Directional dissociation method for biomass fiber cell walls

Abstract

The invention provides a method for directional dissociation of biomass fiber cell walls. The method comprises the following steps: a mechanical pulverization step: carrying out mechanical pulverization treatment on a biomass material to obtain a mechanical pulverization product; a substrate softening step: carrying out softening treatment on the mechanically crushed product by using a basic nutrient solution to obtain a softened substrate; and a biological defibering step: carrying out biological defibering treatment on the softened substrate by utilizing microbial strain fermentation to obtain a biological defibering product. Through a microbial biological defibering technology, directional stripping degradation of biomass fiber cell walls is realized. According to the method, the difficulty that the traditional lignin, hemicellulose and cellulose are mixed and decomposed, and the product utilization rate is low is broken through, so that substances in the biomass fiber cell wall can be utilized to the maximum extent.

Description

Technical Field

[0001] This invention relates to a method for the directional dissociation of biomass fiber cell walls, belonging to the field of biomass processing. Specifically, it relates to a method for the directional dissociation of hemicellulose and partially controlled cellulose in biomass fiber cell walls based on a microbial fermentation-mediated biodegradation mechanism. Background Technology

[0002] Bamboo fiber cell walls are mainly composed of cellulose, hemicellulose, and lignin. Cellulose is the primary skeletal component of the bamboo cell wall, existing in the form of microfibrils, which endow the cell wall with high strength and rigidity. Hemicellulose fills the spaces between the cellulose microfibrils, serving to connect and support the cellulose, while also helping to maintain the stability and flexibility of the cell wall. Lignin, a complex phenolic polymer, permeates the network structure of cellulose and hemicellulose, further enhancing the cell wall's rigidity and resistance to degradation.

[0003] The cell walls of bamboo fibers can be specifically divided into visible layers (wide layers) and dark layers (narrow layers). The difference between them stems from significant variations in the arrangement direction of microfibrils and the content of lignin / hemicellulose. In bamboo fibers, the visible layer is supported by longitudinal cellulose as its core, while the dark layer is reinforced by lignin-hemicellulose as its core. Together, they form the unique multi-layered structure of bamboo fibers. This structure gives bamboo high strength but also results in poor permeability, susceptibility to mold, and difficulty in disintegration.

[0004] Currently, methods for treating bamboo fiber cell walls generally include chemical treatment, mechanical treatment, and enzymatic treatment. Chemical treatment can involve acid-base treatment or organic solvent treatment. Acid-base treatment involves treating bamboo with solutions of different concentrations of acid (such as sulfuric acid, hydrochloric acid, etc.) or alkali (such as sodium hydroxide, etc.) to disrupt the connecting structures between cell walls through chemical reactions, causing cell wall dissociation. For example, soaking bamboo in an appropriate concentration of sodium hydroxide solution at a certain temperature can promote changes in components such as lignin, thereby facilitating fiber dissociation. Organic solvent treatment involves using organic solvents, such as ethanol and acetone, to react with certain components in bamboo, aiding in the separation of cell walls. These organic solvents can dissolve some substances that hinder fiber dissociation, making cell walls easier to separate. However, while damaging lignin and hemicellulose, they also easily damage the chemical structure of cellulose itself (such as causing peeling reactions and excessive hydrolysis), leading to a sharp decrease in the degree of polymerization of cellulose and loss of strength.

[0005] Mechanical processing methods include grinding and high-pressure homogenization. Different types of grinding equipment, such as ball mills and vibratory mills, can be used, controlling appropriate grinding parameters, such as grinding time and rotation speed, to achieve better dissociation effects. High-pressure homogenization involves mixing bamboo material with an appropriate amount of liquid to create a suspension, then passing it through a high-pressure homogenizer to break down the cells under high pressure. High-pressure homogenization generates strong shear forces and pressures, effectively disrupting the cell wall structure and achieving fiber dissociation. However, mechanical processing methods are energy-intensive and rely primarily on random shear forces to break fibers, making it impossible to distinguish between visible and dark layers. This results in severely shortened fiber lengths, producing a large amount of fine debris, destroying the aspect ratio advantage of cellulose as a reinforcing phase, and significantly reducing material performance.

[0006] Enzymatic hydrolysis involves using specific enzymes, such as cellulase and hemicellulase, to act on the cell walls of bamboo fibers. These enzymes specifically break down cellulose and hemicellulose components within the cell walls, leading to the gradual dissociation of the cell walls. By controlling the type, concentration, reaction time, and temperature of the enzymes, the hydrolysis effect can be optimized to achieve a better state of fiber dissociation. Although enzymatic hydrolysis operates under mild conditions, it lacks the synergistic effect of dynamic enzyme production by microorganisms. Enzyme molecules diffuse randomly and attack all accessible substrates. Due to the complexity of the cell walls, enzymes struggle to penetrate efficiently and work synergistically, often resulting in uncontrollable damage to the target cellulose in the exposed layer or failure to dissociate the barrier in the dark layer.

[0007] Given the technical problems existing in the current technology, it is an urgent technical problem to be solved to develop a method that can directionally dissociate the cell wall of biomass fibers. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] In view of the technical problems existing in the prior art, such as: during dissociation, the chemical structure of cellulose itself is easily damaged while destroying lignin and hemicellulose; or the fiber length is severely cut, producing a large amount of fine debris; or the barrier of the visible or dark layer is damaged and cannot be dissociated, the present invention first provides a method for the directional dissociation of biomass fiber cell walls. The method of the present invention effectively solves the two major problems of bamboo material cell wall structure being dense and difficult to directionally penetrate, and lignin, cellulose and hemicellulose being tightly cross-linked and difficult to dissociate individually, thus achieving precise control of bamboo fiber cell wall composition.

