A method for promoting production of gamma-polyglutamic acid by bacillus fermentation using an oxygen carrier

Abstract

This invention relates to the field of microbial fermentation, specifically to a method for promoting the fermentation of Bacillus atrophaeus to produce γ-polyglutamic acid (PGA) using oxygen carriers. This invention involves adding specific alkane oxygen carriers during the initial stage of γ-PGA production via Bacillus atrophaeus NWS-01 fermentation. Hexane, heptane, and hexadecane were selected as effective oxygen carriers, and the optimal single-addition concentration and optimal synergistic addition ratio of each oxygen carrier were determined. This solves the problem of limited γ-PGA yield due to low oxygen transfer efficiency in traditional fermentation processes. Specifically, adding 0.3% heptane alone achieves a γ-PGA yield of 39.4 ± 0.19 g / L, while the synergistic addition of 0.240% hexane, 0.407% heptane, and 1.014% hexadecane results in a maximum yield of 42.1 g / L, representing a 150.6% increase compared to the fermentation system without added oxygen carriers. This method is simple to operate, has high oxygen utilization, low energy consumption, and produces fewer bubbles and lower liquid shear force in the fermentation system, making it suitable for the industrial-scale aerobic high-density fermentation production of γ-PGA.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation, and specifically to a method for promoting the fermentation of Bacillus to produce γ-polyglutamic acid using an oxygen carrier. Background Technology

[0002] γ-Polyglutamic acid (γ-PGA) is a water-soluble polyamino acid synthesized by microorganisms through a ribosome-independent pathway. Its molecular chain is composed of glutamic acid monomers linked by γ-amide bonds. It has excellent properties such as good biocompatibility, biodegradability, high water absorption, strong adhesion and non-toxicity. In the food industry, it can be used as a thickener, preservative and quality improver. In agriculture, it can be used as a water-retaining agent and fertilizer slow-release agent to improve soil fertility and crop stress resistance. It is a bio-based polymer material with great industrialization value.

[0003] In industrial fermentation systems, insufficient dissolved oxygen can cause aerobic bacteria to enter anaerobic metabolic pathways, which not only inhibits the normal growth and reproduction of bacteria, but also hinders the synthesis of γ-PGA, leading to the accumulation of a large amount of by-products, ultimately resulting in low fermentation efficiency and a decline in product yield and quality.

[0004] Traditional γ-PGA fermentation systems primarily rely on physical methods such as mechanical stirring, increased aeration, and increased fermenter pressure to raise dissolved oxygen levels. However, these methods suffer from several technical drawbacks: First, oxygen transfer is slow and oxygen utilization is low. Since the solubility of oxygen in the aqueous phase is already extremely low, simple stirring and aeration only achieve preliminary contact between the gas and liquid phases. Most of the oxygen is not utilized by the cells and is discharged with the exhaust gas, resulting in resource waste. Second, energy consumption is high. Intensive mechanical stirring and high-flow-rate aeration require a large amount of electricity, significantly increasing the energy consumption of γ-PGA fermentation. The production cost is high, which is inconsistent with the trend of green and low-carbon industrial production. Third, high-intensity stirring generates significant liquid shear force, easily causing mechanical damage to bacterial cells, disrupting cell membrane structure and metabolic activity, and thus inhibiting bacterial growth and product synthesis. Fourth, vigorous gas-liquid mixing produces a large amount of stable foam in the fermentation system. This foam not only occupies the effective volume of the fermenter, reducing the liquid loading coefficient, but also causes bacterial cells to overflow with the foam, resulting in cell loss. Simultaneously, the foam hinders gas-liquid mass transfer and heat exchange, disrupting the stability of the fermentation system and increasing the difficulty of controlling the fermentation process. Furthermore, traditional dissolved oxygen regulation methods are less effective in high-density fermentation systems, while high-density fermentation is a key means to achieve high-yield industrial production of γ-PGA. This contradiction severely limits the application of aerobic fermentation strains such as Bacillus atrophaeus, such as the Bacillus atrophaeus NWS-01 previously screened by the applicant, in the industrial production of γ-PGA.

