Method for preparing lignin alkali solution and alkali cellulose from lignocellulose and application of lignin alkali solution and alkali cellulose

Abstract

The invention discloses a method for preparing a lignin alkali solution and alkali cellulose by utilizing lignocellulose and application of the lignin alkali solution and the alkali cellulose. The method provided by the invention comprises the following steps: step 1, carrying out puffing treatment on a lignocellulose raw material to obtain a puffed material; 2, washing the puffed material with water, and carrying out solid-liquid separation to obtain a liquid phase containing xylose and a solid phase material containing lignin and cellulose; step 3, adding a fermentation strain capable of generating ethanol and a sugar source into the xylose-containing liquid phase for fermentation to obtain fermentation liquor, and separating ethanol from the fermentation liquor; 4, the solid-phase material containing lignin and cellulose is dried, and water obtained through optional drying is used for washing in the step 2; 5, adding alkali liquor and ethanol into the solid-phase material containing lignin and cellulose for reaction, and performing solid-liquid separation to obtain an ethanol-containing lignin alkali solution and alkali cellulose; and 6, carrying out ethanol recovery on the ethanol-containing lignin alkali solution to obtain a lignin alkali solution, and reusing the optionally recovered ethanol to the step 5. According to the method, the newly developed saccharomyces cerevisiae strain is used for converting the byproduct xylose liquid in the lignocellulose pretreatment process into the ethanol, and the ethanol is used for preparing the alkali cellulose, so that efficient cyclic utilization of resources is realized, and the production cost is reduced.

Description

Technical Field

[0001] This invention relates to the technical field of biomass conversion and chemical treatment, and more specifically, to a method and application for preparing lignin alkaline solutions and alkali cellulose using lignocellulose. Background Technology

[0002] Lignocellulose, a widely distributed biomass resource in nature, is mainly composed of three components: cellulose, hemicellulose, and lignin. Cellulose is the main structural component of lignocellulose, possessing high crystallinity and good mechanical strength; hemicellulose complements cellulose by providing its non-crystalline properties, giving lignocellulose flexibility; while lignin acts as a natural binder, tightly binding cellulose and hemicellulose together to form a robust cell wall structure. Due to these properties, lignocellulose plays a vital ecological role in nature and is also an important source of energy and materials for humankind.

[0003] In the separation of lignocellulose into its three components, cellulose, hemicellulose, and lignin are extracted separately for further processing and application. This process produces a xylose-rich solution, known as xylose solution. Xylose solution contains a large amount of xylose, a pentose sugar, which can be converted into various valuable chemicals and biofuels, such as ethanol. However, in traditional industrial production, xylose solution is often considered a byproduct and neglected, failing to be fully utilized. This not only wastes resources but also increases production costs. Furthermore, the separated cellulose often requires alkalization to improve its reactivity or solubility for further processing or application. In traditional methods, ethanol production and cellulose alkalization are often two separate processes, leading to inefficient resource utilization and high costs.

[0004] Therefore, there is a need to develop a more economical method for treating lignocellulose that makes fuller use of resources. Summary of the Invention

[0005] To address the problems in existing technologies, this invention proposes a method and application for preparing lignin alkaline solutions and alkali cellulose using lignocellulose. This invention converts xylose solution, a byproduct of lignocellulose pretreatment, into ethanol, and then uses this ethanol to alkalize pretreated materials to obtain alkali cellulose. This method achieves efficient resource recycling and reduces production costs.

[0006] One objective of this invention is to provide a method for preparing lignin alkaline solutions and alkali cellulose using lignocellulose, comprising the following steps:

[0007] Step 1: Puff the lignocellulose raw material to obtain puffed material;

[0008] Step 2: Wash the puffed material with water and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose.

[0009] Step 3: Add fermentation bacteria and sugar source that can generate ethanol to the xylose-containing liquid phase obtained in Step 2 to ferment and obtain fermentation broth. Separate ethanol from the fermentation broth.

[0010] Step 4: Dry the solid material containing lignin and cellulose obtained in Step 2, and optionally use the water obtained from the drying process for washing in Step 2.

[0011] Step 5: Add alkali solution and ethanol to the solid material containing lignin and cellulose obtained in Step 4 to react. After solid-liquid separation, lignin alkali solution and alkali cellulose containing ethanol are obtained.

