阅读说明：本技术 一种降低丁二酸发酵副产物乙酸生成的方法 (Method for reducing generation of acetic acid serving as succinic acid fermentation by-product ) 是由 申乃坤 张红岩 姜明国 王一兵 于 2020-07-15 设计创作，主要内容包括：本发明提供一种降低丁二酸发酵副产物乙酸生成的方法,该方法是在产琥珀酸放线杆菌(<I>Actinobacillus succinogenes</I>)利用不同碳源(葡萄糖、淀粉水解液、纤维素水解液)发酵丁二酸过程中添加一定量的亚硫酸氢钠,可显著降低副产物乙酸的生成。以葡萄糖为原料发酵丁二酸时,添加亚硫酸氢钠后乙酸产量由17.85 g/L降低至8.18 g/L,降幅高达54.17%。本发明的方法解决了微生物发酵产丁二酸过程中,副产物乙酸偏高的问题,降低了丁二酸分离纯化成本,有利于促进微生物发酵法生产丁二酸产业的发展。(The invention provides a method for reducing acetic acid generation of succinic acid fermentation by-products, which is to produce actinobacillus succinogenes (A), (B) Actinobacillus succinogenes ) A certain amount of sodium bisulfite is added in the process of fermenting the succinic acid by utilizing different carbon sources (glucose, starch hydrolysate and cellulose hydrolysate), so that the generation of a byproduct, namely acetic acid can be obviously reduced. When glucose is used as a raw material to ferment the succinic acid, the yield of the acetic acid is reduced from 17.85 g/L to 8.18 g/L after the sodium bisulfite is added, and the reduction amplitude is up to 54.17%. The method solves the problem that the acetic acid as a byproduct is higher in the process of producing the succinic acid by microbial fermentation, reduces the cost of separating and purifying the succinic acid, and is beneficial to promoting the microbial fermentation method to produce the succinic acidThe industry is developed.)

1. The method for reducing the generation of acetic acid which is a by-product of succinic acid fermentation is characterized by comprising the following steps of:

(1) inoculating a single colony of Actinobacillus succinogenes GXAS137 on the plate into a seed culture medium, and culturing at 36-38 ℃ for 16-20 h until the number of bacteria reaches more than 3 hundred million to obtain an expanded culture strain;

(2) performing secondary propagation culture on the strain to be subjected to amplification culture for 8 hours until the number of the strain reaches more than 3 hundred million according to the inoculation amount of 5% (V/V), and obtaining liquid seeds;

(3) inoculating liquid seeds into a fermentation culture medium according to the inoculation amount of 5-15% (V/V), adding 0.05-2 g/L sodium bisulfite 0-12 h after inoculation, adjusting the pH of the fermentation liquor to 6.5-7.0 by using a pH buffering agent, and then filling N₂Or CO₂In the environment, under the conditions of the rotating speed of 100-200 r/min and the temperature of 36-38 ℃, performing batch fermentation or fed-batch fermentation for 30-72 hours to obtain fermentation liquor containing succinic acid, and separating and purifying the fermentation liquor to obtain the succinic acid.

2. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to claim 1, wherein: the adding time of the sodium bisulfite is preferably 6-8 h after inoculation.

3. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to claim 1, wherein: the addition amount of the sodium bisulfite is preferably 0.1-0.5 g/L.

4. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to claim 1, wherein: the seed culture medium comprises the following components in percentage by concentration: glucose 8g/L, yeast powder 12g/L, corn steep liquor 4g/L, NaHCO₃4g/L，NaH₂PO₄9.6g/L，K₂HPO₄1.55g/L。

5. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to claim 1, wherein: the fermentation medium comprises the following components in percentage by concentration: 30-40 g/L of initial total sugar, 5-20 g/L of nitrogen source, 2-10 g/L of monopotassium phosphate, 2-10 g/L of sodium bicarbonate, 0.3-1.0 g/L of calcium chloride and 0.3-1.0 g/L of magnesium chloride.

6. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to claim 1, wherein: in the fed-batch fermentation process, mother liquor with the total sugar concentration of 200-300 g/L is fed in an intermittent flow mode, and the total sugar of fermentation liquor is controlled to be 30-35 g/L.

7. The method for reducing acetic acid production as a by-product of succinic acid fermentation according to claim 5, wherein: the initial total sugar is one or more of saccharides, saccharide-containing raw materials, saccharification liquid of starchy raw materials or hydrolysis liquid of fiber raw materials, and is mixed in any proportion;

the saccharide is one of glucose, sucrose and xylose;

the saccharide-containing raw material is one of cane molasses and xylose mother liquor;

the starchy raw material is one of tapioca flour, corn flour and wheat flour;

the fiber raw material is one of corn straw, bagasse and rice straw.

8. The method for reducing acetic acid production as a by-product of succinic acid fermentation according to claim 5, wherein: the nitrogen source is one or more of corn steep liquor, yeast powder, soybean meal and peanut cake powder which are mixed in any proportion.

