Method for preparing a sugar-containing permeate and method for converting

Abstract

The application provides a preparation method and a conversion method of sugar-containing permeate, and belongs to the field of enzymatic saccharification. The application provides a preparation method of sugar-containing permeate, which comprises the following steps: performing enzymatic hydrolysis on filtrate containing hemicellulose and cellulose by using a biochar-functional bacterial group compound to obtain the sugar-containing permeate; the biochar-functional bacterial group compound comprises biochar and a functional bacterial group loaded on the biochar; the functional bacterial group comprises saccharifying bacteria and electroactive bacteria; and a voltage is applied during the enzymatic hydrolysis. The saccharifying bacteria provide saccharifying enzymes, promote the conversion of cellulose and hemicellulose into reducing sugars such as glucose and xylose, the electroactive bacteria secrete flavin electron shuttles, and jointly act with the biochar conductive interface and the applied microvoltage to improve the electron transfer conditions and improve the enzyme production efficiency of the saccharifying bacteria, thereby improving the hydrolysis of holocellulose.

Description

Technical Field

[0001] This invention relates to the field of enzymatic hydrolysis for sugar production, specifically to a method for preparing and converting a sugar-containing percolate. Background Technology

[0002] Harmonylocellulose, mainly composed of cellulose and hemicellulose, is an important carbon source for preparing mixed sugars from lignocellulosic raw materials such as straw, corn cobs, and bagasse. Existing enzymatic saccharification technologies primarily rely on commercial cellulases and hemicellulases, which often suffer from low sugar concentrations. Summary of the Invention

[0003] This invention provides a method for preparing and converting sugar-containing percolate, and the sugar-containing percolate prepared by this invention has a high sugar content.

[0004] This invention provides a biochar-functional microbial complex, comprising biochar and functional microbial communities supported on the biochar; The functional microbial community includes saccharifying bacteria and electroactive bacteria.

[0005] Preferably, the biochar-functional microbial complex further includes a gel coating the biochar, saccharifying bacteria, and electroactive bacteria.

[0006] Preferably, the ratio of the functional microbial community to biochar is 10-20 mL: 1 g; The volume ratio of saccharifying bacteria to electroactive bacteria in the functional bacterial community is 2-4:1.

[0007] This invention also provides a method for preparing the biochar-functional microbial community complex described in the above technical solution, characterized by comprising the following steps: Functional microbial communities were mixed with biochar for adsorption to obtain a biochar-functional microbial community complex. When the biochar-functional microbial complex also includes a gel, the adsorption process further includes: mixing the adsorbed biochar loaded with functional microbial communities with sodium alginate solution and calcium chloride solution for cross-linking to obtain the biochar-functional microbial complex.

[0008] This invention also provides a method for preparing a sugar-containing percolate, characterized by comprising the following steps: The filtrate containing hemicellulose and cellulose was enzymatically hydrolyzed using a biochar-functional microbial complex to obtain the sugar-containing permeate. The biochar-functional microbial community complex is the biochar-functional microbial community complex described in the above technical solution or the biochar-functional microbial community complex prepared by the preparation method of the above technical solution. A voltage is applied during the enzymatic hydrolysis.

[0009] Preferably, the process is carried out in a permeate bed reactor and includes the following steps: The raw material containing holocellulose is mixed with water and placed above a permeable bed reactor. Then, a fungal spore suspension is sprayed to pre-hydrolyze the raw material, resulting in a filtrate containing hemicellulose and cellulose. The filtrate containing hemicellulose and cellulose flows to the lower layer of the permeate bed reactor, where it undergoes enzymatic hydrolysis under the action of the biochar-functional microbial complex to obtain the sugar-containing permeate.

[0010] Preferably, the fungi include Trichoderma reesei and Aspergillus niger; The concentration of spores in the spore suspension was 1.0 × 10⁻⁶. 6 ~1.0×10 7 spores / mL; the total mass of spores in the spore suspension used for spraying is 5% of the dry weight of the raw material; The dissolved oxygen content of the system during pre-hydrolysis is 0.5~2.0 mg / L.

