Method for increasing yield of lactic acid

Abstract

The invention discloses a method for increasing the yield of lactic acid, which belongs to the technical field of lactic acid production and comprises the following steps: soaking, embedding and fixing, fermenting and post-treating, soaking: mixing the aminated resin with a calcium chloride aqueous solution, stirring at 25-40 DEG C, filtering, and taking filter residues to obtain soaked aminated resin; in the embedding and fixing step, the escherichia coli is subjected to activation and seed culture, a seed solution is centrifuged, and wet thalli are collected; mixing the wet thalli with normal saline, stirring at 20-35 DEG C, adding a sodium alginate aqueous solution and a sodium carbonate aqueous solution, continuously stirring, adding the soaked aminated resin, continuously stirring, adding a calcium chloride aqueous solution, continuously stirring, filtering, and taking filter residues to obtain embedded and immobilized Escherichia coli; when lignocellulose biomass is used as a raw material and Escherichia coli is used for producing D-lactic acid, the yield of D-lactic acid can be increased.

Description

Technical Field

[0001] The present invention relates to the technical field of lactic acid production, and particularly relates to a method for increasing the yield of lactic acid. Background Art

[0002] Lactic acid, also known as 2-hydroxypropanoic acid, is an organic acid widely present in nature and has wide applications in the fields of food, agriculture, medicine, feed, and chemical industry. Since there is 1 chiral carbon atom in the lactic acid molecule, there are 2 isomers of lactic acid, namely D-lactic acid and L-lactic acid. Among them, D-lactic acid is widely used in the production of chiral drugs, pesticide intermediates, and polylactic acid. Since polylactic acid has biodegradability and also has the advantages of high strength, high modulus, and low toxicity, it is considered an ideal material to replace traditional bioplastics. Due to the strong interaction between the poly-D-lactic acid molecular chain and the poly-L-lactic acid molecular chain in the stereocomplex composed of poly-D-lactic acid and poly-L-lactic acid, its melting point is relatively high, about 50 °C higher than that of poly-D-lactic acid or poly-L-lactic acid, and its impact strength is also higher than that of poly-D-lactic acid or poly-L-lactic acid. Furthermore, this has led to a greatly increased production demand for D-lactic acid.

[0003] The production methods of D-lactic acid include chemical synthesis method, enzymatic method, and fermentation method. The chemical synthesis method has high synthesis efficiency, but is costly and toxic; the enzymatic method can obtain D-lactic acid with high optical purity, but the process is complex and the cost is relatively high; the fermentation method has mild production conditions and little environmental pollution, and currently, the D-lactic acid on the market is mainly produced by the fermentation method.

[0004] The fermentation method uses substances such as sugars as carbon sources, plus nitrogen sources and other nutrients, and synthesizes D-lactic acid through microorganisms. According to the different metabolic pathways and products of microorganisms synthesizing D-lactic acid, the fermentation pathways of D-lactic acid can be divided into three types: homolactic fermentation, heterolactic fermentation, and mixed-acid fermentation. In homolactic fermentation, the fermentation pathway used is the glycolysis pathway. In this pathway, lactic acid is the only metabolite of glucose. Through this pathway, 1 mol of glucose can be converted into 2 mol of lactic acid, and the theoretical conversion rate is 100%. However, due to other physiological activities of microorganisms during fermentation, the actual conversion rate cannot reach 100%. When the actual conversion rate is above 80%, it is considered homolactic fermentation; in heterolactic fermentation, the fermentation pathway used is the pentose phosphate pathway. In this pathway, glucose ferments to produce lactic acid, carbon dioxide, and acetic acid (or ethanol). Through this pathway, 1 mol of glucose can be converted into 1 mol of lactic acid; mixed-acid fermentation is a fermentation method of microorganisms performing homolactic fermentation under special circumstances. When the glucose concentration is limited, the fermentation temperature is low, or the pH value changes, microorganisms will perform mixed-acid fermentation. In mixed-acid fermentation, the fermentation pathway used is still the glycolysis pathway, but the metabolic pathway will change, and the main metabolic by-products generated are small-molecule organic acids. Currently, when producing D-lactic acid by the fermentation method, there are a wide variety of fermentation strains used, and the most commonly used fermentation strains are Bacillus sporolactis, Lactobacillus, and Escherichia coli. Among them, both Bacillus sporolactis and Lactobacillus are microorganisms performing homolactic fermentation, and they have the advantages of low energy consumption required for fermentation and high product concentration. However, the lactic acid production capacity of wild-type Bacillus sporolactis and wild-type Lactobacillus is limited. Using molecular means to transform strains has the advantages of strong directivity and the ability to amplify a large number of genes of the target product. However, when Bacillus sporolactis and Lactobacillus transformed by molecular means are fermented, they require high-nutrient culture conditions, and the L-lactic acid dehydrogenase or lactate isomerase in the fermentation will affect the optical purity of the produced D-lactic acid. Escherichia coli is one of the most commonly used engineering strains in the biological field. Its genome sequence has been completely determined, and it also has the advantages of a short fermentation cycle and low nutritional requirements. It is often used in the biological genetic engineering field for gene cloning and vector construction. Studies have found that Escherichia coli with high lactic acid production capacity can be obtained through molecular means.

