Mixed bacteria fermentation liquor and method for preparing lactic acid by pretreating biomass with high lignin content through cooperation of mechanical thread rolling and steam explosion

Abstract

The invention discloses a mixed bacterium fermentation liquid and a method for preparing lactic acid by cooperating with mechanical thread rolling coupled steam explosion pretreatment of high-lignin-content biomass, and the mixed bacterium fermentation liquid comprises a mixed bacterium liquid composed of brevibacillus brevis, bacillus licheniformis, bacillus steterrae, ochrobactrum pseudointermedium and a liquid culture medium suitable for mixed bacterium growth, and fermenting by using the mixed fermentation liquid, carrying out mechanical thread rolling, and then carrying out steam explosion treatment. According to the method disclosed by the invention, the biomass subjected to fermentation pretreatment is more easily permeated by steam, and the energy of steam explosion can more sufficiently act on the material of which the structure is loosened, so that the energy consumption intensity is greatly reduced, and the generation of harmful byproducts in the high-temperature explosion process of lignocellulose, particularly high-lignin-content biomass, can be effectively reduced; the inhibition of subsequent enzymolysis and fermentation processes is obviously reduced, and cost reduction, efficiency improvement and synergetic improvement of product quality are realized.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, specifically to a mixed-culture fermentation broth and a method for preparing lactic acid from high-lignin-content biomass using the mixed-culture fermentation broth in conjunction with mechanical shaving coupled with steam explosion pretreatment. Background Technology

[0002] Lactic acid is a natural organic acid and the most cost-competitive monomer for bioplastics. Its polymer, polylactic acid (PLA), is a biodegradable polymer material used in packaging, textiles, agricultural films, and 3D printing, with a promising market outlook. With the implementation of plastic restriction policies and the expansion of the consumer market, the potential for biodegradable plastics to replace lactic acid is expected to reach tens of millions of tons. Upgrading downstream consumer demand will drive rapid growth in lactic acid demand. Furthermore, using straw as a raw material to replace grain in lactic acid production can further reduce costs and enhance the sustainability of PLA development.

[0003] Currently, enzymatic saccharification remains one of the key steps in the fermentation of lignocellulose into lactic acid. However, the degradation barrier of lignocellulose resists enzymatic hydrolysis, usually requiring appropriate pretreatment methods to improve the hydrolysis effect. Since it does not use chemicals and has a short processing time, steam explosion is one of the most effective pretreatment methods to improve enzymatic hydrolysis. Compared to other methods, steam explosion uses high-pressure steam to penetrate the lignin-carbohydrate complex structure of lignocellulose, causing the material to "explode" due to instantaneous pressure release. This disrupts the lignin barrier, reduces the crystallinity of cellulose, and increases the porosity of the material, thereby improving the enzymatic hydrolysis effect by increasing the accessibility of enzymes to cellulose. However, this method is energy-intensive and produces substances that inhibit the activity of cellulase and subsequent fermentation microorganisms, such as furfural, acetic acid, and phenolic compounds. Furthermore, when the research subject is biomass with high lignin content, such as cotton stalks and wood, the lignin-inhibiting effect (lignin rearrangement and redeposition) may be aggravated, and a more ordered cellulose crystal structure may be formed (the crystallinity of cellulose increases). These factors will further affect the efficiency of subsequent enzymatic hydrolysis and fermentation processes, thereby limiting the application of steam explosion pretreatment technology. Summary of the Invention

[0004] This invention provides a method for preparing lactic acid from high-lignin-content biomass through pretreatment of mixed-culture fermentation broth and synergistic mechanical shredding coupled with steam explosion. The aim is to utilize a novel mixed-culture fermentation broth and the synergistic effect between mixed-culture fermentation, shredding, and steam explosion to impart excellent composition and structural characteristics to high-lignin-content biomass raw materials, thereby effectively improving the efficiency of enzymatic hydrolysis and saccharification, reducing subsequent fermentation inhibition, and reducing the energy consumption of traditional shredding and steam explosion processes.