[0005] To address the low oxygen transfer efficiency in aerobic fermentation, researchers have explored the application of oxygen carriers in microbial fermentation systems. Oxygen carriers are substances that can effectively dissolve oxygen and facilitate oxygen transfer between gas-liquid and liquid-solid phases, significantly improving dissolved oxygen levels and oxygen transfer rates in fermentation systems. Alkane compounds, as hydrophobic oxygen carriers, possess advantages such as high oxygen solubility, rapid oxygen transfer, immiscibility with water, chemical stability, and low toxicity to most microorganisms. Furthermore, they are widely available, inexpensive, and can reduce bubble generation and liquid shear forces in fermentation systems. These factors make them the most promising type of oxygen carrier for aerobic microbial fermentation, and they have been successfully applied in the fermentation processes of yeast, actinomycetes, and other Bacillus strains for enzyme production, antibiotic production, and polyhydroxyalkanoate production, effectively increasing the yield of target products.

[0006] However, the application of alkane oxygen carriers exhibits significant strain and product specificity. Different alkane molecules have different carbon chain lengths and physicochemical properties, resulting in drastically different effects on cell growth, metabolic pathways, and product synthesis of different strains. Even with the same alkane oxygen carrier, different concentrations of addition can have significantly different effects on the cells. Some alkanes can promote product synthesis at low concentrations, but at high concentrations they can be toxic and inhibit cell growth. At the same time, some alkanes, while increasing dissolved oxygen levels and promoting the synthesis of target products, can inhibit cell growth. Currently, research on the application of alkane oxygen carriers in the fermentation of Bacillus atrophaeus to produce γ-PGA is still lacking. There are no reports that clearly identify the appropriate types of alkane oxygen carriers and the optimal concentrations for this type of strain, nor have they elucidated the differential effects of different alkane oxygen carriers on Bacillus atrophaeus cell growth and γ-PGA synthesis. Furthermore, there is no optimization research on the synergistic effects of multiple alkane oxygen carriers, which fails to provide technical support for the efficient aerobic fermentation of Bacillus atrophaeus, such as Bacillus atrophaeus NWS-01 γ-PGA.

[0007] Therefore, screening suitable alkane-based oxygen carriers for Bacillus atrophaeus NWS-01, determining their optimal addition concentration, exploring the synergistic effects of different oxygen carriers and optimizing their ratios, solving the core problem of low oxygen transfer efficiency during the fermentation process of this strain, and achieving a significant increase in γ-PGA yield while reducing fermentation energy consumption and improving the stability of the fermentation system are key to promoting the high-yield industrial application of Bacillus atrophaeus NWS-01 for γ-PGA, and have important theoretical significance and practical application value. Summary of the Invention

[0008] The technical problem to be solved by this invention is to address the shortcomings of existing technologies in the fermentation of Bacillus atrophaeus NWS-01 to produce γ-PGA, which suffers from low oxygen transfer efficiency, limited yield, and high energy consumption. This invention provides a method for promoting the fermentation of Bacillus atrophaeus to produce γ-polyglutamic acid using an oxygen carrier. By screening suitable alkane oxygen carriers and optimizing their addition concentration and ratio, the oxygen transfer efficiency and γ-PGA yield of the fermentation system are improved, while the fermentation energy consumption is reduced.

[0009] To address the aforementioned technical problems, this invention discloses a method for promoting the fermentation of Bacillus atrophus to produce γ-polyglutamic acid using an oxygen carrier.

[0010] The seed culture of Bacillus atrophus was inoculated into the fermentation medium. At the initial stage of fermentation (0 h), an alkane oxygen carrier was added to the fermentation medium once for fermentation culture.

[0011] The atrophic bacillus is Bacillus atrophaeus NWS-01.

[0012] Specifically, this strain is a high-yield, superior γ-PGA strain obtained by the applicant in its previous screening. Detailed information about the strain has been published in Chinese patent CN118421528A. Its fermentation process for producing γ-PGA is a typical aerobic fermentation process. The growth and metabolism of the cells, as well as the biosynthesis of γ-PGA, all require a continuous and sufficient supply of dissolved oxygen. The dissolved oxygen level and oxygen transfer efficiency directly determine the respiration intensity of the cells and the metabolic conversion efficiency of carbon and nitrogen sources, which are the core limiting factors affecting the γ-PGA synthesis efficiency and yield of this strain.

[0013] The alkane oxygen support is any one or a combination of several of n-hexane, n-heptane, and n-hexadecane.

[0014] In some embodiments of the present invention, the alkane oxygen carrier is any one or a combination of several of n-hexane, n-heptane, and n-hexadecane. This invention was first screened by conducting screening experiments on four alkane oxygen carriers (n-hexane, n-heptane, n-dodecane, and n-hexadecane) to clarify the differential effects of each oxygen carrier on the growth of Bacillus atrophaeus NWS-01 cells and the synthesis of γ-PGA.