[0005] Lignocellulosic biomass is widely sourced and inexpensive, and lignocellulosic biomass contains abundant polysaccharides such as cellulose and hemicellulose. Through the microbial fermentation method, lignocellulosic biomass can be converted into high-value-added chemicals, which is of great significance for alleviating the global energy crisis and solving environmental pollution problems. The main components of lignocellulose hydrolysate are sugars such as glucose, xylose, and arabinose, of which about two-thirds are hexoses and about one-third are pentoses. However, most of the existing microorganisms cannot utilize pentoses or have a very low utilization rate of pentoses, which affects the application of lignocellulosic biomass. Through research, it has been found that Escherichia coli obtained by molecular means can utilize both pentoses and hexoses to produce D-lactic acid. Further, during the production of D-lactic acid, low production costs and high production capacity can be achieved simultaneously.

[0006] However, when using Escherichia coli obtained by molecular means to produce D-lactic acid with lignocellulosic biomass as the raw material, the following problems exist: First, since the lignocellulose hydrolysate contains both hexoses and pentoses, Escherichia coli will preferentially utilize hexoses during fermentation and start to utilize pentoses after the hexoses are exhausted. Moreover, there will be a lag period before utilizing pentoses. Compared with only utilizing hexoses and pentoses, the utilization speed is slow, and a part of the pentoses will not be completely utilized, further affecting the yield of D-lactic acid; Second, the fermentation activity of Escherichia coli is sensitive to the concentration of glucose. If the glucose concentration is too high, it will affect the osmotic pressure of Escherichia coli, resulting in a decrease in the fermentation activity of Escherichia coli, further affecting the yield of D-lactic acid. However, if the glucose concentration is low, it is necessary to control the concentration of lignocellulosic biomass, which will also lead to a low yield of D-lactic acid; Third, as the concentration of D-lactic acid produced during fermentation increases, it will have an inhibitory effect on the fermentation activity of Escherichia coli, further affecting the yield of D-lactic acid.

[0007] For the first above-mentioned problem, the most commonly used method at present is to optimize the fermentation conditions and extend the fermentation time. However, since the optimal fermentation temperature of Escherichia coli is 35-37 °C, this optimal fermentation temperature is also suitable for the growth of various miscellaneous bacteria. As the fermentation time extends, the number of miscellaneous bacteria increases, which will affect the fermentation activity of Escherichia coli. Further, it has little effect on increasing the yield of D-lactic acid. For the second above-mentioned problem, the most commonly used method at present is to use calcium alginate hydrogel beads for entrapment and immobilization. Specifically, after mixing an aqueous calcium alginate solution with Escherichia coli, it is dropped into an aqueous calcium chloride solution. Calcium ions in the aqueous calcium chloride solution diffuse into the droplets to form solid calcium alginate hydrogel beads. However, affected by the gel structure of the calcium alginate hydrogel beads, nutrients and fermentation substrates are not easily transferred to the inside of the calcium alginate hydrogel beads, resulting in the fermentation activity of Escherichia coli inside the calcium alginate hydrogel beads being affected. Further, it has little effect on increasing the yield of D-lactic acid. For the third above-mentioned problem, the most commonly used method at present is to use calcium hydroxide as a neutralizing agent in the fermentation. Calcium hydroxide reacts with the produced lactic acid to form calcium lactate. The calcium ions in calcium lactate can also promote the growth of Escherichia coli. However, the lactate ions in calcium lactate will also have a certain inhibitory effect on the fermentation activity of Escherichia coli. Further, it has little effect on increasing the yield of D-lactic acid. Summary of the Invention

[0008] Aiming at the deficiencies of the existing technology, the present invention provides a method for increasing the yield of lactic acid, which can increase the yield of D-lactic acid when using lignocellulosic biomass as a raw material and Escherichia coli for producing D-lactic acid.