[0010] Solution for solving the problem

[0011] This invention provides a method for the directional dissociation of cell walls in biomass fibers, comprising the following steps:

[0012] Mechanical pulverization step: The biomass material is mechanically pulverized to obtain the mechanically pulverized product;

[0013] Substrate softening step: The mechanically pulverized product is softened using a basic nutrient solution to obtain a softened substrate;

[0014] Bioremoval step: The softened substrate is bioremoved by fermentation with microbial strains to obtain bioremoval products.

[0015] According to the method of the present invention, the mechanical crushing process includes coarse crushing and fine crushing, and the product after the fine crushing is graded to obtain a mechanically crushed product with a particle size of 5-50 mm.

[0016] According to the method of the present invention, the basic nutrient solution includes a nitrogen source and a phosphorus source; preferably, the mass ratio of the nitrogen source to the phosphorus source is 3-5:1.

[0017] More preferably, based on the total mass of the mechanically pulverized product as 100%, the amount of nitrogen source added is 1-3%; and the amount of phosphorus source added is 0.3-1%.

[0018] More preferably, the softening substrate has a moisture content of 60-70%.

[0019] According to the method of the present invention, the nitrogen source includes one or a combination of two of urea and yeast extract, and the phosphorus source includes one or a combination of two of potassium dihydrogen phosphate and diammonium hydrogen phosphate.

[0020] According to the method of the present invention, in the bio-degradation step, the amount of microbial strain used is 0.015-0.15% based on the total mass of the mechanically pulverized product as 100%.

[0021] According to the method of the present invention, the microbial strain includes Clostridium species, and preferably, the microbial strain also includes other bacterial groups;

[0022] Based on the total mass of the mechanically pulverized product as 100%, the amount of Clostridium species used is 0.005-0.05%, and the amount of other bacterial groups used is 0.01-0.1%.

[0023] According to the method of the present invention, the Clostridium species include one or more of the following: Clostridium thermophilum, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficile.

[0024] Other microbial groups include one or more of Aspergillus niger, Penicillium, and Bacillus subtilis.

[0025] According to the method of the present invention, the biodegradation treatment includes an aerobic fermentation stage and an anaerobic fermentation stage;

[0026] Preferably, the aerobic fermentation stage includes intermittent aeration under conditions of 25-60℃ and pH 3.0-6.0, and the aerobic fermentation stage lasts for 1-5 days.

[0027] Preferably, the anaerobic fermentation stage includes stopping aeration at a temperature of 25-60℃ and a pH of 4.0-6.5, and the duration of the anaerobic fermentation stage is 7-15 days.

[0028] According to the method of the present invention, the number of bioremoval treatments is two or more, the duration of each bioremoval treatment is 10-18 days, and an intermediate activation treatment is performed between two adjacent bioremoval treatments.

[0029] Preferably, the intermediate activation treatment includes immersing the bioresorption product in a buffer solution at 30-35°C and pH 4.5-5.0 for 2-4 hours; more preferably, the buffer solution includes a citrate-sodium citrate buffer solution.

[0030] According to the method of the present invention, the bioremoval step is followed by a post-processing step, preferably, the post-processing step includes distilling and inactivating the bioremoval product at 100-120°C for 15-30 min.

[0031] The effects of the invention

[0032] This invention utilizes microbial biodegradation technology to achieve the targeted exfoliation and degradation of biomass fiber cell walls. This invention overcomes the difficulties of traditional methods involving the mixing of lignin, hemicellulose, and cellulose, resulting in low product utilization rates, thereby maximizing the utilization of substances within the biomass fiber cell walls.

[0033] This invention creates micro-damage channels through mechanical pulverization, allowing microbial strains to release enzymes that act on exposed specific chemical bonds. Then, through a biodegradation step, based on a microbial fermentation-mediated biodegradation mechanism, it achieves the directional degradation of hemicellulose and the controlled dissociation of some cellulose within the cell walls of bamboo fiber materials. Attached Figure Description

[0034] Figure 1 The image shows a transmission electron microscope (TEM) comparison of the original cell wall layer of the mechanically pulverized product of Example 1 (left), the cell wall product of moso bamboo fiber of Example 1 (middle), and the cell wall product of moso bamboo fiber of Example 2 (right).

[0035] Figure 2 The image shows a comparison of the morphology of a single fiber bundle (left) of the mechanically pulverized product of Example 1, a single fiber bundle (middle) of the bamboo fiber cell wall product of Example 1, and a single fiber bundle (SEM) of the bamboo fiber cell wall product of Example 2, as shown in the scanning electron microscope (SEM) images.

[0036] Figure 3 The image shows a comparison of the cross-sectional morphology of thin-walled cells of the mechanically treated product of Example 3 and the cross-sectional morphology of thin-walled cells of bamboo fiber cell wall product using scanning electron microscopy (SEM).

[0037] Figure 4 The XRD diffraction patterns of the original bamboo fiber cell wall and the directional dissociation of bamboo fiber cell wall products from Examples 1-3 are shown. Detailed Implementation

[0038] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0039] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0040] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0041] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0042] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0043] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0044] This invention first provides a method for the directional dissociation of biomass fiber cell walls, which includes the following steps:

[0045] Mechanical pulverization steps: Biomass materials are mechanically pulverized and then sieved to obtain mechanically pulverized products;

[0046] Substrate softening step: The mechanically pulverized product is softened using a basic nutrient solution to obtain a softened substrate;

[0047] Bioremoval step: The pretreated product is bioremoved by fermentation with microbial strains to obtain the bioremoved product.

[0048] This invention utilizes microbial biodegradation technology to achieve the targeted exfoliation and degradation of biomass fiber cell walls. This invention overcomes the difficulties of traditional methods involving the mixing of lignin, hemicellulose, and cellulose, resulting in low product utilization rates, thus maximizing the utilization of substances within the biomass fiber cell walls.

