Method for saccharifying and pretreating bagasse based on charcoal-reinforced cellulosome synergistic fungal cellulase and application

Abstract

The invention discloses a method for saccharifying and pretreating bagasse based on charcoal-reinforced cellulosome in cooperation with fungal cellulase and application. According to the method, biochar is used for regulating and controlling cellulose degrading bacteria to efficiently synthesize small fibers in a lignocellulose-containing culture medium, and the biochar and low-load fungal cellulase are combined for application, so that efficient saccharification of pretreated bagasse is realized. By introducing the biochar, the metabolic activity of thalli can be enhanced, accumulation of cellulosome is promoted, and the cellulose degradation rate and the sugar yield are remarkably improved through the synergistic effect of the cellulosome and cellulase, so that the efficient sugar production effect is achieved while the usage amount of the cellulase is reduced. The invention provides a novel green, low-carbon and cost-controllable biomass waste saccharification treatment approach, provides an efficient and feasible technical scheme for lignocellulose resource utilization and biomass energy conversion, and has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a method and its application for pretreatment of bagasse based on biochar-enhanced cellulose bodies and synergistic fungal cellulase saccharification. Background Technology

[0002] Lignocellulose is the most abundant renewable biomass resource in nature, and efficient degradation of lignocellulose is a key step in realizing the energy utilization of biomass. Currently, industrial lignocellulose degradation mainly relies on fungal cellulases, but the high cost and large quantity of cellulases lead to high biomass saccharification costs, limiting industrial application. The high proportion of crystalline cellulose in lignocellulose is the main reason why the enzymatic hydrolysis efficiency of fungal cellulase cannot be improved. According to reported literature, certain anaerobic bacteria secrete highly efficient multi-enzyme complexes, namely cellulose bodies, which have the characteristics of anchoring to the surface of lignocellulose, stripping lignocellulose filaments, destroying crystalline cellulose, and improving substrate accessibility, making them highly efficient enzymatic aids for fungal cellulase saccharification. However, the production of cellulose bodies by *Vibrio thermocellulosus* is limited, and improving its synthesis and secretion levels has important theoretical and applied value for the efficient degradation of lignocellulose containing crystalline regions. Summary of the Invention

[0003] The primary objective of this invention is to provide a method for pretreating sugarcane bagasse based on biochar-enhanced cellulose bodies and synergistic saccharification using fungal cellulase. This method utilizes biochar to rapidly increase the yield and activity of cellulose bodies from *Vibrio aceticola*, enhancing its ability to disrupt the structure of crystalline cellulose in lignocellulose. Subsequently, the cellulose bodies produced by the microorganisms are combined with fungal cellulase for saccharification, thereby reducing the amount of fungal cellulase used and lowering saccharification costs. This achieves efficient saccharification of sugarcane bagasse, improving sugar yield and resource utilization efficiency.

[0004] Another object of the present invention is to provide an application of the above-described method.

[0005] This invention is achieved through the following technical solution:

[0006] A method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification includes the following steps:

[0007] (1) Dry, crush and sieve the sugarcane bagasse, treat it with a low eutectic solvent, and then wash and dry the product obtained by treatment to obtain pretreated sugarcane bagasse rich in cellulose.

[0008] (2) Biochar is obtained by pyrolyzing agricultural and forestry waste under an oxygen-deficient environment;

[0009] (3) Use the pretreated sugarcane bagasse obtained in step (1) as the carbon source for the growth of bacteria, and add an appropriate amount of biochar prepared in step (2) to enhance the growth of bacteria and fermentation, so as to fully accumulate the cellulose produced by bacteria.

[0010] (4) After the fermentation in step (3) is completed, a surfactant is added to release cellulose bodies, and fungal cellulase is added at the same time to perform in-situ co-saccharification of the pretreated sugarcane bagasse.

[0011] The drying temperature in step (1) is preferably 70-100 °C; more preferably 80-90 °C.

[0012] The sieve mentioned in step (1) is preferably a 100-200 mesh sieve; more preferably a 100 mesh sieve.

[0013] The eutectic solvent mentioned in step (1) is preferably a mixture of choline chloride, lactic acid and ethylene glycol in a molar ratio of 1:2 to 5:1; more preferably, it is a mixture of choline chloride, lactic acid and ethylene glycol in a molar ratio of 1:5:1.

