A method for synthesizing lactic acid from lignocellulose by using a functionally complementary fungus-bacterium mixed culture system

Abstract

The application discloses a method for synthesizing lactic acid from lignocellulose by using a fungus-bacterium mixed culture system with functional complementation, and comprises the following steps: (1) inoculating activated Trichoderma asperellum into a fermentation medium containing microcrystalline cellulose to perform fermentation, and obtaining a fermentation liquor; and (2) inoculating activated Lactobacillus paracasei into the fermentation liquor obtained in the step (1) to perform anaerobic fermentation and produce lactic acid. When the Lactobacillus paracasei is inoculated when the Trichoderma asperellum is cultured for 40 hours, the highest lactic acid yield is achieved, and reaches 57.59 g / L. The method is currently used to obtain the highest lactic acid yield in the mixed culture fermentation by using microcrystalline cellulose as the only carbon source. Meanwhile, the method can also be applied to a fermentation process for directly synthesizing lactic acid from lignocellulose. When the fermentation medium with corn cobs as the substrate is used to perform fermentation, 9.80 g / L of lactic acid can be produced, the substrate cost for industrial production of lactic acid is greatly reduced, and the method has important application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, specifically relating to a method for synthesizing lactic acid from lignocellulose using a fungal-bacterial mixed system with complementary functions. Background Technology

[0002] Lactic acid is one of the three major organic acids and has been widely used in brewing, medicine, food, cosmetics, cigarettes, leather making and other fields. In addition, lactic acid is also an important bio-based platform compound that can be used as a raw material to manufacture other chemicals, such as polylactic acid, acrylic acid, propionic acid, 2,3-pentanedione, pyruvic acid, propylene, lactate esters (green and environmentally friendly solvents), lactate salts, etc., and has broad application prospects.

[0003] The main industrial production methods for lactic acid include chemical synthesis and microbial fermentation. The former primarily uses acetaldehyde and hydrogen cyanide as raw materials, which react to produce lactic acid, which is then hydrolyzed with H₂SO₄ to generate lactic acid. The latter mainly uses glucose, sucrose, or other substrates as substrates, employing microbial fermentation to produce lactic acid. Chemical synthesis is costly, causes severe environmental pollution, and is difficult to synthesize a single configuration of lactic acid. Microbial fermentation not only overcomes these drawbacks but also offers the following advantages: ① mild fermentation conditions, lower cost, cleaner process, and higher efficiency; ② by selecting suitable strains and substrates, specific optical isomers can be obtained under certain fermentation conditions. Therefore, research on microbial fermentation for lactic acid production is receiving increasing attention.

[0004] Among the many lactic acid-producing microorganisms, *Lactobacillus paracasei* is one of the most promising production strains. However, the current production cost of lactic acid synthesis via microbial fermentation is relatively high, hindering its industrialization. Using inexpensive or waste non-grain biomass resources to replace glucose in lactic acid synthesis not only reuses waste biomass resources but also effectively reduces the cost of raw materials for lactic acid production, which is of great significance for promoting lactic acid bio-fermentation production.

[0005] Lignocellulosic biomass is one of the most important usable energy sources after fossil fuels such as coal, oil, and natural gas, and is considered the world's fourth largest energy source. With the increasing depletion of extractable non-renewable resources, the resource advantages of lignocellulosic biomass raw materials are becoming increasingly apparent. *Trichoderma echinococcus* is a filamentous fungus that can directly utilize lignocellulosic acid as a carbon source for growth and fermentation. *Trichoderma echinococcus* can grow rapidly within 1-2 days of fermentation and secrete large amounts of cellulase, hemicellulase, and β-glucosidase, which can efficiently degrade lignocellulosic raw materials such as corn cobs that have not undergone any chemical or biological treatment, making it extremely valuable in the degradation of lignocellulosic acid.