[0049] The present invention does not specifically limit the biomass used; any biomass commonly used in the art can be used. Specifically, the biomass can be bamboo and / or wood materials. Considering that bamboo materials are more difficult to process and are more suitable for the methods of this application, bamboo materials are preferred as the biomass used in this invention.

[0050] In this invention, the bamboo material is not specifically limited, and may include various bamboo species such as *Phyllostachys edulis*, *Phyllostachys pubescens*, *Phyllostachys liangshanensis*, *Phyllostachys nanmu*, *Phyllostachys pubescens*, *Phyllostachys nigra ... The bamboo species include: silver bamboo, human-faced bamboo, moso bamboo, flowery moso bamboo, flowery bamboo, purple bamboo, spotted bamboo, tortoise-shell bamboo, light bamboo, rigid bamboo, bitter bamboo, golden bamboo, tortoise-patterned bamboo, silver-chain bamboo, jade-edged bamboo, tea-stalk bamboo, arrow bamboo, bubble bamboo, Luohan bamboo, seedling bamboo, goose-feather bamboo, thorny bamboo, yellow bamboo, Chinese bamboo, black bamboo, stone bamboo, lucky bamboo, red bamboo, red-shelled bamboo, early-growing bamboo, large bamboo, water bamboo, bitter bamboo, yellow-stalked black-breasted chicken bamboo, melon bamboo, *Bambusa* species, *Bambusa* species, large green hedge bamboo, *Bambusa* species, and *Bambusa* species. Specifically, the bamboo material of this invention is preferably moso bamboo.

[0051] Mechanical crushing steps

[0052] The mechanical pulverization step of this invention includes mechanically pulverizing biomass materials followed by sieving to obtain the mechanically pulverized product. This invention achieves uniformity of raw materials and increases the contact surface area for microorganisms through the mechanical pulverization step, creating favorable physical conditions for subsequent biological processes.

[0053] In some specific implementations, the mechanical crushing process includes coarse crushing and fine crushing, and the product after fine crushing is graded to obtain mechanically crushed products with a particle size of 5-50 mm.

[0054] This invention employs a step-by-step process of "coarse crushing - fine grinding - sieving and grading" to process biomass materials. For example, a jaw crusher can be used to initially disrupt the macroscopic structure of bamboo materials, followed by fine grinding using a mill, and sieving to ensure particle uniformity, thus obtaining a mechanically pulverized product. This invention utilizes mechanical pulverization to create micro-damage channels and permeable pores for subsequent biodegradation steps, providing pathways for material exchange. The micropores formed by pulverization allow microorganisms to rapidly colonize the cell wall surface, while simultaneously improving the contact efficiency between enzymes and substrates, avoiding uneven local degradation caused by insufficient permeability during biodegradation.

[0055] In this invention, the product after fine crushing and disintegration is subjected to forced classification and recycling through a multi-layer vibrating screening system. The material with a particle size greater than 50 mm is returned to the fine crushing and disintegration stage through the return system; fine powder with a particle size less than 5 mm that is prone to agglomeration and local overheating is removed; and mechanically crushed products with a particle size of 5-50 mm, preferably 10-20 mm, are collected.

[0056] Softening the substrate step

[0057] The substrate softening step of this invention includes softening the mechanically pulverized product using a basic nutrient solution to obtain a softened substrate. This softening process creates an optimal solid-state fermentation environment suitable for the colonization, growth, and function of the target microbial community.

[0058] This invention uses a basic nutrient solution as a nutrient substrate for microbial growth, laying the foundation for subsequent bioremoval steps. Simultaneously, it adjusts the water content to fully penetrate the mechanically pulverized product, disrupting hydrogen bonds between cell walls and keeping the substrate in a moist and soft state. This further weakens the adhesion strength of the dark layer. The moist substrate environment, in conjunction with the microchannels formed by mechanical pulverization, provides a "liquid medium" for microbial attachment to the cell wall surface and enzyme diffusion, avoiding insufficient contact between the mechanically pulverized product and microbial strains in subsequent bioremoval steps due to substrate dryness.

[0059] In some specific implementations, the basic nutrient solution includes a nitrogen source and a phosphorus source. Using nitrogen and phosphorus sources compensates for the natural nitrogen and phosphorus deficiency in biomass materials, providing sufficient growth materials for microorganisms. Specifically, the mass ratio of the nitrogen source to the phosphorus source is 3-5:1; when the mass ratio is 3-5:1, sufficient growth materials can be provided for microorganisms without causing intensified competition among the microbial community or accumulation of metabolic waste due to excessive nutrients.

[0060] Furthermore, the present invention does not impose any particular limitation on the amount of nitrogen and phosphorus sources added, and they can be added as needed. Preferably, based on the total mass of the mechanically pulverized product as 100%, the amount of nitrogen source added is 1-3%; and the amount of phosphorus source added is 0.3-1%.

[0061] The present invention does not specifically limit the nitrogen and phosphorus sources. Specifically, the nitrogen source includes one or a combination of urea and yeast extract, and the phosphorus source includes one or a combination of potassium dihydrogen phosphate and diammonium hydrogen phosphate. In some specific embodiments, the water content of the softened substrate is 60%-70%. When the water content of the softened substrate is 60%-70%, it can further ensure the water supply and aeration space for microbial fermentation; at the same time, the basic nutrient solution can fully penetrate into the mechanically pulverized product, disrupting the hydrogen bonds between cell walls and further weakening the adhesion strength of the dark layer.

[0062] Bio-degradation steps

[0063] This invention utilizes microbial fermentation to biodegrade the pretreated product, thereby obtaining the biodegradation product cellulase.

[0064] Through biodegradation, the microbial strains can precisely target exposed chemical bonds, efficiently degrading the high hemicellulose content in the dark layer, thus exfoliating the dark layer first; exposing the lignin-cellulose structure of the light layer, and further cleaning up the remaining hemicellulose in both the light and dark layers. The microbial strains described in this invention can metabolize and synthesize cellulase and / or hemicellulase during fermentation.