[0011] Preferably, the mass of the biochar-functional microbial complex is 5-15% of the dry weight of the raw material; the volume of the immobilization zone containing the biochar-functional microbial complex accounts for 20-40% of the effective volume of the permeate bed reactor.

[0012] Preferably, the voltage is 0.2~0.6 V.

[0013] The present invention also provides a method for converting sugar-containing percolate, wherein the sugar-containing percolate prepared by the preparation method described above is transported to a completely mixed reactor for conversion.

[0014] Saccharifying bacteria primarily provide saccharifying enzymes such as cellulase, hemicellulase, β-glucosidase, and xylanase, promoting the conversion of cellulose and hemicellulose into reducing sugars such as glucose and xylose. Electroactive bacteria, through secreting flavin-like electron shuttles, work in conjunction with the biochar conductive interface and an applied microvoltage to improve electron transfer conditions, thereby increasing the enzyme production efficiency of saccharifying bacteria and enhancing the hydrolysis of holocellulose. Another portion of electrons flows through riboflavin-like mediators to the extracellular fiber surface, electrodes, or conductive interfaces, thus improving the holocellulose enzymatic hydrolysis interface and increasing the mixed sugar concentration and saccharification yield. Furthermore, this invention relies on the biochar conductive interface to regulate the local redox environment, providing more favorable electron transfer conditions for oxidative lysins such as monooxygenases of cleaving polysaccharides, thereby further promoting the saccharification process and increasing the release of fermentable sugars such as glucose and xylose.

[0015] The LBR dry reactor is suitable for high-solids-load enzymatic hydrolysis, which can increase the sugar concentration, but substrate accumulation leads to localized mass transfer limitation and insufficient enzyme-fiber surface contact. The CSTR wet reactor provides thorough mixing and is suitable for deep enzymatic hydrolysis of oligosaccharides and suspended fine fibers, but wet operation alone can easily dilute the sugar solution. This invention couples LBR dry primary saccharification with CSTR wet secondary saccharification, which can balance the release of high-solids sugars and deep liquid-phase conversion: LBR is responsible for the stratified enzymatic hydrolysis of high-solids-content straw to produce sugar, while CSTR is responsible for the subsequent fermentation conversion of the sugar solution. The two form a continuous reaction system of dry-wet coupling based on the material residence time reflux spray strategy, separating solid straw hydrolysis and liquid-phase sugar fermentation. This not only alleviates the mass transfer limitations and sugar feedback inhibition in the high-solids-content enzymatic hydrolysis process, but also improves the stability and operability of subsequent fermentation. Attached Figure Description

[0016] Figure 1 This is a flowchart of an embodiment of the present invention; Figure 2 The apparatus used in the embodiments. Detailed Implementation

[0017] This invention provides a biochar-functional microbial complex, comprising biochar and functional microbial communities supported on the biochar; The functional microbial community includes saccharifying bacteria and electroactive bacteria.

[0018] In this invention, the biochar-functional microbial complex comprises biochar and functional microbial communities loaded on the biochar; the functional microbial communities include saccharifying bacteria and electroactive bacteria.

[0019] In this invention, the biochar-functional microbial complex preferably further includes a gel coating the biochar, saccharifying bacteria and electroactive bacteria.

[0020] This invention also provides a method for preparing the biochar-functional microbial community complex described in the above technical solution, comprising the following steps: The suspension of functional microbial communities was mixed with biochar for adsorption to obtain the biochar-functional microbial community complex (i.e., the biochar-functional microbial community complex without gel). When the biochar-functional microbial complex also includes a gel, the adsorption process further includes: mixing the adsorbed biochar loaded with functional microbial communities with sodium alginate solution and calcium chloride solution for cross-linking to obtain the biochar-functional microbial complex.

