Method for producing indoleacetic acid through fermentation of Klebsiella variicola and application of fermentation liquor in crop growth promotion
IAA was produced by fermentation of Klebsiella habitat HHXJ2T-1 strain, which solved the problem of low yield of microbial fermentation method, and achieved efficient and environmentally friendly IAA production and crop growth promotion effects.
Patent Information
- Application Number
- CN202411412754.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, the production of indole acetic acid (IAA) by microbial fermentation method is low, which is difficult to meet the demand for large-scale production. The chemical synthesis method has high energy consumption, high cost and pollutes the environment.
The Klebsiella habitat HHXJ2T-1 strain was fermented under specific conditions to produce IAA, using L-tryptophan as a precursor substance to promote plant growth, especially root growth through fermentation broth.
It has achieved high yield IAA production, reduced production costs, reduced fertilizer use, promoted crop rooting and growth, and is environmentally friendly.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial fermentation preparation of plant physiologically active substances, and specifically relates to a method for fermenting a new microbial strain to produce indoleacetic acid and an application method of an indoleacetic acid fermentation broth. Background Art
[0002] Indole-3-acetic acid is the main active ingredient of natural plant auxin and is involved in many processes of plant development. Microorganisms can promote plant growth by secreting indoleacetic acid (hereinafter simply referred to as IAA). IAA can increase the size and distribution of plant roots, which is beneficial for the roots to absorb more nutrients from the soil. Producing plant hormones is one of the main mechanisms of the interaction between microorganisms and host plants. Under the condition of externally adding L-tryptophan, Klebsiella oxytoca HHXJ2T-1 can produce IAA, significantly increase the root length and root mass of vegetables, and can stimulate the germination of cucumber seeds and the development of roots under low-temperature conditions. Studying microorganisms with the ability to secrete IAA, improving the IAA production rate, and scaling up production are beneficial for applying plant growth-promoting bacteria to agricultural production.
[0003] In microorganisms, tryptophan is the precursor substance of its IAA biosynthesis pathway. The biosynthesis pathway of IAA, named after the intermediates of IAA synthesis, can be divided into 5 IAA biosynthesis pathways such as the indolepyruvic acid pathway, the indoleacetamide pathway, the tryptophan side-chain pathway, the tryptamine pathway, and the indoleacetonitrile pathway. Moreover, there may be more than one IAA synthesis pathway in a bacterium.
[0004] Currently, the industrial production method of IAA is mainly chemical synthesis. High temperature and high pressure are required during the production process of IAA, and toxic substances such as formaldehyde and potassium cyanide are used. Moreover, the finished product is expensive. It not only has high energy consumption, but also generates a large amount of harmful waste liquid, causing environmental pollution. Producing IAA by microbial fermentation has the advantages of mild reaction conditions, low energy consumption, no use of toxic and harmful substances, no environmental pollution, and low production cost. Therefore, it has a good market prospect. The IAA content in the fermentation broth produced by the existing microbial fermentation method is very low, all <1000 mg / L, which cannot meet the requirements of large-scale production of IAA by microbial fermentation. There are many types of microbial strains producing IAA at home and abroad, and the metabolic pathways for synthesizing IAA have also been basically explored, but the production scale-up of microbial fermentation synthesis of IAA has not been carried out, and the exploration and design of process parameters and culture medium components during the fermentation process have not been carried out. Summary of the Invention
[0005] The present invention relates to the field of microbial fermentation preparation of plant physiologically active substances, and aims to provide an application of a new microbial strain in the fermentation production of IAA and an application of the fermentation broth in promoting the root growth of crops.
[0006] The new microbial strain is Klebsiella variicola HHXJ2T-1, with the Latin name Klebsiella variicola; it was deposited at the Guangdong Microbial Culture Collection Center on September 2, 2022, at the 5th floor of Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 62759.
[0007] To achieve the above object, the technical solution adopted by the present invention is as follows: S1. Activation of the Klebsiella variicola HHXJ2T-1 strain.
[0008] To achieve the above object, the activation step of the Klebsiella variicola HHXJ2T-1 is as follows: inoculate the Klebsiella variicola HHXJ2T-1 slant strain into the LB liquid medium, and shake culture at 35°C and 200 rpm for 8 hours to obtain the HHXJ2T-1 seed liquid.
[0009] S2. Production of IAA using Klebsiella variicola HHXJ2T-1.
[0010] The inoculation and fermentation steps of the Klebsiella variicola HHXJ2T-1 are as follows: inoculate the seed liquid into the sterilized and cooled fermentation medium, and carry out aerated fermentation at 35°C. Feed supplement is carried out after 3 hours. Ferment at 35°C and pH 7.0 for 48 hours to obtain the fermentation broth containing indoleacetic acid.
[0011] S3. Promote the growth of lettuce using the fermentation broth of Klebsiella variicola HHXJ2T-1.