[0065] In this invention, the microorganisms possess a certain degree of physical penetration and expansion capabilities. First, the microorganisms can enter the dark layer region of the cell wall, rich in hemicellulose / lignin, expanding the structure to create space for enzyme penetration and action. Through metabolism and enzymatic hydrolysis, they soften and partially degrade the dark layer, weakening the interlayer bonding. As the dark layer barrier weakens, the microorganisms can more deeply contact the cellulose in the visible layer and, under controlled conditions, gently dissociate it, aiming to peel off intact fiber bundles rather than completely saccharifying them.

[0066] In some specific implementation schemes, the amount of the microbial strain is 0.015-0.15% based on the total mass of the mechanically crushed product as 100%. This helps to ensure the stable metabolism of the microbial community and the continuous secretion of enzymes during the bioremoval process, and avoids insufficient bioremoval efficiency due to too low an inoculation amount or competitive inhibition of the microbial community due to too high an inoculation amount.

[0067] In some specific embodiments, the microbial strain includes Clostridium species. Preferably, the microbial strain also includes other bacterial groups. The amount of Clostridium species used is 0.005-0.05% based on the mass of the mechanically pulverized product, and the amount of other bacterial groups is 0.01-0.1%. The inventors of this invention have discovered that fermentation using microbial strains can metabolize and synthesize cellulase and / or hemicellulase, achieving the directional degradation of hemicellulose and the controlled degradation of some cellulose in the cell walls of biomass fibers, ultimately leading to the directional dissociation of the biomass fiber cell walls. The related enzyme system released by the Clostridium species can participate in the degradation of hemicellulose and some cellulose in the dark layer, making the structure of the dark layer loose and weakening its cohesive effect; thereby exposing the lignin-cellulose structure of the exposed layer.

[0068] This invention utilizes the targeting ability of Clostridium species to achieve stepwise dismantling of the visible and invisible layers of the cell wall in bamboo fiber materials, overcoming the limitations of traditional processes. Clostridium species can rapidly reproduce and metabolize, producing hemicellulase and cellulase. First, they dismantle the invisible layers in the cell wall of bamboo fiber materials, such as layers S1, S3, and S7. This preferentially degrades the hemicellulose in the invisible layers, disrupting the hemicellulose-lignin cross-linking structure and exposing the cellulose within. The resulting cellulase then partially degrades the cellulose in the invisible layers, loosening its structure and enzymatically breaking it down into monosaccharides.

[0069] Furthermore, Clostridium species can continuously secrete cellulases through reproduction and metabolism, such as endoglucanase, exoglucanase, and / or β-glucosidase. The enzyme activity is dynamically adjusted according to the degradation stage. For example, exoglucanase is preferentially secreted during degradation in the light layer, while endoglucanase activity is enhanced during degradation in the dark layer. In contrast, directly added cellulases are fixed components and cannot adapt to the changes in degradation requirements caused by the differences in the interlayer structure of the cell wall.

[0070] In this invention, *Bacillus subtilis* primarily functions during the intermittent aeration phase of the initial bioremoval process. As an aerobic enzyme-producing strain, it secretes large amounts of hemicellulase, mannosase, and pectinase. These enzymes degrade the intercellular matrix in bamboo cell walls, effectively softening the dense structure of natural bamboo. This opens physical channels for later enzyme penetration into the cell walls, causing microscopic gaps to appear between the dark layers of the cell walls, thus creating prerequisites for deeper dissection by subsequent *Clostridium* strains. *Aspergillus niger* and *Penicillium* further depolymerize the lignocellulose complex by secreting highly active cellulase and xylanase, increasing substrate accessibility.

[0071] In some specific implementations, the Clostridium species include one or more of Clostridium thermophilum, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficile; the other bacterial groups include one or more of Aspergillus niger, Penicillium, and Bacillus subtilis.

[0072] The Bacillus subtilis strain of this invention (accession number CGMCC 1.108) is from the China General Microbiological Culture Collection Center (CGMCC). All Clostridium strains can be purchased from Hangzhou Kenong Agricultural Technology Co., Ltd., and Aspergillus niger and Penicillium can be purchased from Ningbo Taisto Biotechnology Co., Ltd.

[0073] Furthermore, the inventors of this invention have discovered that by dynamically monitoring key parameters (temperature, pH, DO, specific metabolites), intelligently feeding back and regulating environmental conditions (time, temperature, pH, dissolved oxygen, nutrients), guiding the succession of microbial communities and enzyme expression, the temporal and selective degradation and stripping of lignin, hemicellulose, and cellulose can be achieved.

[0074] In some specific implementations, the biodegradation process includes an aerobic fermentation stage and an anaerobic fermentation stage.

[0075] Specifically, the aerobic fermentation stage includes intermittent aeration under conditions of 25-60℃ and pH 3.0-6.0, and the aerobic fermentation stage lasts for 1-5 days.

[0076] In this invention, sterile compressed air can be intermittently introduced into the system to maintain a microaerobic environment. The Clostridium species in the aerobic fermentation stage of this invention can adapt to the environment and proliferate in small quantities, while simultaneously promoting the rapid reproduction of aerobic and facultative anaerobic microorganisms (such as Aspergillus niger and Bacillus subtilis in the auxiliary flora) and the large-scale synthesis and secretion of various hydrolytic enzymes, especially hemicellulase systems. This achieves preferential degradation of dark-layer hemicellulose, disrupting the lignin-hemicellulose cross-linked structure and laying the foundation for subsequent decomposition.

[0077] The inventors of this invention have discovered that intermittent ventilation can ensure oxygen supply while reducing energy consumption. This invention does not impose a particular limitation on the time interval of intermittent ventilation; for example, it can be ventilation for 5-50 minutes followed by a break of 1-30 minutes.