[0009] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A method for increasing the yield of lactic acid, comprising: soaking, entrapment and immobilization, fermentation, and post-treatment; For the soaking, after mixing an amino-functionalized resin with an aqueous calcium chloride solution, stir at 25-40 °C for 5-6 h, filter, and take the filter residue to obtain the soaked amino-functionalized resin; In the soaking, the dosage ratio of the amino-functionalized resin to the aqueous calcium chloride solution is 1 g:7-9 mL; The manufacturer of the amino-functionalized resin is Xi'an LX Technology New Materials Co., Ltd., and the model is LX1000EA; The mass concentration of the aqueous calcium chloride solution is 5%; When storing the soaked amino-functionalized resin, the storage temperature is 4 °C; For the embedding and immobilization, after activating and performing seed culture on Escherichia coli, the seed solution is centrifuged to collect wet bacterial cells. The wet bacterial cells are mixed with physiological saline and stirred at 20 - 35 °C for 10 - 20 min. An aqueous sodium alginate solution and an aqueous sodium carbonate solution are added, and stirring continues for 30 - 40 min. The soaked amino-functionalized resin is added, and stirring continues for 2 - 3 h. An aqueous calcium chloride solution is added, and stirring continues for 2 - 3 h. Then, filtration is carried out, and the filter residue is taken to obtain the embedded and immobilized Escherichia coli. In the embedding and immobilization, the Escherichia coli is obtained as a gift from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and its preservation number is CGMCC 7679. For the activation, Escherichia coli preserved in a glycerol tube at -80 °C is streaked on an activation medium using an inoculation loop, and then incubated in an inverted manner at 37 °C for 18 h to obtain activated colonies. The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, with water as the solvent and a pH value of 7.0. For the seed culture, a single colony is taken from the activated colonies and inoculated into 50 mL of seed medium, and shaken at 37 °C and 100 - 150 r / min for 15 h. Anaerobic conditions are maintained during the shaking culture without passing any air to obtain the seed solution. The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, with water as the solvent and a natural pH value. The mass concentration of the aqueous sodium alginate solution is 1%. The mass concentration of the aqueous sodium carbonate solution is 5%. The mass concentration of the aqueous calcium chloride solution is 5%. The dosage ratio of wet bacterial cells to physiological saline is 1 g:9 - 10 mL. The dosage ratio of wet bacterial cells to the aqueous sodium alginate solution and the aqueous sodium carbonate solution is 1 g:18 - 10 mL:18 - 21 mL. The dosage ratio of wet bacterial cells to the soaked amino-functionalized resin is 1 g:2 - 2.3 g. The dosage ratio of wet bacterial cells to the aqueous calcium chloride solution is 1 g:65 - 70 mL. When storing the embedded and immobilized Escherichia coli, the storage temperature is 4 °C. For the fermentation, the Escherichia coli after entrapment immobilization is added to the fermentation medium, and cultured with shaking at 37 °C and 100 - 150 r / min. An aqueous calcium hydroxide solution is added dropwise to maintain the pH at 7. Anaerobic conditions are maintained during the shaking culture without introducing any air, and the total reducing sugar concentration is monitored in real time. When the total reducing sugar concentration is lower than 3 g / L, the fermentation is terminated to obtain the fermentation product; In the fermentation, the dosage ratio of the Escherichia coli after entrapment immobilization to the fermentation medium is 2 g:30 - 32 mL; The molar concentration of the aqueous calcium hydroxide solution is 3 mol / L; The components of the fermentation medium include: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, 0.072 μg / L boric acid. The solvent is rice straw hydrolysate with a natural pH value; The preparation method of the rice straw hydrolysate is as follows: Wash, dry, and crush the rice straw, and sieve it through a 20 - 30 mesh sieve to obtain rice straw powder; Mix the rice straw powder with a 2% sulfuric acid aqueous solution, and hydrolyze at 121 °C for 1 - 1.5 h. After cooling, add sodium hydroxide to adjust the pH to 4.8 - 5, add Trichoderma reesei cellulase solution, and stir at 50 - 55 °C for 75 - 80 h. Monitor the pH during the stirring process and maintain the pH at 4.8 - 5. After the stirring is completed, filter, take the filtrate, add calcium hydroxide to the filtrate to adjust the pH to 10, stir at 90 °C for 30 - 40 min, filter, take the filtrate, and add a sulfuric acid aqueous solution to adjust the pH to 7 to obtain the rice straw hydrolysate; In the preparation of the rice straw hydrolysate, the dosage ratio of the rice straw powder to the 2% sulfuric acid aqueous solution is 1 g:10 mL; The dosage ratio of the rice straw powder to the Trichoderma reesei cellulase solution is 1 g:7 - 9 mL; The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL; The mass concentration of the sulfuric acid aqueous solution is 10%; The post - treatment includes: ceramic membrane filtration, nanofiltration membrane refining, and ion exchange; For the ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, use a ceramic membrane for filtration, and take the filtrate as the ceramic membrane filtrate; The filtration accuracy of the ceramic membrane is 50 nm; For the nanofiltration membrane refining: Use a nanofiltration membrane to refine the ceramic membrane filtrate, and take the nanofiltration clear phase as the nanofiltrate; The molecular weight cut-off of the nanofiltration membrane is 200 Da; For the ion exchange: successively use D001 strongly acidic styrene-based cation exchange resin and D301 weakly basic acrylic acid-based anion exchange resin to conduct ion exchange on the nanofiltrate, and then perform evaporation and concentration to obtain a lactic acid solution.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the mixed sugar, since Escherichia coli will preferentially utilize easily metabolizable glucose, and start to utilize another sugar after glucose is exhausted and after a lag phase, that is, there is catabolite repression. The generation of catabolite repression is related to the sugar phosphotransferase system. The sugar phosphotransferase system can specifically transport glucose actively across the cell membrane from outside the cell, phosphorylate glucose into glucose-6-phosphate, and then enter the glycolysis pathway. Therefore, the prior art mainly reduces catabolite repression by inhibiting the glucose phosphorylation transport process. In the present invention, by entrapping and immobilizing Escherichia coli on the surface of the aminated resin, the aminated resin can immobilize the extracellular enzymes of Escherichia coli, thereby improving the activity of the extracellular enzymes. Further, it promotes the transport of glucose by Escherichia coli, accelerates the glucose phosphorylation transport process, and further accelerates the subsequent absorption of pentose sugars. In addition, by entrapping and immobilizing Escherichia coli, the generation of miscellaneous bacteria can be avoided; (2) When entrapping and immobilizing Escherichia coli in the present invention, first adsorb calcium ions on the surface of the aminated resin, then mix sodium alginate and sodium carbonate with Escherichia coli, and then mix with the aminated resin adsorbed with calcium ions. The calcium ions on the surface of the aminated resin can bind to alginate and carbonate ions, and bind Escherichia coli to the surface of the aminated resin. In the subsequent process of continuously adding calcium ions, it can promote the entrapment and immobilization of Escherichia coli. The calcium ions binding to alginate can form calcium alginate to entrap Escherichia coli, and the calcium ions binding to carbonate ions can generate calcium carbonate to immobilize Escherichia coli. In the fermentation of Escherichia coli, since both calcium hydroxide and calcium carbonate can act as neutralizing agents, but the neutralizing ability of calcium hydroxide is stronger, therefore, the produced lactic acid preferentially binds to calcium hydroxide. However, as the fermentation progresses, when the product concentration on the surface of Escherichia coli is too high, it will react with calcium carbonate, and calcium carbonate dissolves, releasing calcium ions, thereby increasing the pores and promoting the entry of nutrients and fermentation substrates. The release of calcium lactate avoids the inhibitory effect on Escherichia coli caused by excessive calcium lactate concentration; (3)According to the method of the present invention, when using lignocellulosic biomass as a raw material and Escherichia coli for the production of D-lactic acid, the yield of D-lactic acid can be increased. For the rice straw hydrolysate with a total reducing sugar content of 32.87 g / L, wherein the glucose concentration is 18.03 g / L, the xylose content is 12.32 g / L, and the arabinose content is 2.44 g / L, the fermentation time until the total reducing sugar concentration is lower than 3 g / L is 17.7 - 18.1 h. Continuing the fermentation, the lowest total reducing sugar concentration can reach 1.01 - 1.03 g / L, and the content of D-lactic acid in the fermentation product can reach 28.43 - 28.50 g / L. The optical purity of D-lactic acid in the lactic acid solution obtained after post-treatment is 99.81 - 99.87%. Detailed Embodiments

