A method for two-stage photocatalytic oxidation pretreatment and in-situ detoxification enzymolysis of lignocellulose

Abstract

This invention provides a two-stage photocatalytic oxidation pretreatment and in-situ detoxification enzymatic hydrolysis method for lignocellulose, relating to the field of lignocellulose biomass pretreatment technology. This invention sequentially performs alkaline H2O2 / UV-TiO2 photocatalytic oxidation deconstruction and pH-switched in-situ photocatalytic detoxification in the same whole slurry system. During the in-situ detoxification stage, the addition of new H2O2 is stopped, and photocatalytic oxidation continues using the residual H2O2 from the previous stage and the same immobilized TiO2. This achieves a reduction in residual H2O2 and soluble phenolic and / or furan inhibitors in the whole slurry without pretreatment solid washing or discarding the pretreatment solution. Furthermore, the treated whole slurry is directly connected to cellulase enzymatic hydrolysis, balancing lignocellulose deconstruction, liquid-phase inhibitor control, and retention of usable components in the pretreatment solution. This method shows promising application prospects in the full utilization of lignocellulose components and biorefining processes.

Description

Technical Field

[0001] This invention relates to the field of lignocellulose biomass pretreatment technology, and in particular to a two-stage photocatalytic oxidation pretreatment and in-situ detoxification enzymatic hydrolysis method for lignocellulose. Background Technology

[0002] Lignocellulose biomass is mainly composed of cellulose, hemicellulose, and lignin. Cellulose forms a relatively stable framework structure in the form of microfibrils, while lignin and hemicellulose are distributed around the cellulose, forming a natural anti-degradation barrier through hydrogen bonds, ether bonds, and lignin-carbohydrate complex structures. This structure not only restricts mass transfer and effective contact of cellulase to the cellulose surface, but lignin can also cause non-productive adsorption of cellulase. Therefore, when using lignocellulose to prepare fermentable sugars and further produce fuels or bio-based chemicals, it is usually necessary to pretreat to break down the barrier structure formed by lignin and hemicellulose, thereby improving the accessibility of cellulose and the efficiency of subsequent enzymatic hydrolysis.

[0003] Chinese patent CN 108251467 A discloses a method for improving the enzymatic hydrolysis efficiency of natural lignocellulose through photocatalytic oxidation. The method involves contacting lignocellulose with oxidants such as TiO2, hydrogen peroxide, or persulfate for photocatalytic oxidation treatment, followed by filtration, drying, and enzymatic hydrolysis of the treated sample. However, this method still requires separation of the solid sample after pretreatment, and it does not address the issue of residual oxidants and liquid-phase degradation products affecting cellulase when the pretreatment solution and pretreatment solids are directly incorporated into the subsequent enzymatic hydrolysis as a complete slurry.

[0004] Patent CN 102191279 A discloses a method for biological detoxification of pretreated lignocellulose biomass using fungi, which requires inoculation with microorganisms and cultivation for a certain period of time. Patent CN114317838 A discloses a method for detoxifying lignocellulose hydrolysate by reducing the concentration of inhibitors in the hydrolysate through stripping. These methods can reduce the impact of inhibitors to some extent, but they typically require the introduction of microbial cultivation, gas stripping, or a separate detoxification unit. For systems using H2O2 and TiO2 photocatalysis for lignocellulose pretreatment, removing the pretreatment solution after pretreatment by filtration, centrifugation, and washing can reduce the content of residual H2O2 and soluble inhibitors, but it increases solid-liquid separation and washing operations, and causes the dissolved usable sugars and other usable components in the pretreatment solution to be discharged with the liquid phase. If the entire pulp is directly adjusted to enzymatic hydrolysis conditions without washing, residual H2O2 and soluble degradation products such as phenols and furans may affect cellulase activity. On the other hand, if photocatalytic oxidation treatment continues for a long time under the existing strong alkaline and strong oxidizing conditions, it may increase the risk of further oxidation and loss of carbohydrates.