[0078] In this invention, the anaerobic fermentation stage includes stopping aeration at a temperature of 25-60℃ and a pH of 4.0-6.5, and the duration of the anaerobic fermentation stage is 7-15 days. After aeration is stopped, the residual oxygen in the system is rapidly consumed by microorganisms. By stopping aeration, the residual oxygen in the reactor is rapidly consumed by microorganisms, and when the interstitial oxygen volume concentration in the system naturally decreases to below 0.5%, a strictly anaerobic environment can be formed.

[0079] In an anaerobic environment, the dominant growth of anaerobic Clostridium species and the expression of their unique cellulostomy bodies can be greatly promoted. The enzyme system secreted by Clostridium species has high targeting, and the activity of cellulase system is enhanced, realizing the complete degradation of hemicellulose remaining in the dark layer. As the dark layer is peeled off first, the previously wrapped light layer structure is exposed, creating conditions for subsequent deeper dissociation or cellulose utilization.

[0080] During the anaerobic fermentation stage, the hemicellulose in the dark layer is first efficiently degraded by the hemicellulase system released by microorganisms, making the dark layer structure loose; the lignin-cellulose cross-linked structure of the light layer is exposed, and residual hemicellulose is further cleaned up.

[0081] In some specific implementations, the bioremoval treatment is performed two or more times, with each treatment lasting 10-18 days, and an intermediate activation treatment is performed between adjacent bioremoval treatments. Two or more bioremoval treatments can gradually increase the total hemicellulose dissolution rate while avoiding excessive damage to the cellulose structure from a single treatment.

[0082] In some specific embodiments, the intermediate activation treatment includes immersing the bioresorption product in a buffer solution at 30-35°C and pH 4.5-5.0 for 2-4 hours; more preferably, the buffer solution includes a citrate-sodium citrate buffer solution.

[0083] The bioremoval process generates organic acid metabolites, leading to drastic pH fluctuations and potentially inhibiting residual enzyme activity. The citric acid-sodium citrate buffer system establishes a pH buffer, providing a suitable pH environment for subsequent bioremoval. Simultaneously, the polarity of the citrate ion helps weaken the non-covalent bonds between lignin and cellulose, reducing interfacial binding energy and clearing obstacles for enzyme molecules to enter the cell wall's dark layer. Intermediate activation treatment also washes away metabolic inhibitors accumulated from the first bioremoval process and resets the matrix pH, providing an optimal reaction environment for the second bioremoval.

[0084] It should be noted that when performing two or more bioremoval treatments, since the basal culture medium has been consumed, a substrate softening step is required before the bioremoval treatment. This softening and activation process ensures sufficient basal nutrient solution for subsequent bioremoval. Softening the substrate again replenishes the nutrients needed for subsequent fermentation and inactivates any remaining microorganisms, allowing the secondary bioremoval to proceed effectively.

[0085] Post-processing steps

[0086] In this invention, the method further includes a post-processing step, which includes inactivating the reaction by distillation at a high temperature of 100-120°C for 15-30 minutes, thereby terminating the reaction.

[0087] This invention creates micro-damage channels through mechanical crushing, allowing microorganisms to release enzymes that precisely target exposed chemical bonds. A subsequent biodegradation step effectively solves two major problems: the dense cell wall structure of bamboo makes directional penetration difficult, and the tightly cross-linked lignin, cellulose, and hemicellulose are difficult to dissociate individually. Based on a microbial fermentation-mediated biodegradation mechanism, it achieves the directional degradation of hemicellulose and the controlled degradation of some cellulose within the bamboo fiber cell walls.

[0088] Example

[0089] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0090] Example 1

[0091] Mechanical crushing steps: First, coarsely crush the bamboo using a jaw crusher, controlling the speed at 400 rpm and the crushing time at 5 minutes, until the particle size is 20-100 mm. Then, finely crush it using a grinding mill, controlling the speed at 200 rpm and the crushing time at 15 minutes. Use a standard sieve to screen and collect the mechanically crushed products with a particle size range of 10-20 mm.

[0092] Substrate softening procedure: Place 1 kg of the mechanically pulverized product into a solid-state fermenter. Dissolve 30 g of urea and 5 g of potassium dihydrogen phosphate in 1.58 kg of deionized water and mix thoroughly to prepare a basic nutrient solution. Add the basic nutrient solution to the solid-state fermenter and mix thoroughly with the mechanically pulverized product. The water content of the mechanically pulverized product is measured to be 65%, thus obtaining the softened substrate.

[0093] Inoculation with microorganisms: Dissolve 0.3g of Clostridium thermophilum and 0.5g of Bacillus subtilis in 160g of sterile physiological saline and stir well to prepare a compound bacterial suspension. Inoculate the suspension evenly on the softened substrate surface by spraying and mix thoroughly.

[0094] Bioresorption: The fermenter temperature was set to 30℃. An automatic atomized spray acid (1 mol / L HCl solution) / alkali (2 mol / L NaOH solution) system was used to maintain the pH at 6.5. A sterile air compressor was started, intermittently introducing sterile compressed air (5 minutes on, 30 minutes off) for aerobic fermentation for 3 days. Afterward, aeration was stopped, and the pH was adjusted and maintained at 6.0 using the same automatic atomized spray acid / alkali system. At this point, the interstitial oxygen concentration in the fermenter rapidly dropped below 0.5% within a few hours, entering an anaerobic state. The fermenter temperature was then raised to 50℃, and anaerobic fermentation continued for 10 days to obtain the bioresorption product.

[0095] Post-processing: After fermentation, high-temperature steam is introduced to raise the temperature inside the tank to 100°C and maintain it for 30 minutes. Distillation inactivation is then used to terminate the biodegradation reaction, yielding directionally dissociated bamboo fiber cell wall products.