[0014] The processing temperature in step (1) is preferably 90 to 130°C; more preferably 115 to 130°C.

[0015] The processing time in step (1) is preferably between 2 and 6 hours; more preferably between 2 and 3 hours.

[0016] The cleaning described in step (1) is done using hot water.

[0017] The hot water is preferably hot water at 60-100℃; more preferably hot water at 70-80℃.

[0018] The drying temperature is preferably 40–70°C; more preferably 50–60°C.

[0019] The degree of drying is preferably drying to a constant weight.

[0020] The agricultural and forestry waste mentioned in step (2) is preferably at least one of corn stalks, rice stalks and sugarcane bagasse; more preferably sugarcane bagasse.

[0021] The preferred pyrolysis conditions in step (2) are as follows: the heating rate is 5-15 ℃ / min, and the temperature is raised to 300-700℃ and held for 1-4 h; more preferably, the heating rate is 5-10 ℃ / min, and the temperature is raised to 300℃ and held for 1-2 h; the most preferred condition is the heating rate is 10 ℃ / min, and the temperature is raised to 300℃ and held for 1 h.

[0022] The bacterial strain mentioned in step (3) is a cellulose-degrading bacterium capable of producing cellulose bodies; more preferably, it is a thermophilic anaerobic cellulose-degrading bacterium; most preferably, it is a thermophilic anaerobic bacillus or a thermophilic anaerobic vibrio; and even more preferably, it is one of Acetivibrio thermocellus DSM1313 and Acetivibrio thermocellus ATCC27405.

[0023] The biochar mentioned in step (3) is preferably biochar that can pass through a 100-mesh sieve.

[0024] The biochar concentration in the fermentation system described in step (3) is 0–3.0 g / L; preferably 0.5–2.0 g / L; more preferably 1.0–2.0 g / L.

[0025] In step (3), the concentration of pretreated sugarcane bagasse in the fermentation system is 1-15% by mass (g) to volume (mL); more preferably 3-9% by mass to volume; and most preferably 3-7% by mass to volume.

[0026] The fermentation time in step (3) is preferably 6 to 48 hours; more preferably 18 to 36 hours; and most preferably 24 hours.

[0027] The surfactant mentioned in step (4) is preferably Triton X-100.

[0028] The amount of surfactant added in step (4) is preferably 0.2 to 0.3% by volume; more preferably 0.25%.

[0029] The fungal cellulase mentioned in step (4) is at least one of Trichoderma reesei cellulase crude extract and commercial cellulase.

[0030] The preferred commercial cellulase is Cellic®CTec2.

[0031] The amount of fungal cellulase added in step (4) is 1 to 7.0 FPU / g pretreated sugarcane bagasse; more preferably 4.0 to 6.0 FPU / g pretreated sugarcane bagasse; and most preferably 4.0 to 5.0 FPU / g pretreated sugarcane bagasse.

[0032] The saccharification time in step (4) is 24 to 120 h; more preferably 72 to 96 h; and even more preferably 72 h.

[0033] The above methods are applied in the preparation of cellulose bodies or in the saccharification and fermentation of lignocellulose.

[0034] The lignocellulose is preferably derived from sugarcane bagasse.

[0035] Compared with the prior art, the advantages of this invention are:

[0036] This invention utilizes biochar to regulate the bacterial metabolic environment, significantly enhancing the synthesis and secretion levels of bacterial cellulosomal bodies. This allows for rapid accumulation of cellulosomal bodies in a short period, reducing energy consumption during cultivation. By combining enhanced cellulosomal production with low-load fungal cellulase, the effective breakdown of crystalline cellulose by cellulosomal bodies is fully realized, improving substrate accessibility. Compared to traditional techniques relying solely on fungal cellulase, this invention significantly reduces the amount of exogenous fungal cellulase used and increases reducing sugar yield, achieving synergistic saccharification by bacterial cellulosomal bodies and fungal cellulase. This invention achieves efficient saccharification of biomass waste under green, low-carbon, and cost-controllable conditions, providing an efficient and feasible technical solution for the resource utilization of biomass. Attached Figure Description

[0037] Figure 1 The images show the effects of co-saccharification of Acetivibrio thermocellus DSM1313 with fungal cellulase under different conditions. In the images, A represents the effect of different temperatures on the prepared pyrolytic biochar, B represents the effect of different biochar dosages, C represents the effect of different addition times of Triton and fungal cellulase, D represents the effect of different substrate loadings, and E represents the effect of different fungal cellulase dosages.