[0005] In view of the above-mentioned problems of the prior art, according to one aspect disclosed in this invention, the present invention adopts the following technical solution: A mixed-culture fermentation broth, comprising: Bacillus brevis; Bacillus licheniformis; Sterculia; Pseudomonas intermedia; And liquid culture media suitable for mixed bacterial growth; The mixed fermentation broth is obtained by culturing Bacillus shortiflorus, Bacillus licheniformis, Stellaria media, and Pseudomonas intermedia in a liquid culture medium or by fermenting them separately and then mixing them. The resulting mixed fermentation broth is used as a pretreatment for the preparation of lactic acid from biomass with high lignin content.

[0006] To better realize the present invention, a further technical solution is: Furthermore, the liquid culture medium is LB liquid culture medium.

[0007] Furthermore, it is prepared from Bacillus brevis, Bacillus licheniformis, Sterculia stolonifera, and Pseudomonas intermedius in a fermentation liquid volume ratio of (1-3):1:1:1.

[0008] Furthermore, the OD of the fermentation broths of the aforementioned Bacillus shortbreaden, Bacillus licheniformis, Stellaria media, and Pseudomonas intermedius, which were separately fermented and then mixed, was further measured. 600 The values ​​are all 1.2.

[0009] Furthermore, this invention can also be a method for preparing lactic acid from high-lignin-content biomass using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion pretreatment, comprising: Step 1: Prepare and adjust the high lignin-containing biomass, the above mixed fermentation broth, and Hertz buffer, mix them evenly, and then carry out aerobic fermentation. After fermentation, perform solid-liquid separation and collect the solid and liquid parts separately. Step 2: Wash the solid separated in Step 1 to obtain characteristic biomass A; Step 3: Mechanically shred the biomass of characteristic A to obtain biomass of characteristic B; Step 4: After adding deionized water to the biomass of characteristic B to adjust the initial moisture content, steam explosion treatment is carried out. After the explosion, the residue is collected and dried to obtain pretreated biomass. The pretreated biomass is then crushed. Step 5: Place cellulase in a weakly acidic buffer solution, then place the pulverized pretreated biomass in an enzyme-containing buffer solution for enzymatic hydrolysis to obtain the enzymatic hydrolysate; Step 6: Inoculate the enzymatic hydrolysis substrate with Bacillus coagulans solution activated in mMRS medium, stir and add NaHCO3 to adjust the pH value to 5.5-6.5, and ferment until the lactic acid concentration in the supernatant no longer increases significantly.

[0010] Furthermore, in step 1, the volume ratio of high-lignin biomass, mixed-culture fermentation broth, and Hertz buffer is 1:(0.1-0.5):2.

[0011] Furthermore, in step 1, after the high-lignin biomass, mixed fermentation broth, and Herxol buffer are prepared in proportion, the fermentation concentration is adjusted to 15-35%, and the pH value is adjusted to 7.

[0012] Furthermore, in step 1, the specific process of aerobic fermentation includes: Place the fermentation raw materials in an aerobic fermenter, with the material height above 1 / 2, and maintain at 37℃ for 3 to 10 days. During this process, keep the aeration and stirring continuous, with an aeration rate of 1.0 vvm and a rotation speed of 200 rpm.

[0013] Furthermore, in step 5, the acidic buffer solution is an acetic acid-sodium acetate buffer solution.

[0014] Furthermore, in step 6, the inoculation amount of the fermentation broth is 5-15%.

