A method for producing indole acetic acid by using bacillus amyloliquefaciens stage perfusion fermentation

Abstract

This invention belongs to the field of microbial fermentation technology, specifically relating to a method for producing indoleacetic acid using Bacillus amyloliquefaciens via intermittent perfusion fermentation. The method involves fermenting a seed culture of Bacillus amyloliquefaciens in a fermentation system. When the cell concentration reaches a preset threshold, the fermentation broth is collected. When the volume of the fermentation broth reaches the threshold, collection is stopped, and perfusion is initiated by adding perfusion medium to the fermentation system. Perfusion is stopped once the total volume of the fermentation broth returns to its initial state. The fermentation system continues to ferment, and indoleacetic acid is produced through multiple intermittent perfusion fermentations. This invention employs an intermittent perfusion fermentation mode. By intermittently removing the product fermentation broth and adding fresh medium, a stable supply of nutrients within the fermentation system is maintained, while significantly reducing the accumulation of inhibitory metabolites.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation. Background Technology

[0002] Indole-3-acetic acid (IAA) is a widely distributed natural auxin in plants, playing a crucial regulatory role in plant growth and development. It promotes cell elongation and division, induces adventitious root differentiation, regulates flowering and fruiting processes, and participates in the regulation of plant stress responses. In agricultural production, IAA, as an important plant growth regulator, can effectively increase crop yield, improve the quality of agricultural products, and enhance crop resistance to stress, showing broad application prospects in the cultivation of food crops, cash crops, and horticulture.

[0003] Currently, the industrial production of indoleacetic acid (IAA) mainly relies on two technological pathways: chemical synthesis and microbial fermentation. While chemical synthesis can achieve large-scale production, it has significant limitations: stringent reaction conditions (requiring high temperature, high pressure, or specific catalysts), the potential for toxic and harmful chemical residues during synthesis, low product purity, and the presence of harmful byproducts that contradict the green, low-carbon, and environmentally friendly development principles of modern agriculture, thus limiting its application. In contrast, microbial fermentation, with its advantages of wide availability of raw materials, mild reaction conditions, high product purity, and good environmental compatibility, has become a research hotspot and mainstream development direction for the efficient production of IAA.

[0004] Bacillus amyloliquefaciens, a type of Gram-positive bacterium, is a superior host strain for the microbial fermentation production of indoleacetic acid (IAA). Currently, the processes for producing IAA using Bacillus fermentation mainly focus on batch fermentation and continuous flow fermentation. However, both processes have significant technical drawbacks, hindering the efficient and low-cost production of IAA. 1. In batch fermentation, as the fermentation progresses, nutrients such as carbon and nitrogen sources in the culture medium are continuously consumed, while bacterial metabolites accumulate. High concentrations of metabolic byproducts have a significant feedback inhibitory effect on bacterial growth and IAA synthesis, ultimately leading to a shorter fermentation cycle and difficulty in increasing IAA yield and efficiency, failing to meet the demands of industrial-scale production. 2. Although continuous flow fermentation alleviates the nutrient limitation problem by continuously adding fresh culture medium, some bacteria are lost with the fermentation broth during fermentation, making it difficult to significantly increase the bacterial density in the fermentation system, directly limiting IAA production. 3. Continuous flow fermentation has limited ability to remove inhibitory metabolites accumulated in the fermentation broth, and the product feedback inhibition problem remains unresolved, making it difficult to achieve continuous and efficient synthesis of indoleacetic acid (IAA), resulting in poor fermentation stability. 4. Due to the inherent limitations of the continuous flow fermentation process, some nutrients and precursors (tryptophan) are not fully metabolized and utilized by the cells before being lost with the fermentation broth. This not only wastes nutrients but also further increases the fermentation production cost of IAA, reducing the economic efficiency of the process.

[0005] Therefore, it is necessary to find a way to break through the yield bottleneck of traditional fermentation processes, increase the synthesis and production rate of indoleacetic acid, reduce the cost input in the industrial production process, and improve the economic efficiency and market competitiveness of the entire production process. Summary of the Invention

[0006] In view of the problems of low yield, low productivity, difficulty in continuous production, and low degree of automation in the production of indoleacetic acid using Bacillus amyloliquefaciens in the existing technology, the purpose of this invention is to provide a method for producing indoleacetic acid by staged perfusion fermentation using Bacillus amyloliquefaciens.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for producing indoleacetic acid using Bacillus amyloliquefaciens via staged perfusion fermentation involves fermenting Bacillus amyloliquefaciens seed culture in a fermentation system. When the cell concentration reaches a preset threshold, the fermentation broth is collected. When the volume of the fermentation broth reaches the threshold, the collection of fermentation broth is stopped, and perfusion is initiated to add perfusion culture medium to the fermentation system. Perfusion is stopped once the total volume of the fermentation broth returns to its initial state. The fermentation system continues to ferment, and indoleacetic acid is produced through multiple stages of perfusion fermentation.

[0008] Furthermore, the Bacillus amyloliquefaciens seed culture was fermented in a fermenter until the bacterial cell concentration reached OD500.600 When the pH is 5.0~6.0, the fermentation broth is received through a tangential flow filter. When the volume of the fermentation broth reaches 20%-30% of the initial volume of the fermenter, the receiving of the fermentation broth is stopped, and perfusion is started. The perfusion medium enters the fermenter through a tangential flow filter. Perfusion is stopped when the total volume of the fermentation broth in the fermenter returns to its initial state. The seed liquid in the fermenter continues to ferment. After continuous culture for 3~4 hours, the above operation is repeated. Indoleacetic acid is produced through multiple stages of perfusion fermentation.

[0009] The Bacillus amyloliquefaciens seed culture was inoculated into the fermentation medium in the fermenter at an inoculation rate of 5-10%. The fermentation medium consisted of rice slurry 10-15 g / L, molasses 10-15 g / L, KH2PO4 1-3 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.5-1.5 g / L, MnSO4·H2O 0.1-0.3 g / L, and pH 6.0-7.0. The culture was then sterilized at 121℃ for 20 min.