[0038] Figure 2 Figure showing the comparison of enzymatic hydrolysis results between different fungal cellulase Cellic®CTec2 addition amounts and combined saccharification.

[0039] Figure 3 The figures show the effects of co-saccharification of Acetivibrio thermocellus ATCC27405 with fungal cellulase under different conditions. Among them, A shows the effect of different temperatures on the pyrolytic biochar prepared, B shows the effect of different biochar dosages, C shows the effect of different addition times of Triton and fungal cellulase, D shows the effect of different substrate loadings, and E shows the effect of different fungal cellulase dosages. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0041] The bacteria Acetivibrio thermocellus DSM1313 and Acetivibrio thermocellus ATCC27405 were purchased from the German Microbiology and Cell Culture Collection (DSMZ) of the Leibniz Institute.

[0042] The culture medium for Acetivibrio thermocellus DSM1313 was a modified DSMZ122 medium with the following composition: 5.0 g / L microcrystalline cellulose, 1.50 g / L KH2PO4, 3.93 g / L K2HPO4, 1.30 g / L (NH4)2SO4, 0.10 g / L CaCl2⋅2H2O, 1.00 g / L MgCl2⋅6H2O, 4.50 g / L yeast extract, and 0.001 g / L resazurin. The pH was adjusted to 7.0, and the solvent was deionized water.

[0043] Example 1: Biochar-enhanced Acetivibrio thermocellus DSM1313 combined with low-load fungal cellulase for saccharification pretreatment of bagasse

[0044] (1) Preparation of pretreated bagasse: The bagasse was dried at 85 °C and then screened through a 100-mesh sieve. A eutectic solvent (a homogeneous solvent formed at 80 °C by choline chloride, lactic acid and ethylene glycol in a molar ratio of 1:5:1) was mixed with the screened bagasse at a ratio of 20:1 (v / w). After pretreatment at 130 °C for 3 hours, the bagasse was rinsed with 5 times its volume of hot water at 80 °C and the cellulose-rich residue was collected. The bagasse was dried at 55 °C to constant weight to obtain pretreated bagasse (cellulose content of 59.62% and hemicellulose content of 1.54%).

[0045] (2) Preparation of biochar: After drying the untreated bagasse at 105 °C, the crucible containing the bagasse was wrapped with tin foil and pyrolyzed at 300 °C to 700 °C for 1.0 h with a heating rate of 10 °C / min. Biochar at 300 °C, 400 °C, 500 °C, 600 °C and 700 °C were prepared respectively. After being placed at room temperature, the biochar was passed through a 100-mesh sieve for later use.

[0046] (3) Activation of the strain: Acetivibrio thermocellus DSM1313 was inoculated at a rate of 10.0% (v / v) into an anaerobic serum bottle (total volume 100 mL) containing 50 mL of modified DSMZ122 medium as the working volume. The medium contained 5.0 g / L microcrystalline cellulose as the carbon source. The culture was then incubated at 55 °C and 150 rpm with shaking for 48 h. This process was repeated three times until the strain was stable.

[0047] (4) Preparation of seed culture: After the strain is stabilized, the activated strain is inoculated into DSMZ122 medium containing 5.0 g / L microcrystalline cellulose (Avicel® PH-101, Maclean) as carbon source at an inoculation rate of 10.0% v / v. After culturing in a shaker at 55 ℃ and 150 rpm for 24 h, the seed culture is obtained and stored in a refrigerator at 4 ℃ for later use.

[0048] (5) Saccharification and fermentation: In a modified DSMZ122 medium containing 0–3.0 g / L biochar and 1.0%–15.0% pretreated bagasse loading, 10.0% v / v DSM1313 seed culture was inoculated and cultured at 55 °C and 150 rpm for 6–48 h. Then, 0.25% (v / v) Triton X-100 and 0–5.0 FPU / g substrate commercial fungal cellulase Cellic®CTec2 were added, and saccharification was continued for 72 h.

