阅读说明：本技术 木质纤维素的预处理方法以及其在提高木质纤维素酶解效率中的应用 (Pretreatment method of lignocellulose and application thereof in improving lignocellulose enzymolysis efficiency ) 是由 尚亚平 贾文 于 2018-07-23 设计创作，主要内容包括：本发明公开了一种木质纤维素的预处理方法以及其在提高木质纤维素酶解效率中的应用,木质纤维素的预处理方法包括以下步骤：将木质纤维素进行机械粉碎,在室温20～25℃放置至少24小时晾干,过筛,得到粒径为0.18-0.85mm的木质纤维素颗粒,将所述木质纤维素颗粒放入pH<2.5的无机盐溶液中并于100～160℃保温0.5～3h,得到固液混合物,将固液混合物进行固液分离,得到固体,将所述固体洗涤至中性,洗涤后干燥至少24h,得到预处理后木质纤维素,其中,所述无机盐溶液中无机盐的浓度为0.01～2mol/L。本发明预处理方法所使用的无机盐用量很少,成本降低,节约资源；水洗后金属离子残留量较小,对后续酶解过程的抑制作用较小。本发明预处理方法需采用耐腐蚀设备,常压下即可操作。(The invention discloses a pretreatment method of lignocellulose and application thereof in improving lignocellulose enzymolysis efficiency, wherein the pretreatment method of the lignocellulose comprises the following steps: mechanically crushing lignocellulose, placing the lignocellulose particles at room temperature of 20-25 ℃ for at least 24 hours, airing, sieving to obtain lignocellulose particles with the particle size of 0.18-0.85mm, placing the lignocellulose particles into an inorganic salt solution with the pH of less than 2.5, preserving the heat at 100-160 ℃ for 0.5-3 hours to obtain a solid-liquid mixture, carrying out solid-liquid separation on the solid-liquid mixture to obtain a solid, washing the solid to be neutral, and drying for at least 24 hours after washing to obtain pretreated lignocellulose, wherein the concentration of inorganic salts in the inorganic salt solution is 0.01-2 mol/L. The pretreatment method of the invention uses little inorganic salt, reduces the cost and saves the resources; the residual quantity of metal ions after washing is small, and the inhibition effect on the subsequent enzymolysis process is small. The pretreatment method of the invention needs to adopt corrosion-resistant equipment and can be operated under normal pressure.)

1. A method for pretreating lignocellulose is characterized by comprising the following steps:

mechanically crushing lignocellulose, placing the lignocellulose particles at room temperature of 20-25 ℃ for at least 24 hours, airing, sieving to obtain lignocellulose particles with the particle size of 0.18-0.85mm, placing the lignocellulose particles into an inorganic salt solution with the pH of less than 2.5, preserving the heat at 100-160 ℃ for 0.5-3 hours to obtain a solid-liquid mixture, carrying out solid-liquid separation on the solid-liquid mixture to obtain a solid, washing the solid to be neutral, and drying for at least 24 hours after washing to obtain pretreated lignocellulose, wherein the concentration of inorganic salts in the inorganic salt solution is 0.01-2 mol/L.

2. The pretreatment method according to claim 1, wherein the inorganic salt in the inorganic salt solution is a strong acid weak base salt, and the strong acid weak base salt is a trivalent iron, aluminum, copper or magnesium salt, preferably a trivalent iron and aluminum salt.

3. The pretreatment method according to claim 1 or 2, wherein an acidic solution of an acid equivalent to an inorganic salt in the inorganic salt solution is added so that the inorganic salt solution has a pH of less than 2.5, preferably the pH of the inorganic salt solution is 1-2.

4. The pretreatment method according to claim 3, wherein a ratio of the mass part of the lignocellulose particles having the particle size of 0.18 to 0.85mm to the volume part of the inorganic salt solution is 1: (4-10), wherein the unit of the mass part is g, and the unit of the volume part is mL.

5. The pretreatment method according to claim 2 or 4, wherein the drying temperature is 50 to 80 ℃.

6. The pretreatment method according to claim 5, wherein the lignocellulose is corn cobs.

7. The pretreatment method according to claim 4 or 6, wherein the concentration of the inorganic salt in the inorganic salt solution is 0.01 to 1mol/L, preferably 0.01 to 0.1 mol/L.

8. Pretreated lignocellulose as obtained by the pretreatment method as defined in claim 2, 4 or 6.

9. Use of the pretreated lignocellulose as recited in claim 8 for increasing the efficiency of the lignocellulose enzymolysis.

10. The use according to claim 9, wherein the enzymatic hydrolysis rate of the lignocellulose to glucose is up to 91%.

Technical Field

The invention belongs to the technical field of cellulosic ethanol, and particularly relates to a pretreatment method of lignocellulose and application of the pretreatment method in improving the enzymolysis efficiency of the lignocellulose.

Background

In recent years, the conversion of lignocellulose into liquid fuels, platform chemicals and specialty chemicals has become an international hot topic. Lignocellulose is converted into various sugars by an integrated biological or chemical catalytic conversion technology, and then the sugars are converted into liquid fuel oil such as fuel ethanol and the like and high value-added platform chemicals (5-hydroxymethylfurfural, furan, levulinic acid and the like) by a sugar platform technology, and the refined chemicals, polymers and other energy substances can be prepared by further processing.