[0010] The fermentation conditions for the fermentation system are: temperature 30~37℃, pH 5.5~6.5, and dissolved oxygen ≥30%.

[0011] The perfusion culture medium is formulated as follows: corn steep liquor 3-5 g / L, molasses 5-10 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5-1 g / L, MnSO4·H2O 0.1-0.2 g / L, L-tryptophan 3-5 g / L, pH 5.5-6.5, sterilized at 121℃ for 20 min.

[0012] Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018, accession address Wuhan University, Wuhan, China.

[0013] The seed culture of Bacillus amyloliquefaciens is obtained by inoculating Bacillus amyloliquefaciens strains into an activation medium and culturing the activated bacterial solution, and then inoculating the activated bacterial solution into a seed medium and culturing it to obtain the seed culture. The activation medium consisted of 10-15 g / L peptone, 5-8 g / L yeast extract, and 5-10 g / L NaCl, with a pH of 6.5-7.5, and was sterilized at 121°C for 20 min. The seed culture medium consisted of 10-15 g / L peptone, 5-8 g / L yeast extract, 5-8 g / L NaCl, and pH 6.5-7.5, sterilized at 121℃ for 20 min.

[0014] The fermentation broth from multiple stages of perfusion was collected and mixed, and then subjected to centrifugation, acid precipitation, extraction, concentration, decolorization, and crystallization in sequence to obtain a high-purity indoleacetic acid product. The centrifugation involves centrifuging the collected fermentation broth at a speed of 8000-10000 r / min for 10-15 min to remove the bacterial precipitate, and then collecting the supernatant and combining it with the filtrate. The acid precipitation involves adjusting the pH of the supernatant to 2.0-3.0 and allowing it to stand at 4°C for 2-4 hours to precipitate indoleacetic acid. The extraction process involves adding an equal volume of ethyl acetate to the acid-precipitated mixture for extraction, collecting the organic phase, and repeating the extraction 2-3 times, then combining the organic phases. The concentration process involves concentrating the combined organic phases under reduced pressure at 40-50°C and a vacuum of 0.08-0.09 MPa to obtain a crude paste-like product. The decolorization process involves dissolving the crude product in anhydrous ethanol by heating, adding activated carbon, stirring for 10-15 minutes to decolorize, and then filtering while hot to remove the activated carbon. The crystallization process involves placing the filtrate at 4°C for 12-24 hours to allow crystals to precipitate. The crystals are then collected by filtration, washed 2-3 times with ethanol, and dried to obtain the indoleacetic acid product.

[0015] An apparatus for the method includes a fermenter 1, a perfusion replenishment tank 2, a perfusion receiving tank 3, and a tangential flow filter 10. The tangential flow filter 10 is internally divided into a fermentation broth chamber and a filtration chamber by a hollow fiber membrane 15. The fermentation broth chamber is connected to a fermentation broth inlet 16 and a fermentation broth outlet 17 on one side of the tangential flow filter 10. The filtration chamber is connected to a filtration outlet 18 at the end of the tangential flow filter 10. The fermentation broth inlet 16 is connected to a corresponding port on the fermenter 1 via a first pipeline, which is equipped with a fermentation broth outlet pump 11 and a valve B 7. The fermentation broth outlet 17 is connected to another port on the fermenter 1 via a second pipeline, which is equipped with a valve C 8. The filtration outlet 18 is connected to the perfusion receiving tank 3 via a third pipeline, which is equipped with a valve D 9. Simultaneously, the filtration outlet 18 is connected to the perfusion replenishment tank 2 via a fourth pipeline, which is equipped with a perfusion pump 12 and a valve A 6.

[0016] Specifically, a method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation includes the following steps: I. Strain activation: Inoculate the Bacillus amyloliquefaciens strain into the activation medium and culture it with shaking at 30-37℃ and 150-200 r / min for 12-18 h to obtain the activated bacterial solution; The formulation of the activation medium is as follows: 10-15 g / L peptone, 5-8 g / L yeast extract, 5-10 g / L NaCl, pH 6.5-7.5, sterilized at 121℃ for 20 min.

[0017] Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018, accession address Wuhan University, Wuhan, China.

[0018] II. Seed culture: Inoculate the activated bacterial solution into the seed culture medium at an inoculation rate of 5-10%, and culture with shaking at 30-37℃ and 150-200r / min for 8-12 hours to obtain the seed culture.

[0019] The seed culture medium formula is as follows: peptone 10-15 g / L, yeast powder 5-8 g / L, NaCl 5-8 g / L, pH 6.5-7.5, sterilized at 121℃ for 20 min.

[0020] III. Pre-fermentation culture: Inoculate the seed culture into the fermentation medium of fermenter 1 at an inoculation rate of 5-10%, and carry out fermentation culture. During the process, control the fermentation temperature at 30-37℃. Stabilize the pH of the fermentation system at 5.5-6.5 by automatically adding 1mol / L hydrochloric acid or 1mol / L sodium hydroxide solution. Coordinate the stirring speed and aeration rate to maintain the dissolved oxygen content of the fermentation broth above 30%.

[0021] The fermentation medium formula is as follows: corn steep liquor 10-15 g / L, molasses 10-15 g / L, KH2PO4 1-3 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.5-1.5 g / L, MnSO4·H2O 0.1-0.3 g / L, pH 6.0-7.0; sterilize at 121℃ for 20 min.

[0022] IV. Staged perfusion filtration: [The text abruptly ends here, seemingly mid-sentence fragment.] 600 When the pressure reaches 5.0~6.0, turn on the fermentation broth outlet pump 11 and open control valves B7 and C8 to allow the fermentation broth in fermenter 1 to enter the tangential flow filter 10. The filtration operation pressure is controlled to be ≤0.05MPa by pressure sensor 4. Then, open valve D9 to start tangential flow filtration. The filtrate is collected in the perfusion receiving tank 3 through valve D9.