[0021] In this invention, the preferred ratio of the functional bacterial suspension to biochar is 10-20 mL:1 g, and in specific embodiments of this invention, it can be 12 mL:1 g, 15 mL:1 g, or 18 mL:1 g.

[0022] In this invention, the biochar preferably includes one or more of straw biochar, bamboo charcoal, and sawdust biochar. The particle size of the biochar is preferably 1-5 mm, and in specific embodiments of this invention, it can be 2 mm, 3 mm, or 4 mm. Controlling the particle size between 1 and 5 mm facilitates filling and filtration, and reduces the likelihood of clogging.

[0023] In this invention, the pyrolysis temperature for preparing the biochar is preferably 500-700°C. The biochar preferably comprises pores with a diameter of 5-20 μm. Pores with a diameter of 5-20 μm are suitable for microbial attachment.

[0024] In this invention, the bacteria in the functional bacterial community are preferably in the logarithmic growth phase; the volume ratio of saccharifying bacteria to electroactive bacteria in the functional bacterial community is preferably 2-4:1; the saccharifying bacteria preferably include Bacillus subtilis. The saccharifying bacteria mainly provide saccharifying enzymes such as cellulase, hemicellulase, β-glucosidase, and xylanase, promoting the conversion of cellulose and hemicellulose into reducing sugars such as glucose and xylose; the electroactive bacteria are mainly used to enhance the local electron transport environment: electroactive bacteria can secrete riboflavin electron shuttles, which, in conjunction with biochar, improve the local redox environment, providing more favorable electron transport conditions for oxidative lysins such as polysaccharide monooxygenase, thereby promoting the saccharification process and increasing the release of fermentable sugars such as glucose and xylose.

[0025] In this invention, the adsorption time is preferably 12-24 hours, and in specific embodiments of this invention, it can be 15 hours, 18 hours, or 20 hours.

[0026] In this invention, when the biochar-functional microbial complex further includes a gel, the adsorption process also includes, preferably, the following: cross-linking the adsorbed biochar loaded with functional microbial communities with sodium alginate solution and calcium chloride solution to obtain the biochar-functional microbial complex.

[0027] In this invention, no post-treatment is performed after the adsorption is completed. In this invention, the concentration of the sodium alginate solution is preferably 2-3% (w / v); the mass ratio of biochar to sodium alginate is preferably 2-4:1, and in a specific embodiment of this invention, it can be 3:1.

[0028] In this invention, the mass ratio of sodium alginate to calcium chloride is preferably 1:1.0~2.0, and in a specific embodiment of this invention it can be 1:1.2:.

[0029] In this invention, the cross-linking time is preferably 2-4 hours. The cross-linked gel allows the bacterial cells to stably adhere to the surface and pores of the biochar.

[0030] This invention also provides a method for preparing a sugar-containing percolate, comprising the following steps: The filtrate containing hemicellulose and cellulose was enzymatically hydrolyzed using a biochar-functional microbial complex to obtain the sugar-containing permeate. The biochar-functional microbial community complex is the biochar-functional microbial community complex described in the above technical solution or the biochar-functional microbial community complex prepared by the preparation method described in the above technical solution. A voltage is applied during the enzymatic hydrolysis.

[0031] In this invention, the method for preparing the sugar-containing percolate is preferably carried out in a percolation bed reactor, and preferably includes the following steps: The raw material containing holocellulose is mixed with water and placed above a permeable bed reactor. Then, a fungal spore suspension is sprayed to pre-hydrolyze the raw material, resulting in a filtrate containing hemicellulose and cellulose. The filtrate containing hemicellulose and cellulose flows to the lower immobilization zone of the permeate bed reactor, where it undergoes enzymatic hydrolysis under the action of the biochar-functional microbial complex to obtain the sugar-containing permeate.

[0032] In this invention, the raw material containing hemicellulose is mixed with water and placed above a permeable bed reactor. Then, a fungal spore suspension is sprayed to pre-hydrolyze the raw material to obtain the filtrate containing hemicellulose and cellulose.

[0033] In this invention, the raw materials preferably include straw and / or straw cellulose after lignin removal.