[0011] For a clearer understanding of the technical features, objectives, and effects of the present invention, the detailed embodiments of the present invention are now described.

[0012] Example 1 Preparation of Rice Straw Hydrolysate Wash, dry, and crush rice straw, pass through a 20-mesh sieve to obtain rice straw powder; mix the rice straw powder with a 2% sulfuric acid aqueous solution in a dosage ratio of 1 g:10 mL, hydrolyze at 121 °C for 1 h, after cooling, add sodium hydroxide to adjust the pH to 4.8, add Trichoderma reesei cellulase solution, and the dosage ratio of rice straw powder to Trichoderma reesei cellulase solution is 1 g:8 mL. Stir at 50 °C with a stirring speed of 200 r / min for 75 h. Monitor the pH during the stirring process and maintain the pH at 4.8. After the stirring is completed, filter, take the filtrate, add calcium hydroxide to adjust the pH to 10, stir at 90 °C with a stirring speed of 200 r / min for 30 min, filter, take the filtrate, and add sulfuric acid aqueous solution to adjust the pH to 7 to obtain rice straw hydrolysate; The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL; The mass concentration of the sulfuric acid aqueous solution is 10%; The total reducing sugar content in the rice straw hydrolysate is 32.87 g / L, wherein the glucose concentration is 18.03 g / L, the xylose content is 12.32 g / L, and the arabinose content is 2.44 g / L.

