Method for anaerobic, efficient and continuous production of succinic acid from immobilized escherichia coli

Abstract

The invention discloses a method for anaerobically, efficiently and continuously producing succinic acid by immobilized escherichia coli. Calcium alginate is adopted as an immobilized carrier to embed activated escherichia coli, and immobilized hydrogel beads of 4-5 mm are prepared. The immobilized escherichia coli is inoculated into a fermentation culture medium containing lecithin and can be directly used for anaerobic production of succinic acid. Compared with the traditional free cell process, the fermentation time required by the immobilized cells for achieving the same succinic acid yield as the free cells is shortened by 48 hours. After 7 batches of continuous fermentation, the immobilized carrier is stable in structure, and the yield of succinic acid is increased by 18.70% and reaches 51.61 g / L. The immobilized cell strategy does not need repeated inoculation, the seed culture time is saved, the fermentation period is shortened, moreover, a functional additive lecithin is added into a fermentation culture medium, so that diffusion of products in an embedding environment can be promoted, inhibition of the products on cells is relieved, and the yield is increased. And reference significance is provided for producing chemicals through fermentation of immobilized anaerobic microorganisms in the future.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological fermentation, and particularly relates to a method for continuously producing succinic acid by immobilized Escherichia coli under anaerobic conditions. BACKGROUND

[0002] Succinic acid, also known as succinic acid, is an important C4 platform compound, which has wide application prospects in food, medicine, chemical industry, agriculture and other fields. In the food industry, succinic acid can be used as acidity regulator, flavor enhancer, etc.; in the pharmaceutical field, it is an important intermediate for the synthesis of some drugs; in the chemical industry, succinic acid can be used to produce biodegradable plastics, solvents, coatings, etc.; in the agricultural aspect, its derivatives can be used as plant growth regulators. With the global emphasis on sustainable development and green chemistry, the market demand for succinic acid is increasing, and the technology for efficient production of succinic acid has become a research hotspot.

[0003] Currently, there are still some problems in the production of succinic acid by fermentation using free Escherichia coli. Under anaerobic fermentation conditions, free cells are easily affected by environmental factors such as substrate concentration, product accumulation, pH change, etc., which leads to inhibition of cell growth and metabolism, thereby affecting the yield and production efficiency of succinic acid. In addition, free cells are easily lost during fermentation, making it difficult to achieve continuous fermentation, and frequent seed culture and inoculation operations are required, increasing the production cost and operational complexity. Moreover, after fermentation, it is difficult to separate free cells from the fermentation broth, increasing the difficulty and cost of product separation and purification. In order to solve the above problems, immobilized cell technology emerges as the times require. Immobilized cells are cells fixed on a specific carrier, which can maintain activity and be reused. The existing technology mainly uses sodium alginate, carrageenan, polyacrylamide and other carriers to embed bacteria, and realizes cell immobilization through physical restriction or chemical cross-linking. Compared with free cells, immobilized cells have many advantages. Immobilized cells can increase the density of cells, increase the contact area between cells and substrates, thereby improving the reaction rate and substrate conversion rate. Immobilized carriers can provide a relatively stable microenvironment for cells, reduce the influence of external environmental factors on cells, and enhance the tolerance and stability of cells. Immobilized cells are easy to separate from the fermentation broth, which is convenient for continuous fermentation, improves production efficiency and reduces production cost.

[0004] However, the three-dimensional network structure formed by the immobilized carrier (such as calcium alginate hydrogel beads) may hinder the diffusion of the substrate (such as glucose) to the internal cells, and the efflux efficiency of the product (such as succinic acid) is reduced, which leads to a local concentration that is too high, causing product inhibition. SUMMARY

[0005] The application provides a method for continuously producing succinic acid by anaerobic high-efficiency immobilized Escherichia coli, and functional additives are added to the immobilized carrier to make the substrate more easily diffuse to the surface of the immobilized cells, improve the substrate utilization efficiency, and make the product more easily diffuse out of the hydrogel beads, relieve the inhibition of the product on the cells, improve the fermentation efficiency, and further, the functional additives can interact with the immobilized carrier to enhance the mechanical strength and stability of the carrier.