[0006] Mixed-culture fermentation is a novel fermentation technology that utilizes the synergistic effect of two or more microorganisms to complete a fermentation process. Complex metabolic tasks can be accomplished through the "division of labor" among strains. Currently, the screening and combination of cooperative strains is still a random process, lacking effective theoretical guidance. Furthermore, existing mixed-culture systems cannot effectively coordinate the relationships between strains to achieve optimal ecological levels and maximize their effects, which seriously hinders the development and application of mixed-culture fermentation. By adopting a "learning from nature" strategy, screening natural microbial communities that can directly synthesize lactic acid from lignocellulose, and constructing cross-species functionally complementary mixed-culture fermentation systems through the screening of core functional strains, can help improve the effective cooperation among microbial communities and increase the efficiency of directly synthesizing lactic acid from lignocellulose raw materials through integrated bioprocessing. Summary of the Invention

[0007] To address the high production costs associated with traditional lactic acid fermentation methods that rely on grains or other starchy byproducts, this invention constructs a fungal-bacterial mixed system of *Trichoderma echinococcus* and *Lactobacillus paracasei*, achieving the goal of efficiently synthesizing lactic acid directly from lignocellulosic raw materials. *Trichoderma echinococcus* secretes cellulose and hemicellulose hydrolytic enzymes, efficiently degrading lignocellulosic raw materials; while *Lactobacillus paracasei* cannot utilize cellulose or other polysaccharides, but it can use accumulated cellobiose and glucose as carbon sources. This alleviates the inhibitory effect of cellobiose on cellulose hydrolytic enzymes while simultaneously enabling efficient lactic acid synthesis.

[0008] The present invention discloses a method for synthesizing lactic acid from lignocellulose using a fungal-bacterial mixed system with complementary functions, comprising the following steps:

[0009] (1) The activated Trichoderma hydatids was inoculated into a fermentation medium containing microcrystalline cellulose and fermented for 24-120 h to obtain the fermentation broth;

[0010] (2) Inoculate the activated Lactobacillus paracasei into the fermentation broth obtained in step (1) and ferment the mixture to produce lactic acid.

[0011] When the Trichoderma hygroscopica ferments for 24-120 h as described in step (1), the activated Lactobacillus paracasei from step (2) is inoculated into the fermentation broth obtained in step (1) for anaerobic fermentation to produce lactic acid.

[0012] Preferably, the fermentation time in step (1) is 24-96 h, more preferably 40 h. The activated *Lactobacillus paracasei* is inoculated into the fermentation broth obtained in step (1). The fermentation broth contains a certain amount of glucose, and the activities of cellulase and β-glucosidase are high, enabling continuous degradation of microcrystalline cellulose into glucose. *Lactobacillus paracasei* can then utilize the glucose obtained from this degradation to produce lactic acid. If the inoculation time is too early (24 h), the cellulase activity is low, and it is not utilized for subsequent microcrystalline cellulose degradation. If the inoculation time is too late (72-120 h for *Trichoderma echinococcus* fermentation), the *Trichoderma echinococcus* culture time will be too long, resulting in reduced secretion of cellulase and β-glucosidase, decreased enzyme activity, and the utilization of some of the degraded glucose by *Trichoderma echinococcus*, thus reducing the carbon flux of lactic acid and ultimately leading to a decrease in lactic acid production. Therefore, the inoculation time of *Lactobacillus paracasei* is a crucial step in mixed-culture fermentation for lactic acid production.

[0013] The inoculum size of the activated *Trichoderma hydathodes* in step (1) is 1-5% of the fermentation medium volume; the fermentation medium containing microcrystalline cellulose has the following formula: 0.1-0.5 g / L urea, 1.0-2.0 g / L (NH4)2SO4, 1.0-3.0 g / L KH2PO4, 0.1-0.6 g / L CaCl2, 0.1-0.6 g / L MgSO4·7H2O, 0.002-0.008 g / L FeSO4·7H2O, 0.001-0.003 g / L MnSO4·H2O, 0.001-0.003 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 40-120 g / L microcrystalline cellulose, CaCO3 5~35 g / L, water as solvent, pH adjusted to 5.0-6.0, 121 Sterilize at ℃ for 20 min; the fermentation conditions are: fermentation temperature 28-35 ℃, fermentation time 24-120 h, fermentation pH 5.0-6.0, and rotation speed 0-120 rpm.