[0034] In this invention, the solid content of the mixture obtained by mixing is preferably 20-30%.

[0035] In this invention, the fungi include *Trichoderma reesei* and *Aspergillus niger*. These fungi secrete cellulase, hemicellulase, and related coenzymes to initially disrupt and hydrolyze the fibrous structure of the raw materials.

[0036] In this invention, the total mass of spores in the spore suspension used for spraying is preferably 5% of the dry weight of the raw material.

[0037] In this invention, the concentration of spores in the spore suspension is preferably 1.0 × 10⁻⁶. 6 ~1.0×10 7 The ratio of *Trichoderma reesei* spores to *Aspergillus niger* spores is preferably 2-5:1, but in specific embodiments of this invention, it can be 3:1 or 4:1. *Trichoderma reesei* is mainly responsible for producing cellulase and hemicellulase and is the main enzyme-producing fungus; *Aspergillus niger* mainly produces β-glucosidase, xylanase and other coenzymes, and their proportion should not be too high, otherwise it will easily consume too much sugar or change the pH of the system.

[0038] In this invention, the method for preparing the fungal spore suspension preferably includes the following steps: Trichoderma reesei and Aspergillus niger were activated separately, and then the obtained primary spores were passaged to obtain Trichoderma reesei spores and Aspergillus niger spores, respectively. After diluting Trichoderma reesei spores and Aspergillus niger spores with water, the resulting Trichoderma reesei spore suspension and Aspergillus niger spore suspension were mixed to obtain the spore suspension of the fungus.

[0039] In this invention, the activation temperature is preferably 28°C, and the activation time is preferably 5 to 7 days.

[0040] In this invention, the number of passages is preferably 1 to 2, and the conditions during passage are preferably the same as those during activation.

[0041] Spraying can remove the sugars released from LBR in a timely manner, alleviating the feedback inhibition of enzyme production by fungi and bacteria caused by sugar accumulation. It can also maintain the moisture and mass transfer conditions of LBR materials, allowing the fungal hydrolysis, bacterial saccharification and biochar immobilization reactions in LBR to continue.

[0042] In this invention, the pre-hydrolysis preferably includes a first pre-hydrolysis and a second pre-hydrolysis performed sequentially: The preferred temperature for the first pre-hydrolysis is 30°C. This invention does not impose specific limitations on the amount of spray used or the time for the first decomposition; it is sufficient to allow fungal germination and the establishment of a stable community of functional microorganisms on the carrier and straw surface.

[0043] The preferred temperature for the second pre-hydrolysis is 50°C, and the preferred pH value of the system is 5-5.5. This invention does not have a specific limitation on the time of the second pre-hydrolysis; it can proceed until the pre-hydrolysis is complete.

[0044] In this invention, the dissolved oxygen content of the pre-hydrolysis system is preferably 0.5~2.0 mg / L, and in specific embodiments of this invention, it can be 0.8 mg / L, 1 mg / L, 1.2 mg / L, 1.5 mg / L, or 1.8 mg / L. Controlling the dissolved oxygen content within the above range satisfies the growth and enzyme production needs of aerobic fungi such as *Trichoderma reesei* and *Aspergillus niger*, while also preventing excessive oxygen diffusion to the lower layers.

[0045] The present invention preferably uses the introduction of nanobubbles to supply oxygen in order to maintain the dissolved oxygen level of the system during pre-hydrolysis.

[0046] In this invention, the decomposition is preferably performed by stirring to avoid the raw materials from caking.

[0047] After obtaining the filtrate containing hemicellulose and cellulose, the present invention preferably allows the filtrate containing hemicellulose and cellulose to flow to the lower immobilization zone of the permeate bed reactor, where it undergoes enzymatic hydrolysis under the action of the biochar-functional microbial complex to obtain the sugar-containing permeate.

