A method for producing succinic acid by calcium hydroxide as neutralizer combined with CO2 pulse feedback feeding fermentation

Abstract

The application provides a method for producing succinic acid by using calcium hydroxide as a neutralizing agent and combining with CO2 pulse feedback feeding fermentation, and the method comprises the following steps: after activating a fermentation strain WS100, sequentially performing primary seed culture and secondary seed culture to obtain a secondary seed liquid; and performing fermenter culture on the secondary seed liquid to produce succinic acid, wherein the following operation is performed in the fermenter culture stage: in the initial stage of the fermenter culture, monitoring the pH value of the fermentation liquid, adding a calcium hydroxide solution with a mass fraction of 22-24% as a neutralizing agent until the pH value rises to 7.5-9 to stop adding when the pH value of the fermentation liquid naturally decreases to 5-6.5; then, carbon dioxide gas is introduced until the pH value decreases to 6.5-7 to stop to make the fermentation liquid naturally ferment; and when the continuous accumulation of succinic acid in the fermentation liquid makes the pH value naturally decrease to 5-6.5, the above operation is repeated.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for producing succinic acid by using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback fermentation. Background Technology

[0002] Succinic acid, as a precursor for the synthesis of chemical intermediates, is widely used in food, chemical, pharmaceutical, and biodegradable biomaterials industries. In recent years, through continuous improvement of microbial strains for the bio-fermentation production of succinic acid (Chinese patents CN 104178443A; CN 102827800A), the process for bio-fermentation production of succinic acid is gradually maturing and has been applied industrially. However, some factors, such as cost considerations and the selection of neutralizing agents and product separation and purification issues, still hinder the application of bio-synthetic succinic acid.

[0003] Several neutralizing agents commonly used in organic acid fermentation are also widely used in succinic acid fermentation production. In comparison, magnesium carbonate-based neutralizing agents are generally considered to be the most effective in increasing the fermentation yield and production intensity of succinic acid (Metabolic engineering modification of Escherichia coli for succinic acid synthesis and fermenter scale-up process, Bulletin of Microbiology, 2018, 45(12):2541-2551; Optimization and scale-up of fermentation conditions for succinic acid production by Escherichia coli FMME~N~26, Journal of Process Engineering, 2022, 22(7):854~862). Magnesium carbonate-based neutralizing agents, acting as alkaline reagents, not only neutralize succinic acid produced during fermentation, preventing its toxicity to fermenting organisms, but also promote succinic acid accumulation because magnesium ions are a cofactor for phosphoenolpyruvate carboxylase. The CO2 generated during neutralization is further immobilized by the fermentation intermediates pyruvate / phosphoenolpyruvate, promoting its flow to the succinic acid synthesis pathway and increasing succinic acid yield. However, the disadvantages of magnesium ion neutralizing agents are also obvious. Currently, magnesium carbonate is expensive, making it difficult to compete with sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium carbonate, calcium bicarbonate, and calcium hydroxide. Furthermore, free magnesium ions pose challenges to subsequent product separation and purification processes. As divalent cations, they place a heavy burden on subsequent ion exchange or electrodialysis techniques, resulting in high separation costs.

[0004] Calcium hydroxide, as a traditional neutralizing agent for organic acid fermentation, is characterized by low cost and mature supporting processes. The resulting calcium succinate readily precipitates as calcium sulfate under sulfuric acid conditions, removing calcium ions from the fermentation broth. This simplifies the subsequent product separation and purification process, significantly reducing the production cost of succinic acid. However, using calcium hydroxide as a neutralizing agent in succinic acid fermentation often results in lower fermentation yield and sugar-acid conversion rates compared to other neutralizing agents. While using calcium carbonate or calcium bicarbonate as a neutralizing agent can supplement the CO2 precursor required for succinic acid fermentation, it presents the problem of weak alkalinity, making it difficult to quickly adjust the pH of the fermentation broth.