[0006] The technical scheme adopted by the application is as follows:

[0007] The method for continuously producing succinic acid by anaerobic high-efficiency immobilized Escherichia coli uses calcium alginate as the immobilized carrier, and uses Escherichia coli Suc260 as the seed for anaerobic fermentation to produce succinic acid.

[0008] The diameter of the immobilized Escherichia coli is 4-5 mm, and then the immobilized Escherichia coli is inoculated into a fermentation medium containing 0-0.4% lecithin to produce succinic acid by anaerobic fermentation.

[0009] The immobilized Escherichia coli is inoculated into the fermentation medium, and then is subjected to anaerobic fermentation at 30-38°C for 72-96 h.

[0010] Preferably, the immobilized Escherichia coli is inoculated into the fermentation medium, and then is subjected to anaerobic fermentation at 37°C and 180 rpm for 96 h, and after the fermentation is completed, the immobilized Escherichia coli is transferred into fresh fermentation medium, and is cultured for 96 h in each batch, and the immobilized cells can be reused for more than 7 times.

[0011] Preparation and preparation condition optimization of the immobilized cells

[0012] When the solution containing calcium ions is added to the sodium alginate solution, the molecular chains of the sodium alginate are crosslinked to form a three-dimensional network structure from the original dispersed linear state, and the hydrogel with certain strength and elasticity is formed; the mixture of the E. coli bacterial suspension and the sodium alginate solution is dropped into the calcium chloride solution drop by drop through a syringe at a speed of 70 drops per minute, and the immobilized cell hydrogel beads with a diameter of 4-5 mm are formed; the hydrogel beads are allowed to stand at room temperature for 30 min to stabilize the molding, and then are washed twice with a phosphate buffer with a pH of 7.0, and the immobilized E. coli is obtained.

[0013] The immobilized cell hydrogel beads are formed by using a syringe containing 2% sodium alginate and bacterial suspension mixture to drop the mixture into the CaCl2 solution at a distance of 10 cm from the water surface of the CaCl2 solution at a speed of 70 drops per minute.

[0014] The volume ratio of the E. coli bacterial suspension to the sodium alginate solution can be 1:3-9, and is preferably 1:6.

[0015] OD of the E. coli cell suspension 600 4-5.

[0016] The concentration of the sodium alginate solution is 1-5%, preferably 2-3%.

[0017] Selection of functional additives

[0018] The immobilized E. coli was respectively inoculated into fermentation medium containing different functional additives, and anaerobic bottle fermentation was carried out under anaerobic conditions at 37°C and 180 rpm for 96 h. The production of succinic acid was detected every 24 h.

[0019] The functional additive is at least one of betaine, Tween-80, sodium dodecyl sulfate, lecithin or Triton X-100, preferably lecithin.

[0020] Determination of the concentration of the functional additive

[0021] The immobilized E. coli was respectively inoculated into fermentation medium containing different concentrations of lecithin, and anaerobic bottle fermentation was carried out under anaerobic conditions at 37°C and 180 rpm for 96 h. The production of succinic acid was detected every 24 h.

[0022] The functional additive is 0-0.4% lecithin, preferably the concentration of lecithin is 0.2-0.3%, more preferably the concentration of lecithin is 0.3%.

[0023] Fermentation of immobilized cells

[0024] The immobilized E. coli was washed twice with a phosphate buffer with a pH of 7.0 and then transferred into the fermentation medium. The volume ratio of immobilized E. coli to fermentation medium was 1:5. Every 96 h, the immobilized E. coli was taken out using a sterilized strainer, then washed twice with a phosphate buffer with a pH of 7.0 and transferred into fresh medium. This process was repeated for 7 batches.