[0012] Step Six: The ethanol-containing wood in the solution of this invention is subjected to ethanol recovery to obtain a lignin alkaline solution. Optionally, the recovered ethanol is reused in Step Five.

[0013] The puffed material of the present invention includes xylose, lignin and cellulose.

[0014] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0015] Step 1,

[0016] The lignocellulose raw material is selected from at least one of corn stalks, corn cobs, rice straw, wheat straw, reeds, or bean straw; and / or,

[0017] The puffing process is an instantaneous puffing process performed during screw pressurization; preferably,

[0018] The temperature for the instantaneous expansion treatment of the screw pressurization process is 150℃~230℃; and / or,

[0019] The pressure of the instantaneous expansion treatment of the screw pressurization is 0.4 MPaG to 1.2 MPaG; and / or,

[0020] The instantaneous expansion process of the screw pressurization is 10 min to 40 min.

[0021] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0022] Step two,

[0023] The water washing process uses a solid-liquid mass ratio of 1:0.5-5; and / or,

[0024] The number of water washes is 1-6 times; and / or,

[0025] The solid-liquid separation method is selected from at least one of plate and frame filtration, centrifugal separation, screw extrusion dehydration, and vacuum belt filtration.

[0026] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0027] Step 3,

[0028] The fermentation strain capable of producing ethanol is a Saccharomyces cerevisiae strain, classified as Saccharomyces cerevisiae, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199, deposited on October 14, 2024, and named SEB23.

[0029] This invention uses xylose solution as a fermentation raw material. By adding appropriate microbial strains and nutrients, fermentation is carried out under suitable temperature, pH, and aeration conditions to obtain a fermentation broth containing ethanol. Subsequently, ethanol is separated from the fermentation broth through processes such as distillation to obtain a high-purity ethanol product.

[0030] The method for constructing the Saccharomyces cerevisiae strain of the present invention may include the following steps: expressing SIP18 at a high level in the starting strain to obtain strain SEB23; wherein, the starting strain is specifically strain SEB20, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.22590 (see Chinese patent CN114134058A for details).

[0031] The starting strain highly expresses SIP18 by replacing the endogenous promoter of the SIP18 gene with the TEF1 promoter to achieve high expression of SIP18.

[0032] Preferably, the method for constructing the *Saccharomyces cerevisiae* strain may include the following steps:

[0033] 1) Construct the gRNA plasmid pMEL13-SIP18;

[0034] 2) Construct a repair fragment for high SIP18 expression;

[0035] 3) Prepare Cas9-NAT plasmid;

[0036] 4) Transform the starting strain using CRISPR / Cas9.

[0037] More preferably, the method for constructing the *Saccharomyces cerevisiae* strain may specifically include the following steps:

[0038] Construct the gRNA plasmid pMEL13-SIP18 and the repair fragment for high expression of SIP18; prepare the Cas9-NAT plasmid;

[0039] First, the Cas9-NAT plasmid was introduced into the strain; then, the pMEL13-SIP18 plasmid and the SIP18 high-expression repair fragment were introduced into the strain containing the Cas9-NAT plasmid, and the plasmid was removed from the transformants to obtain the SIP18 high-expression strain SEB23.

[0040] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0041] Step 3,

[0042] The inoculum size of the ethanol-producing fermenting strain is 0.5–2 g cells (dry weight) / L of the fermentation broth; and / or,

[0043] The fermentation temperature is 28℃~40℃; and / or,

[0044] The fermentation time is 48h to 144h; and / or,

[0045] The fermentation pH is 5–7;

[0046] Ethanol can be separated from the fermentation broth by at least one of distillation, membrane separation, or pervaporation.

[0047] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0048] Step 3,

[0049] The sugar source is selected from glucose; preferably, the amount of sugar source added is 40-100 g / L of the fermentation broth.

[0050] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0051] Step 5,

[0052] The alkali in the alkaline solution is selected from at least one of sodium hydroxide or potassium hydroxide; and / or,

[0053] The solvent in the alkaline solution is selected from water, and / or,

[0054] In the reaction system, the mass ratio of ethanol, lignin and cellulose solid phase, alkali and water is (10-26):(30-110):(12-30):(30-110).

[0055] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0056] Step 5,

[0057] The reaction temperature is 25–60 °C; and / or,

[0058] The reaction time is 1.5 to 5 hours.