[0015] Compared with the prior art, the beneficial effects of the present invention are: (1) This invention introduces the concept of mixed microbial fermentation and synergistic mechanical fiber-making before steam explosion. Utilizing the synergistic effect of microorganisms in the mixed fermentation broth and the fiber-making action, it achieves selective removal of lignin from lignocellulosic biomass, transformation of ordered crystalline regions of cellulose into disordered regions, bio-softening of the material flakes, separation of bundled fibers, and fiber finening. This strategy of synergistic fiber-making treatment by mixed microorganisms prioritizes the influence of substrate structure rather than chemical composition, significantly improving material porosity, increasing specific surface area, reducing raw material particle size, and disrupting some crystalline regions, thus laying a good foundation for subsequent processing. Compared to traditional pretreatment methods, the pretreated biomass of this invention is more easily permeated by steam, and the steam explosion... Energy is applied more fully to the loosened material, significantly reducing the energy consumption of mechanical shredding and steam explosion. This effectively reduces the generation of harmful byproducts during the high-temperature explosion of lignocellulose, especially high-lignin-content biomass, and significantly alleviates inhibition during subsequent enzymatic hydrolysis and fermentation. Furthermore, research has shown that traditional steam explosion increases the crystallinity of high-lignin-content biomass, leading to low utilization efficiency and yield in subsequent enzymatic hydrolysis and fermentation. The pretreatment method of this invention, using mixed-culture fermentation coupled with mechanical shredding and steam explosion, significantly reduces the crystallinity of biomass without a significant reduction in cellulose content. This optimization of material composition and structure improves overall saccharification and lactic acid fermentation efficiency. Through process supplementation and optimization, this invention achieves synergistic improvement in cost reduction, efficiency, and product quality, representing a highly efficient, economical, and green technological path for the resource utilization of high-lignin-content biomass.

[0016] (2) The method of the present invention is not only suitable for ordinary lignocellulosic biomass, but also particularly suitable for breaking through the bottleneck of stubborn raw materials with high lignin content and dense structure in this field. No chemical reagents are used in the process (only acid / alkali for adjusting pH value is involved), resulting in low cost due to reduced energy consumption, mild preparation conditions, low toxicity of waste liquid and low treatment difficulty and cost. It has the characteristics of being environmentally friendly and having universality of raw materials.

[0017] (3) The mixed fermentation broth of the present invention, composed of specific strains, can effectively treat different types of lignocellulose biomass raw materials by adjusting the strain ratio and fermentation time in the mixed fermentation broth, achieving selective removal of lignin and optimization of cellulose structure. It has strong adaptability to raw materials, simple fermentation broth preparation, strong practicality, and is easy to promote and industrialize. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only for reference to some embodiments in this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 To illustrate the changes in the chemical composition of cotton stalks before and after pretreatment, Examples 1(2)-(4) and 1(1)-(4) represent the treatment without step (1) and the treatment with step (1), respectively.

[0020] Figure 2A This is a scanning electron microscope image of cotton stalks before pretreatment. Figure 2B The images are scanning electron microscope images of cotton stalks after being treated by Examples 1(2)-(4). Figure 2C The image is a scanning electron microscope image of the cotton stalk after being processed by steps (1)-(4) of Example 1.

[0021] Figure 3 The images show the Fourier transform infrared spectra of cotton stalks before and after pretreatment.

[0022] Figure 4 The images show X-ray diffraction patterns of cotton stalks before and after pretreatment.

[0023] Figure 5 The graph shows the mass fraction of cotton stalks before and after pretreatment (TG-DTG).

[0024] Figure 6 The graph shows the TG-DTG of the mass loss rate of cotton stalks before and after pretreatment. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0026] A mixed-culture fermentation broth, comprising: Bacillus brevis; Bacillus licheniformis; Sterculia; Pseudomonas intermedia; And liquid culture media suitable for mixed bacterial growth; The mixed fermentation broth is obtained by culturing Bacillus shortiflorus, Bacillus licheniformis, Stellaria media, and Pseudomonas intermedia in a liquid culture medium or by fermenting them separately and then mixing them. The resulting mixed fermentation broth is used as a pretreatment for the preparation of lactic acid from biomass with high lignin content.

[0027] The preferred liquid culture medium is LB liquid culture medium.

[0028] It is prepared from Bacillus shortiflorus, Bacillus licheniformis, Sterculia stolonifera, and Pseudomonas intermedia according to the fermentation liquid volume ratio (1-3):1:1:1.

[0029] The OD value of the fermentation broths of Bacillus shortbread, Bacillus licheniformis, Sterculia lysate, and Pseudomonas intermedius was used for the separate fermentation and mixing of the aforementioned Bacillus shortbread, Bacillus licheniformis, Sterculia lysate, and Pseudomonas intermedius fermentation broths. 600 The values ​​are all 1.2.