[0023] V. Post-filtration perfusion feeding: When the filtered volume of the fermentation broth reaches 20%-30% (preferably 25%) of the initial volume of the fermentation tank, stop the fermentation broth outlet pump 11 and simultaneously close valves B7 and D9; then open valve A6 and perfusion pump 12, and the perfusion medium is fed into the fermentation tank 1 from the perfusion replenishment tank 2 through the tangential flow filter 10.

[0024] The perfusion culture medium is formulated as follows: corn steep liquor 3-5 g / L, molasses 5-10 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5-1 g / L, MnSO4·H2O 0.1-0.2 g / L, L-tryptophan 3-5 g / L; pH 5.5-6.5, sterilized at 121℃ for 20 min.

[0025] VI. Termination of Irrigation Process: When the total volume of fermentation liquid in fermenter 1 returns to its initial state, stop the irrigation pump 12 and close valve A 6 to complete a single irrigation.

[0026] VII. Staged Irrigation and Cyclic Culture: After a single irrigation, continue to maintain the fermentation conditions in fermenter 1 at a temperature of 30-37℃, pH 5.5-6.5, and dissolved oxygen ≥30% for 3-4 hours. Then, restart the tangential flow filtration system and repeat the irrigation operations in steps four to six above to carry out intermittent irrigation fermentation. Indoleacetic acid is produced through multiple staged irrigation fermentations.

[0027] 8. Isolation and purification of indoleacetic acid: The fermentation broth discharged during multiple perfusion fermentation processes is collected and subjected to acid precipitation, extraction, concentration, decolorization and crystallization steps in sequence to obtain high-purity indoleacetic acid (IAA) product.

[0028] 1. Acid precipitation: Adjust the pH of the supernatant to 2.0-3.0 and let it stand at 4℃ for 2-4 hours to allow indoleacetic acid to precipitate completely.

[0029] 2. Extraction: Add an equal volume of ethyl acetate to the acid-precipitated mixture for extraction and collect the organic phase; repeat the extraction 2-3 times and combine the organic phases.

[0030] 3. Concentration: The combined organic phases are concentrated under reduced pressure at 40-50℃ and a vacuum of 0.08-0.09MPa to obtain a crude paste.

[0031] 4. Decolorization: Dissolve the crude product in anhydrous ethanol by heating (50-60℃), then add activated carbon (2%-5% of the crude product weight). Stir and decolorize for 10-15 minutes. Filter while hot to remove the activated carbon.

[0032] 5. Crystallization: Place the filtrate at 4℃ for 12-24 hours to allow crystals to precipitate. Collect the crystals by filtration, wash them 2-3 times with a small amount of ice-cold ethanol, and dry them to obtain the indoleacetic acid product.

[0033] Compared with the prior art, the present invention has the following beneficial effects: 1. The advantages of strains lay the foundation for efficient production: This invention uses a self-selected Bacillus amyloliquefaciens strain as the production strain. This strain has both strong indoleacetic acid (IAA) synthesis ability and excellent stress resistance. It can maintain stable metabolic activity during intermittent perfusion fermentation, providing reliable support for efficient IAA synthesis and helping to reduce production costs.

[0034] 2. Optimizing fermentation mode to improve production efficiency: By adopting an intermittent perfusion fermentation mode, the fermentation broth is removed intermittently and fresh culture medium is added, which can maintain a stable supply of nutrients in the fermentation system and significantly reduce the accumulation of inhibitory metabolites. Compared with continuous perfusion fermentation, it can improve the utilization rate of substances in the system, ensure the efficiency of indoleacetic acid synthesis, and further reduce production costs.

[0035] 3. Microbial cell retention technology solves production pain points: To address the problems of easy loss of bacterial cells and nutrients, low bacterial concentration, raw material waste, and insufficient indoleacetic acid (IAA) synthesis efficiency in conventional perfusion fermentation, this invention combines intermittent perfusion fermentation with tangential flow filtration technology to achieve efficient retention of Bacillus amyloliquefaciens, ensuring that the bacterial cells continuously circulate within the fermenter system and participate in IAA production. This reduces the amount of culture medium used while increasing bacterial fermentation concentration and nutrient utilization, achieving efficient and low-cost IAA production. Experimental verification shows that the IAA conversion rate of this invention can reach 73%, significantly higher than traditional batch fermentation and continuous perfusion fermentation, and the fermentation process can operate continuously and stably, effectively solving the technical problem of unstable continuous IAA production.

[0036] 4. Tangential flow filtration ensures continuous fermentation: This invention employs a tangential flow filter, which allows the bacteria in the fermentation broth to form a dynamic circulation between the fermenter and the filter during the staged perfusion filtration process. The flowing fermentation broth can promptly carry away the bacteria attached to the filter membrane surface and return it to the fermenter, avoiding the accumulation of bacteria that could clog the filter and ensuring the continuous and stable operation of the perfusion culture.

[0037] 5. Optimize irrigation flow design to improve production efficiency: This invention optimizes the perfusion flow design: after the fermentation broth completes a single filtration, the perfusion pump is activated, causing the perfusion medium to flow counter-currently along the inner surface of the hollow fiber membrane in the filter, and then enters the fermenter through a valve, forming a backwash enhancement mechanism. This design, while periodically replenishing the fermenter with fresh medium, efficiently removes adsorbed bacteria and impurities from the membrane surface, stabilizes the flux and separation efficiency of the filtration system, and extends the service life of the filter membrane. Furthermore, it promotes the rapid return of retained bacteria from the filter to the fermentation system, allowing them to continue participating in the biosynthesis of indoleacetic acid, thereby significantly improving the overall production efficiency and yield per unit volume of indoleacetic acid.

[0038] 6. Automated optimization of segmental perfusion culture reduces labor costs: By optimizing the operational methods and standardized procedures of staged perfusion culture, a deep integration of the process scheme and the fermenter's automated control system was achieved, establishing an automated staged perfusion culture system. Automated control replaces manual operation, precisely achieving core processes such as determining the timing of perfusion, regulating the perfusion flow rate, and closed-loop management of culture parameters. This significantly reduces the labor intensity of frontline operators and minimizes the costs associated with manual monitoring and operation. Simultaneously, it effectively avoids human error, improves the stability and reproducibility of the culture process, ensures the uniformity of culture product quality, and facilitates a high-efficiency, lean, and intelligent upgrade of the culture production process.