[0059] In the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to the present invention, preferably,

[0060] Step Six,

[0061] The ethanol recovery method is selected from at least one of distillation, membrane separation, and pervaporation separation.

[0062] The second objective of this invention is to provide the application of the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose as described in the first objective of this invention in the resource utilization of lignocellulose.

[0063] The substances and parameters not limited in this invention can be selected according to existing technology, which is a conventional technical means in this field.

[0064] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values; these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein. In the following, various technical solutions can, in principle, be combined with each other to obtain new technical solutions, which should also be considered as specifically disclosed herein.

[0065] This invention utilizes a newly developed strain of *Saccharomyces cerevisiae* to convert xylose solution, a byproduct of lignocellulose pretreatment, into ethanol, which is then used in the preparation of alkali cellulose. Compared with existing technologies, this method has the following advantages:

[0066] Cost reduction: The use of ethanol for alkalization of cellulose through internal recycling reduces costs. Ethanol in the lignin alkali solution can be recovered and reused through methods such as distillation, forming a closed-loop production model.

[0067] Environmentally friendly: Reduces the use and emission of chemicals, lowering the risk of environmental pollution.

[0068] Highly innovative: It proposes an innovative biomass conversion and chemical treatment method that directly produces lignin alkaline solution and alkali cellulose from solid raw materials containing lignin and cellulose, providing new ideas and technical support for the sustainable development of biomass resources.

[0069] Product Uses: Alkali cellulose can react with etherifying agents to form cellulose ethers, which are a wide variety of cellulose derivatives with broad applications.

[0070] Microbial strain preservation information

[0071] The inhibitor-resistant Saccharomyces cerevisiae strain is classified as Saccharomyces cerevisiae, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 32199, deposited on October 14, 2024, and named SEB23. Detailed Implementation

[0072] The present invention will now be described in detail with reference to specific embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the content of the present invention are still within the scope of protection of the present invention.

[0073] It should also be noted that the various specific technical features described in the following embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the various possible combinations will not be described separately in this invention.

[0074] Furthermore, various embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention. The resulting technical solutions are part of the original disclosure of this specification and also fall within the protection scope of the present invention.

[0075] Unless otherwise specified, the raw materials used in the examples and comparative examples are all disclosed in the prior art, such as those that can be directly purchased or prepared according to the preparation methods disclosed in the prior art.

[0076] Preparation Example 1

[0077] Construction of strain SEB23

[0078] 1. Starting strain

[0079] The originating strain is SEB20, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 22590.

[0080] 2. Culture medium

[0081] The culture media used are shown in Table 1. If solid culture media are used, add 2% agar powder before sterilization. The sterilization conditions are 0.1 MPa, 121°C, and 15 min. After the culture medium cools to 50–60°C, add antibiotics.

[0082] Table 1. Culture medium composition

[0083]

[0084] 3. Plasmids, bacterial strains, and primers

[0085] The plasmids and strains used are shown in Table 2; the primers used to construct the gRNA plasmids are shown in Table 3; and the primers used for strain transformation are shown in Table 4.

[0086] Table 2 Plasmid and strain information

[0087]

[0088] (Existing literature [1] Mans R, van Rossum HM, Wijsman M, et al. CRISPR / Cas9: amolecular swiss army knife f or simultaneous introduction of multiple genetic modifications in Saccharomyces cerevisiae [J]. FEMS Yeast Research, 2015, 15(2): fov004.; Existing literature [2] Zhang GC, Kong II, Kim H, et al. Construction of a quadrupleauxotrophic mutant of an industrial polyploid Saccharomyces cerevisiae strain by using RNA-guided Cas9 nuclease[J].Applied and Environmental Microbiology, 2014,80(24):7694-7701.)

[0089] Table 3 Primers for constructing gRNA plasmids

[0090]

[0091] Note: F: upstream primer; R: downstream primer; underline: 20bp recognition sequence.

[0092] The serial number of SIP18 TG F is SEQ ID No. 1, the serial number of SIP18 TG R is SEQ ID No. 2, the serial number of 6006-F is SEQ ID No. 3, and the serial number of 6005-R is SEQ ID No. 4.

[0093] Table 4 Primers required for strain transformation

[0094]

[0095] Note: RF: Repair fragment; Vp: Validation primer; F: Upstream primer; R: Downstream primer

[0096] The serial number of SIP18 RF F is SEQ ID No. 5, the serial number of SIP18 RF R is SEQ ID No. 6, the serial number of SIP18 Vp F is SEQ ID No. 7, and the serial number of SIP18 Vp R is SEQ ID No. 8.