[0049] (6) The saccharification solution was tested according to the method for determining reducing sugars (3,5-dinitrosalicylic acid method) in GB5009.7-2016 National Food Safety Standard. The formula for calculating saccharification efficiency is as follows:

[0050] ;

[0051] Among them, C RS V is the concentration of reducing sugars in the saccharification solution (g / L); V is the volume of the saccharification solution (L); m 木质纤维素 To ensure the quality of pretreated sugarcane bagasse; f 纤维素 f represents the percentage of cellulose. 半纤维素 1.111 is the percentage of hemicellulose; 1.111 is the conversion factor for cellulose to glucose; 1.136 is the conversion factor for hemicellulose to xylose.

[0052] The results are shown below:

[0053] like Figure 1As shown in Figure A, the effects of biochar at different pyrolysis temperatures on the accumulation of reducing sugars during the co-saccharification of DSM1313 and the commercial fungal cellulase Cellic®CTec2 were compared. Except for the type of pyrolysis biochar (biochar at 300 ℃, 400 ℃, 500 ℃, 600 ℃, and 700 ℃), all other conditions were kept consistent. Specifically, the biochar dosage was 1.0 g / L, the pretreated bagasse loading was 5.0% (w / v), and after 24 h of growth on DSM1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic®CTec2 were added, followed by saccharification for another 72 h. The results showed that as the pyrolysis temperature of biochar increased, the effect of biochar in enhancing the combined saccharification of cellulose and fungal cellulase gradually decreased. However, biochar pyrolyzed at 300℃ could increase the accumulation of reducing sugars by about 129.11%, and had the best ability to enhance the combined saccharification of cellulose and fungal cellulase.

[0054] like Figure 1 As shown in Figure B, the effects of different concentrations of 300℃ biochar on the accumulation of reducing sugars during the combined saccharification of DSM1313 and the commercial fungal cellulase Cellic®CTec2 were compared. Except for the biochar dosage (0–3.0 g / L), all other conditions were kept consistent. Specifically, the biochar type was 300℃ pyrolytic biochar, the pretreated bagasse loading was 5.0% (w / v), and after 24 h of growth of DSM1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic®CTec2 were added, followed by saccharification for another 72 h. The results showed that a biochar dosage of 1.0 g / L at 300℃ exhibited the best ability to enhance the combined saccharification of cellulosomal tissue and fungal cellulase, achieving a reducing sugar accumulation of 20.07 g / L and a saccharification efficiency of 59.04%.

[0055] like Figure 1As shown in Figure C, the effects of Triton X-100 and commercial fungal cellulase Cellic® CTec2 addition time on the accumulation of reducing sugars during co-saccharification were compared. Except for the addition time (0–48 h) between Triton and the commercial fungal cellulase, all other conditions were kept consistent. Specifically, the biochar type was pyrolyzed biochar at 300 °C, the biochar dosage was 1.0 g / L, the pretreated bagasse loading was 5.0% (w / v), the Triton X-100 concentration was 0.25% (v / v), and the commercial fungal cellulase Cellic® CTec2 dosage was 5.0 FPU / g substrate, followed by saccharification for 72 h. The results showed that a 24 h addition time for both Triton X-100 and Cellic® CTec2 exhibited the best ability to enhance the co-saccharification of cellulosomal tissue and fungal cellulase, achieving a reducing sugar accumulation of 29.79 g / L and a saccharification efficiency of 87.64%.

[0056] like Figure 1 As shown in Figure D, the effect of pretreated bagasse loading on reducing sugar accumulation during co-saccharification of DSM1313 and the commercial fungal cellulase Cellic®CTec2 was compared. Except for the difference in pretreated bagasse loading (1–15%), all other conditions were kept consistent. Specifically, the biochar type was pyrolytic biochar at 300 °C, and the biochar dosage was 1.0 g / L. After 24 h of growth of DSM1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic®CTec2 were added, and saccharification continued for 72 h. The results showed that as the substrate loading of pretreated bagasse gradually increased, the reducing sugar accumulation gradually increased. A reducing sugar accumulation of 39.03 g / L was achieved under a pretreated bagasse loading of 9.0%. However, the saccharification efficiency reached its maximum at a loading of 5.0%, with an efficiency of 87.64%. Taking all factors into consideration, a pretreatment bagasse loading of 5.0% was selected to obtain the optimal saccharification efficiency.