[0025] The components of the fermentation medium are: 2.6 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, 60 g / L glucose, 48 g / L basic magnesium carbonate as a pH stabilizer, and water as the solvent.

[0026] The application adds different functional additives in the immobilized E. coli fermentation process, and selects a functional additive-lecithin that improves its production performance, establishes a "mass transfer enhancement-carrier protection-metabolic optimization" three-in-one mechanism, and significantly improves the production efficiency and system stability of succinic acid. Its application is not only suitable for anaerobic fermentation system of E. coli, but also provides a general strategy for the immobilized production of other high-product inhibitory metabolites.

[0027] The application has the beneficial effects that: the application uses calcium alginate as an immobilized carrier, and prepares active and stable immobilized E. coli that can be reused by optimizing the ratio of bacterial suspension and sodium alginate. The addition of functional additive lecithin in the fermentation medium can reduce the surface tension of the substrate and the fermentation broth, so that the substrate is more easily diffused to the surface of the immobilized cells, and at the same time, the diffusion of the product in the embedding environment is promoted, and the inhibition of the product on the cells is relieved. Compared with the traditional free cell process, the fermentation time required for the immobilized cells to reach the same succinic acid yield as the free cells is shortened by 48 h. After 7 batches of continuous fermentation, the immobilized carrier structure is stable and the succinic acid yield is increased by 18.70%, which provides a reference for the future use of immobilized anaerobic microorganism fermentation to produce chemicals. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Loss rate of immobilized E. coli after 96 h fermentation under different volume ratios of bacterial solution and sodium alginate solution.

[0029] Figure 2 Glucose concentration change of immobilized E. coli under the condition of adding different functional additives.

[0030] Figure 3 Succinic acid yield of immobilized E. coli under the condition of adding different functional additives.

[0031] Figure 4 Glucose concentration change of immobilized E. coli when adding different concentrations of lecithin.

[0032] Figure 5 Succinic acid yield of immobilized E. coli when adding different concentrations of lecithin.

[0033] Figure 6 Succinic acid yield and yield of immobilized E. coli in different fermentation batches.

[0034] Figure 7 Trend of succinic acid yield of immobilized E. coli in different fermentation batches.

[0035] Figure 8 Comparison of the fermentation results of the 7th batch of immobilized E. coli and free cells. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to specific embodiments.

[0037] Example 1 Preparation of immobilized Escherichia coli and optimization of the volume ratio of bacterial solution to sodium alginate solution

[0038] Pick a single colony of E. coli Suc260 into an LB tube and culture it in a shaking incubator at 37°C for 8-12 hours. Then, inoculate it into a 50 mL LB shake flask with a 2% (v / v) inoculation volume and culture it at 37°C and 180 rpm for 8-12 hours. 600 To 4-5, centrifuge 50mL of bacterial solution at 8000rpm for 5min, discard the supernatant, and then add pH 7.0 phosphate buffer to resuspend the bacterial slurry, repeat twice. Mix the bacterial solution with 2% sodium alginate solution in a volume ratio of 1:3, 1:4, 1:6, and 1:9, and slowly drip the mixture into a plate containing 50mL of 2% (m / v) CaCl2 solution using a 10mL syringe to form immobilized cell hydrogel beads with a diameter of 4-5mm. Let it stand at room temperature for 30min to allow the hydrogel beads to stabilize and then wash twice with pH 7.0 phosphate buffer to obtain immobilized Escherichia coli.

[0039] The immobilized E. coli obtained at different volume ratios were inoculated into the fermentation medium respectively. After 96 hours of anaerobic fermentation, the damage of the carrier was observed and the loss rate was calculated. Figure 1 As shown, a volume ratio of 1:6 resulted in the lowest loss rate of immobilized E. coli. When the volume ratio was higher than 1:6, the number of sodium alginate molecules was relatively small, and the cross-linked structure formed by the reaction with calcium chloride was not dense enough, which damaged the integrity of the hydrogel beads and increased the loss rate. When the volume ratio was lower than 1:6, the diffusion of calcium ions was hindered during the reaction of the mixture with calcium chloride, resulting in uneven cross-linking, cracking, and even breakage of the hydrogel beads, which increased the loss rate.

