Method for producing amino acid by utilizing electrode to regulate fermentation of lysinibacillus

Abstract

The invention discloses a method for producing amino acid by regulating fermentation of lysinibacillus by using an electrode. According to the invention, lysine bacillus is used as a fermentation strain for fermentation production of amino acid. In the fermentation process, a conductive material is used as an electrode to be installed in a fermentation tank, the surface potential and the current density of the electrode are regulated and controlled through an external direct-current power supply, the metabolic pathway of the lysinibacillus is regulated and controlled, and then the fermentation yield of the amino acid is improved. According to the method disclosed by the invention, the fermentation tank is simply improved, and the production efficiency of microbial amino acid fermentation is efficiently improved under lower electric energy consumption, so that the method is a novel microbial amino acid fermentation method.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a method for producing amino acids by regulating lysine bacillus fermentation by utilizing electrodes. Background Art

[0002] Amino acids are essential substances for life activities and have a wide market demand in the fields of medicine, food, feed, and chemical industry. my country is a major producer of amino acids, and the production is increasing year by year. In 2023, the output of amino acids in my country will be about 7.52 million tons. The main amino acid products on the market include glutamic acid, lysine, threonine, methionine, and tryptophan. Among them, lysine and threonine are mainly used for feed additives to improve the protein utilization rate and breeding effect of livestock and poultry. Human medical amino acid products include glutamine, leucine, isoleucine, pharmaceutical grade pyridine, inosine, guanosine, adenosine, proline and other products. Microbial fermentation is the main technology for amino acid production at present. Common amino acid production strains include Corynebacterium glutamicum, Brevibacterium lactofermentum, and Bacillus brevis. After decades of research, the amino acid production efficiency of the above strains has slowed down. In fact, in addition to the above commonly used strains, many other microorganisms in nature also have excellent amino acid potential, so new amino acid fermentation production strains need to be explored. In addition, electrofermentation is an emerging research field. The regulation of the microbial fermentation metabolic pathway and efficiency is achieved by adding electrodes to the fermentation system and adjusting the current of the electrodes. Although electrofermentation technology has been applied in biogas, hydrogen, acetic acid and other fermentation systems, it has not yet been applied to amino acid fermentation. Summary of the invention

[0003] In view of the shortcomings and defects of the prior art, the purpose of the present invention is to provide a method for producing amino acids by fermentation of lysinic Bacillus using electrodes, thereby providing a new technical method for microbial amino acid fermentation.

[0004] The object of the present invention is to provide a method for producing amino acids by fermentation of lysinophilic Bacillus using electrodes, comprising the following steps:

[0005] S1. The seed solution of Bacillus lysinicus was inoculated into the fermentation medium at a volume ratio of 5%, and then placed in a fermentation device;

[0006] S2. The electrodes of the electrode control system are placed in the fermentation device and submerged in the fermentation medium;

[0007] S3. Put the electrode control system in the on state and carry out aerobic fermentation at 30°C and 100 r / min.

[0008] Preferably, the lysinibacillus is spherical lysinibacillus, deformed lysinibacillus or spindle-shaped lysinibacillus.

[0009] Preferably, the fermentation culture medium is composed of: 20.0 g / L glucose, 30.0 g / L beet molasses, 10.0 g / L corn steep liquor powder, 3.0 g / L betaine, 1.5 g / L MgSO4, 20.0 mg / L FeSO4, 450.0 mg / L threonine, 1.0 g / L potassium chloride, 0.6 mg / L biotin, and the solvent is water.

[0010] Preferably, the electrodes consist of a working electrode, a counter electrode and a reference electrode.

[0011] Preferably, the working electrode is a graphite plate, the counter electrode is a platinum wire, and the reference electrode is a silver chloride electrode.

[0012] Preferably, during aerobic fermentation, the electrode control system controls the working electrode potential to be -0.2V or 0.2V.

[0013] Preferably, when the electrode control system controls the working electrode potential to be -0.2 V, the content of Gly, Cys, Ile, Leu and / or Lys produced by lysinophilic Bacillus fermentation is increased.

[0014] Preferably, when the electrode control system controls the working electrode potential to be 0.2 V, the content of Asp, Thr, Ser, Glu, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys and / or Arg produced by lysinophilic Bacillus fermentation is increased.

[0015] Beneficial effects of the present invention:

[0016] 1. The present invention utilizes lysinibacillus for amino acid fermentation. Lysinibacillus has good ecological safety and rich metabolic potential, and can convert nutrients in the culture medium into different types of amino acids and release them into the fermentation liquid. Currently, there are no cases or reports of its use in amino acid fermentation.

