阅读说明：本技术 霍华德氏肠杆菌fx-02发酵生产吲哚乙酸的方法 (Method for producing indoleacetic acid by virtue of Enterobacter hollandii FX-02 fermentation ) 是由 赵国群 徐科玉 于 2021-08-25 设计创作，主要内容包括：本发明属于发酵技术领域,公开了一种霍华德氏肠杆菌FX-02发酵生产吲哚乙酸的方法,将霍华德氏肠杆菌FX-02接种到种子培养基中,获得FX-02种子液,并将获得的FX-02种子液接种到发酵培养基中,进行摇床振荡培养,发酵结束后,即得到含IAA的发酵液。本发明采用霍华德氏肠杆菌FX-02发酵生产吲哚乙酸(IAA)的反应过程条件温和、能耗低、不使用有毒有害物质、没有环境污染,IAA的产量能够满足微生物发酵法生产IAA的要求。(The invention belongs to the technical field of fermentation, and discloses a method for producing indoleacetic acid by fermenting Enterobacter hollandii FX-02. The method for producing the indoleacetic acid (IAA) by fermenting the Enterobacter howsoensis FX-02 has the advantages of mild reaction process conditions, low energy consumption, no use of toxic and harmful substances and no environmental pollution, and the yield of the IAA can meet the requirement of producing the IAA by a microbial fermentation method.)

1. The method for producing the indoleacetic acid by fermenting the Enterobacter hollandii FX-02 is characterized by comprising the following steps of:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and adjusting the pH value to 7.0-7.2;

s2, preparation of a fermentation medium: 10.0-12.0 g/L carbon source, 8.0-10.0 g/L peptone, 10.0g/L tryptophan, 1.2-1.5 g/L inorganic salt, 1.5-1.8 g/L dipotassium hydrogen phosphate and 1L water, and the pH value is adjusted to 7.0-7.2;

s3, preparing FX-02 seed liquid: inoculating Enterobacter hollandii FX-02 into a seed culture medium, and performing shaking culture on a shaking table to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing and cooling the fermentation medium; inoculating FX-02 seed liquid according to the inoculation amount of 5-10% (V/V), carrying out shaking culture on a shaker, and obtaining the IAA-containing fermentation liquid after fermentation is finished.

2. The method for the fermentative production of indoleacetic acid by enterobacter hollandii FX-02 according to claim 1, wherein enterobacter hollandii FX-02 latin is namedEnterobacter hormaechei FX-02, deposited in China center for type culture Collection at 19.4.2021 with the preservation number of CCTCC NO. M2021409.

3. The method for producing indoleacetic acid by fermentation of enterobacter holchenkii FX-02 according to claim 1 or 2, wherein in step S2, the carbon source is glycerol or starch, and the inorganic salt is calcium chloride or magnesium sulfate.

4. The method for the fermentative production of indoleacetic acid from Enterobacter hollandii FX-02 according to claim 1 or 2, wherein in step S3, shaking culture is performed at 30 ℃ and 180r/min for 24 h.

5. The method for producing indoleacetic acid by fermentation of enterobacter holchenkii FX-02 according to claim 1 or 2, characterized in that, in step S4, the fermentation medium is sterilized at 121 ℃ for 20min and cooled to 30 ℃;

and (3) inoculating FX-02 seed solution, and performing shaking culture on a shaking table at the temperature of 28-30 ℃ and the speed of 180-220 r/min for 72-84 h.

6. The method for producing indoleacetic acid by fermenting Enterobacter howsoensis FX-02 according to claim 5, wherein the IAA content in the IAA-containing fermentation liquid obtained in step S4 is 2300-2737.1 mg/L.

Technical Field

The invention belongs to the technical field of fermentation, relates to a method for producing indoleacetic acid by fermentation, and particularly relates to a method for producing indoleacetic acid by fermentation of Enterobacter hollandii FX-02.