[0030] A method for preparing lactic acid from high-lignin-content biomass using mixed-culture fermentation broth coupled with mechanical shredding and steam explosion pretreatment, comprising: Step 1: Prepare and adjust the high-lignin-content biomass, the above-mentioned mixed fermentation broth, and Herxheimer's buffer solution. After mixing evenly, carry out aerobic fermentation. After fermentation, perform solid-liquid separation and collect the solid and liquid fractions separately. The high-lignin-content biomass includes, but is not limited to, any one or a combination of several of the following: cotton stalks, bamboo, coniferous wood, broadleaf wood, mulberry branches, sugarcane bagasse, and reeds. The volume ratio of the high-lignin-content biomass, mixed fermentation broth, and Herxheimer's buffer solution is 1:(0.1-0.5):2; adjust the fermentation concentration to 15-35%; adjust the pH value to 7.

[0031] The specific process of aerobic fermentation is as follows: Place the fermentation raw materials in an 8L aerobic fermentation tank in the laboratory, with the material height above 1 / 2, and keep it at 37℃ for 3 to 10 days. During this process, keep continuous aeration and stirring, with an aeration rate of 1.0 vvm and a rotation speed of 200 rpm.

[0032] Step 2: Wash the solid separated in Step 1 to obtain characteristic biomass A.

[0033] The specific washing process is as follows: The solid part was soaked in a light alkaline solution with a pH of 10-12 adjusted by saturated NaOH solution for 6 hours (wet depigmentation and dissolution of biodegradable lignin), filtered, and then washed with deionized water until neutral.

[0034] Step 3: Mechanically shred the biomass of characteristic A to obtain biomass of characteristic B.

[0035] The yarn rolling process consists of two steps and two rolling operations: first, preliminary separation (coarse rolling) with a roller gap of 0.12–0.15 mm; then, fine rolling with a roller gap of 0.06–0.1 mm. (Linear pressure 40–80 N / mm, roller speed difference 10–20%).

[0036] Step 4: After adding deionized water to the biomass of characteristic B to adjust the initial moisture content, steam explosion treatment is carried out. After the explosion, the residue is collected and dried to obtain pretreated biomass. The pretreated biomass is then crushed.

[0037] The initial moisture content is adjusted to 15-75%. The steam explosion process is as follows: the biomass with the adjusted initial moisture content is placed in a steam explosion machine and held at an explosion pressure of 0.6-1.8 MPa for 120-360 s; the low-temperature drying is drying at 50℃ to obtain pretreated biomass.

[0038] Step 5: Place cellulase in a weakly acidic buffer solution, then place the pulverized pretreated biomass in the enzyme-containing buffer solution for enzymatic hydrolysis until the concentration of fermentable sugar no longer increases significantly, thus obtaining the enzymatic hydrolysis matrix.

[0039] The weakly acidic buffer solution is an acetate-sodium acetate buffer solution (pH=4.8); the pretreated biomass is pulverized to 10-40 mesh; the specific enzymatic hydrolysis process is as follows: the pulverized pretreated biomass is placed in a constant temperature water bath shaker, and hydrolyzed for 48 h at 50℃ and 180 rpm under the conditions of enzyme dosage of 10-50 FPU / g substrate and solid content of 10-30%, to obtain the enzymatic hydrolysis matrix; further, the enzymatic hydrolysis matrix is ​​a mixture of enzymatic hydrolysate and residue after fermentation.

[0040] Fermentable sugars are the sum of glucose, xylose, and arabinose.

[0041] Step 6: Inoculate the enzymatic hydrolysis substrate with Bacillus coagulans solution activated in mMRS medium, stir and add NaHCO3 to adjust the pH value to 5.5-6.5, and ferment until the lactic acid concentration in the supernatant no longer increases significantly.

[0042] NaHCO3 was continuously added at 50°C and 180 rpm with stirring, and fermentation continued for 48–120 h until the lactic acid concentration in the supernatant no longer increased significantly. The concentration of the NaHCO3 solution was 5%, and the amount of NaHCO3 solution added was based on maintaining the required pH value (pH 5.5–6.5) in the fermenter.