[0040] The fermentation medium contains the following components: 2.6 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, 60 g / L glucose, 48 g / L basic magnesium carbonate as a pH stabilizer, and water as the solvent.

[0041] Example 2 Selection and concentration optimization of functional additives

[0042] The method is the same as that in Example 1, except that 6 groups of experiments are set up, and 0.2% of betaine, Tween-80, sodium dodecyl sulfate, lecithin and Triton X-100 are added respectively to the fermentation medium to improve the mass transfer effect, and the group without adding any functional additive is taken as the control group.

[0043] During the cultivation process, samples are taken every 24 h and the glucose concentration and succinic acid yield are determined. When the immobilized E. coli is cultured for 24 h, the addition of betaine, Tween-80, lecithin and Triton X-100 all shows good glucose consumption capacity, especially the experimental group with the addition of lecithin, the glucose consumption rate is 1.17 g / L / h, compared with 0.93 g / L / h of the control group, the glucose consumption rate is increased by 25.81%( Figure 2 ). This shows that lecithin can effectively promote the substrate into the immobilized carrier and improve the substrate consumption rate of the cells. In addition, the experimental group with the addition of lecithin improves the succinic acid production rate, and within 0-96 h, the succinic acid production rate is 0.45 g / L / h, which is increased by 8.89% compared with the control group; especially within the first 24 h, the succinic acid production rate of the experimental group with the addition of lecithin is 0.93 g / L / h, which is increased by 32.86%( Figure 3 ) compared with the control group.

[0044] In order to further improve the substrate consumption rate and product generation rate of the immobilized E. coli, 5 groups of experiments are set up to optimize the addition amount of lecithin, and the addition amount of lecithin is 0, 0.1%, 0.2%, 0.3% and 0.4% respectively.

[0045] When the addition amount of lecithin is 0.3%, the glucose consumption rate and succinic acid production rate of the immobilized E. coli are the highest, which are 0.55 g / L / h and 0.48 g / L / h respectively; compared with the control group, they are increased by 17.02% and 17.07%( Figure 4 and Figure 5 ) respectively. In addition, when no lecithin is added, the succinic acid yield of the immobilized E. coli is 39.72 g / L, and when 0.3% lecithin is added, the succinic acid yield of the immobilized E. coli is 45.88 g / L. This shows that after the addition of lecithin, it is beneficial to the diffusion of the product to the outside of the immobilized carrier, the damage to the cells is reduced, and the production capacity of succinic acid is improved. Within the optimization range, the addition of lecithin can effectively improve the mass transfer without damaging the gel structure or inhibiting the cell activity, while when too much lecithin is added, it will dissolve the sodium alginate gel network, leading to the disintegration of the carrier, or interfere with the integrity of the cell membrane, reducing the activity of the bacterial cells.

[0046] The components in the fermentation medium are: 2.6 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / L H3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / L NaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, 60 g / L glucose, 48 g / L basic magnesium carbonate as pH stabilizer, and water as solvent.

[0047] Example 3 Comparison of fermentation performance of immobilized cells and free cells

[0048] The optimal fermentation conditions obtained in Example 2 were used to verify the repeated use of immobilized cells. The immobilized E. coli was inoculated into the fermentation medium, and every 96 h, the immobilized E. coli was taken out using a sterilized strainer, then washed twice with a phosphate buffer at pH 7.0 and transferred into fresh medium. The immobilized E. coli was repeatedly used for 7 batches. After 7 batches of repeated use, the immobilized E. coli still maintained a good mechanical structure, and the succinic acid yield was stable and showed an upward trend. Figure 6 Because the immobilized cells do not need to undergo the process of cell proliferation again, more carbon sources are used for product synthesis, and the yield of succinic acid is higher. In addition, compared with the traditional free cell process, the immobilized cells shortened the fermentation time by 48 h to reach the same succinic acid yield as the free cells. Figure 7 The immobilized E. coli reached the highest succinic acid yield of 51.61 g / L in the seventh batch. Compared with the free cell fermentation results, the succinic acid yield was increased by 18.70%. Figure 8