[0005] Therefore, further improvements are still needed to find ways to utilize the photocatalysts and residual oxidants already present in the pretreatment stage, without washing the pretreatment solids, discarding the pretreatment liquid, and minimizing the addition of independent detoxification materials and treatment units, to reduce the residual oxidants and soluble inhibitors in the pretreated slurry, and to enable the treated slurry to be directly connected to cellulase hydrolysis. Summary of the Invention

[0006] The present invention provides a two-stage photocatalytic oxidation pretreatment and in-situ detoxification enzymatic hydrolysis method for lignocellulose, comprising the following steps: S1. Alkaline photocatalytic oxidation decomposition: Lignocellulose raw materials are mixed with water to form a slurry. The pH of the slurry is adjusted to 10.0~12.0. H2O2 is added in the presence of an immobilized TiO2 photocatalyst. The total amount of H2O2 used is 0.20~0.70 g / g of oven-dry lignocellulose based on 100% H2O2 mass. The reaction is carried out at 45~70℃ and under UV irradiation at 320~400 nm for 2~6 h to obtain a complete slurry containing pretreated solids and pretreated liquid. S2, pH switching in-situ detoxification: After step S1, the whole slurry is not subjected to solid-liquid separation and water washing. The addition of new H2O2 to the system is stopped. The pH of the whole slurry is adjusted to 6.0~8.5, and the whole slurry continues to be in contact with the immobilized TiO2 photocatalyst used in step S1. It is treated under UV irradiation of 320~400 nm for 0.5~3.0 h to consume the H2O2 remaining in step S1 and to perform photocatalytic oxidation on soluble phenols and / or furan inhibitors in the pretreatment solution. S3. Direct enzymatic hydrolysis: After step S2, without washing the pretreated solid with water or discarding the pretreated liquid, adjust the pH of the system to 4.6~5.2 and the temperature to 45~55℃, and directly add cellulase to carry out enzymatic hydrolysis to obtain an enzymatic hydrolysate containing fermentable sugars.

[0007] Preferably, the immobilized TiO2 photocatalyst in step S1 is a TiO2 film supported on the surface of a glass plate, quartz plate, or hollow glass microspheres. The TiO2 is mainly composed of anatase phase, and no free TiO2 powder is added to the pretreatment slurry. The preparation method of the immobilized TiO2 catalyst is as follows: Clean and dry the glass or quartz plate. Prepare a 100-180 g / L anatase TiO2 aqueous suspension and adjust the pH to 1.0-2.0 with HNO3. Immerse the support in the TiO2 suspension and remove it at 5-20 cm / min. Dry it at 70-90℃, then raise the temperature to 380-450℃ at 2-10℃ / min and hold for 1-3 h. Repeat the above coating, drying, and heat treatment process 2-6 times to obtain an immobilized TiO2 film; or, Hollow glass microspheres were added to a 5–20 g / L TiO2 suspension and contacted with the suspension for 10–60 min under stirring and sonication. The mixture was then filtered, dried, and heat-treated at 380–450 °C for 1–3 h. This process was repeated 2–6 times. Before use, the microspheres were rinsed with water to remove any loosely attached TiO2 particles.

[0008] Preferably, the H2O2 in step S1 is added in 3 to 10 portions, with an interval of 20 to 90 minutes between adjacent additions, and all H2O2 is added in step S1; the pH of the slurry is 10.5 to 11.5, the temperature is 55 to 68°C, the reaction time is 3 to 5 hours, and the total amount of H2O2 used is 0.30 to 0.60 g / g oven-dry lignocellulose.

[0009] Preferably, the pH of step S2 is 6.5~8.0, the temperature is 35~55℃, the UV treatment time is 0.5~2.0 h, and no additional H2O2, persulfate, ozone or hypochlorite is added in step S2; the liquid phase residual H2O2 concentration is not higher than 0.05 g / L as the termination condition.

[0010] In this invention, no water washing operation for removing soluble lignin degradation products is performed between the end of step S1 and the beginning of step S3, and the liquid phase after step S2 treatment and the pretreated solid enter step S3 together.

[0011] Preferably, in step S3, 10-30 FPU / g of substrate cellulase and 5-20 IU / g of substrate β-glucosidase are added, and enzymatic hydrolysis is carried out at 45-55℃ and 80-180 rpm for 24-96 h; the acid used to lower the pH is selected from at least one of HCl, H2SO4, H3PO4, acetic acid, and citric acid.