[0014] Preferably, the concentration of microcrystalline cellulose in the fermentation medium containing microcrystalline cellulose is 80 g / L.

[0015] Preferably, the fermentation conditions in step (1) are: fermentation temperature 30 ℃, fermentation time 40 h, fermentation pH 5.5, and rotation speed 120 rpm.

[0016] The activation medium for *Trichoderma echinococcus* is formulated as follows: 0.1-0.5 g / L urea, 1.0-2.0 g / L (NH4)2SO4, 1.0-3.0 g / L KH2PO4, 0.1-0.6 g / L CaCl2, 0.1-0.6 g / L MgSO4·7H2O, 0.002-0.008 g / L FeSO4·7H2O, 0.001-0.003 g / L MnSO4·H2O, 0.001-0.003 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 10-20 g / L glucose, with water as the solvent, and the pH adjusted to 5.0-6.0. The activation conditions are: 0.5-1.0 mL of *Trichoderma echinococcus* mycelium solution is coated onto PDA medium and incubated at 28-35 ℃ for 72-120 days. h, rinse the colonies in the PDA solid medium with 0.5-1.0 mL of sterile water, and inoculate them into the activation medium, and activate at 28-35 ℃ for 48-96 h;

[0017] The inoculation method of the activated Lactobacillus paracasei in step (2) is as follows: the activated Lactobacillus paracasei is inoculated into the fermentation medium described in step (1) at an inoculation amount of 1-10%; the fermentation conditions are: fermentation temperature of 35-39 ℃, fermentation time of 24-384 h, fermentation pH of 5.0-6.0, and rotation speed of 120-180 rpm.

[0018] Preferably, the fermentation conditions in step (2) are: fermentation temperature 37 ℃, fermentation time 336 h, fermentation pH 5.0, and rotation speed 180 rpm.

[0019] In step 2) of fermentation, 5-35 g / L CaCO3, more preferably 5-15 g / L CaCO3, is added to the culture medium. The optimal pH for *Trichoderma echinococcus* is 5.5, and for *Lactobacillus paracasei*, it is 5.0. Optimizing the CaCO3 concentration allows for optimal pH control of the mixed-culture system. Calcium ions, as trace metal ions, can to some extent increase lactic acid production by *Lactobacillus paracasei*, while *Trichoderma echinococcus* can also utilize the glucose obtained from the degradation of microcrystalline cellulose. Therefore, increasing the calcium ion concentration can inhibit the growth of *Trichoderma echinococcus*, allowing more carbon metabolic flux to flow towards lactic acid synthesis.

[0020] The activation medium formula for *Lactobacillus paracasei* is as follows: 3.0-5.0 g / L yeast extract, 5-10 g / L NaHCO3, 5-10 g / L NaH2PO4·2H2O, 10-20 g / L K2HPO4·3H2O, 2.0-3.0 g / L corn steep liquor, 5-40 g / L CaCO3, with water as the solvent; the activation conditions are as follows: 0.5-1.0 mL of *Lactobacillus paracasei* bacterial suspension is coated onto MRS medium and incubated at 35-39 ℃ for 12-24 h; the colonies in the MRS solid medium are rinsed with 0.5-1.0 mL of sterile water and inoculated onto the activation medium and incubated at 35-39 ℃ and 120-180 rpm for 12-18 h.

[0021] Among them, Trichoderma acicularis ( Trichoderma asperellum The strain, LYS1, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211179 and a deposit date of September 15, 2021. This strain exhibits rapid growth within 1-2 days of fermentation and secretes large amounts of cellulase, hemicellulase, and β-glucosidase. Its β-glucosidase activity is superior to that of *Trichoderma reesei*, effectively addressing the insufficient β-glucosidase secretion in currently commercial strains and efficiently degrading microcrystalline cellulose. Furthermore, based on its high hemicellulase secretion capacity, this strain can also efficiently degrade untreated corn cobs and other lignocellulosic raw materials, demonstrating significant application value in lignocellulosic degradation.