[0043] The inoculum size of the fermentation broth is 5–15% (v / v); the fermentation broth is a Bacillus coagulans broth activated with mMRS medium, and its OD... 600 The value is 1.2. Example 1

[0044] (1) Mix high-lignin biomass (cotton stalks), mixed fermentation broth, and Herxol buffer at a ratio of 1:0.3:2 (w / v / v) (the volume ratio of Bacillus shortbread, Bacillus licheniformis, Sterculia spp., and Pseudomonas intermedius fermentation broth is 3:1:1:1). Then add deionized water to the reaction system to adjust the fermentation concentration to 30%, and continue to finely adjust the pH value to 7 with saturated NaOH solution. Place the well-mixed material in a small aerobic fermenter in the laboratory with continuous aeration and stirring, and ferment aerobically at a fermentation temperature of 37°C for 5 days. During this period, the aeration rate is 1.0 vvm and the rotation speed is 200 rpm. After the fermentation is completed, perform solid-liquid separation and collect the liquid and solid parts. (2) The solid part was soaked in a light alkaline solution with pH adjusted to 10-12 using saturated NaOH solution for 6 h, filtered, and the solid part was further washed with deionized water until neutral to obtain A-type biomass; (3) Place biomass of characteristic A in a spun yarn machine with a linear pressure of 60 N / mm and a roller speed difference of 15%. First, perform preliminary separation (coarse spun yarn) with a roller gap of 0.13 mm. Then, further adjust the roller gap to 0.07 mm and perform fine spun yarn on the preliminarily separated biomass to obtain biomass of characteristic B. (4) The initial moisture content of the B characteristic biomass was 60% and placed in a steam explosion machine. The explosion pressure was set to 1.5 MPa and the pressure holding time was 240 s. After the explosion, the collected residue was dried at 50-60℃ to obtain the pretreated biomass. (5) The pretreated biomass that has been crushed and passed through a 30-mesh sieve is added to an enzyme-containing buffer (pH=4.8) and placed in a constant temperature water bath shaker. Under the conditions of 30 FPU / g substrate of cellulase and 15% solid content, it is hydrolyzed at 50℃ and 180rpm for 48 h to obtain the enzymatically hydrolyzed matrix.

[0045] The supernatant of the enzymatic hydrolysis matrix was filtered through a 0.22 μm filter membrane and the content of fermentable sugars was determined by high performance liquid chromatography. Fermentable sugars are the sum of glucose and xylose. (6) Add Bacillus coagulans to the enzymatic hydrolysate at an inoculum of 10% (v / v), and ferment at 50°C, 180 rpm with continuous addition of NaHCO3 solution for 120 h. Take the fermentation supernatant, filter it through a 0.22 μm filter membrane, and determine the lactic acid content by high performance liquid chromatography. Example 2

[0046] The difference from Example 1 is as follows: (1) High lignin-containing biomass (cotton stalks), mixed fermentation broth, and Herxol buffer were mixed at a ratio of 1:0.3:2 (w / v / v) (the fermentation broth of Bacillus shortbread, Bacillus licheniformis, Sterculia stolonifera, and Pseudomonas intermedius was in a volume ratio of 2:1:1:1). Then, deionized water was added to the reaction system to adjust the fermentation concentration to 30%, and the pH value was further adjusted to 7 with saturated NaOH solution. The mixed material was placed in a small aerobic fermenter in the laboratory with continuous aeration and stirring, and aerobic fermentation was carried out at a fermentation temperature of 37°C for 5 days. During this period, the aeration rate was 1.0 vvm and the rotation speed was 200 rpm. After the fermentation was completed, solid-liquid separation was performed, and the liquid part and solid part were collected. Example 3