Background

Indoleacetic acid (IAA) with molecular formula C₁₀H₉NO₂The pure product is light yellow crystal, sensitive to light and air, easily soluble in anhydrous alcohol, ethyl acetate and dichloroethane, and soluble in diethyl etherAnd acetone, insoluble in benzene, toluene, gasoline, water and chloroform. Indoleacetic acid is a plant growth regulator, can promote cell growth, increase cell volume and weight, and has physiological functions of promoting cell division and differentiation, regulating rooting, etc. The application of IAA can promote the growth of crops and improve the yield of the crops, thereby being widely used in agricultural production.

At present, the industrial production method of the indoleacetic acid is mainly a chemical synthesis method, high temperature and high pressure are required in the production process of the indoleacetic acid, toxic substances such as formaldehyde, potassium cyanide and the like are used, and the price of a finished product is high. Not only has high energy consumption, but also can generate a large amount of harmful waste liquid to cause environmental pollution. The IAA produced by the microbial fermentation method has the advantages of mild reaction conditions, low energy consumption, no use of toxic and harmful substances, no environmental pollution, low production cost and the like, thereby having good market prospect. The IAA content in the fermentation liquor produced by the existing microbial fermentation method is very low and is less than 1000mg/L, and the requirement of the microbial fermentation method for producing IAA in a modeling way cannot be met.

Disclosure of Invention

The invention aims to provide a method for producing indoleacetic acid by fermenting Enterobacter hollandii FX-02, which comprises the steps of inoculating the Enterobacter hollandii FX-02 to a seed culture medium to obtain FX-02 seed liquid, then inoculating the FX-02 seed liquid to a fermentation culture medium for fermentation, wherein after the fermentation is finished, the IAA content in the obtained fermentation liquid can basically meet the requirement of producing the indoleacetic acid by a microbial fermentation method.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for producing indoleacetic acid by Enterobacter hollandii FX-02 fermentation comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and adjusting the pH value to 7.0-7.2;

s2, preparation of a fermentation medium: 10.0-12.0 g/L carbon source, 8.0-10.0 g/L peptone, 10.0g/L tryptophan, 1.2-1.5 g/L inorganic salt, 1.5-1.8 g/L dipotassium hydrogen phosphate and 1L water, and the pH value is adjusted to 7.0-7.2;

s3, preparing FX-02 seed liquid: inoculating Enterobacter hollandii FX-02 into a seed culture medium, and performing shaking culture on a shaking table to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing and cooling the fermentation medium; inoculating FX-02 seed liquid according to the inoculation amount of 5-10% (V/V), carrying out shaking culture on a shaker, and obtaining the IAA-containing fermentation liquid after fermentation is finished.

By way of limitation, the Enterobacter hollandii FX-02 Latin has the name of Enterobacter hormaechei FX-02, is preserved in the China center for type culture Collection at 19.4.2021, with the preservation number of CCTCC NO. M2021409, and the preservation unit address: wuhan, Wuhan university.

As a second limitation, in step S2, the carbon source is glycerol or starch, and the inorganic salt is calcium chloride or magnesium sulfate.

As a third limitation, in step S3, shaking culture was performed at 180r/min for 24 hours at 30 ℃.

As a fourth limitation, in step S4, the fermentation medium is sterilized at 121 ℃ for 20min, cooled to 30 ℃;

and (3) inoculating FX-02 seed solution, and performing shaking culture on a shaking table at the temperature of 28-30 ℃ and the speed of 180-220 r/min for 72-84 h.

As a fifth limitation, in the IAA-containing fermentation liquid obtained in the step S4, the IAA content is 2300-2737.1 mg/L.