[0096] Example 2

[0097] Activation treatment: 500g of the directionally dissociated bamboo fiber cell wall product from Example 1 was used as the starting material. The product was soaked in a citric acid-sodium citrate buffer solution (the mass ratio of citric acid to sodium citrate was approximately 8:13) at 30°C and a concentration of 0.1mol / L and a pH of 5.0 for 4 hours. After soaking, the product was dehydrated to a moisture content of approximately 50% by mechanical pressing to obtain the activated product.

[0098] Substrate softening step: Place the activated product back into the solid-state fermenter. Dissolve 15g of urea and 2.5g of potassium dihydrogen phosphate in 460g of deionized water and mix thoroughly to prepare a basic nutrient solution. Mix the basic nutrient solution and the activated product thoroughly until the water content of the activated product returns to 65%. Sterilize by steaming at 121℃ for 30 minutes to obtain the softened product.

[0099] Inoculation of microorganisms: After cooling the softened product to 40°C, dissolve 0.15g of Clostridium thermophilum and 0.25g of Bacillus subtilis in 80g of sterile physiological saline and stir evenly to prepare a compound bacterial suspension. Inoculate the suspension evenly on the substrate surface by spraying and mix thoroughly.

[0100] Bioresorption: The fermenter temperature was set to 30℃, and the pH was maintained at 6.5 using an automatic atomized spray acid (1 mol / L HCl solution) / alkali (2 mol / L NaOH solution) replenishment system. A sterile air compressor was started, intermittently introducing sterile compressed air (5 minutes on, 30 minutes off) for aerobic fermentation for 3 days. Afterward, aeration was stopped, and the pH was adjusted and maintained at 6.0 using the aforementioned automatic atomized spray acid / alkali replenishment system. At this point, the interstitial oxygen concentration in the fermenter rapidly dropped below 0.5% within a few hours, entering an anaerobic state. The fermenter temperature was then raised to 50℃, and fermentation continued for 10 days to obtain the bioresorption product.

[0101] Post-processing: After fermentation, high-temperature steam is introduced to raise the temperature inside the tank to 100°C and maintain it for 30 minutes to perform inactivation treatment, thereby obtaining the moso bamboo fiber cell wall product with secondary directional dissociation.

[0102] Example 3

[0103] Mechanical crushing steps: First, coarsely crush the bamboo using a jaw crusher, controlling the speed at 300 rpm and the crushing time at 3 minutes, until the particle size is 20-100 mm. Then, finely crush it using a grinding mill, controlling the speed at 300 rpm and the crushing time at 15 minutes. Use a standard sieve to screen and collect the mechanically crushed product with a particle size of 10-20 mm.

[0104] Softening treatment: Place 1 kg of the mechanically pulverized product into a solid-state fermentation tank. Dissolve 30 g of urea and 5 g of potassium dihydrogen phosphate in 1.58 kg of deionized water and mix thoroughly to prepare a basic nutrient solution. Add the basic nutrient solution to the solid-state fermentation tank and mix thoroughly with the mechanically pulverized product. The water content of the mechanically pulverized product is measured to be 65%, thus obtaining the softened substrate.

[0105] Inoculation with microorganisms: Dissolve 0.5g of Clostridium thermophilum and 0.4g of Aspergillus niger in 180g of sterile physiological saline and stir well to prepare a compound bacterial suspension. Inoculate the suspension evenly on the softened substrate surface by spraying and mix well.

[0106] Bioreduction: The fermenter temperature was set to 35℃. An automatic atomized spray system was used to maintain the pH at 6.5, supplementing acid (1 mol / L HCl solution) and alkali (2 mol / L NaOH solution). A sterile air compressor was started, intermittently introducing sterile compressed air (5 minutes on, 30 minutes off) for aerobic fermentation for 3 days. Afterward, aeration was stopped, and the pH was adjusted and maintained at 6.0 using the same automatic atomized spray system. At this point, the interstitial oxygen concentration in the fermenter rapidly dropped below 0.5% within a few hours, entering an anaerobic state. The fermenter temperature was then raised to 50℃, and fermentation continued for 10 days to obtain the bioreduction product.

[0107] Post-processing: After fermentation, high-temperature steam is introduced to raise the temperature inside the tank to 100°C and maintain it for 30 minutes. Distillation inactivation is then used to terminate the biodegradation reaction, yielding directionally dissociated bamboo fiber cell wall products.

[0108] Performance testing

[0109] 1. Observation using scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

[0110] The directed dissociation of bamboo fiber cell wall products in Examples 1-3 was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the results are as follows: Figure 1-3 As shown.

[0111] Scanning electron microscopy and transmission electron microscopy can clearly reveal subtle changes caused by fermentation, such as cell wall rupture, fiber separation, and surface erosion. By observing the cell walls, vessels, and fiber bundles of the microstructure of bamboo materials, the integrity of the fibers and the clarity of cell wall stratification (primary wall and secondary wall) before and after fermentation can be directly compared to determine whether degradation has led to structural loosening.

[0112] Figure 1 The image shows a transmission electron microscope (TEM) comparison of the original cell wall layer of the mechanically pulverized product of Example 1 (left), the cell wall product of moso bamboo fiber of Example 1 (middle), and the cell wall product of moso bamboo fiber of Example 2 (right).