[0022] Among them, Lactobacillus paracasei is classified and named Lactobacillus paracasei ( Lactobacillus paracasei The strain, LYS2, is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20211178 and deposit date of September 15, 2021. This strain exhibits advantages such as high conversion rate, high optical purity, high yield, and low byproducts. It was found to possess a β-glucosidase hydrolase gene within the carbohydrate hydrolase family, and verification showed that it can utilize cellobiose as a carbon source to synthesize lactic acid.

[0023] The strain Lactobacillus paracasei ( Lactobacillus paracasei The screening method for LYS2 is as follows: Weigh an appropriate amount of kimchi broth, dilute it with physiological saline, and pipette 100 mL onto an MRS culture plate. Incubate at 28-37 ℃ for 3-4 days. The resulting colonies are streaked and purified for 5 generations to screen for strains of *Lactobacillus paracasei* capable of producing lactic acid. Lactobacillus paracaseiLYS2. The colonies of strain LYS2 are 2-3 mm in diameter, milky white, slightly convex, round, with a smooth surface and neat edges. The *Lactobacillus paracasei* LYS2 of this invention has a strong lactic acid synthesis capacity, with a lactic acid synthesis yield from glucose reaching up to 1 g / g, demonstrating significant advantages in mixed culture.

[0024] Beneficial effects: Compared with the prior art, the technical advantages of the present invention are as follows:

[0025] (1) Current reports on the production of lactic acid using lignocellulose as a substrate all involve hydrolyzing lignocellulose using acid hydrolysis, alkaline hydrolysis, and enzymatic hydrolysis, and then fermenting the hydrolysate, which is costly. This invention is based on an integrated bioprocessing strategy, which utilizes the functional complementarity between strains through mixed fermentation to directly synthesize lactic acid from lignocellulose raw materials such as microcrystalline cellulose and corn cobs. First, the lignocellulose-degrading strain Trichoderma echinosporum is introduced. After it secretes sufficient hydrolytic enzymes, Lactobacillus paracasei is then introduced to synthesize lactic acid from fermentable sugars.

[0026] (2) When *Trichoderma echinococcus* was cultured for 40 h, the addition of *Lactobacillus paracasei* at an inoculum size of 10% v / v resulted in the highest lactic acid yield, reaching 57.59 g / L. This is also the highest lactic acid yield obtained by mixed-culture fermentation using microcrystalline cellulose as the sole carbon source. At 40 h of culture, *Trichoderma echinococcus* exhibited high cellulase activity in the fermentation broth, effectively degrading microcrystalline cellulose. Subsequently, in the mixed-culture system, cellulase and β-glucosidase maintained high activity and good stability, continuously degrading microcrystalline cellulose into glucose. *Lactobacillus paracasei* could then utilize the degraded glucose to produce lactic acid.

[0027] (3) This method utilizes a mixed microbial system constructed by combining fungi and bacteria across genera. In artificial mixed microbial strategies, multiple microorganisms are fermented in the same system, so the growth conditions between microorganisms must be extremely similar. Previous technical strategies typically construct anaerobic microbial communities for the bioconversion of lignocellulose, and rarely integrate aerobic filamentous fungi with stronger lignocellulose degradation capabilities with anaerobic fermentation microorganisms in the same reactor for the bioconversion of lignocellulose into high-value chemicals. Based on the strong lignocellulose degradation capabilities of filamentous fungi, such as Trichoderma reesei, a fungal-bacterial combination was innovatively adopted. However, since most Trichoderma reesei species have β-glucosidase deficiency, cellobiose accumulation occurs, resulting in feedback inhibition of cellulase. Therefore, how to effectively alleviate cellobiose accumulation and eliminate feedback inhibition of cellulase becomes a major limiting factor in the construction of mixed microbial systems. In this invention, *Trichoderma echinococcus* is selected for the first time as the degrading strain, possessing superior β-glucosidase activity compared to *Trichoderma reesei*, enabling it to degrade microcrystalline cellulose into cellobiose and glucose. Simultaneously, *Lactobacillus paracasei* is selected as the lactic acid synthetase. Although it cannot directly utilize polysaccharides such as microcrystalline cellulose, its ability to directly utilize cellobiose effectively alleviates the inhibition of cellulosic hydrolases by cellobiose accumulation. Furthermore, *Lactobacillus paracasei* LYS2 exhibits a strong lactic acid synthesis capacity, achieving a lactic acid yield of up to 1 g / g from glucose. The mixed-strain system employed in this invention demonstrates a superior ability to synthesize lactic acid from microcrystalline cellulose compared to previously reported levels, such as the 34.7 g / L lactic acid yield reported by Shahab et al. Therefore, this combination helps improve the production efficiency of lactic acid synthesis from microcrystalline cellulose, significantly reducing the cost of industrial lactic acid production and possessing significant application value. Attached Figure Description