[0048] In this invention, the biochar-functional microbial complex is preferably 5-15% of the dry weight of the raw material, and in specific embodiments of this invention, it can be 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13% or 14%; the volume of the immobilization zone accounts for 20-40% of the effective volume of the permeate bed reactor, and in specific embodiments of this invention, it can be 25%, 30% or 35%.

[0049] In this invention, a voltage is applied during the enzymatic hydrolysis; the voltage is preferably 0.2~0.6 V, and in specific embodiments of this invention, it can be 0.3 V, 0.4 V, or 0.5 V. Applying voltage can further enhance electron transfer during the saccharification stage and promote electron flow between electroactive bacteria, flavonoids, and biochar.

[0050] During the operation of the permeate bed reactor, the upper layer is mainly characterized by fungal pre-hydrolysis, while the lower layer is mainly characterized by bacterial deep saccharification and enhanced electron transfer, forming a cross-species synergistic vertical partitioning system of upper-layer fungal cell wall disruption and enzyme production and lower-layer immobilized bacterial saccharification.

[0051] The present invention preferably further includes: refluxing the sugar-containing percolate and spraying it onto the raw material together with other spraying liquids for pre-hydrolysis; the spraying frequency is preferably once every 4 to 8 hours. By periodically spraying low-concentration sugar-containing percolate, the moisture content of the material is adjusted, the mass transfer conditions of the high solids content system are improved, and the released soluble sugars such as glucose and xylose are promptly carried out of the percolation bed reactor, avoiding the inhibition of enzymatic hydrolysis and microbial enzyme production by sugar accumulation.

[0052] The present invention also provides a method for converting sugar-containing percolate, wherein the sugar-containing percolate prepared by the preparation method described above is transported to a completely mixed reactor for conversion.

[0053] In this invention, prior to the conveying process, the sugar-containing percolate is preferably collected and filtered.

[0054] In this invention, the residence time of the sugar-containing percolate during the conversion is preferably 12-24 h, and in specific embodiments of this invention, it can be 15 h, 18 h, or 20 h. This residence time allows the incompletely hydrolyzed oligosaccharides sufficient time to continue saccharification.

[0055] In this invention, the microbial community introduced into the completely mixed reactor preferably includes saccharifying bacteria and enzyme-producing auxiliary bacteria. The microbial community introduced into the CSTR is mainly composed of saccharifying bacteria and enzyme-producing auxiliary bacteria, and fermentation bacteria that consume large amounts of sugar are not introduced to avoid further conversion of the already generated sugar.

[0056] The present invention preferably introduces the corresponding fermentation strain into the CSTR according to the different target products. For example, when used for butanol production, Clostridium acetone-butanol can be introduced.

[0057] In this invention, the preferred temperature of the system during the conversion is 45-50°C, and the preferred pH is 4.8-5.5. The conversion is preferably carried out under stirring conditions, and the preferred stirring speed is 80-150 rpm. Controlling the stirring speed within the above range ensures uniform mixing of the liquid phase while avoiding excessive shear that could affect enzyme activity and cell stability.

[0058] During the conversion, the completely mixed reactor (CSTR) is preferably in a closed state. The closed state reduces the growth of contaminating microorganisms and the aerobic consumption of sugars.

[0059] After the conversion is completed (i.e., after the sugar-containing percolate has been in the CSTR), the present invention preferably discharges the product and then performs filtration, membrane separation or concentration treatment to obtain a sugar-containing diluted solution and a sugar-containing concentrated solution.

[0060] In this invention, the sugar-containing diluent is preferably refluxed and sprayed onto the raw material together with other spraying liquids for pre-hydrolysis.

[0061] In this invention, the mass of the sugar-containing diluent is 30-70% of the mass of the liquid used for spraying.

[0062] Spraying can remove the sugars released from LBR in a timely manner, alleviating the feedback inhibition of enzyme production by fungi and bacteria caused by sugar accumulation. It can also maintain the moisture and mass transfer conditions of LBR materials, allowing the fungal hydrolysis, bacterial saccharification and biochar immobilization reactions in LBR to continue.