[0005] Therefore, there is a need to provide a method for producing succinic acid through fermentation that can both improve the sugar-acid conversion rate of succinic acid fermentation and significantly reduce the fermentation and subsequent separation and purification costs. Summary of the Invention

[0006] The purpose of this invention is to provide a method for producing succinic acid using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback feeding fermentation. Using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback feeding fermentation can improve the sugar-acid conversion rate of succinic acid fermentation while significantly reducing the fermentation and subsequent separation and purification costs.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] This invention provides a method for producing succinic acid using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback fermentation, the method comprising:

[0009] After activating the fermentation strain WS100, it was subjected to primary seed culture and secondary seed culture in sequence to obtain secondary seed liquid;

[0010] The secondary seed culture is fermented in a fermenter to produce succinic acid, wherein the fermenter cultivation stage involves the following operations:

[0011] In the early stage of fermentation in the fermenter, the pH value of the fermentation broth is monitored. When the pH value of the fermentation broth naturally drops to 5-6.5, a 22-24% calcium hydroxide solution is added as a neutralizing agent until the pH value rises to 7.5-9 and then the addition is stopped.

[0012] Then, carbon dioxide gas is introduced until the pH value drops to 6.5-7, at which point the process is stopped to allow the fermentation broth to ferment naturally.

[0013] Repeat the above steps when the pH value naturally drops to 5-6.5 due to the continuous accumulation of succinic acid in the fermentation broth.

[0014] Furthermore, the flow rate of the carbon dioxide gas is 0.1 to 0.5 vvm.

[0015] Furthermore, the basic formula of the culture medium for fermentation in the fermenter is as follows (by mass fraction): 7.5–8.5% glucose and 0.2–0.4% corn steep liquor.

[0016] Further, the secondary seed culture is inoculated into the culture medium in the fermenter at an inoculation rate of 10% to 15%.

[0017] Furthermore, the culture medium in the fermenter contains an initial glucose content of 75-85 g / L, and when the glucose content is lower than 10 g / L during fermenter culture, 15-25 g / L of glucose is added.

[0018] Furthermore, in the fermenter culture, the fermentation temperature is 35-39℃ and the stirring speed is 200-300 r / min.

[0019] Further, the activation includes: culturing the fermentation strain WS100 on LB plates 2 to 3 times, wherein the culture conditions are: culture temperature 35 to 39°C, culture time 12 to 24 hours.

[0020] Furthermore, the conditions for primary seed culture are: 35–39℃, 200–300 r / min shake flask culture for 10–12 h.

[0021] Furthermore, the conditions for the secondary seed culture are: 35-39℃, 200-300r / min shake flask culture for 20-24h; the culture medium obtained from the primary seed culture is inoculated at an inoculation rate of 5%-10%.

[0022] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0023] 1. This invention solves the problem of low succinic acid fermentation yield when calcium hydroxide is used as a neutralizing agent by intermittently pulsedly adding calcium hydroxide and CO2. For example, when adding calcium hydroxide solution as a neutralizing agent at a constant rate, the lack of CO2 supply results in a slow fermentation rate due to the absence of raw materials for the synthesis of oxaloacetic acid from pyruvate / phosphoenolpyruvate. Simultaneously and continuously adding CO2 leads to acidification of the fermentation broth due to carbonic acid production. Furthermore, the added calcium hydroxide and carbonic acid simultaneously generate calcium carbonate precipitate in the fermenter, significantly increasing the amount of calcium hydroxide used. The formation of calcium carbonate further reduces the efficiency of CO2 conversion to succinic acid. Intermittently pulsedly adding calcium hydroxide and CO2 improves the utilization efficiency of both. Results show that compared to the control group with continuous CO2 supply, the fermentation rate and sugar-acid conversion rate of succinic acid are significantly improved.

[0024] 2. This invention uses calcium hydroxide as a neutralizing agent to ferment succinic acid. Carbon dioxide is intermittently introduced into the fermentation broth in gaseous form to supplement the carbon skeleton required for succinic acid synthesis while controlling the pH value of the fermentation broth. Compared with directly using calcium carbonate as a neutralizing agent and carbon skeleton provider, the sugar-acid conversion rate of succinic acid fermentation is increased by 1.03 times.

[0025] 3. This invention uses alternating calcium hydroxide solution and carbon dioxide to control the pH value of the fermentation broth, which can stabilize the pH of the fermentation broth within the range of 5.0 to 9.0. Compared with the control that continuously introduces carbon dioxide, the succinic acid-glycolic acid conversion rate is increased by 1.82 times. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The fermentation process curve is shown in Example 5.

[0028] Figure 2 The fermentation process curve is shown in Comparative Example 2. Detailed Implementation

[0029] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0030] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention are commercially available or obtainable by existing methods.