The principle of the invention is as follows: enterobacter hollandii FX-02 is a bacterial strain which is screened from vegetable rhizosphere soil and has strong IAA production capability. IAA belongs to the secondary metabolite of microorganisms, whereas tryptophan is a precursor for the synthesis of IAA. The concentration of the precursor has a great influence on the secondary metabolite. But the precursor has promotion effect on product synthesis when the concentration of the precursor is lower, and has toxicity and inhibition effect on product synthesis when the concentration of the precursor is higher. Suitable concentrations of the precursors are related to the microbial species. The IAA concentration of most microorganisms is reported to be 0.6-5.0 g/L. Research shows that the pathway for synthesizing IAA by Enterobacter hollandii FX-02 is tryptophan-dependent, and the Enterobacter hollandii FX-02 has strong tryptophan transformation capability and has the tolerance concentration of 10.0g/L to tryptophan. When the fermentation medium contains tryptophan at a high concentration, the Enterobacter hollandii FX-02 can synthesize IAA in a large amount, thereby realizing high yield of IAA. The carbon source, the nitrogen source and the inorganic salt all have obvious influence on the microbial synthesis of IAA. Research shows that the optimum carbon source, nitrogen source and inorganic salt for IAA production by Enterobacter hollandii FX-02 are glycerol, peptone and calcium chloride respectively. In conclusion, the principle of the invention is that the fermentation liquor with high IAA content is finally obtained by using Enterobacter hollandii FX-02 which has stronger IAA production capacity and strong tryptophan transformation capacity and using an optimized culture medium.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

(1) according to the invention, the indole acetic acid IAA is produced by fermenting Enterobacter Howardii FX-02, so that the environmental pollution in the chemical synthesis process of the regulator is reduced, and the method has the advantages of mild reaction conditions, low energy consumption, no use of toxic and harmful substances, no environmental pollution, low production cost and the like;

(2) according to the invention, the IAA is produced by fermenting the Enterobacter hollandii FX-02, the IAA content in the prepared fermentation liquor reaches 2737.1mg/L, and the requirement of producing the IAA by a microbial fermentation method is basically met;

(3) the fermentation medium and the culture conditions provided by the invention are simple, the operation is convenient, the production cost is low, the yield of the heteroauxin is high, the industrial production is easy, the energy is saved, the environment is protected, and the economic and social benefits are huge;

(4) the enterobacter hollandii FX-02 provided by the invention has the advantages of high growth speed, short fermentation period and easiness in large-scale production.

The enterobacter holchenkii FX-02 provided by the invention can be used for producing high-yield indoleacetic acid through fermentation, basically meets the requirement of producing the indoleacetic acid by a microbial fermentation method, and belongs to the technical field of fermentation.

Detailed Description

The present invention is further described with reference to the following examples, but it should be understood by those skilled in the art that the present invention is not limited to the following examples, and any modifications and variations based on the specific examples of the present invention are within the scope of the claims of the present invention.

Example 1A method for producing Indolylacetic acid by Enterobacter hollandii FX-02 fermentation

The embodiment comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and the pH value is adjusted to 7.0;

s2, preparation of a fermentation medium: 10.0g/L of glycerol, 10.0g/L of peptone, 10.0g/L of tryptophan, 1.2g/L of magnesium sulfate, 1.5g/L of dipotassium hydrogen phosphate and 1L of water, and adjusting the pH value to 7.2;

s3, preparing FX-02 seed liquid: inoculating an Enterobacter hollodoides FX-02 slant strain into a seed culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing the fermentation culture medium in autoclave at 121 deg.C for 20min, and cooling to 30 deg.C; inoculating FX-02 seed solution according to the inoculation amount of 5% (V/V), shaking and culturing for 72h at 30 ℃ and 180r/min, and obtaining IAA-containing fermentation liquor with the IAA content of 2349.40mg/L after fermentation is finished.

Example 2 method for producing Indolylacetic acid by Enterobacter hollandii FX-02 fermentation

The embodiment comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and the pH value is adjusted to 7.1;

s2, preparation of a fermentation medium: 12.0g/L of glycerol, 9.0g/L of peptone, 10.0g/L of tryptophan, 1.3g/L of calcium chloride, 1.7g/L of dipotassium hydrogen phosphate and 1L of water, and the pH value is adjusted to 7.0;

s3, preparing FX-02 seed liquid: inoculating an Enterobacter hollodoides FX-02 slant strain into a seed culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing the fermentation culture medium in autoclave at 121 deg.C for 20min, and cooling to 30 deg.C; inoculating FX-02 seed solution according to the inoculation amount of 10% (V/V), shaking and culturing for 80h at 30 ℃ and 180r/min, and obtaining IAA-containing fermentation liquor with the IAA content of 2737.1mg/L after fermentation is finished.

