A method for producing butyric acid by fermentation using lignocellulose as a raw material

Abstract

The present application relates to a kind of lignocellulose as raw material fermentation production butyric acid method, comprising the following steps: (1) lignocellulose raw material is pretreated, enzymolysis, filtration obtains enzymolysis liquid;(2) the enzymolysis liquid is detoxified, separates out the precipitate after pH adjustment as fermentation medium;(3) the seed liquid of bacillus cereus (W1) Bacillus cereus ) W1 is inoculated into fermentation medium in non-strict anaerobic environment, and fermentation production butyric acid.The present application uses lignocellulose as raw material, utilizes bacillus cereus (W1) Bacillus cereus ) W1 in non-strict anaerobic environment fermentation production butyric acid, with butyric acid yield high, product selectivity is high, fermentation cost is low and so on.

Description

Technical Field

[0001] This invention belongs to the field of biomass energy technology, specifically relating to a method for producing butyric acid by fermentation using lignocellulose as a raw material. Background Technology

[0002] Butyric acid is a short-chain 4-carbon fatty acid widely used in the chemical, plastics, textile fiber, food and beverage, and pharmaceutical industries. In the chemical field, its primary use is in the production of cellulose acetate butyrate plastics, which are then used in the manufacture of textile fibers. By introducing butyryl groups into cellulose acetate polymers, the polymers become more flexible and exhibit better resistance to light, low temperatures, and organic solvents. Butyric acid can also be added directly to fibers as an additive to improve their heat and light resistance. Butyric acid derivatives, such as methyl butyrate (with an apple scent), ethyl butyrate (with a pineapple scent), and amyl butyrate (with a pear scent), are used as fragrances and flavorings in food, beverages, and cosmetics.

[0003] In recent years, butyric acid has attracted widespread attention as a feedstock for the production of butanol, a biofuel considered a next-generation liquid fuel following ethanol, which boasts higher energy density and calorific value compared to ethanol. Another potential application of butyric acid lies in the production of biodegradable plastics: butyric acid-rich mixed carboxylic acids can be bioconverted into polyhydroxybutyrate (PHB). PHB is a major component of polyhydroxyalkanoates (PHAs), which are raw materials for the production of biodegradable plastics.

[0004] Currently, the industrial production of butyric acid mainly relies on the butyraldehyde oxidation method, which is based on fossil resources such as petroleum, coal, and natural gas. This method uses butyraldehyde as a raw material, oxygen or air as an oxidant, and a continuous reaction under the action of a catalyst, followed by distillation to obtain butyric acid. Due to its advantages such as readily available and simple raw materials, easy process control, and high product yield, the butyraldehyde oxidation method has become the mainstream method for the commercial production of butyric acid. However, the finite and unsustainable nature of fossil resources and the environmental pollution caused by their extraction and use are forcing people to seek sustainable production methods that can harmonize with nature.

[0005] Microbial fermentation for butyric acid production is a promising alternative technology. Although the current production cost of fermentation is relatively high, with the continuous rise in crude oil prices, the maturation of biomass chemical industry, and the increasing demand for organic and natural products in the food additives, pharmaceuticals, and preservatives sectors, fermentation for butyric acid production will play a more important role in the butyric acid supply market. Under anaerobic conditions, many genera of microorganisms can produce butyric acid, including *Clostridium*, *Butyrivibrio*, *Butyribacterium*, *Sarcina*, *Eubacterium*, *Fusobacterium*, and *Megasphera*.

[0006] Currently, the main factors restricting the development and commercialization of butyric acid production by microbial fermentation are: (1) Low final concentration of butyric acid. In the fermentation system, the growth of butyric acid-producing bacteria is inhibited by the final fermentation product (butyric acid), resulting in a low final concentration of butyric acid in the fermentation broth; (2) High separation cost. The low final concentration of the product in the butyric acid fermentation broth and the presence of byproducts increase the difficulty of separating and purifying butyric acid; (3) High price of fermentation raw materials. Currently, the most commonly used raw materials for butyric acid fermentation are glucose, corn, etc., which are relatively expensive, resulting in high production costs.