[0012] Preferably, the lignocellulose raw material is selected from corn stalks, rice straw, wheat straw, sorghum stalks, sugarcane bagasse, bamboo processing residues, reed stalks and combinations thereof, the particle size of the raw material is 0.15~2.00 mm, and the concentration of the oven-dry raw material in step S1 is 30~150 g / L.

[0013] After one batch of the present invention is completed, the immobilized TiO2 photocatalyst is washed sequentially with 0.05~0.20 mol / L HCl aqueous solution and water, dried, and used for the next batch, and the process is repeated for 2~10 batches.

[0014] This invention also provides an in-situ detoxification method for alkaline H2O2 / UV-TiO2 pretreated lignocellulose pulp, the steps of which are as follows: For the whole slurry containing both pretreated solid and pretreated liquid obtained by UV irradiation with alkaline H2O2 and immobilized TiO2 photocatalyst, solid-liquid separation and water washing are not performed. The addition of new H2O2 is stopped, and the pH of the whole slurry is reduced from 10.0~12.0 in the alkaline pretreatment stage to 6.0~8.5. The whole slurry continues to be in contact with the immobilized TiO2 photocatalyst and is irradiated with 320~400 nm UV for 0.5~3.0 h. Then the whole slurry is adjusted to cellulase hydrolysis conditions and directly hydrolyzed to obtain an enzymatic hydrolysate containing fermentable sugars.

[0015] Preferably, the acid used to lower the pH is selected from at least one of HCl, H2SO4, H3PO4, acetic acid, and citric acid.

[0016] Preferably, no new oxidant is added during the in-situ detoxification stage, and the H2O2 remaining from the alkaline pretreatment stage continues to participate in the oxidation reaction of soluble inhibitors under the action of UV / TiO2.

[0017] Preferably, the pretreatment liquid in the whole slurry after in-situ detoxification treatment does not undergo ion exchange resin, activated carbon adsorption, excessive lime neutralization, or organic solvent extraction, but directly enters the enzymatic hydrolysis step along with the pretreatment solid.

[0018] Preferably, the enzymatic hydrolysate containing fermentable sugars is further used for fermentation by yeast, bacteria, or filamentous fungi to produce ethanol, organic acids, polyols, or other bio-based chemicals.

[0019] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention sets the alkaline photocatalytic oxidation pretreatment and in-situ detoxification of lignocellulose as two consecutive treatment stages within the same whole pulp system, thereby reducing solid-liquid separation and washing operations between pretreatment and enzymatic hydrolysis, and avoiding the physical loss of usable water-soluble components caused by directly discarding the pretreatment liquid, thus providing a process basis for the direct enzymatic hydrolysis of pretreated whole pulp.

[0020] This invention stops adding new H2O2 during the in-situ detoxification stage, allowing the residual H2O2 from the first stage to continue participating in the photocatalytic oxidation reaction under UV and the same immobilized TiO2. This reduces the content of residual H2O2, soluble phenols, and furan inhibitors in the liquid phase while utilizing existing oxidants and photocatalysts, eliminating the need for additional adsorption materials such as activated carbon and ion exchange resins, as well as the need to add new strong oxidants. After the second stage treatment, both residual H2O2 and total phenol content significantly decrease, and the enzymatic hydrolysis performance of the unwashed slurry is correspondingly improved, showing a significant improvement compared to direct enzymatic hydrolysis after the first stage.

[0021] This invention switches the system from the strongly alkaline pretreatment conditions of the first stage to in-situ detoxification conditions at pH 6.0–8.5, allowing residual H₂O₂ and immobilized TiO₂ to continue their liquid-phase oxidation function. This avoids prolonged exposure of lignocellulose to a strongly alkaline oxidizing environment, thus achieving a balance between reducing inhibitors and retaining carbohydrates. The use of immobilized TiO₂ as a photocatalyst prevents free nano-TiO₂ powder from entering the subsequent enzymatic hydrolysis system with the entire slurry. Furthermore, the immobilized catalyst can be separated from the reaction system and reused after treatment, reducing the operational burden of catalyst separation and repeated addition.

[0022] This invention does not require specialized reaction equipment with a specific structure and can be combined with existing UV / TiO2 photocatalytic reaction devices, exhibiting good process adaptability. Detailed Implementation

[0023] In this invention, "complete slurry" refers to a mixed system containing both insoluble pretreated solids and pretreated liquid after pretreatment; "no solid-liquid separation and washing" means that after the first stage, the solids are not separated from the pretreated liquid by filtration, centrifugation, or pressure filtration before washing with water. Acids, water, or buffer solutions added to adjust pH or regulate the content of enzymatically hydrolyzed solids are not considered as washing.