[0057] like Figure 1Figure E in the figure compares the effect of the dosage of the commercial fungal cellulase Cellic® CTec2 on the accumulation of reducing sugars during co-saccharification. Except for the dosage of the commercial fungal cellulase Cellic® CTec2 (0–5.0 FPU / g substrate), all other conditions were kept consistent. Specifically, the biochar type was pyrolytic biochar at 300 °C, and the biochar dosage was 1.0 g / L. After 24 h of growth on DSM1313, 0.25% (v / v) Triton X-100 and the commercial fungal cellulase Cellic® CTec2 were added, and saccharification was continued for another 72 h. The results showed that increasing the dosage of fungal cellulase significantly improved the accumulation of reducing sugars. The saccharification efficiencies obtained with Cellic® CTec2 dosages of 4.0 FPU / g substrate and 5.0 FPU / g substrate were 83.63% and 87.63%, respectively. No significant difference was observed through significance analysis. To minimize the amount of fungal cellulase used, a fungal cellulase dosage of 4.0 FPU / g sugarcane bagasse was selected as the optimal saccharification condition, resulting in a reducing sugar accumulation of 28.43 g / L and a saccharification efficiency of 83.63%.

[0058] Comparative Example 1

[0059] Table 1. Control experiment of biochar-enhanced Acetivibrio thermocellus DSM1313 combined with fungal cellulase for saccharification.

[0060]

[0061] Note: The letters a, b, c, d, and e represent the results of the significance analysis; different letters indicate significant differences.

[0062] Table 1 lists the control experiments of saccharification combined with biochar-enhanced Acetivibrio thermocellus DSM1313 and fungal cellulase. The optimal conditions optimized in Example 1 were used: biochar type was pyrolytic biochar at 300℃, biochar dosage was 1.0 g / L, substrate loading was 5.0% (w / v), and after 24 h of DSM1313 growth, 0.25% (v / v) Triton X-100 and 4.0 FPU / g of commercial fungal cellulase Cellic® CTec2 were added, followed by saccharification for another 72 h. Acetivibrio thermocellus DSM1313 alone pretreated bagasse yielded only 4.04 g / L of reducing sugar (Group 2). Further pretreatment with biochar-enhanced Acetivibrio thermocellus DSM1313 yielded 8.02 g / L of reducing sugar (Group 3). Saccharification of Acetivibrio thermocellus DSM1313 without biochar, combined with fungal cellulase, yielded 20.58 g / L of reducing sugar (Group 4). Pretreatment of sugarcane bagasse with fungal cellulase alone accumulated 4.98 g / L of reducing sugar (Group 5). Further addition of biochar to the fungal cellulase saccharification system accumulated 4.84 g / L of reducing sugar.

[0063] Comparative Example 2

[0064] Figure 2 The enzymatic hydrolysis process of different fungal cellulase Cellic®CTec2 addition amounts and combined saccharification was compared. The fungal cellulase hydrolysis method was kept consistent as follows: 0.1 g of pretreated sugarcane bagasse was mixed with 5.0 mL of DSM122 medium (which did not contain microcrystalline cellulose carbon source), followed by the addition of fungal cellulase Cellic®CTec2 at 10–40 FPU / g substrate. The enzymatic hydrolysis reaction was carried out in a shaking incubator at 55 °C for 72 hours.

[0065] For the combined saccharification group, the supernatant from 24 hours of biochar-enhanced growth of *Acetivibrio thermocellus* DSM1313 was concentrated to 5.0 mL using a 10 kDa ultrafiltration tube. 0.1 g of pretreated sugarcane bagasse was mixed with 5.0 mL of the concentrated supernatant, followed by the addition of 4.0 FPU / g of substrate fungal cellulase Cellic®CTec2. The enzymatic digestion reaction was carried out in a 55°C shaking incubator for 72 hours.

[0066] The results showed that the combined saccharification method using Acetivibrio thermocellus DSM1313 supernatant and fungal cellulase achieved a saccharification efficiency of approximately 83.0% with Cellic®CTec2 at a substrate concentration of only 30.0 FPU / g, while using only 4.0 FPU / g substrate. Therefore, the combined saccharification method can save approximately 6.5 times the amount of fungal cellulase used.