[0047] The difference from Example 1 is as follows: (1) High lignin-containing biomass (cotton stalks), mixed fermentation broth, and Herxol buffer were mixed at a ratio of 1:0.3:2 (w / v / v) (the fermentation broth of Bacillus shortiflorus, Bacillus licheniformis, Sterculia stolonifera, and Pseudomonas intermedius was in a volume ratio of 1:1:1:1). Then, deionized water was added to the reaction system to adjust the fermentation concentration to 30%, and the pH value was further adjusted to 7 with saturated NaOH solution. The mixed material was placed in a small aerobic fermenter in the laboratory with continuous ventilation and stirring, and aerobic fermentation was carried out at a fermentation temperature of 37°C for 5 days. During this period, the ventilation rate was 1.0 vvm and the rotation speed was 200 rpm. After the fermentation was completed, solid-liquid separation was performed, and the liquid part and solid part were collected. Example 4

[0048] The difference from Example 1 is that: (3) the biomass of feature 1 is placed in a spun yarn machine with a linear pressure of 60 N / mm and a roller speed difference of 15%, and is first initially separated (coarse spun yarn) with a roller gap of 0.13 mm. The roller gap is further adjusted to 0.09 mm, and the initially separated biomass is further finely spun to obtain biomass of feature 2. Example 5

[0049] The difference from Example 1 is that: (4) the biomass of Character 2 with an initial moisture content of 60% is placed in a steam blasting machine, the blasting pressure is set to 1.2 MPa, the pressure holding time is 240 s, and blasting is carried out; after the blasting is completed, the collected residue is dried at 50-60℃ to obtain pretreated biomass. Example 6

[0050] The difference from Example 1 is that: (4) the biomass of Character 2 with an initial moisture content of 60% is placed in a steam blasting machine, the blasting pressure is set to 1.8 MPa, the pressure holding time is 240 s, and blasting is carried out; after the blasting is completed, the collected residue is dried at 50-60℃ to obtain pretreated biomass. Example 7

[0051] The difference from Example 1 is that: (4) the biomass of Character 2 with an initial moisture content of 45% is placed in a steam explosion machine, the explosion pressure is set to 1.5 MPa, the pressure holding time is 240 s, and the explosion is carried out; after the explosion, the collected residue is dried at 50-60℃ to obtain pretreated biomass. Example 8

[0052] The difference from Example 1 is that: (5) the pretreated biomass that has been crushed and passed through a 30-mesh sieve is added to an enzyme-containing buffer (pH=4.8) and placed in a constant temperature water bath shaker. Under the conditions of 40 FPU / g substrate of cellulase and 15% solid content, it is hydrolyzed at 50℃ and 180 rpm for 48 h to obtain the enzymatically hydrolyzed matrix. Example 9

[0053] The difference from Example 1 is that: (5) the pretreated biomass that has been crushed and passed through a 20-mesh sieve is added to an enzyme-containing buffer solution (pH=4.8) and placed in a constant temperature water bath shaker. Under the conditions of 30 FPU / g substrate of cellulase and 15% solid content, it is hydrolyzed at 50℃ and 180 rpm for 48 h to obtain the enzymatically hydrolyzed matrix. Example 10

[0054] The difference from Example 1 is that: (5) the pretreated biomass that has been crushed and passed through a 30-mesh sieve is added to an enzyme-containing buffer solution (pH=4.8) and placed in a constant temperature water bath shaker. Under the conditions of 30 FPU / g substrate of cellulase and 20% solid content, it is hydrolyzed at 50℃ and 180 rpm for 48 h to obtain the enzymatically hydrolyzed matrix. Example 11

[0055] The difference from Example 1 is that: (5) the pretreated biomass that has been crushed and passed through a 30-mesh sieve is added to an enzyme-containing buffer solution (pH=4.8) and placed in a constant temperature water bath shaker. Under the conditions of 30 FPU / g substrate of cellulase and 18% solid content, it is hydrolyzed at 50°C and 180 rpm for 48 h to obtain the enzymatically hydrolyzed matrix.

[0056] Comparative Example 1 Instead of mixed-culture fermentation, pretreatment with mechanical shredding and steam explosion was used to obtain pretreated biomass. The pulverized pretreated biomass was then hydrolyzed with cellulase. Lactic acid was prepared by fermentation with Bacillus coagulans using the obtained hydrolysate as a carbon source. This involves steps (3, 4, 5, and 6) in step 1.