[0015] Specifically, n-hexane, n-heptane, and n-hexadecane can all promote the synthesis of γ-PGA. Among them, n-hexadecane is the only oxygen carrier that both promotes product synthesis and is beneficial to cell growth. Although n-hexane and n-heptane can promote production, they have a certain inhibitory effect on cell growth.

[0016] Specifically, n-dodecane has a significant inhibitory effect on the synthesis of γ-PGA. Although it does not inhibit cell growth and can even make cell growth better than the blank control, it cannot be used as an oxygen-producing carrier for the fermentation production of γ-PGA.

[0017] The concentration of n-hexane is 0.1-0.5%, the concentration of n-heptane is 0.2-1.0%, and the concentration of n-hexadecane is 0.2-1.6%.

[0018] In some embodiments of the present invention, when alkane oxygen carriers are added alone, the optimal addition concentrations are 0.2% for n-hexane, 0.3% for n-heptane, and 1% for n-hexadecane. Specifically, when 0.3% n-heptane is added alone, the fermentation yield of γ-PGA can reach 39.4 ± 0.19 g / L, which is the highest yield when only one oxygen carrier is added.

[0019] In some embodiments of the present invention, the alkane oxygen support is n-heptane, or n-hexane + n-heptane + n-hexadecane.

[0020] In some embodiments of the present invention, the concentration of n-heptane added is 0.3%; the concentrations of hexane + n-heptane + n-hexadecane added are 0.240%, 0.407%, and 1.014%, respectively.

[0021] Specifically, the ratio of hexane, n-heptane, and n-hexadecane was obtained through Box-Behnken experimental design and regression fitting optimization. Under this ratio, the fermentation yield of γ-PGA can reach up to 42.1 g / L, which is 150.6% higher than the original fermentation system with 16.8 g / L without oxygen carrier.

[0022] In some embodiments of the present invention, the addition of the alkane oxygen carrier can significantly improve the oxygen transfer rate and oxygen utilization rate of the fermentation system, reduce the energy consumption of the fermentation process, reduce the generation of bubbles in the fermentation system, reduce liquid shear force, and provide a suitable environment for the aerobic high-density fermentation of Bacillus atrophaeus NWS-01.

[0023] The fermentation medium has the following formula: glucose 20-60 g / L, monosodium glutamate 20-60 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 0.3-1 g / L, ammonium sulfate 2-5 g / L, citric acid 0.3-1 g / L, anhydrous magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.01-0.03 g / L, pH 6.2.

[0024] In some embodiments of the present invention, the fermentation medium has the following formulation: glucose 60 g / L, monosodium glutamate 45 g / L, dipotassium hydrogen phosphate 10 g / L, potassium dihydrogen phosphate 1 g / L, ammonium sulfate 5 g / L, citric acid 1 g / L, anhydrous magnesium sulfate 0.1 g / L, manganese sulfate 0.03 g / L, pH 6.2.

[0025] The fermentation culture is carried out under the following conditions: temperature 30~32℃, rotation speed 150~200 rpm, aeration rate 1.0~1.5 vvm, and fermentation cycle 48~60 h.

[0026] In some embodiments of the present invention, the fermentation culture is carried out under the following conditions: temperature 30°C, rotation speed 200 rpm, aeration rate 1.5 vvm, and fermentation cycle 48 h.

[0027] The inoculation dosage is 1~5% v / v.

[0028] In some embodiments of the present invention, the inoculation is performed at a dose of 2% v / v.

[0029] Furthermore, the present invention provides a fermentation additive for promoting the fermentation production of γ-polyglutamic acid by Bacillus atrophus, wherein the fermentation additive is n-heptane, or n-hexane + n-heptane + n-hexadecane.

[0030] The concentration of n-heptane added is 0.3%; the concentrations of hexane, n-heptane, and n-hexadecane added are 0.240%, 0.407%, and 1.014%, respectively.

[0031] Specifically, when promoting the fermentation of Bacillus atrophus to produce γ-polyglutamic acid, the fermentation additive is added to the fermentation system at the initial stage of fermentation (0 h) according to the optimal addition concentration of n-heptane or the synergistic optimal ratio of hexane + n-heptane + n-hexadecane.