[0031] The process of discovering the technical problem of this invention is as follows:

[0032] The inventors of this application, through extensive experiments using calcium hydroxide as a neutralizing agent in succinic acid fermentation, have demonstrated that using calcium hydroxide alone as a neutralizing agent offers a significant advantage in terms of subsequent separation and purification costs, but the fermentation yield is low. Adding calcium carbonate only releases CO2 when the fermentation broth pH is low, but a low pH is detrimental to the growth of fermenting microorganisms. Adding other carbonates leads to high separation and purification costs. Using CO2 to maintain pressure in the fermenter or continuously introducing CO2 gas results in the production of large amounts of insoluble calcium carbonate in the fermenter, hindering subsequent fermentation.

[0033] To overcome the above shortcomings, we propose a novel method for pulsed CO2 supplementation to improve the sugar-acid conversion rate of succinic acid fermentation when using only calcium hydroxide as a neutralizing agent. Compared to the method of continuous CO2 introduction in some patents (Chinese Patent CN 102605009 A), this method supplies CO2 in a pulsed manner based on the pH of the fermentation broth. While maintaining the pH of the fermentation broth within a stable range, this effectively avoids the problem of continuous acidification of the fermentation broth caused by continuous CO2 introduction, which necessitates the continuous addition of calcium hydroxide. Simultaneously, it increases the sugar-acid conversion rate and fermentation rate of succinic acid fermentation.

[0034] The following will provide a detailed description of a method for producing succinic acid using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback fermentation, based on embodiments and experimental data.

[0035] Example 1

[0036] (1) Activation of the strain:

[0037] The succinic acid-producing *Escherichia coli* WS100 was used as the fermentation strain. The construction method of *E. coli* WS100 was as follows: Wild-type *E. coli* W was used as the starting strain (the starting strain was purchased, preservation number ATCC 9637). The lactate dehydrogenase gene (ldh A), alcohol dehydrogenase gene (adh E), pyruvate-formate lyase gene (pflB), pyruvate oxidase gene (poxB), and acetate kinase gene (ackA) were knocked out using the Red homologous recombination system. *E. coli* WS100 was then obtained through anaerobic growth screening. The detailed construction method of *E. coli* WS100 is described in the following literature: Construction and Anaerobic Fermentation of High-Succinic Acid-Producing Recombinant *Escherichia coli*. *Food and Fermentation Industries*. 2013, 31(1):6-10.

[0038] Activate strain WS100 by subculturing it 2-3 times on LB plates at a temperature of 35-39℃ for 12-24 hours.

[0039] (2) Primary seed culture: Pick a single colony from the plate and inoculate it into 50 ml LB medium containing 20 g / L glucose. Incubate at 35-39℃ and 200-300 r / min for 10-12 h.

[0040] (3) Secondary seed culture: Inoculate the primary seed culture at a rate of 5%–10% into the secondary seed culture medium and culture in a shake flask at 35–39℃ and 200–250 r / min for 20–24 h. At this time, the OD of the seed culture is measured. 600 The value should be above 15. The secondary seed culture medium formula is as follows: 40 g / L glucose, 10 g / L yeast extract, 20 g / L tryptone, 1.14 g / L KH2PO4, 0.9 g / L K2HPO4·12H2O, 3.0 g / L (NH4)2SO4, and 0.5 g / L MgSO4·12H2O.

[0041] (4) 10-liter fermenter culture: The basic formula of the fermentation culture medium is: 8% glucose, 0.3% corn steep liquor. The secondary seed culture is inoculated into the fermentation culture medium at a rate of 15%. The initial glucose content of the fermentation medium is 80 g / L. When the glucose content is lower than 10 g / L, 20 g / L glucose is added. The fermentation temperature is 38℃, the stirring speed is 300 r / min, and 23% calcium hydroxide solution is used as a neutralizing agent. In the early stage of fermentation, no neutralizing agent is added, allowing the pH value of the fermentation broth to drop naturally to about 5.0. Then, the peristaltic pump is turned on to manually add calcium hydroxide to raise the pH value of the fermentation broth to about 9.0. Then, the addition of neutralizing agent is stopped immediately, and CO2 gas is introduced (flow rate of 0.2 vvm). When the pH value drops to 7.0, the CO2 introduction is stopped, and the fermentation broth is allowed to ferment naturally. When the succinic acid in the fermentation broth accumulates and the pH value drops naturally to about 5.0, the above operation is repeated.