[0039] 7. Optimized culture medium formulation to reduce raw material costs: This invention systematically optimizes the formulation of fermentation perfusion culture medium. By precisely controlling the amount of L-tryptophan added during staged perfusion, the excessive consumption and waste of precursor substances are effectively avoided while ensuring a sufficient supply of precursors for indoleacetic acid biosynthesis. This significantly reduces raw material production costs and lays a solid foundation for the low-cost, large-scale industrial production of indoleacetic acid. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the apparatus in the irrigation filtration stage according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the apparatus during the irrigation and feeding stage according to an embodiment of the present invention; The components include: 1. Fermentation tank; 2. Perfusion replenishment tank; 3. Perfusion receiving tank; 4. Pressure sensor; 5. Fermentation tank controller; 6. Valve A; 7. Valve B; 8. Valve C; 9. Valve D; 10. Tangential flow filter; 11. Fermentation broth outlet pump; 12. Perfusion pump; 13. Liquid level contact gauge; 14. Perfusion control level electrode. Figure 3 This is a diagram showing the working flow direction of the tangential flow filter during the irrigation filtration stage. Figure 4 This is a diagram showing the working flow direction of the tangential flow filter during the irrigation and feeding stage. Among them, 15 is hollow fiber membrane; 16 is fermentation broth inlet; 17 is fermentation broth outlet; and 18 is filtrate outlet. Detailed Implementation

[0041] Unless otherwise specified, the conventional microbiological experimental methods, such as culture medium sterilization and aseptic operation, involved in this invention shall be performed in accordance with conventional techniques in the field. The pH value during fermentation can be adjusted by automatically adding hydrochloric acid or sodium hydroxide solution, and the dissolved oxygen level can be controlled by adjusting the aeration rate and stirring speed. The staged perfusion volume can be adjusted by the output signal from the level gauge inside the fermenter.

[0042] The invention will now be further described with reference to the accompanying drawings.

[0043] like Figures 1 to 4 As shown, the tangential flow filter 10 is internally divided into a fermentation broth chamber and a filtrate chamber by a hollow fiber membrane 15. The fermentation broth chamber is connected to the fermentation broth inlet 16 and fermentation broth outlet 17 on one side of the tangential flow filter 10, and the filtrate chamber is connected to the filtrate outlet 18 at the end of the tangential flow filter 10. The fermentation broth inlet 16 is connected to the corresponding port on the fermenter 1 through a first pipeline, which is equipped with a fermentation broth outlet pump 11 and valve B 7. The fermentation broth outlet 17 is connected to another port on the fermenter 1 through a second pipeline, which is equipped with valve C 8. The filtrate outlet 18 is connected to the perfusion receiving tank 3 through a third pipeline, which is equipped with valve D 9. At the same time, the filtrate outlet 18 is connected to the perfusion replenishment tank 2 through a fourth pipeline, which is equipped with a perfusion pump 12 and valve A 6.

[0044] The working principle of the tangential flow filter is as follows: the fermentation broth flows parallel to the outer surface of the hollow fiber membrane in a tangential direction. Part of the broth permeates through the membrane layer and enters the inner cavity of the membrane to form filtrate, which is then collected and flows into the receiving tank. The concentrated fermentation broth retained is returned to the fermenter to achieve circulation. This tangential flow mode can effectively alleviate the problems of concentration polarization on the filter membrane surface and filter cake accumulation, and significantly improve the stability of the filtration process and the continuous operation time.

[0045] The hollow fiber membrane is made of hydrophilic modified multi-channel polytetrafluoroethylene (PTFE) hollow fiber membrane, which has excellent chemical stability and resistance to fluid shock. The filter membrane has a precise pore size of 0.22μm. Relying on the high efficiency of tangential flow mass transfer, it can efficiently retain Bacillus amyloliquefaciens cells in continuous fermentation operation, while ensuring that small molecule metabolites such as indoleacetic acid can quickly pass through the membrane layer, thus achieving efficient separation of cells and target metabolites.

[0046] The tangential flow filter and the fermenter are sealed together via a sterile quick-connect fitting. All connecting pipes and the tangential flow filter are sterilized by steam at 121°C for 20 minutes before being connected to the fermentation system under aseptic conditions, meeting the stringent quality control requirements of the aseptic fermentation process.

[0047] To improve the operational stability and filtration flux of membrane modules, this invention optimizes the perfusion flow direction to construct an online backwashing system for the filter membrane. After the fermentation broth completes a single filtration, the perfusion medium is flushed against the inner surface of the hollow fiber membrane in the tangential flow filter, and then returned to the fermenter through a valve. This backwashing design can efficiently remove adsorbed bacteria and retain impurities on the filter membrane surface, stabilize the flux and separation efficiency of the filtration system, and extend the service life of the filter membrane. At the same time, it can quickly return the bacteria retained in the filter to the fermentation system, allowing them to continuously participate in the biosynthesis of indoleacetic acid, ultimately significantly improving the overall production efficiency and fermentation yield per unit volume of indoleacetic acid.

[0048] Example 1 A method for producing indoleacetic acid using Bacillus perfusion fermentation, the specific steps of which are as follows: S1. Strain activation: Bacillus amyloliquefaciens was inoculated into activation medium and cultured with shaking at 35℃ and 150 rpm for 18 h to obtain activated bacterial solution. The activation medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min. Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018, accession address Wuhan University, Wuhan, China.

[0049] S2. Seed culture: The activated bacterial solution was inoculated into the seed culture medium at a 5% inoculation rate and cultured with shaking at 30℃ and 150 rpm for 12 h to obtain the seed culture. The seed culture medium formula was: 10 g / L peptone, 5 g / L yeast extract, 8 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min.