[0028] Figure 1 To optimize the effect of adding different CaCO3 concentrations on lactic acid yield in the co-culture system;

[0029] Figure 2 To optimize the effect of different inoculation times of Lactobacillus paracasei on lactic acid production in a co-culture system;

[0030] Figure 3 To optimize the effect of different microcrystalline cellulose concentrations on lactic acid yield in the co-culture system;

[0031] Figure 4 Three-dimensional plots and contour plots showing the effects of different microcrystalline cellulose concentrations and CaCO3 concentrations on lactic acid production;

[0032] Figure 5 Three-dimensional plots and contour plots showing the effects of different microcrystalline cellulose concentrations and Lactobacillus paracasei inoculation times on lactic acid production;

[0033] Figure 6 Three-dimensional plots and contour plots showing the effects of different Lactobacillus paracasei inoculation times and CaCO3 concentrations on lactic acid production;

[0034] Figure 7 To determine the yield of lactic acid under optimal co-culture fermentation conditions;

[0035] Figure 8 To optimize the lactic acid yield under different corn cob concentrations during co-culture fermentation. Detailed Implementation

[0036] Trichoderma is classified and named Trichoderma acicularis ( Trichoderma asperellum The strain number is LYS1.

[0037] Lactobacillus paracasei is classified as Lactobacillus paracasei ( Lactobacillus paracasei The strain number is LYS2, which comes from the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211178.

[0038] Example 1: Effect of different CaCO3 concentrations in the culture medium of a mixed bacterial system on the final lactic acid yield.

[0039] (1) Take 1 mL of Trichoderma hygroscopica solution and coat it onto PDA solid medium (46 g / L potato dextrose agar medium, 20 g / L agar) and incubate at 30 ℃ for 120 h. Rinse the colonies in the PDA solid medium with 1 mL of sterile water and inoculate them onto activation medium. Activate at 30 ℃ for 48 h. The activation medium formula is: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L MnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, 10 g / L glucose, with water as the solvent, and adjust the pH to 5.5.

[0040] (2) Take 1 mL of Lactobacillus paracasei culture and coat it in MRS medium (10 g / L peptone, 5 g / L beef extract powder, 4 g / L yeast powder, 20 g / L glucose, 1 g / L Tween-80, 2 g / L K2HPO4, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L MgSO4·7H2O, 0.05 g / L MgSO4·4H2O, 20 g / L agar) and incubate at 37 ℃ for 24 h. Rinse the colonies in the MRS solid medium with 1 mL of sterile water and inoculate it into an activation culture medium and incubate at 37 ℃ and 180 rpm for 18 h.

[0041] The activation culture medium formula is: 5.0 g / L yeast extract, 10 g / L NaHCO3, 9.6 g / L NaH2PO4·2H2O, 15.5 g / L K2HPO4·3H2O, 2.5 g / L corn steep liquor, 30 g / L CaCO3, with water as the solvent.

[0042] (3) The activated Trichoderma hydathodes was inoculated into the fermentation medium at an inoculation rate of 4% v / v and fermented at 30 ℃ and 120 rpm for 48 h to obtain the fermentation broth;

[0043] The fermentation medium formula was as follows: 0.3 g / L urea, 1.4 g / L (NH4)2SO4, 2.0 g / L KH2PO4, 0.3 g / L CaCl2, 0.3 g / L MgSO4·7H2O, 0.005 g / L FeSO4·7H2O, 0.00156 g / L MnSO4·H2O, 0.0014 g / L ZnSO4·7H2O, 0.002 g / L CoCl2, with a microcrystalline cellulose concentration of 80 g / L. The solvent was water, the pH was adjusted to 5.5, and the medium was sterilized at 121℃ for 20 min.