[0097] The remaining unspecified raw materials, processes, instruments, and operating methods are all conventional commercially available materials or conventional technologies in this field.

[0098] 4. Construction of strain SEB23

[0099] 4.1 Construction of gRNA plasmid pMEL13-SIP18

[0100] 1) Constructing double-stranded gRNA fragments

[0101] Using the genome information of the model yeast S288c as a reference, the base sequence of the promoter region of the SIP18 gene was searched on the Saccharomyces Genome Database (SGD https: / / www.yeastgenome.org / ). This sequence was input into the CRISPOR (CRISPOR(tefor.net)) website to obtain the gRNA recognition sequence (20 bp). Primer pairs SIP18 TG F / R (sequences shown in Table 3) containing the 20 bp recognition sequence and 50 bp homologous arms were synthesized. The two single-stranded primers were diluted to 10 μM with sterile water, mixed in equal volumes, heated at 95°C for 5 min, and cooled to room temperature to obtain the double-stranded gRNA fragment.

[0102] 2) Amplifying the linear backbone of the gRNA plasmid

[0103] The linear backbone of the gRNA plasmid was amplified using pMEL13 plasmid as a template. The PCR reaction system and conditions are shown in Table 5. The linear backbone of the gRNA plasmid was obtained after purification of the PCR product.

[0104] Table 5. PCR amplification of gRNA linear backbone

[0105]

[0106] The template plasmid was digested using FastDigest DpnI. The digestion system and reaction conditions are shown in Table 6. The amount of DpnI used was determined by the amount of plasmid (1 μg plasmid plus 1 μL DpnI). The gRNA linear backbone was obtained after purification.

[0107] Table 6. GRNA linear backbone template digestion system

[0108]

[0109] 3) Gibson connects gRNA fragments to the linear backbone.

[0110] The gRNA fragment and pMEL13 linear backbone were ligated via homologous recombination. The Gibson ligation reaction system and conditions are shown in Table 7. 5 μL of ligation buffer was transformed into *E. coli*, and the bacterial culture was plated on LB+Kan plates and incubated overnight at 37°C. Transformants were inoculated into test tubes containing 5 mL of LB+Kan liquid medium and incubated for 12–16 h (160 rpm, 37°C). The bacterial cells were collected, and the gRNA plasmid was extracted. Sequencing confirmed the correct gRNA plasmid pMEL13-SIP18.

[0111] Table 7 Gibson linkage reaction system

[0112]

[0113] 4.2 Constructing the Repair Fragment

[0114] The endogenous promoter of SIP18 was replaced with the TEF1 promoter to achieve high expression of SIP18. Using genomic DNA from strain SEB20 as a template, the repair fragment containing the TEF1 promoter sequence and homologous arms was amplified using primers SIP18 RF F and SIP18 RF R (primer sequences are shown in Table 4, and TEF1 promoter sequences are shown in Table 8). The PCR reaction system and conditions are shown in Table 9. The PCR product was purified and used for transformation.

[0115] Table 8 TEF1 Starter Sequence

[0116]

[0117] P TEF1 Its serial number is SEQ ID No. 9.

[0118] Table 9. PCR Amplification Repair Fragments

[0119]

[0120] 4.3 Preparation of Cas9-NAT plasmid

[0121] E. coli containing the Cas9-NAT plasmid were inoculated into 5 mL of LB+NAT liquid medium and cultured at 37°C with shaking at 160 rpm for 12–16 h. The bacterial cells were collected by centrifugation, and the Cas9-NAT plasmid was extracted.

[0122] 4.4 CRISPR / Cas9 Conversion

[0123] (1) Cas9-NAT plasmid was introduced into Saccharomyces cerevisiae strains via lithium acetate conversion.

[0124] a. Activate the SEB20 strain by streaking it onto a 2% YPD plate, and inoculate an appropriate amount of the bacterial cells into 5 mL of 2% YPD liquid medium. Incubate at 30°C and 160 rpm for 16 h with shaking.

[0125] b. Inoculate 2 mL of bacterial culture into 100 mL of 2% YPD culture medium and incubate at 30°C and 160 rpm for 2–3 hours. When OD 600 When the concentration reaches 0.2-0.3, centrifuge to collect all bacterial cells. Wash the bacterial precipitate twice with sterile water and resuspend it in 300 μL of sterile water. Place it on ice for later use.