[0113] Depend on Figure 1 It can be seen that the cell walls of natural bamboo fiber materials have a dense and intact structure. In their natural state, the visible and dark layers are tightly adhered, dense, and without gaps. In Example 1, tiny gaps began to appear between the dark layers of the cell walls in the directionally dissociated bamboo fiber cell wall product. This is sufficient to indicate that the intermittent aeration during the bioremoval process promoted the large-scale production of hemicellulase, mannosase, and pectinase by the aerobic strain *Bacillus subtilis*; the anaerobic environment of the bioremoval process activated *Clostridium thermocellum*, which, utilizing its previously accumulated enzyme system and its own secreted cellulase, precisely acted on the interlayer cellulose and hemicellulose, preferentially degrading the dark layers in the structure. This led to the cell walls undergoing layered peeling from the inside. In Example 2, the gaps and cracks between the dark layers of the cell walls in the directionally dissociated bamboo fiber cell wall product became more numerous, and the gaps were significantly widened and lengthened. Many dark layers were completely degraded, causing adjacent visible layers to lose their original support, thus transforming the physical structure of the fiber from rigid and dense to highly porous.

[0114] Figure 2 The image shows a comparison of the morphology of the mechanically pulverized product of Example 1 (left), the directionally dissociated bamboo fiber cell wall product of Example 1 (middle), and the directionally dissociated bamboo fiber cell wall product of Example 2 using scanning electron microscopy (SEM).

[0115] Depend on Figure 2 It can be seen that although the mechanically crushed product of Example 1 has caused some mechanical damage and slight fuzzing on the surface through mechanical crushing, the fibers are still tightly bound together by lignin and hemicellulose between fibers and between sublayers inside the fibers.

[0116] In Example 1, the fiber bundles of the directionally dissociated bamboo fiber cell wall products underwent significant fibrillation and lamellar disintegration. This caused the tightly packed fiber bundles to become fluffy, and numerous finer filaments were dissociated and released.

[0117] In Example 2, the directionally dissociated bamboo fiber cell wall product exhibited larger and more numerous voids and cracks, resulting in a looser overall cell wall structure and a significantly intensified degree of dissociation. The separation between cell wall layers created distinct cavities, and the layered peeling structure was very prominent, demonstrating the cumulative effect and deep processing capability of the method of this invention. Furthermore, the directionally dissociated bamboo fiber cell wall product of Example 2 selectively removed the loosely structured, amorphous cellulose portion, leaving behind a cellulose skeleton with higher crystallinity and purity.

[0118] Figure 3 The image shows a comparison of the cross-sectional morphology of thin-walled cells of the mechanically treated product of Example 3 and the cross-sectional morphology of thin-walled cells of bamboo fiber cell wall product using scanning electron microscopy (SEM).

[0119] Depend on Figure 3 As can be seen, the cell structure of the directionally dissociated bamboo fiber cell wall product in Example 3 was severely damaged. The cell wall became rough and porous, and a large number of ruptures and collapses occurred, leaving only a fragmented skeleton.

[0120] Therefore, the method of this invention can not only achieve layered dissociation, but also achieve "porous" modification of the cell wall. Figure 1-3 It can be seen that a large number of pores appeared in the solid part of the bamboo fiber cell wall, and the thin-walled cell structure was also effectively degraded. This indicates that the method of the present invention can be used to prepare biomass materials with different microstructures, such as layered separated fibers, porous biological scaffolds, and hydrogels.

[0121] 2. Nanoindentation (DSI) test

[0122] Nanoindentation technology can obtain mechanical property parameters such as hardness and elastic modulus of bamboo materials by measuring the load-displacement curve during the process of a probe being pressed into the surface of a bamboo material sample. This reflects the influence of the fermentation process on the microscopic mechanical properties of bamboo materials, and further helps to analyze the correlation between changes in cell wall structure and pore structure and mechanical properties.

[0123] The sample surface was observed using the optical microscope integrated into the nanoindenter. The mechanically pulverized product from Example 1 and the directionally dissociated bamboo fiber cell wall products from Examples 1-3 were selected for nanoindentation testing. At least five different regions were selected for each sample, and five points were tested in each region to ensure statistical representativeness of the data. Continuous stiffness measurement mode was activated, with an initial load of 50 μN, applied at a rate of 10 μN / s to the maximum load of 1000 μN, held for 5 seconds, and then unloaded. The load-displacement curve and stiffness change data were recorded, and the results are shown in Table 1.

[0124] Table 1

[0125]

[0126] As shown in Table 1, the directional dissociation of the cell wall layer by microorganisms significantly reduces its mechanical strength. Furthermore, the decrease in internal cellulose crystallinity and the reduction in lignin and polysaccharide components during cell wall degradation also significantly affect the reduction in micromechanical strength. The dark layer of the cell wall, due to the degradation of lignin and hemicellulose and the local decrease in cellulose crystallinity, exhibits a directional decrease in micromechanical strength. This contrasts with the light layer, where only a portion of the cellulose crystallinity is reduced, resulting in a moderate decrease in micromechanical strength. This approach preserves the main fibrous skeletal structure while reducing the difficulty of subsequent processing.

[0127] 3. XRD diffraction test

[0128] XRD diffraction tests were performed on the mechanically pulverized product of Example 1 and the directionally dissociated bamboo fiber cell wall products of Examples 1-3 using a Bruker D8 ADVANCE X-ray diffractometer (Germany). The crystallinity was calculated according to the following formula, and the results are as follows: Figure 4 As shown in Table 2.

[0129] CrI (%) = (I 002 -I am ) / I 002 ×100%

[0130] Among them, CrI, I 002 and I am These represent the relative crystallinity, the diffraction intensity of the 002 plane (2θ=22°), and the diffraction intensity of the amorphous region (2θ=18°), respectively.

[0131] Table 2

[0132]

[0133] Depend on Figure 4As shown in Table 2, the method of the present invention can effectively regulate the crystalline structure of bamboo fiber cell walls. After the initial bioresorption treatment in Example 1, the crystallinity significantly increased from the original 58% to 81.7%, showing an upward trend. This is because the enzyme system secreted by microorganisms preferentially acts on the loosely structured amorphous regions in the bamboo fiber cell walls, hydrolyzing and removing hemicellulose and cellulose amorphous chain segments, thereby increasing the relative proportion of crystalline regions in the remaining components, which macroscopically manifests as an increase in crystallinity.