9. The method for reducing the generation of acetic acid as a by-product of succinic acid fermentation according to any one of claims 1 to 8, wherein the method comprises the following steps: the pH buffering agent is one or more of basic magnesium carbonate, sodium carbonate, magnesium hydroxide and sodium hydroxide which are mixed in any proportion, and the total concentration is 20-80 g/L.

Technical Field

The invention belongs to the technical field of industrial microbial fermentation, and particularly relates to a method for reducing the generation of a fermentation byproduct of offspring.

Background

Succinic acid (b)Succinicacid) Also known as succinic acid (butanedioic acid) It is a common natural organic acid, which was first isolated from amber by Georgius in 1546 and is thus named. The molecular formula of succinic acid is C₄H₆O₄Molecular weight 118.09, picric acid, relative density 1.572, melting point 188 ℃, boiling point 235 ℃ (decomposition temperature).

Succinic acid is an important C4 platform compound, is a basic raw material of important bulk chemicals such as1, 4-butanediol, tetrahydrofuran, gamma-butyrolactone, N-methylpyrrolidone, adipic acid and the like and special chemicals, is widely applied to industries such as food, plastics, medicines, spices and the like, and can replace benzene to synthesize more than 250 chemical products. The most promising field is synthetic plastics, which are the main raw materials for synthesizing biodegradable polymer materials such as polybutylene succinate (PBS), polyethylene glycol succinate (PES), polypropylene glycol succinate (PPS), and the like.

At present, the main production method of succinic acid is a chemical method synthesis method by using petroleum as a raw material, which not only needs to consume a large amount of non-renewable petrochemical raw materials and has higher production cost, but also has serious environmental pollution.

Compared with the chemical synthesis method, the microbial fermentation method has the following advantages: (1) the raw material is cheap and renewable biomass resource; (2) can absorb a large amount of CO in the fermentation process₂The environment is protected; (3) the fermentation condition is mild. Therefore, microbial fermentation has attracted attention in recent years, and has become a hot spot of research at home and abroad in recent years.

Actinobacillus succinogenes: (Actinobacillus succinogenes) The strain has the advantages of high yield, utilization of various carbon sources, strong tolerance and the like, and is one of the most potential industrial strains at present. But is currently limitedA. succinogenesThe large-scale production of the succinic acid has high production cost and difficult separation and purification, the high acetic acid (which can account for about 1/3 of the yield of the succinic acid) serving as a byproduct is one of the limiting factors, and the generation of a large amount of acetic acid not only reduces the yield of the succinic acid and the utilization rate of raw materials, but also increases the later separation and purification cost of the succinic acid. Thus, by-productsThe higher acetic acid becomes one of the limiting factors of the industrial production of the succinic acid by the microbial fermentation method.

At present, in order to reduce the generation of byproduct acetic acid, physical and chemical mutagenesis, metabolic engineering transformation and other means are generally adopted to breed excellent strains, or the concentration of a carbon source is increased and CO is increased₂Ventilation of (1), addition of neutral red or H₂As a fermentation process control technology such as an electronic carrier and the like, the metabolic flux of a carbon source in a cell flows to a product succinic acid more, and the generation of a byproduct acetic acid can also be reduced, but the reduction amplitude is limited and generally does not exceed 20%.

Disclosure of Invention

Aiming at the defect of high yield of acetic acid as a by-product in the existing fermentation production of succinic acid, the invention provides a method for reducing the generation of acetic acid as a by-product in succinic acid fermentation.

The invention is realized by the following technical scheme:

a method for reducing the generation of acetic acid as a by-product of succinic acid fermentation comprises the following steps:

(1) inoculating a single colony of Actinobacillus succinogenes GXAS137 on the plate into a seed culture medium, and culturing at 36-38 ℃ for 16-20 h until the number of bacteria reaches more than 3 hundred million to obtain an expanded culture strain;

(2) performing secondary propagation culture on the strain to be subjected to amplification culture for 8 hours until the number of the strain reaches more than 3 hundred million according to the inoculation amount of 5% (V/V), and obtaining liquid seeds;

(3) inoculating liquid seeds into a fermentation culture medium according to the inoculation amount of 5-15% (V/V), adding 0.05-2 g/L sodium bisulfite 0-12 h after inoculation, adjusting the pH of the fermentation liquor to 6.5-7.0 by using a pH buffering agent, and then filling N₂Or CO₂In the environment, under the conditions of the rotating speed of 100-200 r/min and the temperature of 36-38 ℃, performing batch fermentation or fed-batch fermentation for 30-72 hours to obtain fermentation liquor containing succinic acid, and separating and purifying the fermentation liquor to obtain the succinic acid.

The strain used in the present invention is Actinobacillus succinogenes (R) ((R))Actinobacillus succinogenes) GXAS137, the collection unit is called China center for type culture Collection, and the collection number is: CC (challenge collapsar)TCC M2011399, with preservation date of 2011, 11/18.

According to the invention, sodium bisulfite is added in the process of producing succinic acid by fermenting actinobacillus succinogenes, so that sodium bisulfite and an intermediate metabolite acetaldehyde are combined into insoluble sulfonated hydroxy acetaldehyde, and acetaldehyde cannot be converted into acetic acid as a hydrogen acceptor, thereby reducing the generation of a byproduct acetic acid, improving the purity of succinic acid and reducing the cost of extracting succinic acid by subsequent purification.