[0007] Lignocellulose is the most abundant renewable resource in nature, used to produce energy products such as fuel ethanol and biodiesel, and is also a major raw material for chemical preparation and papermaking. Lignocellulose is mainly composed of cellulose, hemicellulose, and lignin. Its complex chemical structure limits its efficient utilization, therefore pretreatment is necessary to remove lignin and hydrolyze cellulose and hemicellulose into fermentable sugars, thereby improving the degradation and conversion rate of lignocellulose. Taking corn stalks as an example, cellulose and hemicellulose, two polysaccharides, account for more than 60% of the stalks, with hemicellulose making up about one-third of the total sugars. Therefore, whether the hydrolysis products of hemicellulose can be fermented to produce the target product is crucial for the utilization of lignocellulose. However, most wild fungal species cannot simultaneously utilize glucose and xylose, resulting in the waste of xylose. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a method for producing butyric acid through fermentation using lignocellulose as a raw material. This invention utilizes lignocellulose as a raw material and employs Bacillus cereus W1 to ferment and produce butyric acid under a non-strictly anaerobic environment, offering advantages such as high butyric acid yield, high product selectivity, and low fermentation cost.

[0009] This invention provides a method for producing butyric acid by fermentation using lignocellulose as a raw material, comprising the following steps:

[0010] (1) The lignocellulose raw material is pretreated, enzymatically hydrolyzed, and filtered to obtain the enzymatic hydrolysate;

[0011] (2) Detoxify the enzyme hydrolysate, separate the precipitate, and adjust the pH to use as a fermentation medium;

[0012] (3) The seed culture of Bacillus cereus W1 was inoculated into the fermentation medium and fermented to produce butyric acid under a non-strict anaerobic environment.

[0013] The lignocellulose raw material mentioned in step (1) is at least one of straw, sawdust, etc. containing cellulose, hemicellulose and lignin, preferably straw, and more preferably corn straw.

[0014] The purpose of the pretreatment in step (1) is to break down the stubborn structure of the lignocellulose raw material, exposing the cellulose from the encapsulation of hemicellulose and lignin, thereby increasing the accessibility of cellulase and improving the enzymatic hydrolysis efficiency. The pretreatment in this invention can be a conventionally used pretreatment method, preferably dilute acid pretreatment or dilute acid steam explosion pretreatment. Specifically, the dilute acid steam explosion pretreatment involves: pulverizing the lignocellulose raw material to 0.5-5 cm, adding an acid solution at a ratio of 1 g: 2-5 mL to wet it, with an acid solution concentration of 0.5 wt%-5 wt%, preferably 2.0 wt%-3.0 wt%, and then introducing it into the retention chamber of a steam explosion device. The solution is maintained at a temperature of 120-200℃, preferably 120-160℃, and a pressure of 0.4-1.2 MPa, preferably 0.8-1.0 MPa, for 10-30 minutes, followed by instantaneous pressure release to obtain the steam explosion pretreated material. The acid is at least one of sulfuric acid, hydrochloric acid, and nitric acid, preferably sulfuric acid.

[0015] Step (1) After pretreatment, the material is prepared into a solution with a substrate concentration of 8wt%-20wt%, where substrate concentration refers to the percentage of solid mass to total system mass. Cellulase is then added for enzymatic hydrolysis. The cellulase is any enzyme protein or mixture of enzyme proteins capable of hydrolyzing cellulose and / or hemicellulose into monosaccharides, including four types of enzyme protein components: cellulase exonuclease, cellulase endonuclease, β-glucosidase, and xylanase. The cellulase can be a commercial enzyme preparation or produced by in-situ fermentation using enzyme-producing strains. During enzymatic hydrolysis, the amount of cellulase added is 20-50 FPIU / g cellulose, the pH value is 4.5-5.5, the temperature is 45-55℃, the stirring rate is 50-300 r / min, and the hydrolysis time is 24-72 h.

[0016] The enzymatic hydrolysate obtained by filtration in step (1) is mainly a mixed sugar solution produced by the hydrolysis of cellulose and hemicellulose, which mainly contains pentose sugars such as xylose, hexose sugars such as glucose, and some other substances produced by hydrolysis.

[0017] The detoxification process described in step (2) employs an alkaline detoxification method. Specifically, the pH of the enzymatic hydrolysate is adjusted to 10-11 using an alkali, and then treated at 40-60℃ for 0.5-4.0 hours. After centrifugation to separate the precipitate, a detoxified solution is obtained. The alkali used is at least one of Ca(OH)2, NaOH, etc.

[0018] Step (2) Adjust the pH to 6.0-9.0, preferably 5.0-7.0, and use it as the fermentation medium.