[0063] The purpose of the Completely Mixed Reactor (CSTR) is not to continue processing large amounts of solid straw, but to further saccharify the sugar solution carried out by the LBR spray, which contains soluble oligosaccharides, cellobiose, and a small amount of fine fiber particles in the liquid phase. This process further converts them into fermentable sugars such as glucose and xylose, increasing the proportion of soluble sugars and C6 and C5 sugars, reducing the accumulation of sugar solution in the LBR, and improving the continuous operating efficiency of the entire system (LBR+CSTR).

[0064] This invention achieves partitioned hydrolysis and saccharification of solid-phase substrates through vertical spatial stratification: the upper layer utilizes nanobubbles for oxygen supply and is inoculated with aerobic fungi producing cellulase and hemicellulase. The fungal hyphae's penetrating ability and extracellular enzyme secretion capacity initiate the initial cell wall disruption and hydrolysis of straw fiber structure. The lower layer contains immobilized biochar carriers loaded with synergistic saccharifying enzyme-producing bacteria and electroactive bacteria, further promoting the conversion of cellulose and hemicellulose into fermentable sugars such as glucose and xylose. The electroactive bacteria secrete flavin-like electron shuttles such as riboflavin and FMN, and regulate the local redox environment through the biochar's conductive interface, providing electron transfer conditions for oxidative lysins such as monooxygenases of cleavable polysaccharides, thereby enhancing the depolymerization of recalcitrant polysaccharides. During the reaction, leachate is periodically sprayed, maintaining suitable moisture and mass transfer conditions for the high-solids-content material and promptly removing soluble sugars into the CSTR for subsequent fermentation or conversion, mitigating the feedback inhibition of sugar accumulation on the enzymatic hydrolysis process. Overall, this system integrates fungal pre-hydrolysis, bacterial deep saccharification, biochar immobilization, enhanced electron transfer in electroactive bacteria, and dry-wet coupling reaction into the same process, achieving synergistic enhancement of enzymatic hydrolysis for saccharification of high-solids straw in different zones.

[0065] The preparation and conversion methods of the sugar-containing percolate provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Figure 1 Flowchart for an embodiment; Figure 2 The apparatus used in the embodiments.

[0067] Sources and preparation of microbial agents: The saccharifying bacteria was Bacillus subtilis DSM 10: The purchased lyophilized Bacillus subtilis DSM 10 bacterial powder was reconstituted with physiological saline, streaked onto LB agar plates, and incubated at 37℃ for 18–24 h. Single colonies were picked and inoculated into LB liquid medium, and incubated at 37℃ and 150 rpm for 12–18 h to obtain the primary seed culture. The LB liquid medium consisted of: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.0, and sterilized at 121℃ for 20 min. The primary seed culture was transferred to CMC-xylan composite induction medium at a 5% inoculum volume and cultured at 37℃ and 150 rpm for 36 h to obtain saccharified bacterial culture. The CMC-xylan composite induction medium consisted of: sodium carboxymethyl cellulose 10 g / L, xylan 5 g / L, peptone 5 g / L, yeast extract 2 g / L, KH2PO4 1 g / L, MgSO4·7H2O 0.5 g / L, NaCl 0.5 g / L, and pH 7.0. After incubation, the OD600 of the bacterial culture was controlled between 0.8 and 1.0.

[0068] The electroactive bacteria used was the publicly available Shewanella oneidensis MR-1, which was inoculated into LB liquid medium and cultured at 30℃ and 150 rpm until the OD600 reached 0.8~1.0 before use.

[0069] Example 1 Using delignified corn stalk cellulose as raw material, the material was pulverized to 1 mm and then loaded into an LBR infiltration bed reactor. The solids content was adjusted to 20% by spraying water (no further water spraying was performed). A mixed spore solution of *Trichoderma reesei* and *Aspergillus niger* (spore ratio 3:1) was inoculated onto the upper layer, with a spore concentration of 1.0 × 10⁻⁶. 7 The inoculum size is 5% of the dry weight of the straw. Low-intensity oxygen supply through nanobubbles keeps the dissolved oxygen in the upper layer below 1.0 mg / L, allowing the fungi to maintain a good state of growth and enzyme production.