[0126] c. Boil salmon sperm DNA (ssDNA) at 100℃ for 5 minutes, then place on ice for later use;

[0127] d. Add 60% PEG4000 (115 μL), 4M lithium acetate solution (5 μL), ssDNA (10 μL), Cas9-NAT plasmid (100 ng) and yeast cells (50 μL) to a 1.5 mL centrifuge tube in sequence, mix well and heat shock at 42 °C for 40 min.

[0128] e. Centrifuge at 8,000×g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and incubate at 30℃ and 160 rpm for 2–3 h.

[0129] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, and resuspend the cells in 1 mL of sterile water. Spread 100 μL of the bacterial culture onto a 2% YPD+NAT plate and incubate at 30°C for 2–3 days.

[0130] g. Streak the transformants on 2% YPD+NAT plates. Transformants that grow contain the Cas9-NAT plasmid.

[0131] (2) High expression of SIP18

[0132] a. The strain SEB20Cas9 containing the Cas9-NAT plasmid was streaked onto a 2% YPD+NAT plate for activation. An appropriate amount of bacterial cells was inoculated into 5 mL of 2% YPD+NAT liquid medium and cultured at 30℃ and 160 rpm for 16 h with shaking.

[0133] b. Inoculate 2 mL of bacterial culture into 100 mL of 2% YPD+NAT culture medium and incubate at 30°C and 160 rpm for 2–3 hours. When OD 600 When the concentration reaches 0.2-0.3, centrifuge to collect all bacterial cells. Wash the bacterial precipitate twice with sterile water and resuspend it in 300 μL of sterile water. Place it on ice for later use.

[0134] c. Boil salmon sperm DNA (ssDNA) at 100℃ for 5 minutes, then place on ice for later use;

[0135] d. In a 1.5 mL centrifuge tube, add 60% PEG4000 (240 μL), 4M lithium acetate solution (9 μL), ssDNA (25 μL), pMEL13-SIP18 plasmid (600 ng), and repair fragment (1.5–2.5 μg) in sequence. Add sterile water to make up to 351 μL, add yeast cells (50 μL), mix well, and heat shock at 42 °C for 40 min.

[0136] e. Centrifuge at 8,000×g for 1 min, discard the supernatant, wash the cells twice with sterile water, add 1 mL of 2% YPD culture medium, and incubate at 30℃ and 160 rpm for 2–3 h.

[0137] f. Centrifuge at 8,000×g for 1 min, discard the supernatant, resuspend the cells in 150 μL of sterile water, spread all the bacterial culture on 2% YPD+NAT+G418 plates, incubate at 30℃ for 2-3 days, and pick transformants for colony PCR verification.

[0138] 4.5 Colony PCR Validation

[0139] a. Add 95 μL of 1% SDS, 5 μL of 4M lithium acetate solution and an appropriate amount of bacterial cells to a 1.5 mL centrifuge tube in sequence, vortex and incubate at 75 °C for 10 min;

[0140] b. Add 300 μL of anhydrous ethanol to the centrifuge tube and vortex; centrifuge at 13000 rpm at room temperature for 3 min, discard the supernatant, and dry at 37°C for 10 min with the cap open.

[0141] c. Add 100 μL of sterile water, vortex, centrifuge at 13000 rpm at room temperature for 1 min, and retain the supernatant;

[0142] d. Measure the concentration of the supernatant, adjust the OD to 10-20 ng / μL, take 1 μL as a template for PCR verification, and the PCR reaction system and reaction conditions are shown in Table 10.

[0143] e. 1.5% agarose gel electrophoresis (100V, 30min) was used to verify the target band, and the PCR products with the correct bands were sequenced for confirmation.

[0144] Table 10 Colony PCR Validation System

[0145]

[0146] 4.6 Plasmid Removal

[0147] The specific steps for removing the Cas9-NAT plasmid and pMEL13-SIP18 plasmid from the correct transformant are as follows:

[0148] a. Streak the correct transformants on a 2% YPD plate, pick a small number of cells and seed them in 10 mL of 2% YPD medium, and incubate at 30°C for 1 day.

[0149] b. Take 200 μL of bacterial culture and dilute it 10 μL. 5 Double the amount of bacterial culture, spread 100 μL of the culture onto a 2% YPD plate, and incubate for 1–2 days.