[0019] The *Bacillus cereus* W1 strain described in step (3) was deposited at the China Center for Type Culture Collection (CCTCC) on July 21, 2022, with accession number CCTCC NO: M 20221147. Under a microscope, *Bacillus cereus* W1 cells are rod-shaped, 4-7 μm long, Gram-positive, and facultative anaerobic, capable of growth under non-strictly anaerobic conditions. The optimal growth pH is 4.5-9.0, and the optimal temperature is 20-40℃. It shows weakly positive results for arginine dihydrolase, pyruvate VP reaction, gelatin hydrolysis, and starch hydrolysis. This strain can utilize any one or a combination of glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol, and sorbitol.

[0020] The preparation of the Bacillus cereus W1 seed culture in step (3) uses seed culture media commonly used in the art, preferably RCM medium. Specifically, the preserved Bacillus cereus W1 is inoculated onto a solid plate medium and activated at 28-42℃ for 12-48 hours. Then, activated single colonies are picked and inoculated into the seed culture medium and incubated statically at 28-42℃ for 12-48 hours.

[0021] The seed liquid described in step (3) is inoculated into the fermentation medium at a volume ratio of 2%-20%, preferably 5%-15%.

[0022] The fermentation described in step (3) can be carried out using any of the currently known fermentation methods, such as stirred tank fermentation, in-situ extraction fermentation, and gas in-situ extraction fermentation.

[0023] The non-strict anaerobic fermentation described in step (3) involves deoxygenation for the first 24 hours, preferably the first 2 hours, of fermentation, after which deoxygenation ceases until fermentation ends. Deoxygenation can be achieved by introducing N2, adding an oxygen absorber, etc. Anaerobic fermentation involves deoxygenation throughout the entire fermentation process.

[0024] The fermentation conditions for step (3) are as follows: fermentation temperature is 28-42℃, preferably 32-38℃; the initial pH is natural, and the pH is controlled to 4.0-9.0 after the logarithmic phase, preferably 5.0-7.0; the stirring speed is 10-100 r / min, preferably 30-80 r / min; the fermentation time is 72-120 h, preferably 24-72 h. At least one of NaOH, KOH, etc., can be used to adjust the pH.

[0025] Step (3) After 24 hours of fermentation, a nitrogen source is added. The nitrogen source can be at least one of ammonium acetate, ammonium chloride and ammonium sulfate, preferably ammonium acetate. The concentration of the added nitrogen source is 5-30 g / L, preferably 10-20 g / L. The addition rate is 0.5-5 mL / h, preferably 1-3 mL / h.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention uses lignocellulose as raw material, which is pretreated, enzymatically hydrolyzed and detoxified to obtain a sugar-containing enzymatic hydrolysate, which is used as a fermentation culture medium to produce butyric acid by fermentation with Bacillus cereus W1 in a non-strict anaerobic environment. The butyric acid content in the product is not less than 75%, which has the advantages of high butyric acid yield and high product selectivity. Moreover, it solves the problem of high cost caused by the consumption of grain as fermentation raw material.

[0028] (2) Bacillus cereus W1 can use sugars with different carbon numbers, such as glucose and xylose in the enzymatic hydrolysate, as substrates for fermentation. It also has strong oxygen tolerance, which reduces the deoxygenation process of traditional anaerobic fermentation. It does not require full deoxygenation, thus avoiding the risks of poor cell growth and fermentation failure caused by incomplete deoxygenation and reducing operating energy consumption.

[0029] (3) By adding a nitrogen source during the fermentation process, the present invention achieves a higher selectivity for butyric acid in the product and further increases the proportion of butyric acid in the product. Detailed Implementation

[0030] The technical solution and its effects of the present invention will be further described in detail below with reference to specific embodiments. The embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures, but the scope of protection of the present invention is not limited to the following embodiments. In the present invention, wt% is a mass fraction.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0032] The lignocellulose raw material used in this embodiment of the invention is corn stalk, which contains 38.2 wt% cellulose, 22.1 wt% hemicellulose, 20.2 wt% lignin, and 3.9 wt% ash. It is crushed to a particle size of 0.5-2.0 cm by a pulverizer.

[0033] This invention utilizes liquid chromatography (LC) to analyze the components in the fermentation broth. The LC analysis conditions were as follows: Instrument: Agilent 1260; Column: BIO-RAD HPX-87H Ion Exclusion Column; Detector: Refractive index detector (RID); Mobile phase: 0.005 mol / L H₂SO₄ aqueous solution; Flow rate: 0.7 mL / min; Column temperature: 65℃; RID temperature: 40℃; Injection volume: 5 μL. The dry matter concentration was determined using a Sartorius HG63 moisture analyzer via the drying method.