[0013] Example 2 Embedding, Fermentation, and Post-treatment 1. Soaking: Mix the aminated resin with a calcium chloride aqueous solution in a dosage ratio of 1 g:7 mL, stir at 25 °C with a stirring speed of 100 r / min for 5 h, filter, take the filter residue to obtain the soaked aminated resin, and store the soaked aminated resin at 4 °C; The manufacturer of the aminated resin is Xi'an BlueSail New Materials Co., Ltd., and the model is LX1000EA; The mass concentration of the calcium chloride aqueous solution is 5%; 2. Embedding and immobilization: Use an inoculation loop to streak Escherichia coli preserved in a -80 °C glycerol tube on the activation medium, and then incubate it upside down at 37 °C for 18 h; Inoculate a single colony into 50 mL of the seed medium, and shake culture it at 37 °C and 100 r / min for 15 h. Keep anaerobic during the shaking culture without passing any air to obtain the seed solution. Centrifuge the seed solution and collect the wet bacterial cells; Mix the wet bacterial cells with physiological saline at a dosage ratio of 1 g:9 mL, stir at a stirring speed of 20 r / min at 20 °C for 10 min, add the sodium alginate aqueous solution and the sodium carbonate aqueous solution. The dosage ratio of the wet bacterial cells to the sodium alginate aqueous solution and the sodium carbonate aqueous solution is 1 g:18 mL:18 mL, continue stirring for 30 min, add the soaked aminated resin. The dosage ratio of the wet bacterial cells to the soaked aminated resin is 1 g:2 g, continue stirring for 2 h, add the calcium chloride aqueous solution. The dosage ratio of the wet bacterial cells to the calcium chloride aqueous solution is 1 g:65 mL, continue stirring for 2 h, filter, and take the filter residue to obtain the embedded and immobilized Escherichia coli. Place the embedded and immobilized Escherichia coli at 4 °C for storage; The Escherichia coli was obtained as a gift from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and the preservation number is CGMCC 7679; The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0; The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural; The mass concentration of the sodium alginate aqueous solution is 1%; The mass concentration of the sodium carbonate aqueous solution is 5%; The mass concentration of the calcium chloride aqueous solution is 5%; 3. Fermentation: The immobilized Escherichia coli is added to the fermentation medium. The dosage ratio of the immobilized Escherichia coli to the fermentation medium is 2 g:30 mL. It is cultured with shaking at 37 °C and 100 r / min. An aqueous calcium hydroxide solution is added dropwise to maintain the pH at 7. Anaerobic conditions are maintained during the shaking culture without introducing any air, and the total reducing sugar concentration is monitored in real time. When the total reducing sugar concentration is lower than 3 g / L, the fermentation is terminated to obtain the fermentation product; The components of the fermentation medium include: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L iron(III) chloride hexahydrate, 0.3 μg / L cobalt(II) chloride hexahydrate, 0.15 μg / L copper(II) chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese(II) chloride tetrahydrate, 0.072 μg / L boric acid. The solvent is the rice straw hydrolysate obtained in Example 1, and the pH value is natural; The molar concentration of the aqueous calcium hydroxide solution is 3 mol / L; 4. Post-treatment: (1) Ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, it is filtered using a ceramic membrane, and the filtrate is taken as the ceramic membrane filtrate; The filtration accuracy of the ceramic membrane is 50 nm; (2) Nanofiltration membrane refining: The ceramic membrane filtrate is refined by nanofiltration using a nanofiltration membrane, and the nanofiltration clear phase is taken as the nanofiltrate; The cut-off molecular weight of the nanofiltration membrane is 200 Da; (3) Ion exchange: The nanofiltrate is subjected to ion exchange using D001 strongly acidic styrene-based cation exchange resin and D301 weakly basic acrylic-based anion exchange resin in sequence, and then evaporated and concentrated to obtain a lactic acid solution.

[0014] Example 3 Embedding, Fermentation and Post-treatment 1. Soaking: The amidated resin and the aqueous calcium chloride solution are mixed according to a dosage ratio of 1 g:9 mL, and stirred at a stirring speed of 300 r / min at 40 °C for 6 h, filtered, and the filter residue is taken to obtain the soaked amidated resin. The soaked amidated resin is stored at 4 °C; The manufacturer of the amidated resin is Xi'an BlueSail New Materials Co., Ltd., and the model is LX1000EA; The mass concentration of the aqueous calcium chloride solution is 5%; 2. Embedding and immobilization: Use an inoculation loop to streak Escherichia coli preserved in a glycerol tube at -80°C on an activation medium, and then incubate it in an inverted position at 37°C for 18 h; Inoculate a single colony into 50 mL of seed medium and shake culture at 37°C and 150 r / min for 15 h. Maintain anaerobic conditions during the shaking culture without passing any air to obtain a seed solution. Centrifuge the seed solution and collect the wet bacterial cells; Mix the wet bacterial cells with physiological saline at a dosage ratio of 1 g:10 mL, stir at a stirring speed of 60 r / min at 35°C for 20 min, add an aqueous sodium alginate solution and an aqueous sodium carbonate solution. The dosage ratio of wet bacterial cells to the aqueous sodium alginate solution and the aqueous sodium carbonate solution is 1 g:10 mL:21 mL, continue stirring for 40 min, add the soaked amino-functionalized resin. The dosage ratio of wet bacterial cells to the soaked amino-functionalized resin is 1 g:2.3 g, continue stirring for 3 h, add an aqueous calcium chloride solution. The dosage ratio of wet bacterial cells to the aqueous calcium chloride solution is 1 g:70 mL, continue stirring for 3 h, filter, and take the filter residue to obtain the embedded and immobilized Escherichia coli. Store the embedded and immobilized Escherichia coli at 4°C; The Escherichia coli was obtained as a gift from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and the preservation number is CGMCC 7679; The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0; The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural; The mass concentration of the aqueous sodium alginate solution is 1%; [[ID=,10]]The mass concentration of the aqueous sodium carbonate solution is 5%; The mass concentration of the aqueous calcium chloride solution is 5%; 3. Fermentation: Add the embedded and immobilized Escherichia coli to the fermentation medium. The dosage ratio of the embedded and immobilized Escherichia coli to the fermentation medium is 2 g:32 mL, shake culture at 37°C and 150 r / min, add an aqueous calcium hydroxide solution dropwise to maintain the pH at 7. Maintain anaerobic conditions during the shaking culture without passing any air, and monitor the total reducing sugar concentration in real time. When the total reducing sugar concentration is lower than 3 g / L, end the fermentation to obtain the fermentation product; The components of the fermentation medium include: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, 0.072 μg / L boric acid. The solvent is the rice straw hydrolysate obtained in Example 1, and the pH value is natural; The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L; 4. Post-treatment: (1) Ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, use a ceramic membrane for filtration, and take the filtrate as the ceramic membrane filtrate; The filtration accuracy of the ceramic membrane is 50 nm; (2) Nanofiltration membrane refinement: Use a nanofiltration membrane to refine the ceramic membrane filtrate, and take the nanofiltration clear phase as the nanofiltrate; The cut-off molecular weight of the nanofiltration membrane is 200 Da; (3) Ion exchange: Sequentially use D001 strong acidic styrene-based cation exchange resin and D301 weak basic acrylic-based anion exchange resin to perform ion exchange on the nanofiltrate, and then perform evaporation and concentration to obtain a lactic acid solution.