[0044] (4) Taking a mixed culture time of 48 h as an example, the activated Lactobacillus paracasei was inoculated into the fermentation broth obtained in step (3) at 10% v / v. Five groups of fermentation media were set up, with CaCO3 concentrations of 0 g / L, 5 g / L, 15 g / L, 25 g / L, 35 g / L, 37 ℃, 180 rpm for 336 h.

[0045] During the cultivation process, samples were taken every 24 hours to measure lactic acid production. The highest lactic acid production (34.69 g / L) was observed when the CaCO3 concentration reached 15 g / L. Figure 1When the CaCO3 concentration increases, its calcium ion concentration and the pH of the culture medium have a significant impact on the mixed bacterial system. The optimal pH for *Trichoderma echinococcus* is 5.5, and for *Lactobacillus paracasei*, it is 5.0. Optimizing the CaCO3 concentration allows for optimal pH control of the mixed bacterial system. Calcium ions, as trace metal ions, can to some extent increase lactic acid production in *Lactobacillus paracasei*, while *Trichoderma echinococcus* can also utilize glucose obtained from the degradation of microcrystalline cellulose. Therefore, increasing the calcium ion concentration can inhibit the growth of *Trichoderma echinococcus*, directing more carbon metabolic flux towards lactic acid synthesis. Furthermore, the cellulase secreted by *Trichoderma echinococcus* is highly stable in the fermentation broth and can continuously degrade microcrystalline cellulose to glucose, supplying the growth of lactic acid bacteria and lactic acid synthesis.

[0046] Example 2: Effect of different inoculation times of Lactobacillus paracasei on final lactic acid yield

[0047] The method is the same as in Example 1, except that the CaCO3 concentration is 15 g / L and the inoculation method is to inoculate the fermentation broth obtained in step (3) at an inoculation amount of 10% v / v. The difference is that five groups of experiments are set in step (3), with fermentation times of 24 h, 48 h, 72 h, 96 h, and 120 h, respectively.

[0048] During the cultivation process, samples were taken every 24 hours to determine the lactic acid production. When *Trichoderma echinococcus* was cultured for 48 hours, the addition of *Lactobacillus paracasei* resulted in the highest lactic acid production, reaching 38.51 g / L. Figure 2 In this system, cellulase secreted by *Trichoderma echinococcus* effectively degrades microcrystalline cellulose. Subsequently, in the mixed-culture system, cellulase and β-glucosidase maintain high enzyme activities, continuously degrading microcrystalline cellulose into glucose. *Lactobacillus paracasei* can then utilize the glucose obtained from degrading lignocellulose to produce lactic acid. Inoculation too early (24 h) results in low cellulase activity, preventing its utilization for subsequent microcrystalline cellulose degradation. Inoculation too late (72-120 h) leads to excessively long *Trichoderma echinococcus* culture times, decreasing cellulase and β-glucosidase activities, and some of the glucose obtained from degradation is also utilized by *Trichoderma echinococcus*, thus reducing the carbon flux for lactic acid synthesis and ultimately decreasing lactic acid production.

[0049] Example 3: Effect of different microcrystalline cellulose concentrations on final lactic acid yield

[0050] The method is the same as in Example 2, except that the inoculation time of Lactobacillus paracasei is 48 h, and the inoculation method is to inoculate 10% v / v into the fermentation broth obtained in step (3), with a CaCO3 concentration of 15 g / L; the difference is that in step (3), four groups of experiments were set up, with microcrystalline cellulose concentrations of 60 g / L, 80 g / L, 100 g / L, and 120 g / L, respectively. When the microcrystalline cellulose concentration was 60 g / L, the lactic acid yield was 40.03 g / L ( Figure 3 The highest lactic acid concentration (46.86 g / L) was obtained from mixed-culture fermentation when the microcrystalline cellulose concentration was 80 g / L. The lactic acid concentration was 27.23 g / L when the microcrystalline cellulose concentration was 100 g / L, and 18.64 g / L when the concentration was 120 g / L. This demonstrates that *Lactobacillus paracasei* alone cannot ferment microcrystalline cellulose, but a mixed-culture system with complementary functions can produce 46.86 g / L of lactic acid from 80 g / L microcrystalline cellulose.