[0017] 2. The present invention uses an electrode control system to regulate the redox environment inside and outside the microbial cells during the fermentation process, and regulates the potential or current density on the electrode surface through a DC power supply or a constant potential instrument to change the redox state of coenzymes such as NAD / NADH, NADP / NADPH in the fermentation microbial cells, thereby changing the rate and pathway of electron transfer and metabolic synthesis. Under different potentials or currents, the type and concentration of amino acids released by lysine Bacillus are also different. Therefore, the current / voltage can be adjusted according to the characteristics of the strain and production needs to achieve efficient fermentation production of specific amino acids, providing a new and effective method for effectively improving the fermentation yield of amino acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure shows the effect of the electrode system on the fermentation of L-lysine by spherical lysinibacillus when the potential of the working electrode is 0.2V; wherein the solid line curve is the concentration change curve of L-lysine when the potential of the working electrode is 0.2V, and the dotted line curve is the concentration change curve of L-lysine when no power is applied (control).

[0019] Figure 2 The figure shows the effect of the electrode system on the growth of spherical lysine Bacillus when the potential of the working electrode is 0.2V; wherein the solid line curve is the concentration change curve of spherical lysine Bacillus when the potential of the working electrode is 0.2V, and the dotted line curve is the concentration change curve of spherical lysine Bacillus when no power is applied (control).

[0020] Figure 3 The effect of different electrode potentials on the secretion of amino acids by Bacillus sphaeroides. DETAILED DESCRIPTION

[0021] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0022] The following examples are sphaericus Lysinibacillus sphaericus C3-41 donated by the Wuhan Institute of Virology, Chinese Academy of Sciences, and have been published in Hu, X., Fan, W., Han, B., et al., Complete genome sequence of the mosquitocidal bacterium Bacillus sphaericus C3-41 and comparison with those of closely related Bacillus species. J. Bacteriol. (2008) 190: 2892-2902.

[0023] The Lysinibacillus sphaericus in the following examples can be replaced by other types of Lysinibacillus, including but not limited to Lysinibacillus varians, Lysinibacillus fusiformis, and the like.

[0024] The potentiostat in the following embodiments may be replaced by a DC power supply.

[0025] Example 1

[0026] The spherical lysine Bacillus Lysinibacillus sphaericus C3-41 (stored in a glycerol tube) was activated and spread on an LB plate to form a single colony. The single colony was inoculated and cultured overnight in LB culture medium to prepare a seed solution. The seed solution (OD 600 =1.0) was inoculated at a volume ratio of 5% in a 200 mL reagent bottle containing 120 mL of fermentation culture medium (20.0 g / L glucose, 30.0 g / L beet molasses, 10.0 g / L corn steep liquor powder, 3.0 g / L betaine, 1.5 g / L MgSO4, 20.0 mg / L FeSO4, 450.0 mg / L threonine, 1.0 g / L potassium chloride, 0.6 mg / L biotin, and the solvent was water), and aerobic fermentation was carried out at 30°C and 100 r / min (magnetic rotor).

[0027] The reagent bottle used for fermentation is equipped with an electrode control system, which mainly includes three parts: a constant potential instrument, a wire, and electrodes (including a working electrode, a counter electrode, and a reference electrode). The wire connects the electrode to the constant potential instrument. The connection between the wire and the electrode is treated with a waterproof coating for corrosion prevention. When working, the electrode is placed in the fermentation device and submerged in the fermentation culture medium.

[0028] Graphite plates (12 cm 2 ) as the working electrode, platinum wire as the counter electrode, and the reference electrode as the silver chloride electrode (Ag / AgCl / Cl - ), all three electrodes were fixed on the bottle cap with a gas permeable filter membrane (pore size of 0.2 microns). During the fermentation process, a constant potential instrument was used to control the working electrode potential to 0.2 V (relative to the reference electrode). In the control group, electrodes of the same material were also set, but no constant potential was applied, so that it was in an open circuit state.

[0029] This example focuses on the effect of the electrode system on the L-lysine fermentation capacity of spherical lysine Bacillus. During the fermentation process, samples were taken at a fixed time every day and the concentration of L-lysine was analyzed by high performance liquid chromatography, and the OD 600 Reflects the growth of bacteria.

[0030] like Figure 1 As shown in the figure, when the potential of the working electrode is 0.2V, the electrode can significantly improve the fermentation efficiency of L-lysine, and its highest concentration of 1.57g / L appears on the third day of fermentation, which is 40.2% higher than the concentration of the control group (1.12g / L). On the other hand, there is no significant difference in the bacterial concentration between the treatment group and the control group ( Figure 2 ), indicating that the electrode system improves the efficiency of L-lysine synthesis by changing the distribution of metabolic flux within a single cell, rather than increasing the concentration of L-lysine by promoting cell growth. This also means that more carbon sources are used for the synthesis of L-lysine.