[0012] The application of the fermentation broth of Klebsiella variicola HHXJ2T-1 in promoting the growth of lettuce: determine the indoleacetic acid content in the fermentation broth containing indoleacetic acid by high performance liquid chromatography, and sequentially dilute the fermentation broth into liquid agents with indoleacetic acid concentrations of 20, 30, and 40 mg / kg. During the root growth and planting process of crops, treat them in the form of leaf spraying. The first treatment starts 7 days after the seedling transplantation, and one treatment is carried out every 7 days for a total of 3 times. After 30 days, measure the fresh weight of the above-ground part and the dry weight of the underground part of each treatment group.
[0013] The method for fermenting and producing IAA using Klebsiella variicola HHXJ2T-1 provided by the present invention has the following beneficial effects compared with the prior art: (1) The culture medium and culture conditions are simple, the operation is easy, and the production cost is low; (2) IAA has high yield, is easy to produce on a large scale, is energy-saving and environmentally friendly, and has huge economic and social benefits; (3) It can effectively promote the rooting and growth of crops, reduce the use of chemical fertilizers, and increase crop yields. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 shows the colony morphology of Klebsiella exogena HHXJ2T-1 on Axubei medium (nitrogen-free culture medium) Figure 2 is a phenotype diagram of IAA-treated lettuce in Example 2 DETAILED DESCRIPTION
[0015] The present invention provides a method for producing indoleacetic acid by fermentation of a strain of Klebsiella spp. and application of the fermentation liquid in promoting the growth of crops. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
[0016] Example 1 Application of Klebsiella exogena HHXJ2T-1 in fermentation production of IAA 1. Materials and equipment Strain: Klebsiella variicola HHXJ2T-1, Latin name Klebsiella variicola; deposited on September 2, 2022 at Guangdong Microbiological Culture Collection Center, Building 59, 5th Floor, No. 100, Xianlie Middle Road, Guangzhou, with the deposit number GDMCC No: 62759. Fermentation equipment: 5-liter fully automatic fermentation tank.
[0017] 2. Operation methods and steps (1) Seed liquid preparation: Inoculate the Klebsiella exogena HHXJ2T-1 slant strain into LB liquid medium and culture at 35°C and 200 rpm for 8 hours. The composition of the seed medium is: peptone 10 g / L, yeast powder 10 g / L, NaCl 5 g / L, pH 7.0-7.2.
[0018] (2) Inoculation and fermentation: Sterilize the culture medium for fermentation at 121°C for 20 min and then cool it to 35°C. Inoculate the seed liquid into the sterilized and cooled culture medium for fermentation (7 - 10 g / L glycerol, 3.0 - 7.0 g / L yeast powder, 2.0 - 5.0 g / L dry corn steep liquor, 0.2 - 0.5 g / L ammonium sulfate, 10.0 g / L L-tryptophan, 0.2 - 1.0 g / L anhydrous magnesium sulfate, 5 - 10 g / L potassium dihydrogen phosphate, 0.2 - 1 g / L sodium citrate, 1 L water, adjust the pH value to 7.0 - 7.2). After aerated fermentation for 3 h, add the following substances: 400 mL of 60 - 70% glycerol and 150 mL of 20 - 25% ammonia water. Maintain the temperature at 35°C, pH 7.0, stirrer speed in series with dissolved oxygen, and the dissolved oxygen is automatically controlled > 20%, and the tank pressure is 0.04 - 0.06 Mpa. Ferment for 48 hours to obtain a fermentation broth containing 2.58 - 2.97 g / Kg of IAA.
[0019] Example 2: Application of the fermentation broth containing IAA in promoting crop growth 1. Materials Strain: Klebsiella oxytoca HHXJ2T-1 Rooting material: Lettuce (Xiangzhilong Italian lettuce for all seasons) Planting medium: Substrate soil Planting container: Cultivation pot with a volume of 2 L 2. Operation methods and steps (1) Preparation of the fermentation broth containing IAA bacterial liquid: The same as in Example 1.
[0020] (2) Normal sowing and seedling raising of lettuce (3) Treatment: Determine the IAA content in the microbial bacterial liquid containing IAA by high performance liquid chromatography. Dilute the fermentation broth into use solutions with IAA concentrations of 20, 30, and 40 mg / kg in sequence, and set the sterilized culture medium for fermentation as the control. Set parallel for each concentration and treatment, and treat in the form of leaf spraying. Start the first treatment 7 days after seedling transplantation, and then treat once every 7 days for a total of 3 times. Measure the fresh weight of the above-ground part and the dry weight of the underground part of each treatment group after 30 d.
[0021] 3. Results
[0022] Compared with the control, treatments with various concentrations of IAA could significantly increase the fresh weight of the above-ground parts of lettuce (P = 0.000) and the dry weight of the underground parts (P = 0.000). According to the above test results, Klebsiella pneumoniae HHXJ2T-1 provided by the present invention has a strong ability to produce IAA, and the fermented liquid containing IAA can promote the growth of crops, especially induce the growth of roots. Therefore, it has good application prospects in promoting crop growth.