[0049] Anaerobic fermentation is usually slow because the energy produced by anaerobic respiration is less, and the growth and metabolism of microorganisms are relatively slow. Compared with the existing technology of anaerobic fermentation using free cells, the immobilized cell strategy does not need to be inoculated repeatedly, saves the seed culture time, and shortens the fermentation cycle. However, local product accumulation in the immobilized carrier can easily lead to carrier swelling or disintegration. By adding functional additives, the diffusion of products in the embedded environment can be promoted, and the inhibition of products on cells can be alleviated, providing a reference for future production of chemicals using immobilized anaerobic microorganisms.​

Claims

1. A method for anaerobic, efficient and continuous production of succinic acid by immobilized Escherichia coli, characterized in that: Calcium alginate was used as an immobilized carrier, and Escherichia coli Suc260 was used as a seed for anaerobic fermentation production of succinic acid. E. coli cells were cultured to prepare a bacterial suspension, which was then evenly mixed with the immobilized carrier to prepare immobilized E. coli. The immobilized E. coli had a diameter of 4-5 mm and was then inoculated into a fermentation medium containing 0-0.4% functional additives for anaerobic fermentation to produce succinic acid.

2. The method according to claim 1, wherein The immobilized Escherichia coli was inoculated into the fermentation medium and fermented under anaerobic conditions at 30-38°C for 72-96 h.

3. The method according to claim 1, wherein The functional additive is at least one of betaine, Tween-80, sodium lauryl sulfate, lecithin or Triton X-100.

4. The method according to claim 1, wherein The immobilization method of the immobilized Escherichia coli comprises the following steps: picking a single colony of Escherichia coli into an LB test tube and culturing it for 8-12 hours, then inoculating it into an LB shake flask and culturing it at 37°C and 180 rpm for 8-12 hours; uniformly mixing the bacterial solution with a sodium alginate solution, slowly dripping the mixture into a 2% CaCl2 solution using a syringe to form immobilized cell hydrogel beads with a diameter of 4-5 mm, standing the beads at room temperature for 30 minutes to stabilize the hydrogel beads, and then washing them twice with a phosphate buffer solution with a pH of 7.0 to obtain the immobilized Escherichia coli.

5. The method according to claim 4, characterized in that The volume ratio of the bacterial liquid to the sodium alginate solution is 1:3-9.

6. The method according to claim 5, characterized in that The concentration of the sodium alginate solution is 1-5%.

7. The method according to claim 5, characterized in that The OD of the bacterial solution 600 It is 4~5.

8. The method according to claim 3, characterized in that 0.2-0.3% of functional additives are added to the fermentation medium.

9. The method according to claim 2, wherein The fermentation medium composition was: 2.6 g / L (NH4)2HPO4, 0.87 g / L NH4H2PO4, 0.15 g / L KCl, 0.37 g / L MgSO4·7H2O, 2.4 g / L FeCl3·6H2O, 0.1 g / LH3BO3, 0.3 g / L CoCl2·6H2O, 0.15 g / L CuCl2·2H2O, 0.5 g / L ZnCl2·4H2O, 0.5 g / LNaMoO4·2H2O, 0.5 g / L MnCl2·4H2O, 60 g / L glucose, 48 g / L basic magnesium carbonate as a pH stabilizer, and water as the solvent.

10. The method according to claim 4, wherein The immobilized cell hydrogel beads were formed by injecting a mixture of 2% sodium alginate and bacterial suspension into the syringe at a distance of 10 cm from the surface of the CaCl2 solution at a rate of 70 drops / min.

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