[0034] The seed culture medium (RCM) formula is as follows: 10 g / L peptone, 10 g / L beef meal, 3 g / L yeast extract, 5 g / L glucose, 1.0 g / L soluble starch, 5.0 g / L sodium chloride, 3.0 g / L sodium acetate, and 0.5 g / L L-cysteine ​​hydrochloride. Sterilize at 121°C for 15 min.

[0035] Example 1

[0036] (1) The crushed corn stalks were soaked in a 2wt% dilute sulfuric acid aqueous solution at a solid-liquid ratio of 1g:3mL for 30 minutes, and then placed into the retention chamber of a steam explosion device. The pressure was maintained at 150℃ and 0.9MPa for 30 minutes, followed by instantaneous depressurization and explosion to obtain corn stalks pretreated by dilute acid steam explosion. The corn stalks were mainly composed of xylose, xylooligosaccharides, xylan, cellulose, and lignin, with a dry matter concentration of 32%. The contents of xylose, xylooligosaccharides, and xylan were 19.11% (relative to dry matter), and the cellulose content was 39.7% (relative to dry matter). The pretreated corn stalks were adjusted to a substrate concentration of 12.6% by adding water. The cellulase used was Novozymes Ctec2, with a cellulase addition of 30 FPIU / g cellulose. The enzymatic hydrolysis pH was 5.0, and the enzymatic hydrolysis was carried out at 50℃ and 150r / min for 48 hours. The hydrolysate was then filtered to obtain the enzymatic hydrolysate. The glucose concentration was found to be 62.5 g / L and the xylose concentration was 14.7 g / L.

[0037] (2) The enzymatic hydrolysate was detoxified using an alkaline method, that is, the pH was adjusted to 10 with Ca(OH)2 and placed at 60℃ for 1 hour to detoxify. After separating the precipitate, the detoxified liquid was obtained. Before fermentation, the pH of the detoxified liquid was adjusted to 6.5-7.0 as the fermentation medium.

[0038] (3) Bacillus cereus W1 was activated on solid RCM agar plates and cultured at 35℃ for 24 h. Then, activated single colonies were picked and inoculated into seed culture medium without deoxygenation and cultured at 35℃ for 24 h to obtain seed liquid. The seed liquid was inoculated into fermentation medium at a volume ratio of 10% for fermentation using a vertical stirred tank at a stirring speed of 50 r / min. Nitrogen gas was introduced during the first 2 hours of fermentation under non-strict anaerobic conditions, and no N2 deoxygenation was required thereafter. The fermentation temperature was 35℃, and the pH was kept at natural. After 24 hours of fermentation, the pH was controlled to 6.5±0.2 using NaOH. After 24 hours of fermentation, ammonium acetate solution was added at a concentration of 20 g / L at a flow rate of 1.0 mL / h. Fermentation was completed after 72 h to obtain a fermentation broth containing butyric acid and acetic acid, with butyric acid concentration of 19.23 g / L (79.17%) and acetic acid concentration of 20.83%.

[0039] Example 2

[0040] (1) Same as Example 1.

[0041] (2) The enzymatic hydrolysate was detoxified using an alkaline method, that is, the pH was adjusted to 11 with Ca(OH)2 and placed at 40℃ for 4 hours for detoxification. After separating the precipitate, the detoxified liquid was obtained. Before fermentation, the pH of the detoxified liquid was adjusted to 6.2-6.5 as the fermentation medium.

[0042] (3) Bacillus cereus W1 was activated on solid RCM agar plates and cultured at 35°C for 24 h. Then, activated single colonies were picked and inoculated into seed culture medium without deoxygenation and cultured at 35°C for 24 h to obtain seed culture. The seed culture was inoculated into fermentation medium at a volume ratio of 15% for fermentation using a vertical stirred tank at a stirring speed of 100 r / min. Nitrogen gas was introduced during the first 2 hours of fermentation under non-strict anaerobic conditions, and no further deoxygenation was required. The fermentation temperature was 28°C, and the pH was kept at natural. After 24 hours of fermentation, the pH was controlled to 7.0 ± 0.2 using potassium hydroxide. After 24 hours of fermentation, ammonium acetate solution was added at a concentration of 10 g / L at a flow rate of 2.0 mL / h. Fermentation was completed after 72 h to obtain a fermentation broth containing butyric acid and acetic acid, with butyric acid concentration of 16.07 g / L (77.43%) and acetic acid concentration of 22.57%.