[0015] Comparative Example 1 This comparative example is a modification based on the technical solution of Example 2. The specific modification is as follows: In the first-step soaking step, deionized water is used to replace the calcium chloride aqueous solution in equal volume.

[0016] The remaining technical solutions are the same as those in Example 2.

[0017] Comparative Example 2 This comparative example is a modification based on the technical solution of Example 2. The specific modification is as follows: Omit the first-step soaking step, and omit the addition of the amino-functionalized resin after soaking in the second-step embedding and fixing step. Specifically, change the second-step embedding and fixing step to: Use an inoculation loop to streak Escherichia coli preserved in a glycerol tube at -80°C on an activation medium, and then incubate it upside down at 37°C for 18 h; inoculate a single colony into 50 mL of seed medium, and shake culture it at 37°C and 100 r / min for 15 h. Keep anaerobic during the shaking culture without passing any air to obtain a seed solution. Centrifuge the seed solution to collect the wet cells; mix the wet cells with physiological saline at a dosage ratio of 1 g:10 mL, stir at a stirring speed of 20 r / min at 20°C for 10 min, add an aqueous sodium alginate solution and an aqueous sodium carbonate solution. The dosage ratio of the wet cells to the aqueous sodium alginate solution and the aqueous sodium carbonate solution is 1 g:18 mL:18 mL, continue to stir for 30 min, add an aqueous calcium chloride solution. The dosage ratio of the wet cells to the aqueous calcium chloride solution is 1 g:65 mL, continue to stir for 2 h, filter, and take the filter residue to obtain the entrapped and immobilized Escherichia coli. Place the entrapped and immobilized Escherichia coli at 4°C for storage; The Escherichia coli was obtained as a gift from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and the preservation number is CGMCC 7679; The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0; The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L iron(III) chloride hexahydrate, 0.1 μg / L cobalt(II) chloride hexahydrate, 0.1 μg / L copper(II) chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese(II) chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural; The mass concentration of the aqueous sodium alginate solution is 1%; The mass concentration of the aqueous sodium carbonate solution is 5%; The mass concentration of the aqueous calcium chloride solution is 5%.

[0018] The rest of the technical solutions are the same as those in Example 2.

[0019] Comparative Example 3 This comparative example is a modification based on the technical solution of Example 2. The specific modification is as follows: In the second step of the entrapment and immobilization step, the addition of the aqueous sodium carbonate solution is omitted, that is, the second step of entrapment and immobilization is changed to: Use an inoculation loop to streak Escherichia coli preserved in a glycerol tube at -80°C on an activation medium, and then incubate it in an inverted position at 37°C for 18 h; inoculate a single colony into 50 mL of seed medium, and incubate it with shaking at 37°C and 100 r / min for 15 h. Maintain anaerobic conditions during the shaking culture without introducing any air to obtain a seed solution. Centrifuge the seed solution to collect the wet bacterial cells; mix the wet bacterial cells with physiological saline at a dosage ratio of 1 g:10 mL, stir at a stirring speed of 20 r / min at 20°C for 10 min, add an aqueous sodium alginate solution. The dosage ratio of the wet bacterial cells to the aqueous sodium alginate solution is 1 g:18 mL, continue stirring for 30 min, add the soaked amino-functionalized resin. The dosage ratio of the wet bacterial cells to the soaked amino-functionalized resin is 1 g:2 g, continue stirring for 2 h, add an aqueous calcium chloride solution. The dosage ratio of the wet bacterial cells to the aqueous calcium chloride solution is 1 g:65 mL, continue stirring for 2 h, filter, and collect the filter residue to obtain the entrapped and immobilized Escherichia coli. Store the entrapped and immobilized Escherichia coli at 4°C; The Escherichia coli was obtained as a gift from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and the preservation number is CGMCC 7679; The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.0; The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural; The mass concentration of the aqueous sodium alginate solution is 1%; The mass concentration of the aqueous calcium chloride solution is 5%.