[0067] Example 2: Biochar-enhanced Acetivibrio thermocellus ATCC27405 combined with low-load fungal cellulase for saccharification pretreatment of bagasse.

[0068] (1) Preparation of pretreated bagasse: The bagasse was dried at 85 °C and then screened through a 100-mesh sieve. A eutectic solvent (a homogeneous solvent formed at 80 °C by choline chloride, lactic acid and ethylene glycol in a molar ratio of 1:5:1) was mixed with the screened bagasse at a ratio of 20:1 (v / w). After pretreatment at 130 °C for 3 hours, the bagasse was rinsed with 5 times its volume of hot water at 80 °C and the cellulose-rich residue was collected. The bagasse was dried at 55 °C to constant weight to obtain pretreated bagasse (cellulose content of 59.62% and hemicellulose content of 1.54%).

[0069] (2) Preparation of biochar: After drying the untreated bagasse at 105 °C, the crucible containing the bagasse was wrapped with tin foil and pyrolyzed at 300 °C to 700 °C for 1.0 h with a heating rate of 10 °C / min. Biochar at 300 °C, 400 °C, 500 °C, 600 °C and 700 °C were prepared respectively. After being placed at room temperature, the biochar was passed through a 100-mesh sieve for later use.

[0070] (3) Activation of the strain: Acetivibrio thermocellus ATCC27405 was inoculated at a rate of 10.0% (v / v) into an anaerobic serum bottle (total volume 100 mL) containing 50 mL of modified DSMZ122 medium as the working volume. The medium contained 5.0 g / L microcrystalline cellulose as the carbon source. The culture was then incubated at 55 °C and 150 rpm with shaking for 48 h. This process was repeated three times until the strain was stable.

[0071] (4) Preparation of seed culture: After the strain is stabilized, the activated strain is inoculated into DSMZ122 medium containing 5.0 g / L microcrystalline cellulose (Avicel® PH-101, Maclean) as carbon source at an inoculation rate of 10.0% v / v. After culturing in a shaker at 55 ℃ and 150 rpm for 24 h, the seed culture is obtained and stored in a refrigerator at 4 ℃ for later use.

[0072] (5) Saccharification and fermentation: In a modified DSMZ122 medium containing 0–3.0 g / L biochar and 1.0%–15.0% pretreated sugarcane bagasse load, 10.0% v / v ATCC27405 seed culture was inoculated and cultured at 55 °C and 150 rpm for 6–48 h. Then, 0.25% (v / v) Triton X-100 and 0–5.0 FPU / g substrate commercial fungal cellulase Cellic®CTec2 were added, and saccharification was continued for 72 h.

[0073] (6) The saccharification solution was determined according to the method for determining reducing sugars (3,5-dinitrosalicylic acid method) in GB5009.7-2016 National Food Safety Standard. The saccharification efficiency was calculated as described in Example 1.

[0074] The results are as follows:

[0075] like Figure 3 As shown in Figure A, the effects of biochar at different pyrolysis temperatures on the accumulation of reducing sugars during co-saccharification by Acetivibrio thermocellus ATCC27405 and the commercial fungal cellulase Cellic® CTec2 were compared. Except for the type of pyrolysis biochar (biochar at 300 ℃, 400 ℃, 500 ℃, 600 ℃, and 700 ℃), all other conditions were kept consistent. Specifically, the biochar dosage was 1.0 g / L, the pretreated bagasse loading was 5.0 % (w / v), and after 24 h of growth on DSM 1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic® CTec2 were added, followed by saccharification for another 72 h. The results showed that as the pyrolysis temperature of biochar increased, the effect of biochar in enhancing the combined saccharification of cellulose and fungal cellulase gradually decreased. However, biochar pyrolyzed at 300 °C could increase the accumulation of reducing sugars by about 111.55%, and had the best ability to enhance the combined saccharification of cellulose and fungal cellulase.