[0024] The immobilized TiO2 catalyst preparation method used in the following examples is as follows: Clean and dry a glass or quartz plate. Prepare a 150 g / L anatase TiO2 aqueous suspension, adjust the pH to 1.5 with HNO3, immerse the support in the TiO2 suspension, and then remove it at 12 cm / min. Dry it at 80℃, then raise the temperature to 400℃ at 5℃ / min and hold for 2 h. Repeat the above coating, drying, and heat treatment process four times to obtain an immobilized TiO2 film; or, Hollow glass microspheres were added to a 15 g / L TiO2 suspension and contacted for 35 min under stirring and sonication. The mixture was then filtered, dried, and heat-treated at 400℃ for 2 h. This process was repeated 4 times. Before use, the microspheres were rinsed with water to remove any loosely attached TiO2 particles.

[0025] Example 1 Crush the corn stalks to 60-80 mesh, wash with water, and dry at 60℃ to constant weight. Weigh 10.0 g of oven-dried corn stalks, add 250 mL of deionized water, and adjust the pH to 11.0 using NaOH aqueous solution.

[0026] Four layers of anatase TiO2 film glass plates were placed in a 500 mL photocatalytic reactor, and TiO2-coated hollow glass microspheres were added. A UV-A light source with a main emission wavelength of 365 nm and a rated power of 100 W was used, with the distance between the light source and the light-receiving surface of the immobilized TiO2 catalyst being 8 cm. The dimensions of the immobilized TiO2 glass plates were 5 cm × 10 cm, and both sides of each glass plate were coated with TiO2 film, with an average TiO2 loading of 0.80 ± 0.05 mg / cm³. 2 The reactor is equipped with four glass plates, with a total coating area of ​​400 cm². 2 The effective light-receiving area directly exposed to UV radiation is 200 cm². 2 The amount of TiO2-coated hollow glass microspheres added is 8.0 g / L of slurry, and the TiO2 loading is 8.0 ± 0.5 wt% based on the total mass of the TiO2-coated hollow glass microspheres.

[0027] The total amount of pure H2O2 used was 0.60 g / g of oven-dried corn stalks. A 30 wt% H2O2 aqueous solution was used, with H2O2 added in six portions at 0 h, 0.5 h, 1.0 h, 1.5 h, 2.0 h, and 2.5 h, respectively, with the same amount added each time. The total reaction time for the first stage was 4.0 h, and the reaction temperature was 60℃. During the reaction, NaOH aqueous solution was used to maintain the pH at 10.8–11.2.

[0028] After the first stage, stop adding H2O2 and do not perform filtration, centrifugation, or washing. Keep the immobilized TiO2 in contact with the entire slurry, slowly adjust the pH of the slurry to 7.0 with an aqueous HCl solution, adjust the temperature to 50℃, and continue 365 nm UV irradiation and stirring for 1.5 h. Take liquid phase samples every 30 min to determine residual H2O2; if the residual H2O2 is still higher than 0.05 g / L after 1.5 h, continue UV treatment and retest every 30 min until it is no higher than 0.05 g / L. The total time of the second stage should not exceed 3.0 h.

[0029] After the second stage, the immobilized TiO2 support was removed, but the liquid phase was not discarded. The entire slurry was adjusted to pH 4.8 and replenished with 0.2 mol / L sodium acetate buffer to bring the substrate concentration to 2 wt%. Cellulase at 20 FPU / g substrate (based on the initial oven-dry lignocellulosic raw material mass) and β-glucosidase at 12 IU / g substrate were added, and enzymatic hydrolysis was performed at 50℃ and 100 rpm for 72 h. After filtration, the glucose, xylose, and arabinose content of the hydrolysate was determined.

[0030] After the second stage of in-situ detoxification treatment, the residual H2O2 decreased from 0.43 g / L to 0.031 g / L, and the total phenols decreased from 635 mg GAE / L to 245 mg GAE / L, indicating that the in-situ detoxification treatment can effectively reduce residual oxidants and soluble inhibitory components.