[0134] After the secondary bioresorption treatment in Example 2, the crystallinity actually decreased significantly from 81.7% to 48.7%. This indicates that after a large amount of amorphous regions were removed, the continuous and in-depth bioresorption process began to affect the crystalline regions of cellulose, disrupting part of the crystal lattice structure and leading to a decrease in crystallinity. Therefore, the method of the present invention can not only remove amorphous components but also "activate" the crystalline structure of cellulose according to different treatment depths. This is of great significance for improving the efficiency of subsequent enzymatic hydrolysis and saccharification or the accessibility of chemical modification reactions.

[0135] The comparison between Example 1 and Example 3 shows that by adjusting the fermentation conditions and strains, the degree of degradation of the amorphous and crystalline regions can be precisely controlled, thereby achieving customized control of the crystallinity of the final product.

[0136] Therefore, this invention, through a step-by-step and controllable biodegradation process, can selectively achieve the dual effects of "purification and crystallization" or "crystal breaking and activity enhancement" as needed.

[0137] 4. Detection of cellulose, hemicellulose, and lignin

[0138] The contents of cellulose, hemicellulose and lignin were tested according to the standard NY / T3494-2019 "Determination of Cellulose, Hemicellulose and Lignin in Agricultural Biomass Raw Materials", and the results are shown in Table 3 below.

[0139] Table 3

[0140]

[0141] As shown in Table 3, the method of the present invention can achieve directional and controllable dissociation of biomass fiber cell wall components. After the initial biodegradation in Example 1, the hemicellulose content decreased significantly, while the decrease in cellulose content was relatively small. This demonstrates that the method of the present invention has high selectivity, preferentially degrading the more loosely structured and easily enzymatically hydrolyzed hemicellulose and amorphous cellulose in the initial stage of degradation, thereby preferentially destroying the cell wall's "adhesion and buffer layer" (i.e., the dark layer).

[0142] Comparing the results of Examples 1 and 2, after the secondary bioresorption treatment, hemicellulose was further removed, and the degree of cellulose degradation also increased. This indicates that by adjusting the number of bioresorption cycles, the depth of dissociation can be controlled, achieving a gradual and controllable stripping of the cellulose skeleton.

[0143] Example 3 used different auxiliary strains and process parameters to demonstrate that by adjusting the strain combination, inoculation ratio and fermentation conditions, the relative contents of cellulose, hemicellulose and lignin in the final product can be effectively controlled to meet the needs of different subsequent applications.

[0144] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0145] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for the directional dissociation of cell walls in biomass fibers, characterized in that, Includes the following steps: Mechanical pulverization step: The biomass material is mechanically pulverized to obtain the mechanically pulverized product; Substrate softening step: The mechanically pulverized product is softened using a basic nutrient solution to obtain a softened substrate; Bioremoval step: The softened substrate is bioremoved by fermentation with microbial strains to obtain bioremoval products.

2. The method according to claim 1, characterized in that, The mechanical crushing process includes coarse crushing and fine crushing, and the product after the fine crushing is graded to obtain mechanically crushed products with a particle size of 5-50 mm.

3. The method according to claim 1 or 2, characterized in that, The basic nutrient solution includes a nitrogen source and a phosphorus source; preferably, the mass ratio of the nitrogen source to the phosphorus source is 3-5:

1. More preferably, based on the total mass of the mechanically pulverized product as 100%, the amount of nitrogen source added is 1-3%; and the amount of phosphorus source added is 0.3-1%. More preferably, the softening substrate has a moisture content of 60-70%.

4. The method according to claim 3, characterized in that, The nitrogen source includes one or a combination of two of urea and yeast extract, and the phosphorus source includes one or a combination of two of potassium dihydrogen phosphate and diammonium hydrogen phosphate.

5. The method according to claims 1-4, characterized in that, In the bio-degradation step, the amount of microbial strain used is 0.015-0.15%, based on the total mass of the mechanically pulverized product as 100%.

6. The method according to any one of claims 1-5, characterized in that, The microbial strains include Clostridium species, and preferably, the microbial strains also include other bacterial groups; Based on the total mass of the mechanically pulverized product as 100%, the amount of Clostridium species used is 0.005-0.05%, and the amount of other bacterial groups used is 0.01-0.1%.

7. The method according to claim 6, characterized in that, The Clostridium species include one or more of the following: Clostridium thermofibrinolyticum, Clostridium xantholyticum, Clostridium fibrinolyticum, and Clostridium difficileum. Other microbial groups include one or more of Aspergillus niger, Penicillium, and Bacillus subtilis.

8. The method according to any one of claims 1-7, characterized in that, The biodegradation process includes an aerobic fermentation stage and an anaerobic fermentation stage; Preferably, the aerobic fermentation stage includes intermittent aeration under conditions of 25-60℃ and pH 3.0-6.0, and the aerobic fermentation stage lasts for 1-5 days. Preferably, the anaerobic fermentation stage includes stopping aeration at a temperature of 25-60℃ and a pH of 4.0-6.5, and the duration of the anaerobic fermentation stage is 7-15 days.

9. The method according to claim 8, characterized in that, The number of biological de-removal treatments is two or more, and the duration of each biological de-removal treatment is 10-18 days. An intermediate activation treatment is performed between two adjacent biological de-removal treatments. Preferably, the intermediate activation treatment includes immersing the bioresorption product in a buffer solution at 30-35°C and pH 4.5-5.0 for 2-4 hours; more preferably, the buffer solution includes a citrate-sodium citrate buffer solution.

10. The method according to any one of claims 1-9, characterized in that, The bioremoval step is followed by a post-processing step. Preferably, the post-processing step includes distilling and inactivating the bioremoval product at 100-120°C for 15-30 minutes.

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