[0032] Beneficial effects:

[0033] 1. This invention is the first to screen out the alkane oxygen carriers suitable for the fermentation of Bacillus atrophaeus NWS-01 to produce γ-PGA as n-hexane, n-heptane, and n-hexadecane, and clarifies the optimal single addition concentration and the optimal ratio of synergistic addition of each oxygen carrier. At the same time, it clarifies that n-dodecane is not suitable as an oxygen carrier for promoting the production of γ-PGA by this strain, providing a new technical approach for the fermentation production of γ-PGA.

[0034] 2. The method of the present invention significantly improves the oxygen transfer rate of the fermentation system by 15% and the oxygen utilization rate by 50% by adding alkane oxygen carriers, reduces the fermentation energy consumption by 10%, reduces the bubble generation in the fermentation system by 13%, and reduces the liquid shear force by 12%, making it suitable for aerobic high-density fermentation of Bacillus atrophaeus NWS-01.

[0035] 3. In this invention, the yield of γ-PGA can reach 39.4±0.19 g / L when 0.3% n-heptane is added alone, and the yield of γ-PGA can reach up to 42.1 g / L when the optimal ratio of oxygen carrier is added in combination. Compared with the original system without oxygen carrier, the yield is increased by 150.6%, which greatly improves the fermentation yield of γ-PGA.

[0036] 4. The second-order empirical regression model established in this invention, which correlates γ-PGA yield with the concentrations of three oxygen carriers, exhibits high goodness of fit. It can be used for the analysis and prediction of γ-PGA fermentation processes, providing a theoretical basis for process optimization in industrial fermentation production. Verification experiments were conducted on the regression model using three parallel fermentation experiments with the addition ratios of 0.240% hexane, 0.407% heptane, and 1.014% hexadecane. The γ-PGA yields were 41.0 g / L, 41.3 g / L, and 42.1 g / L, respectively. The maximum difference between the experimental values ​​and the model prediction (41.5 g / L) was only 1.4%, indicating that the model has high reliability and can guide industrial fermentation production.

[0037] 5. The method of the present invention is simple to operate, and the oxygen carrier is added in a one-time manner, without the need for complicated feedstock operations. It is easy to achieve industrial scale-up and has good prospects for industrial application. Attached Figure Description

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0039] Figure 1 To investigate the effects of adding different concentrations of n-hexane on the growth (OD) of Bacillus atrophaeus NWS-01 cells. 660 The influence of γ-PGA synthesis on the synthesis of γ-PGA is shown in the figure.

[0040] Figure 2 To investigate the effects of adding different concentrations of n-heptane on the growth (OD) of Bacillus atrophaeus NWS-01 cells. 660 The influence of γ-PGA synthesis on the synthesis of γ-PGA is shown in the figure.

[0041] Figure 3To investigate the effects of adding different concentrations of n-dodecane on the growth (OD) of Bacillus atrophaeus NWS-01 cells. 660 The influence of γ-PGA synthesis on the synthesis of γ-PGA is shown in the figure.

[0042] Figure 4 To investigate the effects of adding different concentrations of n-hexadecane on the growth (OD) of Bacillus atrophaeus NWS-01 cells. 660 The influence of γ-PGA synthesis on the synthesis of γ-PGA is shown in the figure.

[0043] Figure 5 The response surface methodology diagram shows the effect of different oxygen carriers on γ-PGA production. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0045] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0046] In the following embodiments, the detailed information of Bacillus atrophaeus NWS-01 has been disclosed in Chinese Patent CN118421528A.

[0047] In the following examples, w / v means g / 100 mL.

[0048] Example 1: Screening of Monoalkane Oxygen Supports and Determination of Optimal Addition Concentration

[0049] After activating Bacillus atrophaeus NWS-01, a single colony was picked and inoculated into fresh LB liquid medium, and cultured overnight at 30°C and 200 rpm to obtain seed culture.

[0050] Four alkane oxygen carriers—n-hexane, n-heptane, n-dodecane, and n-hexadecane—were selected and added at different concentrations to the fermentation medium for γ-PGA production at the initial stage of fermentation (0 h). A blank control group without added oxygen carriers was set up, and each group was tested in triplicate. The activated seed culture was inoculated at a rate of 2% v / v into the above-mentioned fermentation mediums containing different oxygen carriers (60 g / L glucose, 45 g / L monosodium glutamate, 10 g / L dipotassium hydrogen phosphate, 1 g / L potassium dihydrogen phosphate, 5 g / L ammonium sulfate, 1 g / L citric acid, 0.1 g / L anhydrous magnesium sulfate, 0.03 g / L manganese sulfate, pH 6.2). Aerobic fermentation was carried out under the following conditions: temperature 30℃, agitator speed 200 rpm, aeration rate 1.5 vvm, and fermentation period 48 h. The fermentation conditions were kept consistent across all experimental groups.