Example 3 method for producing Indolylacetic acid by Enterobacter hollandii FX-02 fermentation

The embodiment comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and the pH value is adjusted to 7.1;

s2, preparation of a fermentation medium: 12.0g/L of starch, 8.0g/L of peptone, 10.0g/L of tryptophan, 1.5g/L of calcium chloride, 1.6g/L of dipotassium hydrogen phosphate and 1L of water, and the pH value is adjusted to 7.2;

s3, preparing FX-02 seed liquid: inoculating an Enterobacter hollodoides FX-02 slant strain into a seed culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing the fermentation culture medium in autoclave at 121 deg.C for 20min, and cooling to 30 deg.C; inoculating FX-02 seed solution according to the inoculation amount of 8% (V/V), shaking and culturing for 84h at 28 ℃ at 200r/min, and obtaining IAA-containing fermentation liquor with the IAA content of 2668.5mg/L after fermentation is finished.

Example 4A method for producing Indolylacetic acid by Enterobacter hollandii FX-02 fermentation

The embodiment comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and the pH value is adjusted to 7.2;

s2, preparation of a fermentation medium: 10.0g/L of starch, 8.0g/L of peptone, 10.0g/L of tryptophan, 1.2g/L of calcium chloride, 1.8g/L of dipotassium hydrogen phosphate and 1L of water, and the pH value is adjusted to 7.1;

s3, preparing FX-02 seed liquid: inoculating an Enterobacter hollodoides FX-02 slant strain into a seed culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing the fermentation culture medium in autoclave at 121 deg.C for 20min, and cooling to 30 deg.C; inoculating FX-02 seed solution according to the inoculation amount of 6% (V/V), shaking and culturing for 75h at 29 ℃ and 220r/min, and obtaining the IAA-containing fermentation liquor with the IAA content of 2300mg/L after fermentation is finished.

Example 5A method for producing Indolylacetic acid by Enterobacter hollandii FX-02 fermentation

The embodiment comprises the following steps:

s1, preparation of a seed culture medium: 10.0g/L of peptone, 5.0g/L, NaCl 10.0.0 g/L of yeast extract powder and 1L of water, and the pH value is adjusted to 7.2;

s2, preparation of a fermentation medium: 11.0g/L of glycerol, 10.0g/L of peptone, 10.0g/L of tryptophan, 1.5g/L of magnesium sulfate, 1.6g/L of dipotassium hydrogen phosphate and 1L of water, and the pH value is adjusted to 7.0;

s3, preparing FX-02 seed liquid: inoculating an Enterobacter hollodoides FX-02 slant strain into a seed culture medium, and carrying out shaking culture at 30 ℃ and 180r/min for 24h to obtain FX-02 seed liquid;

s4, IAA fermentation: sterilizing the fermentation culture medium in autoclave at 121 deg.C for 20min, and cooling to 30 deg.C; inoculating FX-02 seed solution according to the inoculum size of 7% (V/V), shaking and culturing for 82h at 28 ℃ and 180r/min, and obtaining IAA-containing fermentation liquor with the IAA content of 2562.8mg/L after fermentation is finished.

Example 6 Effect of Tryptophan on the production of Indolylacetic acid by Enterobacter hollandii FX-02

The procedure of this example was substantially the same as that of example 1, except that the amount of tryptophan was varied, and IAA-containing fermentation broth samples N1-N5 were obtained by performing the procedure of example 1 using tryptophan at different concentrations to verify the effect of tryptophan on the production of indole acetic acid by Enterobacter holchenii FX-02, wherein the amounts of tryptophan and the IAA content in the samples obtained by varying the amounts of tryptophan are specifically shown in Table 1.

TABLE 1 Tryptophan amount and IAA content in the samples obtained

As is clear from Table 1, different concentrations of tryptophan were used for the preparations of samples N1 to N5 and example 1, respectively, indicating that the highest IAA content was obtained in the IAA-containing fermentation broth at a tryptophan concentration of 10.0 g/L.