[0150] c. Resuspend a small number of cells in 200 μL of sterile water (pick 5-8 single bacteria from each transformant), and spot 2 μL of each bacterial suspension onto 2% YPD, 2% YPD+G418, and 2% YPD+NAT plates respectively, and incubate at 30℃ for 1-2 days; strains that can only grow on 2% YPD plates have successfully had their plasmids removed. The final SIP18 high-expression strain SEB23 is obtained.

[0151] Example 1

[0152] A method for preparing lignin alkaline solution and alkali cellulose using lignocellulose includes the following steps:

[0153] Step 1: The lignocellulose raw material is subjected to screw compression instantaneous puffing treatment to obtain puffed material; wherein, the temperature of the screw compression instantaneous puffing treatment is 165℃; the pressure of the screw compression instantaneous puffing treatment is 0.6MPaG; and the time of the screw compression instantaneous puffing treatment is 30min.

[0154] Step 2: Wash the puffed material with water at a solid-liquid mass ratio of 1:0.9, wash 3 times, and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose.

[0155] Step 3: Add ethanol-producing fermentation bacteria and glucose to the xylose-containing liquid phase obtained in Step 2 for fermentation. Add glucose to a concentration of 40 g / L for the fermentation broth. The inoculum size of the fermentation bacteria is 1 g cells (dry weight) / L for the fermentation broth. The fermentation temperature is 35℃. The fermentation time is 96 h. The fermentation pH is 6. The resulting fermentation broth contains approximately 18 g / L of ethanol. Ethanol is then separated from the fermentation broth by distillation. The fermentation strain is the *Saccharomyces cerevisiae* strain from Preparation Example 1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199 and accession name SEB23.

[0156] Step 4: Dry the solid material containing lignin and cellulose from Step 2, and use the water obtained from the drying process for washing in Step 2.

[0157] Step 5: Add sodium hydroxide aqueous solution and ethanol to the solid phase material containing lignin and cellulose from Step 4 to carry out the reaction. The mass ratio of ethanol, solid phase of lignin and cellulose, sodium hydroxide and water is 15:55:19:55. After solid-liquid separation, a lignin alkaline solution containing ethanol and alkali cellulose are obtained.

[0158] Step 6: The ethanol-containing lignin base solution is distilled to recover the ethanol, and the recovered ethanol is reused in Step 5.

[0159] Example 2

[0160] A method for preparing lignin alkaline solution and alkali cellulose using lignocellulose includes the following steps:

[0161] Step 1: The lignocellulose raw material is subjected to screw compression instantaneous puffing treatment to obtain puffed material; wherein, the temperature of the screw compression instantaneous puffing treatment is 150℃; the pressure of the screw compression instantaneous puffing treatment is 0.8MPaG; and the time of the screw compression instantaneous puffing treatment is 40min.

[0162] Step 2: Wash the puffed material with water at a solid-liquid mass ratio of 1:2; wash 3 times and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose.

[0163] Step 3: Add ethanol-producing fermentation bacteria and glucose to the xylose-containing liquid phase obtained in Step 2 for fermentation. Add glucose to a concentration of 50 g / L for the fermentation broth. The inoculum size of the fermentation bacteria is 1.4 g cells (dry weight) / L for the fermentation broth. The fermentation temperature is 28℃. The fermentation time is 138 h. The fermentation pH is 6. The resulting fermentation broth contains ethanol. Ethanol is then separated from the fermentation broth by distillation. The fermentation strain is the *Saccharomyces cerevisiae* strain from Preparation Example 1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199 and accession name SEB23.

[0164] Step 4: Dry the solid material containing lignin and cellulose from Step 2, and use the water obtained from the drying process for washing in Step 2.

[0165] Step 5: Add a mixture of sodium hydroxide solution and ethanol to the solid phase material containing lignin and cellulose from Step 4 to carry out the reaction. The mass ratio of ethanol, solid phase of lignin and cellulose, sodium hydroxide and water is 16:76:20:72. After solid-liquid separation, a lignin alkaline solution containing ethanol and alkali cellulose are obtained.

[0166] Step 6: The ethanol-containing lignin base solution is distilled to recover the ethanol, and the recovered ethanol is reused in Step 5.