[0043] Example 3

[0044] (1) Same as Example 1.

[0045] (2) The enzymatic hydrolysate was detoxified using an alkaline method, that is, the pH was adjusted to 11 with Ca(OH)2 and placed at 50℃ for 2.0 h for detoxification. After separating the precipitate, the detoxified liquid was obtained. Before fermentation, the pH of the detoxified liquid was adjusted to 7.0-7.5 as the fermentation medium.

[0046] (3) Bacillus cereus W1 was activated on solid RCM agar plates and cultured at 35℃ for 24 h. Then, activated single colonies were picked and inoculated into seed culture medium without deoxygenation and cultured at 35℃ for 24 h to obtain seed culture. The seed culture was inoculated into fermentation medium at a volume ratio of 10% for fermentation using a vertical stirred tank at a stirring speed of 50 r / min. Nitrogen gas was introduced during the first 2 hours of fermentation under non-strict anaerobic conditions, and no N2 deoxygenation was required thereafter. The fermentation temperature was 38℃, and the pH was kept at natural. After 24 hours of fermentation, the pH was controlled to 6.5±0.2 using NaOH. After 24 hours of fermentation, ammonium acetate solution was added at a concentration of 15 g / L at a flow rate of 1.5 mL / h. Fermentation was completed after 72 h to obtain a fermentation broth containing butyric acid and acetic acid, with butyric acid concentration of 18.91 g / L (78.76%) and acetic acid concentration of 21.24%.

[0047] Example 4

[0048] Similar to Example 1, the difference lies in that the cellulase used in step (1) was prepared on-site by fermentation using a proprietary strain, Trichoderma reesei PB3, deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M2021474. The cellulase dosage was 30 FPIU / g cellulose, the pH for enzymatic hydrolysis was 5.0, and hydrolysis was carried out at 50℃ and 150 r / min for 72 h to obtain the hydrolysate. The glucose concentration was 60.1 g / L, and the xylose concentration was 12.2 g / L. In the fermentation broth after fermentation, the butyric acid concentration was 18.16 g / L, accounting for 78.3%, and the acetic acid concentration was 21.7%.

[0049] Example 5

[0050] Same as Example 1, except that the nitrogen source added during the fermentation process in step (3) was replaced with ammonium chloride. In the fermentation broth after fermentation, the concentration of butyric acid was 17.79 g / L, accounting for 77.62%, and the proportion of acetic acid was 22.38%.

[0051] Example 6

[0052] Same as Example 1, except that no nitrogen source was added during the fermentation process in step (3). In the fermentation broth after fermentation, the concentration of butyric acid was 16.58 g / L, accounting for 70.63%, and the concentration of acetic acid was 29.37%.

[0053] Comparative Example 1

[0054] Same as Example 1, except that nitrogen gas was introduced throughout the fermentation process in step (3). After fermentation, the butyric acid concentration in the fermentation broth was 18.71 g / L, accounting for 75.55%, and the acetic acid concentration was 24.45%.

[0055] Comparative Example 2

[0056] Same as Example 1, except that pH was not controlled during fermentation in step (3). In the fermentation broth after fermentation, butyric acid concentration was 7.36 g / L, accounting for 77.23%, and acetic acid accounted for 22.77%.

[0057] Comparative Example 3

[0058] Same as Example 1, except that the hydrolysate in step (2) was not detoxified. The butyric acid concentration in the fermentation broth after fermentation was 1.07 g / L.

Claims

1. A method for producing butyric acid by fermentation using lignocellulose as a raw material, characterized by The method comprises the following steps: (1) pretreating and enzymatically hydrolyzing the lignocellulose raw material to obtain an enzymatic hydrolysate by filtration; (2) detoxifying the enzymatic hydrolysate, adjusting the pH after separating the precipitate to obtain a fermentation medium; (3) Bacillus cereus ( Bacillus cereus W1 seed culture was inoculated into fermentation medium and fermented to produce butyric acid under a non-strict anaerobic environment; The aforementioned Bacillus cereus ( Bacillus cereus W1 was deposited at the China Center for Type Culture Collection on July 21, 2022, with accession number CCTCC NO: M 20221147.

2. The method of claim 1, wherein: The lignocellulose raw material in step (1) is at least one of straw and sawdust containing cellulose, hemicellulose and lignin.