[0051] After fermentation is complete, the cell concentration (OD) of each experimental group is measured. 660 ) and γ-PGA production.

[0052] The results are as follows Figures 1-4 As shown, Figure 1 The optimal concentration of hexane was 0.2% (w / v), at which γ-PGA synthesis was significantly increased, but cell growth was inhibited compared to the control. 660 The decrease was significant with increasing n-hexane concentration; Figure 2 The optimal concentration of heptane was 0.3% (w / v), at which the yield of γ-PGA reached 39.4±0.19 g / L, the highest value when a single oxygen carrier was added. However, the cell growth was inhibited compared to the blank control. Figure 3 At all concentrations, dodecane significantly inhibited γ-PGA synthesis. Although the bacterial growth was better than the blank control, it could not be used as an oxygen-producing carrier. Figure 4 The optimal concentration of hexadecane is 1% (w / v). At this concentration, not only is the synthesis of γ-PGA significantly increased, but the cell growth is also better than that of the blank control, thus achieving a dual promotion of product synthesis and cell growth.

[0053] To improve the synthesis yield of γ-PGA, n-hexane, n-heptane, and n-hexadecane were selected as suitable oxygen carriers, and further optimization was carried out by synergistic addition of the three.

[0054] Example 2: Optimization of the synergistic addition concentration of three oxygen carriers

[0055] 1. Experimental Design: A Box-Behnken experimental design was adopted, with the concentrations of n-hexane (X1), n-heptane (X2), and n-hexadecane (X3) as the factors of investigation, as shown in Table 1. Each factor was set with 3 levels (-1, 0, +1), where the levels of n-heptane were 0.1%, 0.3%, and 0.5% (w / v), the levels of n-hexane were 0.1%, 0.2%, and 0.3% (w / v), and the levels of n-hexadecane were 0.5%, 1.0%, and 1.5% (w / v). The yield of γ-PGA was used as the response value, and the center point was set with 3 replicates.

[0056] Referring to the fermentation process of Example 1, this example uses the Box-Behnken experimental design and OD... 660 The yield results of γ-PGA are shown in Table 2.

[0057] Table 1. Factors and levels in the Box-Behnken experimental design

[0058]

[0059] Table 2 Box-Behnken Experimental Design and Results

[0060]

[0061] 2. Regression Model Establishment: Statistica 8.0 software was used to perform regression fitting on the 15 sets of experimental data in Table 2 to establish a second-order empirical regression model of γ-PGA yield and the concentration of the three oxygen carriers, and analysis of variance was performed.

[0062] Specifically, the experimental data were fitted using the 3**(kp) and Box-Behnken Design module in the Experimental Design module of Statistica 8.0 software to obtain a second-order empirical model of the relationship between the target response value and each factor (as shown in the following formula).

[0063] Y = 33.6458 + 3.5325X1 + 1.9675X1 2 +0.1500X2+2.1725X2 2 +0.1025X3+0.8913X3 2

[0064] In the formula, Y is the concentration of γ-PGA (g / L), and X1, X2 and X3 are n-hexane, n-heptane and n-hexadecane (%, w / v), respectively.

[0065] The results of the ANOVA and regression analysis are shown in Table 3. The p-values ​​of the quadratic terms (n-hexane Q, n-heptane Q, n-hexadecane Q) and the linear term (n-hexane L) for each factor in the model are all less than 0.05, indicating a significant impact. However, the interaction term is not reflected in the equation, indicating that the interaction between X1, X2, and X3 has no significant effect on the yield of γ-PGA. The ANOVA results show that the coefficient of determination R0 of this equation is... 2 The corrected coefficient of determination (Adj.R) is 0.9846. 2 The value of 0.9731 indicates that the model fits the actual situation well and can reflect the influence of various factors on γ-PGA yield. It can be used for the analysis and prediction of γ-PGA fermentation. Figure 5 The response surface methodology diagram shows the effect of different oxygen carriers on γ-PGA production.

[0066] Table 3 Regression results of the Box-Behnken experimental design

[0067]

[0068] 3. Determination of the optimal ratio: Based on the regression model coefficients and response surface analysis, the optimal ratio of the three oxygen carriers for synergistic addition was determined to be 0.240% (w / v) of n-hexane, 0.407% (w / v) of n-heptane, and 1.014% (w / v) of n-hexadecane. The model predicted that the yield of γ-PGA under this ratio would be 41.5 g / L.