[0167] Example 3

[0168] A method for preparing lignin alkaline solution and alkali cellulose using lignocellulose includes the following steps:

[0169] Step 1: The lignocellulose raw material is subjected to screw compression instantaneous puffing treatment to obtain puffed material; wherein, the temperature of the screw compression instantaneous puffing treatment is 200℃; the pressure of the screw compression instantaneous puffing treatment is 0.5MPaG; and the time of the screw compression instantaneous puffing treatment is 15min.

[0170] Step 2: Wash the puffed material with water at a solid-liquid mass ratio of 1:3; wash 3 times and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose.

[0171] Step 3: Add ethanol-producing fermentation bacteria and glucose to the xylose-containing liquid phase obtained in Step 2 for fermentation. Add glucose to a concentration of 60 g / L for the fermentation broth. The inoculum size of the fermentation bacteria is 2 g cells (dry weight) / L for the fermentation broth. The fermentation temperature is 40℃. The fermentation time is 55 h. The fermentation pH is 6.5. The resulting fermentation broth contains ethanol. Ethanol is then separated from the fermentation broth by distillation. The fermentation strain is the *Saccharomyces cerevisiae* strain from Preparation Example 1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199 and accession name SEB23.

[0172] Step 4: Dry the solid material containing lignin and cellulose from Step 2, and use the water obtained from the drying process for washing in Step 2.

[0173] Step 5: Add a mixture of sodium hydroxide solution and ethanol to the solid material containing lignin and cellulose from Step 4 to carry out the reaction. The mass ratio of ethanol, solid phase of lignin and cellulose, sodium hydroxide and water is 12:40:13:40. After solid-liquid separation, a lignin alkaline solution containing ethanol and alkali cellulose are obtained.

[0174] Step 6: The ethanol-containing lignin base solution is distilled to recover the ethanol, and the recovered ethanol is reused in Step 5.

[0175] Example 4

[0176] A method for preparing lignin alkaline solution and alkali cellulose using lignocellulose includes the following steps:

[0177] Step 1: The lignocellulose raw material is subjected to screw compression instantaneous puffing treatment to obtain puffed material; wherein, the temperature of the screw compression instantaneous puffing treatment is 190℃; the pressure of the screw compression instantaneous puffing treatment is 0.9MPaG; and the time of the screw compression instantaneous puffing treatment is 25min.

[0178] Step 2: Wash the puffed material with water at a solid-liquid mass ratio of 1:4; wash twice and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose.

[0179] Step 3: Add ethanol-producing fermentation bacteria and glucose to the xylose-containing liquid phase obtained in Step 2 for fermentation. Add glucose to a concentration of 60 g / L for the fermentation broth. The inoculum size of the fermentation bacteria is 0.8 g cells (dry weight) / L for the fermentation broth. The fermentation temperature is 30℃. The fermentation time is 110 h. The fermentation pH is 5.5. An ethanol-containing fermentation broth is obtained. Ethanol is then separated from the fermentation broth by distillation. The fermentation strain is the *Saccharomyces cerevisiae* strain from Preparation Example 1, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199 and accession name SEB23.

[0180] Step 4: Dry the solid material containing lignin and cellulose from Step 2, and use the water obtained from the drying process for washing in Step 2.

[0181] Step 5: Add a mixture of sodium hydroxide solution and ethanol to the solid material containing lignin and cellulose from Step 4 to carry out the reaction. The mass ratio of ethanol, solid phase of lignin and cellulose, sodium hydroxide and water is 24:100:30:100. After solid-liquid separation, a lignin alkaline solution containing ethanol and alkali cellulose are obtained.

[0182] Step 6: The ethanol-containing lignin base solution is distilled to recover the ethanol, and the recovered ethanol is reused in Step 5.

[0183] The lignin yield and alkali cellulose yield in the lignin alkaline solutions obtained in Examples 1-4 above are summarized in Table 11, as follows:

[0184] Table 11

[0185] Example Lignin yield in lignin alkaline solution Alkali cellulose yield Example 1 74.1％ 160.2％ Example 2 72.2％ 179.9％ Example 3 70.1％ 200.1％ Example 4 68.3％ 219.8％

[0186] in:

[0187] Lignin yield in lignin alkaline solution = mass of lignin in lignin alkaline solution / mass of lignin in straw;

[0188] Alkali cellulose yield = mass of alkali cellulose obtained in the experiment / mass of cellulose contained in the straw.