[0057] Comparative Example 2 Instead of mixed-culture fermentation and mechanical shredding, pretreatment was performed directly using steam explosion to obtain pretreated biomass. The pulverized pretreated biomass was then enzymatically hydrolyzed using cellulase. Lactic acid was prepared by fermentation with Bacillus coagulans using the obtained enzymatically hydrolyzed substrate as a carbon source. This corresponds to steps (4, 5, and 6) in step 1.

[0058] Comparative Example 3 Without any pretreatment, the high-lignin-content biomass (cotton stalks) was directly hydrolyzed with cellulase; the resulting enzymatically hydrolyzed matrix was used as a carbon source for fermentation with Bacillus coagulans to prepare lactic acid. This is equivalent to steps (5 and 6) in step 1.

[0059] Example Sample Testing: The contents of glucose, xylose, and lactic acid obtained from enzymatic hydrolysis and fermentation were determined using liquid chromatography. The yield of lactic acid and the sugar-acid conversion rate were calculated. Three replicates were set up for each example and comparative cotton stalk-based enzymatic hydrolysis and fermentation experiment, and the average value was taken. The results are as follows: project Fermentable sugar concentration / g / L Acid production rate / g / (L·h) Sugar-acid conversion rate / % Example 1 67.8 0.809 85.9 Example 2 65.7 0.777 85.1 Example 3 63.3 0.745 84.7 Example 4 66.8 0.806 86.9 Example 5 49.9 0.549 79.2 Example 6 37.5 0.410 78.7 Example 7 66.7 0.801 86.5 Example 8 69.2 0.837 87.1 Example 9 67.5 0.809 86.3 Example 10 The concentration was higher at 74.9, but liquefaction was incomplete. 0.894 86.0 Example 11 73.6 Complete liquefaction 0.919 89.9 Comparative Example 1 64.8 0.759 84.0 Comparative Example 2 35.5 0.625 79.4 Comparative Example 3 14.2 0.313 69.1 In summary, the functional microbial ensemble of this invention offers comprehensive functional coverage with virtually no weaknesses. It encompasses all key functional modules of lignocellulose degradation—lignin depolymerization / modification, hemicellulose degradation, cellulose degradation, and aromatic debris removal—solving the potential problem of insufficient kinetics in initial lignin depolymerization in existing three-strain systems. This microbial ensemble exhibits synergistic effects, forming an efficient metabolic cycle and functional redundancy. For example, in the core cellulose degradation stage, lichens and Bacillus brevis constitute functional redundancy, making the system more robust. Even if the activity of one strain is temporarily low, another can take its place, ensuring the stability of the core function. The mixed strain of this invention has great application potential, capable of independently processing untreated or poorly pretreated natural lignocellulose raw materials, and has broad application prospects in areas such as efficient composting, straw return to the field, and biomass saccharification.

[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0061] In this specification, the terms "one embodiment," "another embodiment," "embodiment," etc., refer to specific features, structures, or characteristics described in connection with that embodiment, which are included in at least one embodiment described in the general description of this application. The appearance of the same term in multiple places in the specification does not necessarily refer to the same embodiment. Furthermore, when a specific feature, structure, or characteristic is described in connection with any embodiment, it is intended to suggest that implementing such a feature, structure, or characteristic in conjunction with other embodiments also falls within the scope of this invention.

[0062] Although the invention has been described herein with reference to several illustrative embodiments, it should be understood that many other modifications and implementations can be devised by those skilled in the art, which will fall within the scope and spirit of the principles disclosed herein. More specifically, various variations and modifications can be made to the components and / or layout of the subject matter combination within the scope of the disclosure and claims. Besides variations and modifications to the components and / or layout, other uses will be apparent to those skilled in the art.