[0070] The lower layer is filled with straw biochar immobilized microbial agent (3mm particle size biochar is mixed with a mixed microbial suspension and adsorbed at 35℃ for 12 hours, wherein the concentration of the mixed microorganisms in the mixed microbial suspension is 10). 8 The biochar and mixed bacteria were used in a ratio of 1 g to 15 mL (CFU / mL, with a volume ratio of saccharifying bacteria to electroactive bacteria of 4:1) for enzymatic hydrolysis. The amount added was 10% of the dry weight of the straw. Graphite electrodes were placed on both sides of the immobilization zone and a micro voltage of 0.3 V was applied to promote electron transfer in the bacteria during the saccharification stage.

[0071] The LBR operating temperature is controlled at 50℃ and the pH at 5.2. The reflux liquid (i.e., the filtrate obtained from enzymatic hydrolysis) is sprayed every 6 hours, with each spray volume being 8% of the effective volume of the LBR. The soluble sugars, oligosaccharides, and fine fiber particles carried out by the spray liquid (reducing sugar yield 83.6%) are fed into the CSTR (no additional bacteria are added to the CSTR; the reaction is carried out solely by the bacteria and enzymes carried out by the spray liquid). The CSTR temperature is 48℃, pH 5.0, stirring speed is 120 rpm, and residence time is 18 hours, for further post-saccharification.

[0072] After membrane separation, the sugar concentrate from the CSTR enters the subsequent purification process, while the low-sugar permeate is refluxed back to the LBR for further spraying. After 5 days of operation, the reducing sugar yield reached 92.4%, and the C6 sugar:C5 sugar ratio was 1.68, indicating that the high-solids enzymatic hydrolysis of the LBR and the post-saccharification of the CSTR liquid phase achieved a good wet-dry coupling effect.

[0073] Example 2 A vertical LBR infiltration bed reactor with an effective volume of 20 L was constructed, with a height-to-diameter ratio of 2:1. The top was equipped with a nanobubble air inlet and a spray nozzle, while the bottom was equipped with a leachate collection port and a return port. A 3 mm aperture stainless steel screen was installed in the middle and lower part to support the upper layer of solid straw and ensure that the leachate flows smoothly downward.

[0074] The lower layer is a 1.5 L biochar immobilization zone, filled with straw biochar immobilization agent (a mixture of 3 mm straw biochar, saccharifying bacteria, and electroactive bacteria was adsorbed for 18 h, then cross-linked with 2.5% sodium alginate and 3.0% calcium chloride (based on 100 g of 3 mm biochar, 200 mL of 2.5 wt% sodium alginate solution was added; subsequently, it was immersed in 400 mL of 3.0 wt% calcium chloride solution for cross-linking), so that the bacteria are stably attached to the surface and pores of the biochar. Specifically, 3 mm biochar and the mixed bacterial suspension were mixed and adsorbed at 35℃ for 12 hours, with the mixed bacterial concentration in the suspension being 10... 8 CFU / mL, the ratio of biochar to mixed bacteria is 1 g: 15 mL, and the volume ratio of saccharifying bacteria to electroactive bacteria in the mixed bacteria is 4:1.

[0075] Graphite electrodes are installed on both sides of the immobilization zone, and a micro voltage of 0.3 V is applied during operation.