[0020] The rest of the technical solutions are the same as those in Example 2.

[0021] Comparative Example 4 This comparative example is a modification based on the technical solution of Example 2. The specific modification is as follows: Omit the first step of soaking and the second step of entrapment and immobilization, and use an equal mass of wet bacterial cells instead of the entrapped and immobilized Escherichia coli in the third step of fermentation. The preparation method of the wet bacterial cells is as follows: Use an inoculation loop to streak Escherichia coli preserved in a glycerol tube at -80°C on an activation medium, and then incubate it in an inverted position at 37°C for 18 h; inoculate a single colony into 50 mL of seed medium and shake culture it at 37°C and 100 r / min for 15 h. Maintain anaerobic conditions during the shake culture without passing any air to obtain a seed solution. Centrifuge the seed solution and collect the wet bacterial cells.

[0022] The remaining technical solutions are the same as those in Example 2.

[0023] Test Example 1 Statistically analyze the fermentation time in the fermentation steps of Examples 2 - 3 and Comparative Examples 1 - 4, that is, the fermentation time from the start of fermentation until the total reducing sugar concentration is lower than 3 g / L. The statistical results are as follows:

[0024] Test Example 2 In the fermentation steps of Examples 2 - 3 and Comparative Examples 1 - 4, after the total reducing sugar concentration is lower than 3 g / L, continue the fermentation and monitor the total reducing sugar concentration in real time, and record the lowest total reducing sugar concentration. The recording results are as follows:

[0025] And after the total reducing sugar concentration no longer changes and the calcium lactate in the fermentation product is converted into lactic acid, detect the D-lactic acid content. The detection results are as follows:

[0026] Test Example 3 Detect the optical purity of D-lactic acid in the lactic acid solution obtained in the post-treatment steps of Examples 2 - 3 and Comparative Examples 1 - 4. The detection results are as follows:

[0027] It can be seen from the results of Test Example 1 and Test Example 2 that compared with Example 2, Comparative Examples 1 - 4 all have problems of slow fermentation speed, incomplete fermentation, and low D-lactic acid production.

Claims

1. A method for increasing lactic acid production, characterized in that, Including: Soaking, embedding and fixing, fermentation, post-treatment; For the soaking, after mixing the aminated resin with an aqueous calcium chloride solution, stir at 25 - 40 °C, filter, and take the filter residue to obtain the soaked aminated resin; For the embedding and fixing, after activating and performing seed culture on Escherichia coli, centrifuge the seed liquid to collect the wet bacterial cells; mix the wet bacterial cells with physiological saline, stir at 20 - 35 °C, add an aqueous sodium alginate solution and an aqueous sodium carbonate solution, continue stirring, add the soaked aminated resin, continue stirring, add an aqueous calcium chloride solution, continue stirring, filter, and take the filter residue to obtain the embedded and fixed Escherichia coli; In the embedding and fixing, the Escherichia coli is obtained as a gift from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, and the preservation number is CGMCC 7679; For the fermentation, add the embedded and fixed Escherichia coli into the fermentation medium, shake culture at 37 °C and 100 - 150 r / min, add an aqueous calcium hydroxide solution to maintain the pH at 7, keep anaerobic during the shake culture without passing any air, and monitor the total reducing sugar concentration in real time. When the total reducing sugar concentration is lower than 3 g / L, end the fermentation to obtain the fermentation product; The solvent of the fermentation medium is the hydrolysate of rice straw.

2. The method for increasing lactic acid production according to claim 1, wherein In the soaking, the dosage ratio of the aminated resin to the aqueous calcium chloride solution is 1 g:7 - 9 mL; The manufacturer of the aminated resin is Xi'an Lanhxiao Technology New Materials Co., Ltd., and the model is LX1000EA; The mass concentration of the aqueous calcium chloride solution is 5%; When storing the soaked aminated resin, the storage temperature is 4 °C.

3. The method for increasing lactic acid production according to claim 1, wherein In the embedding and fixing, for the activation, use an inoculation loop to streak the Escherichia coli preserved in a -80 °C glycerol tube on the activation medium, and then incubate it upside down at 37 °C for 18 h to obtain the activated colonies; The components of the activation medium include: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, 20 g / L xylose, 20 g / L agar, the solvent is water, and the pH value is 7.

0.