[0189] As can be seen from the results in Table 11, the present invention utilizes a newly developed Saccharomyces cerevisiae strain to convert xylose solution, a byproduct of lignocellulose pretreatment, into ethanol, and then uses this ethanol in the preparation of alkali cellulose, thereby achieving efficient recycling of resources and reducing production costs.

[0190] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

[0191] All publications, patent applications, patents, and other references mentioned in this specification are incorporated herein by reference. Unless otherwise defined, all technical and scientific terms used in this specification have the meanings commonly understood by those skilled in the art. In case of conflict, the definitions in this specification shall prevail.

[0192] When this specification uses the prefixes “known to those skilled in the art,” “prior art,” or similar terms to derive materials, substances, methods, steps, apparatus, or components, the objects derived from such prefixes cover those commonly used in the art at the time of this application, but also include those that are not currently commonly used but will become generally recognized in the art as suitable for similar purposes.

[0193] In the context of this specification, except where expressly stated otherwise, any matters or issues not mentioned shall apply directly to those known in the art without any modification.

Claims

1. A method for preparing lignin alkaline solution and alkali cellulose using lignocellulose, characterized in that, Includes the following steps: Step 1: Puff the lignocellulose raw material to obtain puffed material; Step 2: Wash the puffed material with water and separate the solid and liquid phases to obtain a liquid phase containing xylose and a solid phase containing lignin and cellulose. Step 3: Add fermentation bacteria and sugar source that can generate ethanol to the xylose-containing liquid phase obtained in Step 2 to ferment and obtain fermentation broth. Separate ethanol from the fermentation broth. Step 4: Dry the solid material containing lignin and cellulose obtained in Step 2, and optionally use the water obtained from the drying process for washing in Step 2. Step 5: Add alkali solution and ethanol to the solid material containing lignin and cellulose obtained in Step 4 to react. After solid-liquid separation, lignin alkali solution and alkali cellulose containing ethanol are obtained. Step 6: The ethanol-containing lignin base solution is subjected to ethanol recovery to obtain a lignin base solution. Optionally, the recovered ethanol is reused in Step 5.

2. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 1, The lignocellulose raw material is selected from at least one of corn stalks, corn cobs, rice straw, wheat straw, reeds, or bean straw; and / or, The puffing process is an instantaneous puffing process performed during screw pressurization; preferably, The temperature for the instantaneous expansion treatment of the screw pressurization process is 150℃~230℃; and / or, The pressure of the instantaneous expansion treatment of the screw pressurization is 0.4 MPaG to 1.2 MPaG; and / or, The instantaneous expansion process of the screw pressurization is 10 min to 40 min.

3. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step two, The water washing process uses a solid-liquid mass ratio of 1:0.5-5; and / or, The number of water washes is 1-6 times; and / or, The solid-liquid separation method is selected from at least one of plate and frame filtration, centrifugal separation, screw extrusion dehydration, and vacuum belt filtration.

4. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 3, The fermentation strain capable of producing ethanol is a Saccharomyces cerevisiae strain, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.32199, deposited on October 14, 2024, and named SEB23.

5. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 3, The inoculum size of the ethanol-producing fermenting strain is 0.5–2 g cells (dry weight) / L of the fermentation broth; and / or, The fermentation temperature is 28℃~40℃; and / or, The fermentation time is 48h to 144h; and / or, The fermentation pH is 5–7; Ethanol can be separated from the fermentation broth by at least one of distillation, membrane separation, or pervaporation.

6. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 3, The sugar source is selected from glucose; preferably, the amount of sugar source added is 40-100 g / L of the fermentation broth.

7. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 5, The alkali in the alkaline solution is selected from at least one of sodium hydroxide or potassium hydroxide; and / or, The solvent in the alkaline solution is selected from water; and / or, In the reaction system, the mass ratio of ethanol, lignin and cellulose solid phase, alkali and water is (10-26):(30-110):(12-30):(30-110).

8. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step 5, The reaction temperature is 25–60 °C; and / or, The reaction time is 1.5 to 5 hours.

9. The method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to claim 1, characterized in that: Step Six, The ethanol recovery method is selected from at least one of distillation, membrane separation, and pervaporation separation.

10. The application of the method for preparing lignin alkaline solution and alkali cellulose using lignocellulose according to any one of claims 1-9 in the resource utilization of lignocellulose.

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