As a further improvement of the invention, the adding time of the sodium bisulfite is preferably 6-8 h after inoculation.

In a further improvement of the invention, the addition amount of the sodium bisulfite is preferably 0.1-0.5 g/L.

The adding time and the adding amount of the sodium bisulfite are important factors influencing the fermentation and acetic acid production of the actinobacillus succinogenes GXAS137, the sodium bisulfite has certain toxic effect on cells, and if the adding time is too early, the growth of thalli is inhibited, so the growth amount of the thalli is influenced, and the generation of succinic acid is further influenced; if the sodium bisulfite is added too late, acetic acid as a by-product is already produced in a large amount. Similarly, if the addition amount of sodium bisulfite is too low, the intermediate product acetaldehyde forms less insoluble precipitate, and a large amount of acetic acid is still generated; when the addition amount of the sodium bisulfite is too much, the sodium bisulfite has toxic action on bacterial strains, and can reduce the activity of cells and even die the cells, thereby leading to lower concentration of the fermentation product butane. According to the invention, through tests, the optimal adding time and the optimal adding amount of the sodium bisulfite are respectively 6-8 h and 0.1-0.5 g/L after inoculation.

As a further improvement of the invention, the components and concentrations of the seed culture medium are as follows: glucose 8g/L, yeast powder 12g/L, corn steep liquor 4g/L, NaHCO₃4g/L，NaH₂PO₄9.6g/L，K₂HPO₄1.55g/L。

As a further improvement of the invention, the fermentation medium comprises the following components in percentage by weight: 30-40 g/L of initial total sugar, 5-20 g/L of nitrogen source, 2-10 g/L of monopotassium phosphate, 2-10 g/L of sodium bicarbonate, 0.3-1.0 g/L of calcium chloride and 0.3-1.0 g/L of magnesium chloride.

As a further improvement of the invention, in the fed-batch fermentation process, mother liquor with total sugar concentration of 200-300 g/L is fed in intermittently, and the total sugar content of the fermentation liquor is controlled to be 30-35 g/L.

In a further improvement of the present invention, the initial total sugar (i.e., carbon source) is one or more of a saccharide, a saccharide-containing material, a saccharified solution of a starchy material, or a hydrolyzed solution of a fibrous material, and is mixed at an arbitrary ratio;

the saccharide is one of glucose, sucrose and xylose;

the saccharide-containing raw material is one of cane molasses and xylose mother liquor;

the starchy raw material is one of tapioca flour, corn flour and wheat flour;

the fiber raw material is one of corn straw, bagasse and rice straw.

As a further improvement of the invention, the nitrogen source is one or a mixture of more of corn steep liquor, yeast powder, soybean meal and peanut cake powder in any proportion.

As a further improvement of the invention, the pH buffering agent is one or more of basic magnesium carbonate, sodium carbonate, magnesium hydroxide and sodium hydroxide which are mixed in any proportion, and the total concentration is 20-80 g/L.

The invention has the beneficial effects that:

the invention obviously reduces the generation of the byproduct acetic acid by adding a certain amount of sodium bisulfite, overcomes the defect that the generation of the byproduct in the succinic acid fermentation is reduced by breeding excellent strains or regulating and controlling fermentation conditions in the prior art, but the reduction range is limited, has important significance for improving the utilization rate of raw materials and reducing the later-stage succinic acid separation and purification cost, and is beneficial to promoting the development of the biomass succinic acid industry.

Drawings

FIG. 1 is a graph showing the yields of succinic acid and its by-product, acetic acid, obtained by fermentation with different carbon sources and addition of sodium bisulfite in example 9 of the present invention, wherein CK is a control group and NaHSO₃For adding NaHSO₃And (4) grouping.

FIG. 2 is a schematic diagram of the metabolic pathway of Actinobacillus succinogenes of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples.

The following examples are presented to analyze the succinic acid and acetic acid production as follows:

sample treatment: centrifuging the fermentation liquid at 12000r/min at room temperature for 10min, collecting supernatant, filtering with sterile filter membrane with pore diameter of 0.22 μm, and detecting the concentration of succinic acid and residual reducing sugar in the fermentation liquid by High Performance Liquid Chromatography (HPLC).

Organic acid determination, HPLC method, Daian Utimat3000, autosampler, chromatographic column, Rezex ROA-organic acid 300 × 7.8.8 mm, mobile phase 2.5 mmol/L H₂SO₄pH2.5, column temperature 45 deg.C, sample introduction amount 10uL, flow rate 0.6mL/min, and ultraviolet detector wavelength 210 nm.

Biomass determination: the measurement was carried out at 660nm using a Spectrophotometer (DU 800 UV/VIS Spectrophotometer, Beckman, USA), the sample being treated with 0.2M HCl to dissolve the MgCO present₃Centrifuging at 12000r/min for 10min, and washing with distilled water for three times to remove pigment and impurities.