[0051] Example 4: Optimization of the response surface methodology for lactic acid synthesis using microcrystalline cellulose in a mixed bacterial system.

[0052] Experimental analysis revealed three factors influencing lactic acid yield: microcrystalline cellulose concentration, CaCO3 concentration, and sequential inoculation time. To achieve the highest lactic acid yield, Design Expert software was used for response surface methodology optimization, selecting these three conditions. Microcrystalline cellulose concentration (A), CaCO3 concentration (B), and sequential inoculation time (C) were selected as variables, with lactic acid yield (Y) as the response value. Each factor had three levels, represented by -1, 0, and 1. Multiple regression analysis was performed based on the experimental results to derive the empirical relationship between lactic acid yield (Y) and each factor.

[0053] Figure 4 , Figure 5 , Figure 6 The response surface methodology includes a 3D surface plot and contour plot. The surface plot represents the interaction between two single factors. In the surface plot, the red dot in the center represents the highest lactic acid concentration, which gradually decreases from the center outwards. The highest lactic acid yield is achieved when the microcrystalline cellulose concentration is 80 g / L and the CaCO3 concentration is 15 g / L. The highest lactic acid yield is also achieved when the MCC concentration is 80 g / L and the lactic acid bacteria inoculation time is 48 h. The highest lactic acid yield is obtained when the CaCO3 concentration is 15 g / L and the lactic acid bacteria inoculation time is 48 h.

[0054] Analysis using Design Expert software predicted that the highest lactic acid yield (53.92 g / L) would be achieved under the following conditions: microcrystalline cellulose concentration of 82.69 g / L, CaCO3 concentration of 19.33 g / L, and Lactobacillus paracasei inoculation time of 40 h. Validation using these predicted fermentation conditions yielded a final lactic acid yield of 57.59 g / L. Figure 7 The experimental results were found to be basically consistent with the model predictions.

[0055] Example 5: Effect of different corn cob concentrations on final lactic acid yield

[0056] The method is the same as in Example 3, except that the inoculation time of Lactobacillus paracasei is 48 h, and the inoculation method is to inoculate the fermentation broth obtained in step (3) at an inoculation amount of 10% v / v, with a CaCO3 concentration of 15 g / L; the difference is that in step (3), four groups of experiments are set up, with corn cobs replacing the microcrystalline cellulose in the fermentation medium, and the corn cob concentrations are 60 g / L, 80 g / L, 100 g / L, and 120 g / L, respectively.

[0057] During the cultivation process, samples were taken every 24 hours to measure lactic acid production. When the corn cob concentration was 60 g / L, the lactic acid production was 3.68 g / L. Figure 8 When the corn cob concentration was 80 g / L, the highest lactic acid concentration was obtained from mixed fermentation, which was 9.80 g / L; when the corn cob concentration was 100 g / L, the lactic acid concentration was 4.13 g / L; and when the corn cob concentration was 120 g / L, the lactic acid concentration was 4.49 g / L.

[0058] Experiments have shown that using a mixed microbial system, 80 g / L corn cobs can produce 9.80 g / L lactic acid, demonstrating the feasibility of using a mixed microbial system of Trichoderma echinosporum and Lactobacillus paracasei to directly synthesize lactic acid from lignocellulose raw materials, which is expected to reduce the cost of fermentation raw materials.