Claims

1. A mixed-culture fermentation broth, characterized in that, include: Bacillus brevis; Bacillus licheniformis; Sterculia; Pseudomonas intermedia; And liquid culture media suitable for mixed bacterial growth; The mixed fermentation broth is obtained by culturing Bacillus shortiflorus, Bacillus licheniformis, Stellaria media, and Pseudomonas intermedia in a liquid culture medium or by fermenting them separately and then mixing them. The resulting mixed fermentation broth is used as a pretreatment for the preparation of lactic acid from biomass with high lignin content.

2. The mixed-culture fermentation broth according to claim 1, characterized in that, The liquid culture medium is LB liquid culture medium.

3. The mixed-culture fermentation broth according to claim 1, characterized in that, It is prepared from Bacillus shortiflorus, Bacillus licheniformis, Sterculia stolonifera, and Pseudomonas intermedia according to the fermentation liquid volume ratio (1-3):1:1:

1.

4. The mixed-culture fermentation broth according to claim 1, characterized in that, The OD value of the fermentation broths of Bacillus shortbread, Bacillus licheniformis, Sterculia lysate, and Pseudomonas intermedius was used for the separate fermentation and mixing of the aforementioned Bacillus shortbread, Bacillus licheniformis, Sterculia lysate, and Pseudomonas intermedius fermentation broths. 600 The values ​​are all 1.

2.

5. A method for preparing lactic acid from high-lignin-content biomass using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion pretreatment, characterized in that... include: Step 1: Prepare and adjust the high lignin-containing biomass, the mixed fermentation broth according to any one of claims 1-4, and Hertz buffer, mix them evenly, and then carry out aerobic fermentation. After the fermentation is completed, perform solid-liquid separation and collect the solid part and liquid part respectively. Step 2: Wash the solid separated in Step 1 to obtain characteristic biomass A; Step 3: Mechanically shred the biomass of characteristic A to obtain biomass of characteristic B; Step 4: After adding deionized water to the biomass of characteristic B to adjust the initial moisture content, steam explosion treatment is carried out. After the steam explosion is completed, the residue is collected and dried to obtain pretreated biomass. The pretreated biomass is then crushed. Step 5: Place cellulase in a weakly acidic buffer solution, then place the pulverized pretreated biomass in an enzyme-containing buffer solution for enzymatic hydrolysis to obtain the enzymatic hydrolysate; Step 6: Inoculate the enzymatic hydrolysis substrate with Bacillus coagulans solution activated in mMRS medium, stir and add NaHCO3 to adjust the pH value to 5.5-6.5, and ferment until the lactic acid concentration in the supernatant no longer increases significantly.

6. The method for preparing lactic acid from high-lignin-content biomass by pretreatment using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion, as described in claim 5, is characterized in that... In step 1, the volume ratio of high lignin-containing biomass, mixed fermentation broth, and Hertz buffer is 1:(0.1-0.5):

2.

7. The method for preparing lactic acid from high-lignin-content biomass by pretreatment using mixed-culture fermentation broth in conjunction with mechanical skeining coupled with steam explosion, as described in claim 5 or 6, is characterized in that... In step 1, the high-lignin biomass, mixed fermentation broth, and Hertz buffer are prepared in proportion, and the fermentation concentration is adjusted to 15-35%, and the pH value is adjusted to 7.

8. The method for preparing lactic acid from high-lignin-content biomass by pretreatment using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion, as described in claim 5, is characterized in that... In step 1, the specific process of aerobic fermentation includes: Place the fermentation raw materials in an aerobic fermenter, with the material height above 1 / 2, and maintain at 37℃ for 3 to 10 days. During this process, keep the aeration and stirring continuous, with an aeration rate of 1.0 vvm and a rotation speed of 200 rpm.

9. The method for preparing lactic acid from high-lignin-content biomass by pretreatment using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion, as described in claim 5, is characterized in that... In step 5, the acidic buffer solution is an acetic acid-sodium acetate buffer solution.

10. The method for preparing lactic acid from high-lignin-content biomass by pretreatment using mixed-culture fermentation broth in conjunction with mechanical shredding coupled with steam explosion, as described in claim 5, is characterized in that... In step 6, the inoculation amount of the fermentation broth is 5-15%.

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