[0031] Example 2 The difference from Example 1 is that: the total amount of H2O2 used in the first stage is 0.50 g / g of dry corn stalks, which is added in 8 equal amounts in 8 separate additions with an interval of 30 min between two adjacent additions; the reaction time in the first stage is 4.5 h; the remaining steps are the same as in Example 1.

[0032] Example 3 The difference from Example 1 is that: in the first stage, the pH is maintained at 10.5~10.8, the temperature is 60℃, and the total amount of H2O2 used is 0.45 g / g; in the second stage, the pH is adjusted to 7.5 and UV treatment is performed at 45℃ for 2.0 h; the remaining steps are the same as in Example 1.

[0033] Example 4 The difference from Example 1 is that the lignocellulose raw material is rice straw, the particle size is 0.25~0.50 mm, the dry raw material concentration is 50 g / L, and the total amount of H2O2 used is 0.50 g / g; the remaining steps are the same as in Example 1.

[0034] Example 5 The difference from Example 1 is that the lignocellulose raw material is wheat straw, the particle size of the crushed material is 0.18~0.50 mm, the temperature of the first stage is 60℃, and the total amount of H2O2 used is 0.45 g / g; the remaining steps are the same as in Example 1.

[0035] Example 6 The difference from Example 1 is that the lignocellulose raw material is sugarcane bagasse with a particle size of 0.25~1.00 mm, the dry raw material concentration is 80 g / L, and the total amount of H2O2 used is 0.55 g / g; the pH of the second stage is 6.5; the remaining steps are the same as in Example 1.

[0036] Comparative Example 1 The difference from Example 1 is that: after the first stage, the in-situ pH switching detoxification in step S2 is not performed. Instead, the entire slurry is directly adjusted to pH 4.8 and cellulase and β-glucosidase are added for enzymatic hydrolysis; the remaining steps are the same as in Example 1.

[0037] Comparative Example 2 The difference from Example 1 is that: after the first stage, a 100 mesh filter is used to separate the pretreated solids, and the solids are washed with deionized water until the pH reaches 6.5~7.5, and all the pretreated liquid is discarded; step S2 is not performed; the washed solids are enzymatically hydrolyzed according to the enzymatic hydrolysis conditions of Example 1.

[0038] Comparative Example 3 The difference from Example 1 is that: after the first stage, the addition of H2O2 is stopped, but the pH is not switched. The pH of the whole slurry is maintained at 10.8~11.2 and UV irradiation continues for 1.5 h. Then the pH is adjusted to 4.8 for enzymatic hydrolysis. The remaining steps are the same as in Example 1.

[0039] Comparative Example 4 The difference from Example 1 is that: after the first stage, the pH of the whole slurry was adjusted to 7.0, but in the second stage, the UV light source was turned off, and the mixture was stirred at 50°C for 1.5 h, and then the pH was adjusted to 4.8 for enzymatic hydrolysis; the remaining steps are the same as in Example 1.

[0040] Test results: Table 1. Pretreatment structural changes and enzymatic hydrolysis results (n=3, mean ± standard deviation)

[0041] Note: Glucose yield = total glucose mass in the system at the end of enzymatic hydrolysis / theoretical glucose mass that can be converted from dextran in the initial oven-dry feed × 100%.

[0042] Table 2. Content of residual H2O2 and inhibitors in the liquid phase at the end of the first stage and before enzymatic digestion (n=3, mean ± standard deviation)

[0043] Note: S1 in Comparative Examples 1-4 is the same as in Example 1, so it is indicated by "—", and the values ​​are the same as in Example 1.

[0044] Table 3. Cyclic performance of immobilized TiO2 in Example 1 (n=3, mean ± standard deviation)

[0045] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A two-stage photocatalytic oxidation pretreatment and in-situ detoxification enzymatic hydrolysis method for lignocellulose, characterized in that, Includes the following steps: S1. Alkaline photocatalytic oxidation decomposition: Lignocellulose raw materials are mixed with water to form a slurry. The pH of the slurry is adjusted to 10.0~12.