[0042] (5) Detection and techniques for relevant parameters during fermentation:

[0043] ① Biomass measurement: Take 1 mL of fermentation broth and neutralize the calcium hydroxide in the fermentation broth with 1 mL of 6 mol / L hydrochloric acid. Then dilute the fermentation broth with pure water 2 to 30 times. Use a spectrophotometer to measure the OD value of the diluted fermentation broth at a wavelength of 600 nm.

[0044] ② Glucose measurement: Take 1 mL of fermentation broth, centrifuge at 8000 r / min for 1 min, take the supernatant, dilute it appropriately, and measure it using a biosensor SBA 40D.

[0045] ③ Measurement of succinic acid: The fermentation broth was treated with 6% sulfuric acid solution at a volume ratio of 1:1, allowed to stand for several minutes, centrifuged at 10000 r / min for 10 min, and diluted according to the growth status before measuring the succinic acid content using high-performance liquid chromatography (HPLC). The HPLC measurement conditions were as follows: column: Bio-Rad HPX 87H; mobile phase: 5 mmol / L H2SO4 solution (filtered through a 0.22 μm membrane to remove impurities and degassed by sonication for 30 min before use); flow rate: 0.5 mL / min; column temperature: 45℃; detector: PDA; detection wavelength: 210 nm; injection volume: 10 μL.

[0046] The sugar-acid conversion rate (%) is calculated according to formula (1):

[0047]

[0048] Where ρ is the succinic acid concentration in g / L; V is the total volume of the fermentation broth in L; and m is the total sugar consumption in g. After fermentation, the succinic acid concentration was measured to be 52.76 g / L, with a conversion rate of 69.55%.

[0049] Examples 2 to 11

[0050] Examples 2 through 11 employed similar fermentation conditions and experimental methods to Example 1. The difference lay in the following: when the pH of the fermentation broth naturally decreased to a specific slightly acidic pH (e.g., pH 5, 5.5, 6.0, 6.5), calcium hydroxide was added to raise the pH to an alkaline pH (e.g., pH 7.5, 8.0, 8.5, 9.0). The addition of neutralizing agent was then immediately stopped, and CO2 gas was simultaneously introduced (flow rates of 0.1, 0.2, 0.3, 0.4, 0.5 vvm). Once the pH decreased to a slightly neutral pH (e.g., pH 6, 5, 7), CO2 introduction was stopped, and the fermentation broth was allowed to ferment naturally. When the succinic acid in the fermentation broth accumulated and the pH naturally decreased to a specific slightly acidic pH (e.g., pH 5.0, 5.5, 6.0, 6.5), calcium hydroxide was added again, and the above operation was repeated. Specific parameters are shown in Table 1.

[0051] Comparative Example 1

[0052] The same strain, fermentation conditions, and experimental methods as in Example 1 were used. The difference was that CO2 gas was not introduced during the entire fermentation process, and 8% calcium carbonate powder was used as a neutralizing agent. The results are shown in Table 2.

[0053] Comparative Example 2

[0054] The same strain, fermentation conditions, and experimental methods as in Example 1 were used. The difference was that CO2 gas was not introduced during the entire fermentation process. The fermentation results are shown in Table 2, and the fermentation progress curve is shown in [Figure 1]. Figure 2 .

[0055] Comparative Example 3

[0056] The same strain, fermentation conditions, and experimental methods as in Example 1 were used. The difference was that CO2 gas was introduced at a constant rate (flow rate of 0.5 vvm) throughout the entire fermentation process. The fermentation results are shown in Table 2, and the fermentation progress curves are shown in [Figure 1]. Figure 2 .

[0057] Experiment Example 1, Result Determination

[0058] The yield of succinic acid and the sugar-acid conversion rate in each embodiment and comparative example are shown in Table 1 and Table 2.

[0059] Table 1. Experimental results of Example 1, Implementation 11

[0060]

[0061] As can be seen from Table 1, the pH control method in Example 5 was optimal, achieving a succinic acid yield of 85.65 g / L and a sugar-acid conversion rate of 96.75%. The fermentation process curve is shown in Table 1. Figure 1 Alternating the use of calcium hydroxide solution and carbon dioxide to control the pH value of the fermentation broth can stabilize the pH value of the fermentation broth within the range of 5.5-7.5.