[0023] The above embodiments further illustrate the above content of the present invention, but this should not be construed as limiting the scope of the above subject matter of the present invention to the above embodiments. All technologies implemented based on the above content fall within the protection scope of the present invention.
Claims
1. A strain of Klebsiella oxytoca HHXJ2T-1, characterized in that, The described Klebsiella variicola was deposited at the Guangdong Microbial Culture Collection Center, 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou on September 2, 2022. Its Latin name is Klebsiella variicola, and the deposit number is GDMCC No: 62759.
2. Application of the Klebsiella variicola HHXJ2T-1 described in claim 1 in the production of indole-3-acetic acid.
3. A method for producing indoleacetic acid, characterized in that, The method includes fermenting and culturing the Klebsiella variicola HHXJ2T-1 described in claim 1, selecting the optimal culture medium and culture conditions, and collecting the culture product indole-3-acetic acid.
4. The method according to claim 3, characterized in that, It includes the following steps: S1. Prepare the seed medium: 10.0 g / L peptone, 5.0 g / L yeast extract powder, 10.0 g / L NaCl, 1 L water, and adjust the pH value to 7.0 - 7.2; S2. Prepare the initial fermentation medium for a 5 L fermenter: 7 - 10 g / L glycerol, 3.0 - 7.0 g / L yeast powder, 2.0 - 5.0 g / L corn steep liquor dry powder, 0.2 - 0.5 g / L ammonium sulfate, 10.0 g / L L-tryptophan, 0.2 - 1.0 g / L anhydrous magnesium sulfate, 5 - 10 g / L potassium dihydrogen phosphate, 0.2 - 1 g / L sodium citrate, 1 L water, adjust the pH value to 7.0 - 7.2, and the initial filling volume of the 5 L tank is 1.5 L; S3. Prepare the HHXJ2T-1 seed liquid: Inoculate the slant strain of Klebsiella variicola HHXJ2T-1 into the LB liquid medium, and shake culture at 35°C and 200 rpm for 8 hours to obtain the HHXJ2T-1 seed liquid; S4. IAA fermentation: After sterilizing the fermentation medium at 121°C for 20 min and then cooling it to 35°C, inoculate the seed liquid into the sterilized and cooled fermentation medium (7 - 10 g / L glycerol, 3.0 - 7.0 g / L yeast powder, 2.0 - 5.0 g / L corn steep liquor dry powder, 0.2 - 0.5 g / L ammonium sulfate, 10.0 g / L L-tryptophan, 0.2 - 1.0 g / L anhydrous magnesium sulfate, 5 - 10 g / L potassium dihydrogen phosphate, 0.2 - 1 g / L sodium citrate, 1 L water, adjust the pH value to 7.0 - 7.2). After aeration fermentation for 3 h, add the following substances: 400 ml of 60 - 70% glycerol and 150 ml of 20 - 25% ammonia water; maintain the temperature at 35°C, pH 7.0, stirrer speed in series with dissolved oxygen, and the dissolved oxygen is automatically controlled > 20%, and the tank pressure is 0.04 - 0.06 Mpa. Ferment for 48 hours to obtain a fermentation broth containing 2.58 - 2.97 g / Kg of indole-3-acetic acid.
5. Application of fermentation broth containing indoleacetic acid in promoting crop growth, characterized in that, It includes the IAA fermentation broth obtained by fermenting and culturing the Klebsiella variicola HHXJ2T-1 described in claim 4.
6. Use of the fermentation broth containing indoleacetic acid according to claim 5 in promoting crop growth, characterized in that, The required materials include: the IAA fermentation broth obtained by fermenting Klebsiella variicola HHXJ2T-1, Xiangzhilong Four Seasons Italian lettuce, substrate soil, and a cultivation pot with a volume of 2 L.
7. Use of the fermentation broth containing indoleacetic acid according to claim 5 in promoting crop growth, characterized in that, The specific operation method and steps include: S1. Preparation of the fermentation broth bacterial solution containing IAA: as described in claim 4; S2. Normal sowing and seedling raising of lettuce; S3. IAA treatment: Determine the content of indole acetic acid in the microbial bacterial solution containing indole acetic acid by high performance liquid chromatography. Dilute the fermentation broth successively into the use solutions with indole acetic acid concentrations of 20, 30, and 40 mg / kg, and set the sterilized culture medium used for fermentation as the control. Set up parallels for each concentration and treatment, and treat in the form of leaf spraying. Start the first treatment 7 days after the seedling transplantation, and then treat once every 7 days for a total of 3 times. Measure the fresh weight of the above-ground part and the dry weight of the underground part of each group of treatments after 30 days.
8. Use of the fermentation broth containing indoleacetic acid according to claim 7 in promoting crop growth, characterized in that, Applying the fermentation broth containing indole acetic acid during crop rooting and planting can effectively promote crop rooting and growth, reduce the amount of chemical fertilizer used, and increase crop yield.