[0050] S3. Early fermentation culture: The seed culture was inoculated into the fermentation medium of a 5L fermenter 1 at a 5% inoculation rate. The liquid level was detected by the liquid level gauge 13. The initial liquid volume was 4L. After inoculation, fermentation was carried out for 6 hours, and the fermentation temperature was controlled at 35℃. The pH of the fermentation system was stabilized at 6.5 by automatically adding 1mol / L hydrochloric acid solution. The stirring speed and aeration rate were adjusted in a coordinated manner to maintain the dissolved oxygen content of the fermentation broth above 30%.

[0051] The fermentation medium formula is as follows: corn steep liquor 10 g / L, molasses 10 g / L, KH2PO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.1 g / L, pH 7.0; sterilized at 121℃ for 20 min.

[0052] S4. Staged perfusion filtration: bacterial cell concentration OD 600 When the pressure reaches 6.0, the perfusion program is set in the fermentation operating system, the fermentation broth outlet pump 11 of the tangential filtration system is turned on, and the control valves B7 and C8 are opened to allow the fermentation broth in the fermenter 1 to enter the tangential flow filter 10. The filtration operation pressure is controlled to be ≤0.05MPa by the pressure sensor 4. Then, the tangential flow filtration is started by opening the valve D9, and the filtrate is collected in the perfusion receiving tank 3 through the valve D9.

[0053] S5. Post-filtration feeding: When the fermentation broth reaches a filtration volume of 1L, the fermentation system identifies the liquid level signal through the perfusion control level electrode 14, automatically stops the fermentation broth outlet pump 11, and closes valves B7 and D9. Then, it opens valve A6 and the perfusion pump 12, allowing the perfusion medium to be fed into the fermenter 1 from the perfusion replenishment tank 2 through the tangential flow filter 10.

[0054] The perfusion medium was formulated as follows: corn steep liquor 3 g / L, molasses 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.2 g / L, L-tryptophan 4 g / L; pH 6.5, sterilized at 121℃ for 20 min.

[0055] S6. Irrigation process terminated: When the total volume of fermentation liquid in fermenter 1 returns to 4L, the liquid level contact level gauge 13 outputs a signal to fermenter controller 5, and fermenter controller 5 issues a command to stop perfusion pump 12 and close valve A 6, completing a single perfusion.

[0056] S7. Staged perfusion and circulation culture: After a single perfusion, the fermentation temperature of fermenter 1 is maintained at 35°C. The pH is stabilized at 6.5 by automatically adding 1 mol / L hydrochloric acid solution, and the dissolved oxygen content is controlled at ≥30%. After culturing for another 3 hours, the tangential flow filtration system of steps S4-S6 above is restarted, and the above perfusion operation is repeated for intermittent perfusion automatic culture.

[0057] Indoleacetic acid was detected using high-performance liquid chromatography (HPLC), with the specific chromatographic conditions set as follows: The chromatographic column used was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was methanol-0.1% phosphoric acid aqueous solution (volume ratio 50:50); the flow rate was 1.0 mL / min; the column temperature was 30℃; and the detection wavelength was 280 nm.

[0058] S8. Isolation and purification of indoleacetic acid: The fermentation broth from multiple batches was collected, and the final indoleacetic acid (IAA) concentration was 658 mg / L, with an IAA conversion rate of 65%. Hydrochloric acid was slowly added to the supernatant to adjust the pH to 2.5, and the mixture was allowed to stand at 4°C for 3 hours to allow IAA to precipitate completely. The acid-precipitated mixture was extracted with an equal volume of ethyl acetate, and the organic phase was collected. This extraction was repeated three times, and the organic phases were combined. The combined organic phases were concentrated under reduced pressure at 50°C and 0.08 MPa to obtain a crude paste. The crude product was dissolved in anhydrous ethanol at 60°C, and activated carbon (3% of the crude product weight) was added. The mixture was stirred and decolorized for 15 minutes, and the activated carbon was removed by hot filtration. The filtrate was allowed to stand at 4°C for 12 hours to precipitate crystals. The crystals were collected by filtration, washed three times with a small amount of ice-cold ethanol, and dried to obtain the final IAA product with a purity of 92.5%.

[0059] Example 2 A method for producing indoleacetic acid using Bacillus perfusion fermentation, the specific steps of which are as follows: S1. Strain activation: Bacillus amyloliquefaciens was inoculated into activation medium and cultured with shaking at 35℃ and 150 rpm for 18 h to obtain activated bacterial solution. The activation medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min. Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018, accession address Wuhan University, Wuhan, China.

[0060] S2. Seed culture: The activated bacterial solution was inoculated into the seed culture medium at an inoculum volume of 5%, and cultured with shaking at 35℃ and 150 rpm for 12 h to obtain the seed culture. The seed culture medium formula was: 10 g / L peptone, 5 g / L yeast extract, 8 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min.

[0061] S3. Early fermentation culture: The seed culture was inoculated into the fermentation medium of a 5L fermenter 1 at a 5% inoculation rate. The liquid level was detected by the liquid level gauge 13. The initial liquid volume was 4L. After inoculation, fermentation was carried out for 6 hours, and the fermentation temperature was controlled at 35℃. The pH of the fermentation system was stabilized at 6.0 by automatically adding 1mol / L sodium hydroxide solution. The stirring speed and aeration rate were adjusted in a coordinated manner to maintain the dissolved oxygen content of the fermentation broth above 30%.

[0062] The fermentation medium formula is as follows: corn steep liquor 10 g / L, molasses 10 g / L, KH2PO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.1 g / L, pH 7.0; sterilized at 121℃ for 20 min.

[0063] S4. Staged perfusion filtration: bacterial cell concentration OD 600 When the pressure reaches 6.0, the perfusion program is set in the fermentation operating system, the fermentation broth outlet pump 11 of the tangential filtration system is turned on, and the control valves B7 and C8 are opened to allow the fermentation broth in the fermenter 1 to enter the tangential flow filter 10. The filtration operation pressure is controlled to be ≤0.05MPa by the pressure sensor 4. Then, the tangential flow filtration is started by opening the valve D9, and the filtrate is collected in the perfusion receiving tank 3 through the valve D9.