[0076] Corn stalks crushed to 2 mm were loaded into the LBR (Liquid Crystal Burger), with a top layer loading of 2.0 kg and an initial solids content adjusted to 28%. Then, a mixed spore solution of *Trichoderma reesei* and *Aspergillus niger* was evenly sprayed in, with a spore concentration of 1.0 × 10⁻⁶. 7The inoculum density was 5% of the dry weight of the straw (3:1 ratio of *Trichoderma reesei* to *Aspergillus niger* spores per mL). For the first 24 hours after startup, the temperature was controlled at 30°C, with no high-flow-rate spraying, primarily to allow fungal germination and biochar formation. Afterward, the temperature was increased to 50°C, the pH was maintained at 5.2, and nanobubbles were used to maintain dissolved oxygen in the upper layer at 1.0 mg / L. Once in normal operation, 500 mL of reflux liquid was sprayed every 6 hours, flowing from top to bottom through the straw layer and biochar immobilization zone, and collected from the bottom. The reactor was stirred at a low speed for 10 minutes per hour at 60 rpm to prevent straw compaction and maintain the stratified structure as much as possible. After 5 days of continuous operation, the sugar solution was discharged (reducing sugar yield 83.2%), and the solid residue remained in the LBR for the next feeding and spraying cycle.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biochar-functional microbial community complex, characterized in that, Includes biochar and functional microbial communities supported on the biochar; The functional microbial community includes saccharifying bacteria and electroactive bacteria.

2. The biochar-functional microbial community complex according to claim 1, characterized in that, The biochar-functional microbial complex also includes a gel encapsulating the biochar, saccharifying bacteria, and electroactive bacteria.

3. The biochar-functional microbial community complex according to claim 1, characterized in that, The ratio of the functional microbial community to biochar is 10-20 mL: 1 g; The volume ratio of saccharifying bacteria to electroactive bacteria in the functional bacterial community is 2-4:1; The saccharifying bacteria include Bacillus subtilis.

4. The method for preparing the biochar-functional microbial community complex according to any one of claims 1 to 3, characterized in that, Includes the following steps: The suspension of functional microbial communities was mixed with biochar for adsorption to obtain a biochar-functional microbial community complex. When the biochar-functional microbial complex also includes a gel, the adsorption process further includes: mixing the adsorbed biochar loaded with functional microbial communities with sodium alginate solution and calcium chloride solution for cross-linking to obtain the biochar-functional microbial complex.

5. A method for preparing a sugar-containing percolate, characterized in that, Includes the following steps: The filtrate containing hemicellulose and cellulose was enzymatically hydrolyzed using a biochar-functional microbial complex to obtain the sugar-containing permeate. The biochar-functional microbial community complex is the biochar-functional microbial community complex according to any one of claims 1 to 3 or the biochar-functional microbial community complex prepared by the preparation method according to claim 4. A voltage is applied during the enzymatic hydrolysis.

6. The preparation method according to claim 5, characterized in that, This is carried out in a permeate bed reactor and includes the following steps: The raw material containing holocellulose is mixed with water and placed above a permeable bed reactor. Then, a fungal spore suspension is sprayed to pre-hydrolyze the raw material, resulting in a filtrate containing hemicellulose and cellulose. The filtrate containing hemicellulose and cellulose flows to the lower layer of the permeate bed reactor, where it undergoes enzymatic hydrolysis under the action of the biochar-functional microbial complex to obtain the sugar-containing permeate.

7. The preparation method according to claim 5 or 6, characterized in that, The fungi include Trichoderma reesei and Aspergillus niger; The concentration of spores in the spore suspension was 1.0 × 10⁻⁶. 6 ~1.0×10 7 spores / mL; the total mass of spores in the spore suspension used for spraying is 5% of the dry weight of the raw material; The dissolved oxygen content of the system during pre-hydrolysis is 0.5~2.0 mg / L.

8. The preparation method according to claim 5, characterized in that, The mass of the biochar-functional microbial complex is 5-15% of the dry weight of the raw material; the volume of the immobilization zone containing the biochar-functional microbial complex accounts for 20-40% of the effective volume of the permeate bed reactor.

9. The preparation method according to claim 5, characterized in that, The voltage is 0.2~0.6 V.

10. A method for converting sugar-containing permeate, characterized in that, The sugar-containing percolate prepared by the preparation method according to any one of claims 5 to 9 is transported to a fully mixed reactor for conversion.

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