Claims

1. A method for synthesizing lactic acid from lignocellulose using a fungal-bacterial mixed system with complementary functions, characterized in that, Includes the following steps: (1) The activated Trichoderma hydatids was inoculated into a fermentation medium containing microcrystalline cellulose and fermented for 40 h to obtain the fermentation broth; (2) Inoculate the activated Lactobacillus paracasei into the fermentation broth of step (1) and anaerobic ferment to produce lactic acid; The Trichoderma mentioned in step (1) is Trichoderma acicularis ( Trichoderma asperellum LYS1, with accession number CCTCCNO: M 20211179; the concentration of microcrystalline cellulose in the fermentation medium containing microcrystalline cellulose is 82.69 g / L; In step (1), the amount of activated Trichoderma hygroscopicum inoculated is 1-5% of the fermentation medium volume; The fermentation conditions for step (1) are: fermentation temperature 28-35 ℃, fermentation pH 5.0-6.0, and rotation speed 0-120 rpm; The *Lactobacillus paracasei* mentioned in step (2) is *Lactobacillus paracasei* (… Lactobacillus paracasei LYS2 has been deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20211178 and deposit date of September 15, 2021. In step (2) during fermentation, 19.33 g / L CaCO3 was added to the culture medium; The inoculation method of activated Lactobacillus paracasei in step (2) is as follows: Activated Lactobacillus paracasei is inoculated into the fermentation broth obtained in step (1) at an inoculation amount of 1-10% v / v; The fermentation conditions for step (2) are: fermentation temperature 35-39 ℃, fermentation time 24-384 h, fermentation pH 5.0-6.0, and rotation speed 120-180 rpm.

2. The method according to claim 1, characterized in that, The fermentation medium containing microcrystalline cellulose in step (1) is formulated as follows: 0.1-0.5 g / L urea, 1.0-2.0 g / L (NH4)2SO4, 1.0-3.0 g / L KH2PO4, 0.1-0.6 g / L CaCl2, 0.1-0.6 g / L MgSO4·7H2O, 0.002-0.008 g / L FeSO4·7H2O, 0.001-0.003 g / L LmnSO4·H2O, 0.001-0.003 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 40-120 g / L microcrystalline cellulose, CaCO3 5~35 g / L, with water as the solvent, and the pH adjusted to 5.0-6.

0.

3. The method according to claim 1, characterized in that, Step (1) The activation conditions for *Trichoderma echinococcus* are as follows: Take 0.5-1.0 mL of *Trichoderma echinococcus* mycelium solution and coat it onto PDA medium, then incubate at 28-35 ℃ for 72-120 h; rinse the colonies in the PDA solid medium with 0.5-1.0 mL of sterile water, and inoculate them onto activation medium, then activate at 28-35 ℃ for 48-96 h; The formulation of the *Trichoderma echinococcus* seed activation medium is: 0.1-0.5 g / L urea, 1.0-2.0 g / L (NH4)2SO4, 1.0-3.0 g / L KH2PO4, 0.1-0.6 g / L CaCl2, 0.1-0.6 g / L MgSO4·7H2O, 0.002-0.008 g / L FeSO4·7H2O, 0.001-0.003 g / L LnSO4·H2O, 0.001-0.003 g / L ZnSO4·7H2O, 0.001-0.003 g / L CoCl2, 10-20 g / L glucose, water as solvent, adjust pH to 5.0-6.

0.

4. The method according to claim 1, characterized in that, The activation conditions for *Lactobacillus paracasei* in step (2) are as follows: 0.5-1.0 mL of *Lactobacillus paracasei* bacterial suspension is coated onto MRS medium and cultured at 35-39 ℃ for 12-24 h; the colonies in the MRS medium are rinsed with 0.5-1.0 mL of sterile water and inoculated onto an activation culture medium, then cultured at 35-39 ℃ and 120-180 rpm for 12-18 h; the MRS medium consists of: 5-10 g / L peptone, 3-6 g / L beef extract powder, 3-5 g / L yeast extract, 10-20 g / L glucose, 1.0-2.0 g / L Tween-80, 1.0-3.0 g / L K2HPO4, 3.0-6.0 g / L sodium acetate, 1.0-3.0 g / L triammonium citrate, 0.1-0.3 g / L MgSO4·7H2O, and 0.01-0.1 g / L... MgSO4·4H2O, 10-20 g / L agar; Lactobacillus paracasei activation medium: 3.0-5.0 g / L yeast extract, 5-10 g / L NaHCO3, 5-10 g / L NaH2PO4·2H2O, 10-20 g / L K2HPO4·3H2O, 2.0-3.0 g / L corn steep liquor, 5-40 g / L CaCO3, solvent: water.

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