[0064] S5. Post-filtration feeding: When the fermentation broth reaches a filtration volume of 1L, the fermentation system identifies the liquid level signal through the perfusion control level electrode 14, automatically stops the fermentation broth outlet pump 11, and closes valves B7 and D9. Then, it opens valve A6 and the perfusion pump 12, allowing the perfusion medium to be fed into the fermenter 1 from the perfusion replenishment tank 2 through the tangential flow filter 10.

[0065] The perfusion culture medium was formulated as follows: corn steep liquor 3 g / L, molasses 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.2 g / L, L-tryptophan 4 g / L; pH 6.5, sterilized at 121℃ for 20 min.

[0066] S6. Irrigation process terminated: When the total volume of fermentation liquid in fermenter 1 returns to 4L, the liquid level contact level gauge 13 outputs a signal to fermenter controller 5, and fermenter controller 5 issues a command to stop perfusion pump 12 and close valve A 6, completing a single perfusion.

[0067] S7. Staged perfusion and circulation culture: After a single perfusion, the fermentation temperature of fermenter 1 is maintained at 35℃. The pH is stabilized at 6.0 by automatically adding 1mol / L sodium hydroxide solution, and the dissolved oxygen content is controlled at ≥30%. After culturing for another 3 hours, the tangential flow filtration system of steps S4-S6 is restarted, and the above perfusion operation is repeated for intermittent perfusion automatic culture.

[0068] Indoleacetic acid was detected using high-performance liquid chromatography (HPLC), with the specific chromatographic conditions set as follows: The chromatographic column used was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was methanol-0.1% phosphoric acid aqueous solution (volume ratio 50:50); the flow rate was 1.0 mL / min; the column temperature was 30℃; and the detection wavelength was 280 nm.

[0069] S8. Isolation and purification of indoleacetic acid: The fermentation broth from multiple production runs was collected, yielding 710 mg / L of indoleacetic acid (IAA), with an IAA conversion rate of 68%. Hydrochloric acid was slowly added to the supernatant to adjust the pH to 2.5, and the mixture was allowed to stand at 4°C for 3 hours to allow IAA to precipitate completely. An equal volume of ethyl acetate was added to the acid-precipitated mixture for extraction, and the organic phase was collected. This extraction was repeated three times, and the organic phases were combined. The combined organic phases were concentrated under reduced pressure at 50°C and a vacuum of 0.08 MPa to obtain a crude paste. The crude product was dissolved in anhydrous ethanol at 60°C, and activated carbon (3% of the crude product weight) was added. The mixture was stirred and decolorized for 15 minutes, and the activated carbon was removed by hot filtration. The filtrate was allowed to stand at 4°C for 12 hours to precipitate crystals. The crystals were collected by filtration, washed three times with a small amount of ice-cold ethanol, and dried to obtain the final IAA product with a purity of 93.2%.

[0070] Example 3 A method for producing indoleacetic acid using Bacillus perfusion fermentation, the specific steps of which are as follows: S1. Strain activation: Bacillus amyloliquefaciens was inoculated into activation medium and cultured with shaking at 35℃ and 150 rpm for 18 h to obtain activated bacterial solution. The activation medium formula was: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min. Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018.

[0071] S2. Seed culture: The activated bacterial solution was inoculated into the seed culture medium at a 5% inoculation rate and cultured with shaking at 37℃ and 150 rpm for 12 h to obtain the seed culture. The seed culture medium formula was: 10 g / L peptone, 5 g / L yeast extract, 8 g / L NaCl, pH 7.5, sterilized at 121℃ for 20 min.

[0072] S3. Early fermentation culture: The seed culture was inoculated into the fermentation medium of a 5L fermenter 1 at a 5% inoculation rate. The liquid level was detected by the liquid level gauge 13. The initial liquid volume was 4L. After inoculation, fermentation was carried out for 6 hours, and the fermentation temperature was controlled at 35℃. The pH of the fermentation system was stabilized at 5.5 by automatically adding 1mol / L hydrochloric acid solution. The stirring speed and aeration rate were adjusted in a coordinated manner to maintain the dissolved oxygen content of the fermentation broth above 30%.

[0073] The fermentation medium formula is as follows: corn steep liquor 10 g / L, molasses 10 g / L, KH2PO4 1 g / L, K2HPO4·3H2O 2 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.1 g / L, pH 7.0; sterilized at 121℃ for 20 min.

[0074] S4. Staged perfusion filtration: bacterial cell concentration OD 600 When the pressure reaches 6.0, the perfusion program is set in the fermentation operating system, the fermentation broth outlet pump 11 of the tangential filtration system is turned on, and the control valves B7 and C8 are opened to allow the fermentation broth in the fermenter 1 to enter the tangential flow filter 10. The filtration operation pressure is controlled to be ≤0.05MPa by the pressure sensor 4. Then, the tangential flow filtration is started by opening the valve D9, and the filtrate is collected in the perfusion receiving tank 3 through the valve D9.

[0075] S5. Post-filtration feeding: When the fermentation broth reaches a filtration volume of 1L, the fermentation system identifies the liquid level signal through the perfusion control level electrode 14, automatically stops the fermentation broth outlet pump 11, and closes valves B7 and D9. Then, it opens valve A6 and the perfusion pump 12, allowing the perfusion medium to be fed into the fermenter 1 from the perfusion replenishment tank 2 through the tangential flow filter 10.

[0076] The perfusion culture medium was formulated as follows: corn steep liquor 3 g / L, molasses 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, MnSO4·H2O 0.2 g / L, L-tryptophan 4 g / L; pH 6.5, sterilized at 121℃ for 20 min.

[0077] S6. Irrigation process terminated: When the total volume of fermentation liquid in fermenter 1 returns to 4L, the liquid level contact level gauge 13 outputs a signal to fermenter controller 5, and fermenter controller 5 issues a command to stop perfusion pump 12 and close valve A 6, completing a single perfusion.