[0031] Example 2

[0032] The spherical lysine Bacillus Lysinibacillus sphaericus C3-41 stored in a glycerol tube was activated and spread on an LB plate to form a single colony. The single colony was inoculated and cultured overnight in LB culture medium to prepare a seed solution. The seed solution (OD 600 =1.0) was inoculated at a volume ratio of 5% in a 200 mL reagent bottle containing 120 mL of fermentation culture medium (20.0 g / L glucose, 30.0 g / L beet molasses, 10.0 g / L corn steep liquor powder, 3.0 g / L betaine, 1.5 g / L MgSO4, 20.0 mg / L FeSO4, 450.0 mg / L threonine, 1.0 g / L potassium chloride, 0.6 mg / L biotin, and the solvent was water), and aerobic fermentation was carried out at 30°C and 100 r / min (magnetic rotor).

[0033] The reagent bottle used for fermentation is equipped with an electrode control system, which mainly includes three parts: a constant potential instrument, a wire, and electrodes (including a working electrode, a counter electrode, and a reference electrode). The wire connects the electrode to the constant potential instrument. The connection between the wire and the electrode is treated with a waterproof coating for corrosion prevention. When working, the electrode is placed in the fermentation device and submerged in the fermentation culture medium.

[0034] Graphite plates (12 cm 2 ) as the working electrode, platinum wire as the counter electrode, and the reference electrode is Ag / AgCl / Cl - The three electrodes were fixed on the bottle cap with a gas permeable filter membrane (0.2 micron pore size). During the fermentation process, a constant potential instrument was used to control the working electrode potential to -0.2V or 0.2V (relative to the reference electrode). In the control group, electrodes of the same material were also set, but no constant potential was applied, so that it was in an open circuit state.

[0035] like Figure 3As shown in the figure, when different electrode potentials are used, the redox environment in the culture system will change, and the corresponding amino acid fermentation products of microorganisms will also change accordingly. The 16 amino acids in the supernatant sample were analyzed using an amino acid analyzer. Compared with the control, the concentrations of 14 of them (Asp, Thr, Ser, Glu, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, Arg) increased significantly under the regulation of the 0.2V electrode, and the five amino acids Gly, Cys, Ile, Leu, and Lys increased under -0.2V conditions. The above results show that applying a constant potential helps to improve the synthesis of certain types of amino acids in bacteria, and different potentials have different effects on the amino acid metabolic pathways and cell growth rates of bacteria. Therefore, the improvement of the fermentation yield of specific amino acids can be achieved by changing the electrode potential.

Claims

1. A method for producing amino acids by fermentation of lysinibacillus using electrodes, characterized in that: The following steps are involved: S1. The seed solution of Bacillus lysinicus was inoculated into the fermentation medium at a volume ratio of 5%, and then placed in a fermentation device; S2. The electrodes of the electrode control system are placed in the fermentation device and submerged in the fermentation medium; S3. Put the electrode control system in the on state and carry out aerobic fermentation at 30°C and 100 r / min.

2. The method according to claim 1, characterized in that The lysinibacillus is spherical lysinibacillus, deformed lysinibacillus or spindle-shaped lysinibacillus.

3. The method according to claim 1, characterized in that The fermentation culture medium is composed of: 20.0 g / L glucose, 30.0 g / L beet molasses, 10.0 g / L corn steep liquor powder, 3.0 g / L betaine, 1.5 g / L MgSO4, 20.0 mg / L FeSO4, 450.0 mg / L threonine, 1.0 g / L potassium chloride, 0.6 mg / L biotin, and the solvent is water.

4. The method according to claim 1, characterized in that The electrode consists of a working electrode, a counter electrode and a reference electrode.

5. The method according to claim 4, characterized in that The working electrode is a graphite plate, the counter electrode is a platinum wire, and the reference electrode is a silver chloride electrode.

6. The method according to claim 1, characterized in that During aerobic fermentation, the electrode control system controls the working electrode potential to be -0.2V or 0.2V.

7. The method according to claim 6, characterized in that When the electrode control system controls the working electrode potential to be -0.2V, the content of Gly, Cys, Ile, Leu and / or Lys produced by lysinophilic Bacillus fermentation is increased.

8. The method according to claim 6, characterized in that When the electrode control system controls the working electrode potential to be 0.2V, the content of Asp, Thr, Ser, Glu, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys and / or Arg produced by lysinophilic Bacillus fermentation is increased.

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