bacillus siamensis) is inoculated into LB liquid culture medium. The liquid fermentation conditions are as follows: the fermentation temperature is 29°C, the rotation speed is 180r / min, and the fermentation time is 36h. After the fermentation is completed, the Siamese Bacillus TWS79 bacterial cell is obtained by centrifugation.
[0050] The Bacillus siamese TWS79 cells were resuspended in sterile water to prepare a bacterial suspension, i.e., a microbial agent, with an effective viable count of ≥ 0.2 × 10 8 cfu / ml.
[0051] Solid microbial agents or bio-organic fertilizers or compound microbial fertilizers prepared by adsorption, mixing and solid fermentation of microbial agents are called microbial fertilizers.
[0052] The application of microbial agents in reducing the effective cadmium content in soil and the cadmium content in rice is in farmland with mild heavy metal pollution. The microbial agents are applied by root irrigation and / or spraying and / or flushing on the soil.
[0053] The application of any one or more of Bacillus siamensis TWS79 and / or Bacillus siamensis TWS79 cells and / or microbial agents and / or microbial fertilizers in straw degradation, cellulase production and extracellular polysaccharide production, and its application scenario is farmland with mild heavy metal pollution.
[0054] Example 3
[0055] The content of this embodiment is basically the same as that of embodiment 1, except that the method for preparing Bacillus siamensis TWS79 bacterial cells is prepared by using Bacillus siamensis TWS79.
[0056] The invention discloses a Siamese Bacillus TWS79 bacterial cell prepared by Siamese Bacillus TWS79 (Bacillus siamensis), and a preparation method of Siamese Bacillus TWS79 bacterial cell. Siamese Bacillus TWS79 (Bacillus siamensis) is inoculated into LB liquid culture medium. The liquid fermentation conditions are as follows: the fermentation temperature is 30°C, the rotation speed is 190r / min, and the fermentation time is 48h. After the fermentation is completed, the Siamese Bacillus TWS79 bacterial cell is obtained by centrifugation.
[0057] The Bacillus siamese TWS79 cells were resuspended in sterile water to prepare a bacterial suspension, i.e., a microbial agent, with an effective viable count of ≥ 0.2 × 10 8 cfu / ml.
[0058] Solid microbial agents or bio-organic fertilizers or compound microbial fertilizers prepared by adsorption, mixing and solid fermentation of microbial agents are called microbial fertilizers.
[0059] The application of microbial agents in reducing the effective cadmium content in soil and the cadmium content in rice is in farmland with mild heavy metal pollution. The microbial agents are applied by root irrigation and / or spraying and / or flushing on the soil.
[0060] The application of any one or more of Bacillus siamensis TWS79 and / or Bacillus siamensis TWS79 cells and / or microbial agents and / or microbial fertilizers in straw degradation, cellulase production and extracellular polysaccharide production, and its application scenario is farmland with mild heavy metal pollution.
[0061] The following is a test of microbial agents on reducing the effective cadmium content in soil and rice:
[0062] The potted plant test was conducted in the greenhouse of Hunan Tewos Ecological Co., Ltd. in Suining County, Hunan Province, and the test period was from May 2024 to October 2024. The pH of the potted soil was 5.32, the total cadmium content was 0.472 mg / kg, and the effective cadmium content was 0.124 mg / kg.
[0063] The experiment set up T1 (control treatment, clean water), T2 (0.1% straw organic fertilizer by soil weight), T3 (TWS79 bacterial agent prepared in Example 2, with an effective viable count of 1.0×10 8 cfu / ml), T4 (TWS79 bacterial agent prepared in Example 2 + 0.1% straw organic fertilizer by soil weight), and 5 replicates for each treatment. Before planting, the fertilizer was mixed with the soil, and the bacterial agent was applied with water, 6 ml per pot. After completion, routine maintenance was performed. After rice was harvested, the effective cadmium content in the soil was measured ( Figure 5 ), cadmium content in rice ( Figure 6 ).
[0064] It can be seen from this that adding microbial agents can reduce the effective cadmium content in soil and the cadmium content in rice, and the effect is better when applied with straw organic fertilizer.
[0065] Based on the above examples and comprehensive analysis of the test results, strain TWS79 is able to produce cellulase and extracellular polysaccharides, and has the function of reducing the effective cadmium content in soil and the cadmium content in rice.
[0066] Components not described in detail herein are prior art.
[0067] Although the specific embodiments of the present invention are described in detail above, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in this field without departing from the purpose of the present invention, and modifications or deformations without creative labor are still within the protection scope of the present invention.
Claims
1. A strain of Bacillus siamese TWS79, characterized in that: The Siamese Bacillus subtilis TWS79 (Bacillus siamensis) was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 25, 2024, with the deposit number CGMCC No.32347.
2. The Siamese Bacillus TWS79 bacterial cell prepared according to the Siamese Bacillus TWS79 (Bacillus siamensis) described in claim 1.
3. The method for preparing the siam bacillus TWS79 cell according to claim 2, characterized in that: Bacillus siamensis TWS79 was inoculated into LB liquid culture medium. The liquid fermentation conditions were as follows: fermentation temperature was 28-30°C, rotation speed was 170-190 r / min, and fermentation time was 24-48 h. After the fermentation was completed, Bacillus siamensis TWS79 cells were obtained by centrifugation.
4. A microbial agent containing the siam bacillus TWS79 bacterial cell according to claim 2 or 3, characterized in that: The Siamese Bacillus TWS79 cells are resuspended in sterile water to prepare a bacterial suspension, i.e., the microbial agent, with an effective viable count of ≥ 0.2×10 8 cfu / ml.
5. A microbial fertilizer containing the microbial agent according to claim 4, characterized in that: The microbial agent is a solid microbial agent or a biological organic fertilizer or a compound microbial fertilizer prepared by the microbial agent through adsorption, mixing and solid fermentation, that is, the microbial fertilizer.
6. Use of the microbial agent according to claim 4 in reducing the effective cadmium content in soil and rice.
7. Use of any one or more of the Bacillus siamensis TWS79 according to claim 1 and / or the Bacillus siamensis TWS79 cells according to claim 2 and / or the microbial agent according to claim 4 and / or the microbial fertilizer according to claim 5 in straw degradation, cellulase production and exopolysaccharide production.
8. A method for applying Bacillus siamensis TWS79, characterized in that: The method comprises irrigating the roots and / or spraying and / or flushing the soil with the Bacillus siamensis TWS79 described in claim 1 and / or the Bacillus siamensis TWS79 bacteria described in claim 2 and / or the microbial agent described in claim 4 and / or the microbial fertilizer described in claim 5.
9. The use according to claim 6 or 7, characterized in that: The application scenario is farmland that is slightly contaminated by heavy metals.