[0078] S7. Staged perfusion and circulation culture: After a single perfusion, the fermentation temperature of fermenter 1 is maintained at 35℃. The pH is stabilized at 5.5 by automatically adding 1mol / L hydrochloric acid solution, and the dissolved oxygen content is controlled at ≥30%. After culturing for another 3 hours, the tangential flow filtration system of steps S4-S6 is restarted, and the above perfusion operation is repeated for intermittent perfusion automatic culture.

[0079] Indoleacetic acid was detected using high-performance liquid chromatography (HPLC), with the specific chromatographic conditions set as follows: The chromatographic column used was a C18 reversed-phase column (4.6 mm × 250 mm, 5 μm); the mobile phase was methanol-0.1% phosphoric acid aqueous solution (volume ratio 50:50); the flow rate was 1.0 mL / min; the column temperature was 30℃; and the detection wavelength was 280 nm.

[0080] S8. Isolation and purification of indoleacetic acid: The fermentation broth from multiple batches was collected, and the final indoleacetic acid (IAA) concentration was 736 mg / L, with an IAA conversion rate of 73%. Hydrochloric acid was slowly added to the supernatant to adjust the pH to 2.5, and the mixture was allowed to stand at 4°C for 4 hours to allow IAA to precipitate completely. The acid-precipitated mixture was extracted with an equal volume of ethyl acetate, and the organic phase was collected. This extraction was repeated three times, and the organic phases were combined. The combined organic phases were concentrated under reduced pressure at 50°C and 0.08 MPa to obtain a crude paste. The crude paste was dissolved in anhydrous ethanol at 60°C, and activated carbon (3% of the crude product weight) was added. The mixture was stirred and decolorized for 15 minutes, and the activated carbon was removed by hot filtration. The filtrate was allowed to stand at 4°C for 12 hours to precipitate crystals. The crystals were collected by filtration, washed three times with a small amount of ice-cold ethanol, and dried to obtain the final IAA product with a purity of 96.3%.

[0081] Comparative Example 1 The traditional batch fermentation method for producing indoleacetic acid involves the following steps: D1. Strain activation and D2. Seed culture steps are the same as in Example 3.

[0082] D3. Traditional batch fermentation: The seed culture was inoculated at a rate of 5% into the fermentation medium (same as in Example 3) in a 5L fermenter, with an initial volume of 4L. After inoculation, fermentation was carried out first until the cell concentration (OD) was reached. 600When the pH reached 6.0, sterilized tryptophan solution was added to the fermenter to achieve a tryptophan concentration of 1 g / L in the fermentation broth. The fermentation temperature was maintained at 35°C, and the pH was stabilized at 5.5 by automatically adding 1 mol / L hydrochloric acid solution. The dissolved oxygen content was controlled to be ≥30%. Samples were taken periodically to detect key parameters such as indoleacetic acid (IAA) concentration and cell concentration in the fermentation broth to ensure a stable and controllable fermentation process. The final IAA content in the fermentation broth was 363 mg / L, with a conversion rate of 36%.

[0083] D4. Isolation and purification of indoleacetic acid: After collecting the fermentation broth, the same indoleacetic acid separation and purification steps as in Example 3 were used to obtain the indoleacetic acid product. The product purity was 89.1%.

[0084] Comparative Example 2 The specific steps of the continuous perfusion fermentation method for producing indoleacetic acid are as follows: D1. Strain activation and D2. Seed culture steps are the same as in Example 3.

[0085] D3. Conventional continuous perfusion culture: The seed culture was inoculated into the fermentation medium (same as in Example 3) of a 5L fermenter at an inoculation rate of 5%, with an initial liquid volume of 4L. After inoculation, fermentation culture was carried out first, and the bacterial cell concentration was increased to OD100. 600 When the culture medium reached pH 6.0, a continuous perfusion program was set in the fermentation operating system. The perfusion pump and fermentation broth outlet pump were turned on, and the pump speed was set to pump the perfusion medium in (the same as in Example 3). At the same time, the fermentation broth outlet pump was turned on to export an equal amount of fermentation broth to maintain a constant volume of fermentation broth, thus achieving continuous culture. During fermentation, the fermentation temperature was maintained at 35°C, and the pH of the fermentation system was stabilized at 5.5 by automatically adding 1 mol / L hydrochloric acid solution, and the dissolved oxygen content was controlled to be ≥30%. During the culture period, samples were taken daily at regular intervals to detect key parameters such as the concentration of indoleacetic acid (IAA) and cell concentration in the fermentation broth, ensuring that the fermentation process was stable and controllable. The final IAA content in the fermentation broth was 593 mg / L, with a conversion rate of 59%.

[0086] D4. Isolation and purification of indoleacetic acid: The combined perfusion culture and fermentation broths were then purified using the same indoleacetic acid separation and purification steps as in Example 3 to obtain the indoleacetic acid product. The product purity was 90.5%.

[0087] Table 1 shows a comparison of the indicators of Examples 1-3 and Comparative Examples 1-2.

[0088] Table 1

[0089] IAA conversion rate calculation: IAA conversion rate is the ratio of the total IAA mass in the final fermentation broth to the total amount of tryptophan used in the fermentation, i.e.: IAA conversion rate = total IAA generated / amount of tryptophan used ×.

[0090] As shown in Table 1, this invention employs an intermittent perfusion fermentation mode. By intermittently removing the fermentation broth and replenishing it with fresh culture medium, a stable supply of nutrients within the fermentation system can be maintained, while significantly reducing the accumulation of inhibitory metabolites. Compared to continuous perfusion fermentation, this improves the utilization rate of substances within the system and ensures the efficiency of indoleacetic acid synthesis. This invention combines intermittent perfusion fermentation with tangential flow filtration technology to achieve efficient retention of Bacillus amyloliquefaciens, ensuring that the bacteria continuously circulate within the fermenter system and participate in indoleacetic acid production. This can increase the bacterial fermentation concentration and nutrient utilization rate while reducing the amount of culture medium used, achieving efficient and low-cost production of indoleacetic acid. In Example 3 of this invention, the indoleacetic acid conversion rate reaches 73%, significantly higher than Comparative Examples 1 and 2, and the fermentation process can operate continuously and stably, effectively solving the technical problem of unstable continuous production of indoleacetic acid.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation, characterized in that: Bacillus amyloliquefaciens seed culture was fermented in a fermentation system. When the cell concentration reached a preset threshold, the fermentation broth was collected. When the volume of the fermentation broth reached the threshold, the collection of fermentation broth was stopped, and perfusion was started to add perfusion medium to the fermentation system. Perfusion was stopped after the total volume of the fermentation broth returned to its initial state. The fermentation system was allowed to continue fermentation. Indoleacetic acid was produced through multiple stages of perfusion fermentation.

2. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The Bacillus amyloliquefaciens seed culture was fermented in a fermenter until the bacterial cell concentration reached OD500. 600 When the pH is 5.0~6.0, the fermentation broth is received through a tangential flow filter. When the volume of the fermentation broth reaches 20%-30% of the initial volume of the fermenter, the receiving of the fermentation broth is stopped, and perfusion is started. The perfusion medium enters the fermenter through a tangential flow filter. Perfusion is stopped when the total volume of the fermentation broth in the fermenter returns to its initial state. The seed liquid in the fermenter continues to ferment. After continuous culture for 3~4 hours, the above operation is repeated. Indoleacetic acid is produced through multiple stages of perfusion fermentation.

3. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The fermentation conditions for the fermentation system are: temperature 30~37℃, pH 5.5~6.5, and dissolved oxygen ≥30%.

4. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The perfusion culture medium is formulated as follows: corn steep liquor 3-5 g / L, molasses 5-10 g / L, KH2PO4 1-2 g / L, MgSO4·7H2O 0.5-1 g / L, MnSO4·H2O 0.1-0.2 g / L, L-tryptophan 3-5 g / L, pH 5.5-6.5, sterilized at 121℃ for 20 min.

5. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: Bacillus amyloliquefaciens is deposited at the China Center for Type Culture Collection and is classified as Bacillus amyloliquefaciens (B. amyloliquefaciens). Bacillus amyloliquefaciens SY-SF-01, accession number CCTCC NO: M 2018264, accession date May 14, 2018, accession address Wuhan University, Wuhan, China.

6. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The Bacillus amyloliquefaciens seed culture was inoculated into the fermentation medium in the fermenter at an inoculation rate of 5-10%. The fermentation medium consisted of rice slurry 10-15 g / L, molasses 10-15 g / L, KH2PO4 1-3 g / L, K2HPO4·3H2O 2-5 g / L, MgSO4·7H2O 0.5-1.5 g / L, MnSO4·H2O 0.1-0.3 g / L, and pH 6.0-7.

0. The culture was then sterilized at 121℃ for 20 min.

7. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The seed culture of Bacillus amyloliquefaciens is obtained by inoculating Bacillus amyloliquefaciens strains into an activation medium and culturing the activated bacterial solution, and then inoculating the activated bacterial solution into a seed medium and culturing it to obtain the seed culture. The activation medium consisted of 10-15 g / L peptone, 5-8 g / L yeast extract, and 5-10 g / L NaCl, with a pH of 6.5-7.5, and was sterilized at 121°C for 20 min. The seed culture medium consisted of 10-15 g / L peptone, 5-8 g / L yeast extract, 5-8 g / L NaCl, and pH 6.5-7.5, sterilized at 121℃ for 20 min.

8. The method for producing indoleacetic acid using Bacillus amyloliquefaciens stage perfusion fermentation according to claim 1, characterized in that: The fermentation broth from multiple stages of perfusion was collected and mixed, and then subjected to centrifugation, acid precipitation, extraction, concentration, decolorization, and crystallization in sequence to obtain a high-purity indoleacetic acid product. The centrifugation involves centrifuging the collected fermentation broth at a speed of 8000-10000 r / min for 10-15 min to remove the bacterial precipitate, and then collecting the supernatant and combining it with the filtrate. The acid precipitation involves adjusting the pH of the supernatant to 2.0-3.0 and allowing it to stand at 4°C for 2-4 hours to precipitate indoleacetic acid. The extraction process involves adding an equal volume of ethyl acetate to the acid-precipitated mixture for extraction, collecting the organic phase, and repeating the extraction 2-3 times, then combining the organic phases. The concentration process involves concentrating the combined organic phases under reduced pressure at 40-50°C and a vacuum of 0.08-0.09 MPa to obtain a crude paste-like product. The decolorization process involves dissolving the crude product in anhydrous ethanol by heating, adding activated carbon, stirring for 10-15 minutes to decolorize, and then filtering while hot to remove the activated carbon. The crystallization process involves placing the filtrate at 4°C for 12-24 hours to allow crystals to precipitate. The crystals are then collected by filtration, washed 2-3 times with ethanol, and dried to obtain the indoleacetic acid product.

9. An apparatus used in the method of claim 1, characterized in that: The system includes a fermenter (1), a perfusion replenishment tank (2), a perfusion receiving tank (3), and a tangential flow filter (10). The tangential flow filter (10) is internally divided into a fermentation liquid chamber and a filtration liquid chamber by a hollow fiber membrane (15). The fermentation liquid chamber is connected to the fermentation liquid inlet (16) and fermentation liquid outlet (17) on one side of the tangential flow filter (10), and the filtration liquid chamber is connected to the filtration liquid outlet (18) at the end of the tangential flow filter (10). The fermentation liquid inlet (16) is connected to the corresponding end of the fermenter (1) via a first pipeline. The first pipeline is connected to the fermentation broth outlet pump (11) and valve B (7). The fermentation broth outlet (17) is connected to another port on the fermentation tank (1) through the second pipeline. The second pipeline is equipped with valve C (8). The filtrate outlet (18) is connected to the perfusion receiving tank (3) through the third pipeline. The third pipeline is equipped with valve D (9). At the same time, the filtrate outlet (18) is connected to the perfusion replenishment tank (2) through the fourth pipeline. The fourth pipeline is equipped with perfusion pump (12) and valve A (6).

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