4. The method for increasing lactic acid production according to claim 1, characterized in that, In the embedding and fixing, for the seed culture, pick a single colony from the activated colonies and inoculate it into 50 mL of the seed medium, shake culture at 37 °C and 100 - 150 r / min for 15 h, keep anaerobic during the shake culture without passing any air to obtain the seed liquid; The components of the seed medium include: 20 g / L xylose, 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 1.5 μg / L ferric chloride hexahydrate, 0.1 μg / L cobalt chloride hexahydrate, 0.1 μg / L copper chloride dihydrate, 0.1 μg / L zinc chloride, 0.1 μg / L sodium molybdate dihydrate, 0.2 μg / L manganese chloride tetrahydrate, 0.05 μg / L boric acid, the solvent is water, and the pH value is natural.

5. The method for increasing lactic acid production according to claim 1, wherein In the embedding and fixing, the mass concentration of the aqueous sodium alginate solution is 1%; The mass concentration of the aqueous sodium carbonate solution is 5%; The mass concentration of the aqueous calcium chloride solution is 5%.

6. The method for increasing lactic acid production according to claim 1, characterized in that, In the embedding and fixation, the dosage ratio of wet bacteria to physiological saline is 1 g: 9 - 10 mL; The dosage ratio of wet bacteria to sodium alginate aqueous solution and sodium carbonate aqueous solution is 1 g: 18 - 10 mL: 18 - 21 mL; The dosage ratio of wet bacteria to the amino-functionalized resin after soaking is 1 g: 2 - 2.3 g; The dosage ratio of wet bacteria to calcium chloride aqueous solution is 1 g: 65 - 70 mL; When storing the embedded and fixed Escherichia coli, the storage temperature is 4 °C.

7. The method for increasing lactic acid production according to claim 1, wherein In the fermentation, the dosage ratio of the embedded and fixed Escherichia coli to the fermentation medium is 2 g: 30 - 32 mL; The molar concentration of the calcium hydroxide aqueous solution is 3 mol / L.

8. The method for increasing lactic acid production according to claim 1, wherein In the fermentation, the components of the fermentation medium include: 0.87 g / L ammonium dihydrogen phosphate, 2.63 g / L diammonium hydrogen phosphate, 0.18 g / L magnesium sulfate heptahydrate, 0.15 g / L betaine hydrochloride, 2.4 μg / L ferric chloride hexahydrate, 0.3 μg / L cobalt chloride hexahydrate, 0.15 μg / L copper chloride dihydrate, 0.3 μg / L zinc chloride, 0.3 μg / L sodium molybdate dihydrate, 0.5 μg / L manganese chloride tetrahydrate, 0.072 μg / L boric acid, and the solvent is rice straw hydrolysate with a natural pH value.

9. The method for increasing lactic acid production according to claim 8, characterized in that, In the preparation of the rice straw hydrolysate in the fermentation, the preparation method is as follows: Wash, dry, and crush the rice straw, sieve it through a 20 - 30 mesh sieve to obtain rice straw powder; Mix the rice straw powder with a 2% sulfuric acid aqueous solution, hydrolyze it at 121 °C for 1 - 1.5 h, after cooling, add sodium hydroxide to adjust the pH to 4.8 - 5, add Trichoderma reesei cellulase solution, stir at 50 - 55 °C for 75 - 80 h, monitor the pH during the stirring process and maintain the pH at 4.8 - 5, after the stirring ends, filter, take the filtrate, add calcium hydroxide to the filtrate to adjust the pH to 10, stir at 90 °C for 30 - 40 min, filter, take the filtrate, and add a sulfuric acid aqueous solution to adjust the pH to 7 to obtain the rice straw hydrolysate; In the preparation of the rice straw hydrolysate, the dosage ratio of rice straw powder to the 2% sulfuric acid aqueous solution is 1 g: 10 mL; The dosage ratio of rice straw powder to Trichoderma reesei cellulase solution is 1 g: 7 - 9 mL; The enzyme activity of the Trichoderma reesei cellulase solution is 1 FPU / mL; The mass concentration of the sulfuric acid aqueous solution is 10%.

10. The method for increasing lactic acid production according to claim 1, characterized in that, The post-treatment includes: ceramic membrane filtration, nanofiltration membrane refining, and ion exchange; The ceramic membrane filtration: After converting calcium lactate in the fermentation product into lactic acid, use a ceramic membrane for filtration, and take the filtrate as the ceramic membrane filtrate; The filtration accuracy of the ceramic membrane is 50 nm; The nanofiltration membrane refining: Use a nanofiltration membrane to refine the ceramic membrane filtrate, and take the nanofiltration clear phase as the nanofiltrate; The retention molecular weight of the nanofiltration membrane is 200 Da; The ion exchange: Use D001 strong acidic styrene-based cation exchange resin and D301 weak basic acrylic-based anion exchange resin in sequence to perform ion exchange on the nanofiltrate, and then perform evaporation and concentration to obtain a lactic acid solution.

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