[0076] like Figure 3As shown in Figure B, the effects of different concentrations of 300 °C biochar on the accumulation of reducing sugars during the combined saccharification of ATCC27405 and the commercial fungal cellulase Cellic®CTec2 were compared. Except for the biochar dosage (0–3.0 g / L), all other conditions were kept consistent. Specifically, the biochar type was 300 °C pyrolytic biochar, the pretreated bagasse loading was 5.0% (w / v), and after 24 h of growth on DSM1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic®CTec2 were added, followed by saccharification for another 72 h. The results showed that a biochar dosage of 2.0 g / L at 300 °C exhibited the best ability to enhance the combined saccharification of cellulosomal tissue and fungal cellulase, achieving a reducing sugar accumulation of 15.09 g / L and a saccharification efficiency of 44.39%.

[0077] like Figure 3 As shown in Figure C, the effects of the addition time of Triton X-100 and the commercial fungal cellulase Cellic® CTec2 on the accumulation of reducing sugars during co-saccharification were compared. Except for the addition time (0–48 h) between Triton and the commercial fungal cellulase, all other conditions were kept consistent. Specifically, the biochar type was pyrolyzed biochar at 300 °C, the biochar dosage was 2.0 g / L, the loading of pretreated bagasse was 5.0% (w / v), the concentration of Triton X-100 was 0.25% (v / v), and the dosage of the commercial fungal cellulase Cellic® CTec2 was 5.0 FPU / g substrate, followed by saccharification for 72 h. The results showed that the addition time of Triton X-100 and the commercial fungal cellulase Cellic® CTec2 at 36 h exhibited the best ability to enhance the co-saccharification of cellulosomal tissue and fungal cellulase, achieving a reducing sugar accumulation of 18.16 g / L and a saccharification efficiency of 53.41%.

[0078] like Figure 3As shown in Figure D, the effect of pretreated bagasse loading on reducing sugar accumulation during co-saccharification of DSM1313 and the commercial fungal cellulase Cellic®CTec2 was compared. Except for the difference in pretreated bagasse loading (1–15%), all other conditions were kept consistent. Specifically, the biochar type was pyrolytic biochar at 300℃, and the biochar dosage was 2.0 g / L. After 36 h of growth of DSM1313, 0.25% (v / v) Triton X-100 and 5.0 FPU / g of the substrate commercial fungal cellulase Cellic®CTec2 were added, and saccharification continued for another 72 h. The results showed that as the substrate loading of pretreated bagasse gradually increased, the reducing sugar accumulation gradually increased. A reducing sugar accumulation of 22.16 g / L was achieved under a pretreated bagasse loading of 9.0%. However, the saccharification efficiency reached its maximum at a loading of 3.0%, with a saccharification efficiency of 60.08%. Taking all factors into consideration, a pretreatment bagasse loading of 3.0% was selected to obtain the optimal saccharification efficiency.

[0079] like Figure 3 Figure E shows the effect of the dosage of the commercial fungal cellulase Cellic® CTec2 on the accumulation of reducing sugars during co-saccharification. Except for the dosage of the commercial fungal cellulase Cellic® CTec2 (0–7.0 FPU / g substrate), all other conditions were kept consistent. Specifically, the biochar type was pyrolytic biochar at 300℃, and the biochar dosage was 2.0 g / L. After 36 h of growth on DSM1313, 0.25% (v / v) Triton X-100 and the commercial fungal cellulase Cellic® CTec2 were added, and saccharification was continued for another 72 h. The results showed that increasing the dosage of fungal cellulase significantly improved the accumulation of reducing sugars. The saccharification efficiencies obtained with Cellic® CTec2 dosages of 6.0 FPU / g substrate and 7.0 FPU / g substrate were 85.82% and 86.24%, respectively. No significant difference was observed through significance analysis. To minimize the amount of fungal cellulase used, a fungal cellulase dosage of 6.0 FPU / g sugarcane bagasse was selected as the optimal saccharification condition, resulting in a reducing sugar accumulation of 17.17 g / L and a saccharification efficiency of 85.82%.

[0080] Comparative Example 3

[0081] Table 2. Control experiment of biochar-enhanced Acetivibrio thermocellus ATCC27405 combined with fungal cellulase for saccharification.

[0082]

[0083] Note: The letters a, b, c, d, and e represent the results of the significance analysis; different letters indicate significant differences.