[0069] Example 3: Validation Experiment of the Regression Model

[0070] According to the optimal ratio determined in Example 2, at the initial stage of fermentation (0h), 0.240% n-hexane, 0.407% n-heptane, and 1.014% n-hexadecane were added to the fermentation medium at one time. Bacillus atrophaeus NWS-01 was inoculated at 2% v / v for aerobic fermentation culture. Three parallel experiments were set up, and the fermentation conditions were the same as in Example 1.

[0071] After fermentation, the yield of γ-PGA was measured. The results showed that the yields of γ-PGA in the three parallel experiments were 41.0 g / L, 41.3 g / L, and 42.1 g / L, respectively. The maximum difference between the experimental values ​​and the model predictions was 1.4%, indicating that the model fit was good and the optimal ratio has practical application value.

[0072] Example 4: Comparison of post-fermentation effects after optimization

[0073] Referring to the fermentation process of Example 1, the fermentation results of the blank control group without oxygen carrier, the experimental group with 0.3% n-heptane alone, and the experimental group with the optimal ratio of oxygen carrier were compared. The results are as follows: the γ-PGA yield of the blank control group was 16.8 g / L; the γ-PGA yield of the group with 0.3% n-heptane alone was 39.4±0.19 g / L, which was about 134.5% higher than that of the blank control group; the highest γ-PGA yield of the group with the optimal ratio of oxygen carrier was 42.1 g / L, which was 150.6% higher than that of the blank control group. In addition, the oxygen transfer rate of the fermentation system increased by 15% and the oxygen utilization rate reached 50%, the fermentation energy consumption was reduced by 10%, the bubble generation of the fermentation system was reduced, the bubble density was reduced by 13%, and the liquid shear force was reduced by 12%.

[0074] This invention provides a method for promoting the fermentation of Bacillus atrophus to produce γ-polyglutamic acid using an oxygen carrier. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for promoting the fermentation of Bacillus to produce γ-polyglutamic acid using an oxygen carrier, characterized in that, The seed culture of Bacillus atrophus was inoculated into the fermentation medium. At the initial stage of fermentation (0 h), an alkane oxygen carrier was added to the fermentation medium once for fermentation culture. The alkane oxygen support is any one or a combination of several of n-hexane, n-heptane, and n-hexadecane.

2. The method according to claim 1, characterized in that, The atrophic Bacillus is Bacillus atrophaeus NWS-01.

3. The method according to claim 1, characterized in that, The concentration of n-hexane added is 0.1~0.5%, the concentration of n-heptane added is 0.2~1.0%, and the concentration of n-hexadecane added is 0.2~1.6%.

4. The method according to claim 1, characterized in that, The alkane oxygen support is n-heptane, or n-hexane + n-heptane + n-hexadecane.

5. The method according to claim 4, characterized in that, The concentration of n-heptane added is 0.3%; the concentrations of hexane, n-heptane, and n-hexadecane added in the combination of hexane, n-heptane, and n-hexadecane are 0.240%, 0.407%, and 1.014%, respectively.

6. The method according to claim 1, characterized in that, The fermentation medium has the following formula: glucose 20-60 g / L, monosodium glutamate 20-60 g / L, dipotassium hydrogen phosphate 1-10 g / L, potassium dihydrogen phosphate 0.3-1 g / L, ammonium sulfate 2-5 g / L, citric acid 0.3-1 g / L, anhydrous magnesium sulfate 0.1-0.2 g / L, manganese sulfate 0.01-0.03 g / L, pH 6.

2.

7. The method according to claim 1, characterized in that, The fermentation culture is carried out under the following conditions: temperature 30~32℃, rotation speed 150~200 rpm, aeration rate 1.0~1.5 vvm, and fermentation cycle 48~60 h.

8. The method according to claim 1, characterized in that, The inoculation dosage is 1~5% v / v.

9. A fermentation additive for promoting the fermentation of Bacillus atrophus to produce γ-polyglutamic acid, characterized in that, The fermentation additive is n-heptane, or n-hexane + n-heptane + n-hexadecane.

10. The fermentation additive according to claim 9, characterized in that, The concentration of n-heptane added is 0.3%; the concentrations of hexane, n-heptane, and n-hexadecane added in the combination of hexane, n-heptane, and n-hexadecane are 0.240%, 0.407%, and 1.014%, respectively.

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