Example 7 comparative experiment

Comparative example 1

Adopting a method of transferring a culture dish to shake flask liquid culture, firstly preparing a culture medium by using 200g of peeled potatoes and 20g of glucose, adding distilled water to 1L, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20 min; after inoculation, the culture is continuously fermented for 10 days at the temperature of 30 ℃, the rotating speed of 130r/min and under the condition of no light. Separating fermented mycelium and bacterial liquid by suction filtration, extracting at a ratio of ethyl acetate to bacterial liquid of 1:1 for 3 times. The ethyl acetate extracts are combined, concentrated and evaporated to dryness, and then the volume is determined to be 3mL by using chromatographic grade methanol. The parameters measured by the high performance liquid chromatography method comprise: the chromatographic column is a Venusil ASB-C18 column (4.6 mm. times.250 mm, 5 μm); the mobile phase is water-acetonitrile (v: v, 65: 35); the detection wavelength is 280nm, the flow rate is 1mL/min, and the column temperature is 25 ℃. Through measurement and calculation, the content of the 3-indoleacetic acid in the fermentation liquor reaches 1.8mg/L, and is marked as D1.

Comparative example 2

(1) Preparing a seed solution: inoculating Enterobacter (Enterobactersp.) LY6 slant strain into LB liquid culture medium, culturing at 35 deg.C under shaking at 150r/min, inoculating into seed fermentor containing LB liquid culture medium at 3% inoculum size (V/V) when absorbance of the bacterial solution reaches 0.7 at 600nm wavelength, fermenting at 35 deg.C under stirring speed of 180r/min with sterile air, and fermenting for 7 hr to obtain secondary seed solution. The LB medium comprises the following components: tryptone 10 g.L-1, yeast extract 5 g.L-1, NaCl 10 g.L-1, pH 7.2;

(2) inoculation and fermentation: sterilizing the culture medium for fermentation at 121 ℃ for 20min, cooling to 37 ℃, inoculating the secondary seed solution into the sterilized and cooled culture medium for fermentation (the culture medium for fermentation comprises, by weight, 0.8% of tryptone, 0.4% of yeast extract, 0.7% of NaCl0.7%, 2% of glucose and 1L, pH 7.2.2%), and supplementing the following substances after 12h of ventilation fermentation: fructose 0.7%, L-tryptophan 0.12%, sodium pyruvate 0.02%, and copper sulfate 0.01%; maintaining the temperature at 36 deg.C, pH7.2, stirring speed at 160r/min, fermentation tank pressure at 0.07Mpa, dissolved oxygen at 50mg/kg, and fermenting for 40 h. Adding stabilizer after fermentation to obtain fermentation liquid containing indoleacetic acid 575.2 mg/kg, labeled as D2.

Comparative example 3

The comparative example is substantially the same as the step of example 1, except that the carbon source in the step S1 is molasses, and after the fermentation is finished, the IAA content in the IAA-containing fermentation liquid is 1727.4mg/L and is marked as D3.

Comparative example 4

This comparative example is essentially the same as example 1 except that the nitrogen source in step S1 was ammonium chloride and that after the end of the fermentation, the IAA content in the IAA-containing fermentation broth was 623.7mg/L, designated D1.

Table 2 shows the results of comparing the IAA content in the IAA-containing fermentation broths obtained in comparative examples 1 to 4 and example 1, respectively.

TABLE 2 comparative results

As shown in Table 2, in example 1, the IAA content in the IAA-containing fermentation broth obtained in example 1 was much higher than that in comparative examples D1 to D2, i.e., in the production of IAA by other microbial fermentation methods, indicating that high production of IAA was possible by Enterobacter hollandii FX-02; example 1 comparison with comparative examples D3-D4 shows that when producing indoleacetic acid by fermentation of Enterobacter hollandii FX-02, glycerol is used as a carbon source and magnesium sulfate is used as an inorganic salt, the IAA content in the prepared IAA-containing fermentation liquid is high.

Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.