[0084] Table 2 lists the control experiments of saccharification using biochar-enhanced Acetivibrio thermocellus ATCC27405 in combination with fungal cellulase. The optimal conditions optimized in Example 1 were used, namely, pyrolytic biochar at 300 °C, biochar dosage of 2.0 g / L, and substrate loading of 3.0% (w / v). After 36 h of growth of DSM1313, 0.25% (v / v) Triton X-100 and 6.0 FPU / g of the commercial fungal cellulase Cellic® CTec2 were added, and saccharification was continued for another 72 h. Saccharification pretreatment of bagasse with Acetivibrio thermocellus ATCC27405 alone yielded only 2.04 g / L of reducing sugar (Group 2). Further addition of biochar-enhanced Acetivibrio thermocellus ATCC27405 to pretreat bagasse yielded 4.02 g / L of reducing sugar (Group 3). Saccharification of Acetivibrio thermocellus ATCC27405 without biochar, combined with fungal cellulase, yielded 14.58 g / L of reducing sugar (Group 4). Pretreatment of sugarcane bagasse with fungal cellulase alone resulted in the accumulation of 6.52 g / L of reducing sugar (Group 5). Further addition of biochar to the fungal cellulase saccharification system resulted in the accumulation of 6.84 g / L of reducing sugar.

[0085] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification, characterized in that... Includes the following steps: (1) Dry, crush and sieve the sugarcane bagasse, treat it with a low eutectic solvent, and then wash and dry the product obtained by treatment to obtain pretreated sugarcane bagasse rich in cellulose. (2) Biochar is obtained by pyrolyzing agricultural and forestry waste under an oxygen-deficient environment; (3) Use the pretreated sugarcane bagasse obtained in step (1) as the carbon source for the growth of bacteria, and add an appropriate amount of biochar prepared in step (2) to enhance the growth of bacteria and fermentation, so as to fully accumulate the cellulose produced by bacteria. (4) After the fermentation in step (3) is completed, a surfactant is added to release cellulose bodies, and fungal cellulase is added at the same time to perform in-situ co-saccharification of the pretreated sugarcane bagasse.

2. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The eutectic solvent mentioned in step (1) is a mixture of choline chloride, lactic acid and ethylene glycol in a molar ratio of 1:2 to 5:

1. The agricultural and forestry waste mentioned in step (2) is at least one of corn stalks, rice stalks and sugarcane bagasse.

3. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The drying temperature described in step (1) is 70–100 °C; The sieve mentioned in step (1) is a 100-200 mesh sieve; The cleaning described in step (1) is a hot water cleaning process; The drying temperature described in step (1) is 40 to 70°C.

4. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The processing temperature described in step (1) is 90–130°C; The processing time described in step (1) is between 2 and 6 hours; The pyrolysis conditions described in step (2) are as follows: the heating rate is 5-15 ℃ / min, and the temperature is raised to 300-700 ℃ and held for 1-4 h.

5. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The bacterial strain mentioned in step (3) is a cellulose-degrading bacterium capable of producing cellulose bodies; The surfactant mentioned in step (4) is Triton X-100; The fungal cellulase mentioned in step (4) is at least one of Trichoderma reesei cellulase crude extract and commercial cellulase.

6. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 5, characterized in that: The bacterial strain mentioned in step (3) is a thermophilic anaerobic cellulose-degrading bacterium; The commercial cellulase mentioned is Cellic®CTec2.

7. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The biochar mentioned in step (3) is biochar that can pass through a 100-mesh sieve; The biochar concentration in the fermentation system described in step (3) is 0–3.0 g / L; In step (3), the concentration of pretreated sugarcane bagasse in the fermentation system is 1-15% by mass-volume ratio; The amount of surfactant added in step (4) is 0.2% to 0.3% by volume. The amount of fungal cellulase added in step (4) is 1 to 7.0 FPU / g of pretreated sugarcane bagasse.

8. The method for pretreating bagasse based on biochar-reinforced cellulose bodies and synergistic fungal cellulase saccharification according to claim 1, characterized in that: The fermentation time described in step (3) is 6 to 48 hours; The saccharification time in step (4) is 24 to 120 h.

9. The application of the method according to any one of claims 1 to 8 in the preparation of cellulose bodies or in the saccharification and fermentation of lignocellulose.

10. The application according to claim 9, characterized in that: The lignocellulose mentioned is derived from sugarcane bagasse.

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