[0062] Table 2 Comparison of fermentation results between Example 5 and Comparative Example 3

[0063]

[0064] In Comparative Example 1, the succinic acid content in the fermentation broth was 37.13 g / L, and the sugar-acid conversion rate was 47.62%, which was lower than the corresponding data in all embodiments of the present invention. Example 5 showed the best fermentation results, with the sugar-acid conversion rate increasing by 1.03 times compared to Comparative Example 1.

[0065] In Comparative Example 2, the succinic acid content in the fermentation broth was 15.11 g / L, and the sugar-acid conversion rate was 24.84%, which was lower than the corresponding data in all examples. Among them, Example 5 showed a sugar-acid conversion rate that was 2.89 times higher than that in Comparative Example 2.

[0066] Compared with Comparative Example 3, the sugar-acid conversion rate of succinic acid fermentation in Example 5 was 1.82 times higher than that of the control (Comparative Example 3) with continuous CO2 supplementation.

[0067] In summary, the present invention overcomes the low efficiency of existing strains using calcium hydroxide as a neutralizing agent to produce succinic acid through fermentation. It provides a method for producing succinic acid by combining calcium hydroxide as a neutralizing agent with CO2 pulse feedback feeding, offering a new approach to simplifying the process and synthesizing succinic acid at low cost.

[0068] This invention provides a general pH control method for fumaric acid and malic acid fermentation. Many organic acid fermentations involve CO2 fixation, but CO2 replenishment often conflicts with the addition of alkalis such as calcium hydroxide. Based on pH measurements of the fermentation broth, this invention provides a useful approach to effectively control the pH of the fermentation broth and improve CO2 utilization by periodically and cyclically adding alkali and CO2, thereby increasing the sugar-acid conversion rate in the fermentation of related organic acids.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0070] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0071] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0072] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for producing succinic acid using calcium hydroxide as a neutralizing agent combined with CO2 pulse feedback fermentation, characterized in that, The method includes: After activating the fermentation strain WS100, it was subjected to primary seed culture and secondary seed culture in sequence to obtain secondary seed liquid; The secondary seed culture is fermented in a fermenter to produce succinic acid, wherein the fermenter cultivation stage involves the following operations: In the early stage of fermentation in the fermenter, the pH value of the fermentation broth is monitored. When the pH value of the fermentation broth naturally drops to 5-6.5, a 22%-24% calcium hydroxide solution is added as a neutralizing agent until the pH value rises to 7.5-9 and then the addition is stopped. Then, carbon dioxide gas is introduced until the pH value drops to 6.5-7, at which point the process is stopped to allow the fermentation broth to ferment naturally. When the pH value naturally drops to 5-6.5 due to the continuous accumulation of succinic acid in the fermentation broth, repeat the above operation. The method for constructing the fermentation strain WS100 includes: Using wild-type Escherichia coli W as the starting strain, the lactate dehydrogenase gene ldh A, alcohol dehydrogenase gene adh E, pyruvate formate lyase gene pflB, pyruvate oxidase gene poxB, and acetate kinase gene ackA were knocked out respectively using the Red homologous recombination system, and then Escherichia coli WS100 was obtained by screening through anaerobic growth. The flow rate of the carbon dioxide gas is 0.1–0.5 vvm; The culture medium in the fermenter contains an initial glucose content of 75-85 g / L. During the fermenter culture, when the glucose content is lower than 10 g / L, 15-25 g / L of glucose is added. In the fermenter culture, the fermentation temperature is 35-39℃ and the stirring speed is 200-300 r / min.

2. The method according to claim 1, characterized in that, The basic formula of the culture medium for fermentation in the fermenter is as follows (by mass fraction): 7.5%–8.5% glucose and 0.2%–0.4% corn steep liquor.

3. The method according to claim 1, characterized in that, The secondary seed culture was inoculated into the culture medium in the fermenter at an inoculation rate of 10% to 15%.

4. The method according to claim 1, characterized in that, The activation includes: culturing the fermentation strain WS100 on LB plates 2 to 3 times, with the culture conditions being: culture temperature 35 to 39°C and culture time 12 to 24 h.

5. The method according to claim 1, characterized in that, The conditions for primary seed culture are: 35–39℃, 200–300 r / min shake flask culture for 10–12 h.

6. The method according to claim 1, characterized in that, The conditions for the secondary seed culture are: 35-39℃, 200-300 r / min shake flask culture for 20-24 h; the culture medium obtained from the primary seed culture is inoculated at an inoculation rate of 5%-10%.

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