3. The method of claim 2, wherein: The lignocellulose raw material in step (1) is straw.

4. The method of claim 3, wherein: The lignocellulose raw material in step (1) is corn straw.

5. The method of claim 1, wherein: The pretreatment in step (1) adopts dilute acid pretreatment or dilute acid steam explosion pretreatment; the acid is at least one of sulfuric acid, hydrochloric acid and nitric acid.

6. The method of claim 5, wherein: The acid is sulfuric acid.

7. The method of claim 1 or 5, wherein: The pretreatment adopts dilute acid steam explosion pretreatment, specifically: the lignocellulose raw material is crushed to 0.5-5 cm, 1 g:2-5 mL of acid solution is added for wetting, the acid solution has a concentration of 0.5wt%-5wt%, is fed into a retainer of a steam explosion device, is maintained at a temperature of 120-200℃ and a pressure of 0.4-1.2 MPa for 10-30 min, and is instantaneously released by pressure relief to obtain a steam explosion pretreated material.

8. The method of claim 7, wherein: The acid solution has a concentration of 2.0wt%-3.0wt%, is fed into a retainer of a steam explosion device, is maintained at a temperature of 120-160℃ and a pressure of 0.8-1.0 MPa for 10-30 min.

9. The method of claim 1, wherein: The pretreated material in step (1) is prepared into a feed liquid with a substrate concentration of 8wt%-20wt%, and then cellulase is added for enzymatic hydrolysis.

10. The method of claim 1, wherein: The cellulase is an enzyme protein or enzyme protein mixture for hydrolyzing cellulose and / or hemicellulose into monosaccharides, which includes four types of enzyme protein components: cellulose exohydrolase, cellulose endohydrolase, beta-glucosidase and xylanase. The cellulase is a commercial enzyme preparation or is produced by in-situ fermentation of a cellulase-producing strain.

11. The method of claim 1, 9 or 10, wherein: In the enzymatic hydrolysis process, the cellulase is added in an amount of 20-50 FPIU / g cellulose, the pH value of the enzymatic hydrolysis is 4.5-5.5, the temperature is 45-55℃, the stirring rate is 50-300 r / min, and the enzymatic hydrolysis time is 24-72 h.

12. The method of claim 1, wherein: The detoxification in step (2) adopts an over-alkali method, specifically: the enzymatic hydrolysate is adjusted to a pH of 10-11 by an alkali, is treated at 40-60℃ for 0.5-4.0 h, and is centrifuged to separate the precipitate to obtain a detoxified feed liquid; the alkali is at least one of Ca(OH)2 and NaOH.

13. The method of claim 1, wherein: The pH is adjusted to 6.0-9.0 to obtain a fermentation medium.

14. The method of claim 1, wherein: Step (3) the Bacillus cereus (W1) Bacillus cereus The preparation of W1 seed liquid is inoculating preserved Bacillus cereus W1 into solid plate medium, culturing at 28-42°C for 12-48 h, then picking the activated single colony and inoculating into seed medium, and culturing at 28-42°C for 12-48 h.

15. The method of claim 1 or 14, wherein: The seed liquid in step (3) is inoculated into the fermentation medium at a volume ratio of 2%-20%.

16. The method of claim 15, wherein: The seed liquid in step (3) is inoculated into the fermentation medium at a volume ratio of 5%-15%.

17. The method of claim 1, wherein: The non-strict anaerobic fermentation in step (3) is deoxygenated within the first 24 hours of fermentation, and is not deoxygenated thereafter until the end of fermentation; the anaerobic fermentation is deoxygenated throughout the fermentation.

18. The method of claim 17, wherein: The non-strict anaerobic fermentation in step (3) is deoxygenated within the first 2 hours of fermentation.

19. The method of claim 1, wherein: The fermentation temperature is 28-42℃, the initial pH is natural, the pH is controlled at 4.0-9.0 after logarithmic phase, the stirring speed is 10-100 r / min, and the fermentation time is 72-120 h.

20. The method of claim 19, wherein: The fermentation temperature is 32-38℃; the pH is controlled at 5.0-7.0 after logarithmic phase; and the stirring speed is 30-80 r / min.

21. The method of claim 1, wherein: The nitrogen source is at least one of ammonium acetate, ammonium chloride and ammonium sulfate.

22. The method of claim 21, wherein: The nitrogen source is ammonium acetate.

23. The method of claim 21 or 22, wherein: The concentration of the nitrogen source is 5-30 g / L, and the flow rate is 0.5-5 mL / h.

24. The method of claim 23, wherein: The concentration of the nitrogen source is 10-20 g / L, and the flow rate is 1-3 mL / h.