0. H2O2 is added in the presence of an immobilized TiO2 photocatalyst. The total amount of H2O2 used is 0.20~0.70 g / g of oven-dry lignocellulose based on 100% H2O2 mass. The reaction is carried out at 45~70℃ and under UV irradiation at 320~400 nm for 2~6 h to obtain a complete slurry containing both pretreated solids and pretreated liquid. S2, pH switching in-situ detoxification: After step S1, stop adding new H2O2, adjust the pH of the whole slurry to 6.0~8.5, so that the whole slurry continues to be in contact with the immobilized TiO2 photocatalyst used in step S1, and is treated under UV irradiation of 320~400 nm for 0.5~3.0 h. S3. Direct enzymatic hydrolysis: After step S2, adjust the pH of the system to 4.6~5.2 and the temperature to 45~55℃, add cellulase to carry out enzymatic hydrolysis, and obtain an enzymatic hydrolysate containing fermentable sugars.

2. The method according to claim 1, characterized in that, The immobilized TiO2 photocatalyst includes a support and a TiO2 film loaded on the surface of the support, wherein the support is selected from at least one of a glass plate, a quartz plate, or hollow glass microspheres.

3. The method according to claim 2, characterized in that, When the support is a glass plate or a quartz plate, the preparation method of the immobilized TiO2 photocatalyst includes: preparing an anatase TiO2 aqueous suspension of 100-180 g / L, adjusting the pH to 1.0-2.0 with HNO3, immersing the support in the TiO2 aqueous suspension and then removing it at 5-20 cm / min, drying it at 70-90℃, then heating it to 380-450℃ at 2-10℃ / min and holding it at that temperature for 1-3 h, and repeating the coating, drying and heat treatment 2-6 times; when the support is hollow glass microspheres, adding hollow glass microspheres to a TiO2 suspension of 5-20 g / L at a ratio of 50-150 g / L, contacting it under stirring and ultrasonic conditions for 10-60 min, separating and drying it, then heat-treating it at 380-450℃ and holding it at that temperature for 1-3 h, and repeating the above treatment 2-6 times.

4. The method according to claim 1, characterized in that, In step S1, H2O2 is added in 3 to 10 portions, with an interval of 20 to 90 minutes between adjacent additions; the pH of the slurry is 10.5 to 11.5, the temperature is 55 to 68°C, the reaction time is 3 to 5 hours, and the total amount of H2O2 used is 0.30 to 0.60 g / g oven-dried lignocellulose.

5. The method according to claim 1, characterized in that, In step S2, the pH of the slurry is 6.5~8.0, the temperature is 35~55℃, the UV treatment time is 0.5~2.0 h, and step S2 ends when the residual H2O2 concentration in the liquid phase is no higher than 0.05 g / L.

6. The method according to claim 1, characterized in that, In step S3, add 10-30 FPU / g cellulase and 5-20 IU / g β-glucosidase to the solid treated with absolute intervention, and enzymatically hydrolyze for 24-96 h at 45-55℃ and 80-180 rpm.

7. The method according to claim 1, characterized in that, The lignocellulose raw material is selected from at least one of corn stalks, rice straw, wheat straw, sorghum stalks, sugarcane bagasse, bamboo processing residues, and reed stalks. The particle size of the lignocellulose raw material is 0.15~2.00 mm, and the concentration of the oven-dry lignocellulose raw material in step S1 is 30~150 g / L.

8. The method according to claim 1, characterized in that, After one processing batch is completed, the immobilized TiO2 photocatalyst is washed sequentially with 0.05~0.20 mol / L HCl aqueous solution and water, dried, and then used for the next processing batch. The immobilized TiO2 photocatalyst can be reused for 2~10 batches.

9. A method for in-situ detoxification of alkaline H2O2 / UV-TiO2 pretreated lignocellulose pulp, characterized in that, The pH of the whole slurry, which contains both pretreated solid and pretreated liquid, was reduced from 10.0-12.0 to 6.0-8.5 after pretreatment with alkaline H2O2 and immobilized TiO2 photocatalyst under UV irradiation. Then, the slurry was irradiated with UV at 320-400 nm for 0.5-3.0 h. Subsequently, the whole slurry was adjusted to cellulase hydrolysis conditions and directly hydrolyzed to obtain an enzymatic hydrolysate containing fermentable sugars.

10. The in-situ detoxification method according to claim 9, characterized in that, The acid used to lower the pH of the entire slurry is selected from at least one of HCl, H2SO4, H3PO4, acetic acid, and citric acid.

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