Generally, the biocatalytic conversion process of lignocellulose mainly comprises the following four steps: pretreatment, hydrolysis, fermentation and product recovery. In order to increase the efficiency of the lignocellulose biotransformation, an appropriate pretreatment process is selected to reduce the biomass recalcitrance (the recalcitrance is mainly derived from the structure of plant cell walls consisting of cross-linked polymer networks, glycoproteins and lignin), change the physical structure and chemical composition of a substrate, increase the accessible surface area of the substrate, and improve the enzymatic hydrolysis reaction rate and the enzymatic hydrolysis conversion rate in the subsequent hydrolysis process. The pretreatment method of lignocellulose includes mechanical method, physical-chemical method, biological method and the like. Researches show that the pretreatment effect can be enhanced to a certain extent by the pretreatment of strong acid and weak base salt, but higher metal ion concentration is needed, which results in the improvement of pretreatment cost and the increase of the cost of washing pretreated materials on one hand, and on the other hand, residual metal ions also have an inhibiting effect on the enzymolysis process of pretreated substrates, so that a pretreatment method which can reduce the use amount of inorganic salt and achieve the ideal pretreatment effect is needed to be found.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a lignocellulose pretreatment method, the pretreated lignocellulose is suitable for high-solid-concentration enzymolysis fermentation, the higher ethanol concentration is obtained, and the rectification cost is reduced.

The invention also aims to provide application of the pretreated lignocellulose obtained by the pretreatment method in improving the enzymolysis efficiency of the lignocellulose.

The purpose of the invention is realized by the following technical scheme.

A method for pretreating lignocellulose, comprising the steps of:

mechanically crushing lignocellulose, placing the lignocellulose particles at room temperature of 20-25 ℃ for at least 24 hours, airing, sieving to obtain lignocellulose particles with the particle size of 0.18-0.85mm, placing the lignocellulose particles into an inorganic salt solution with the pH of less than 2.5, preserving the heat at 100-160 ℃ for 0.5-3 hours to obtain a solid-liquid mixture, carrying out solid-liquid separation on the solid-liquid mixture to obtain a solid, washing the solid to be neutral, and drying for at least 24 hours after washing to obtain pretreated lignocellulose, wherein the concentration of inorganic salts in the inorganic salt solution is 0.01-2 mol/L.

In the above technical scheme, the inorganic salt in the inorganic salt solution is a strong acid weak base salt.

In the above technical solution, the strong acid and weak base salt is trivalent iron salt, aluminum salt, copper salt or magnesium salt, preferably trivalent iron salt and trivalent aluminum salt.

In the technical scheme, the pH value of the inorganic salt solution is enabled to be less than 2.5 by adding an acid solution of the same acid radical as the inorganic salt in the inorganic salt solution.

In the technical scheme, the pH of the inorganic salt solution is 1-2, and preferably the pH is less than or equal to 1.

In the above technical solution, the ratio of the mass part of the lignocellulose particles with the particle size of 0.18-0.85mm to the volume part of the inorganic salt solution is 1: (4-10), wherein the unit of the mass part is g, and the unit of the volume part is mL.

In the technical scheme, the drying temperature is 50-80 ℃.

In the above technical scheme, the lignocellulose is corncob.

In the technical scheme, the concentration of the inorganic salt in the inorganic salt solution is 0.01-1 mol/L, and preferably 0.01-0.1 mol/L.

The pretreated lignocellulose obtained by the pretreatment method.

An enzymolysis method of lignocellulose comprises the following steps:

uniformly mixing the pretreated lignocellulose, the cellulase and the buffer solution, and performing enzymolysis for 24-96 hours in a shaking table with the temperature of 40-55 ℃ and the rpm of 100-200, wherein the ratio of the mass part of the pretreated lignocellulose to the volume part of the buffer solution to the volume part of the cellulase is 1 (1-100): (0.1-0.5), wherein the unit of the mass part is g, and the unit of the volume part is mL.

In the technical scheme, the buffer solution is a sodium citrate buffer solution or a citric acid buffer solution.

In the technical scheme, the concentration of the buffer solution is 0.5-1 mol/L.

In the technical scheme, the ratio of the mass part of the pretreated lignocellulose to the volume part of the buffer solution is 1 (3-50), and preferably 1 (5-20).

In the technical scheme, the pH value of the buffer solution is 4.0-5.5.

The pretreated lignocellulose obtained by the pretreatment method is applied to improving the enzymolysis efficiency of the lignocellulose.

In the technical scheme, the enzymolysis rate of the lignocellulose for hydrolyzing into glucose is up to 91%.

The enzymolysis method is applied to improving the enzymolysis efficiency of the lignocellulose.

In the technical scheme, the enzymolysis rate of the lignocellulose for hydrolyzing into glucose is up to 91%.

Compared with the prior art, the invention has the following beneficial technical effects:

the pretreatment method of the invention uses little inorganic salt, reduces the cost and saves the resources; the residual quantity of metal ions after washing is small, and the inhibition effect on the subsequent enzymolysis process is small. The pretreatment method of the invention needs to adopt corrosion-resistant equipment and can be operated under normal pressure.

The metal ions in the inorganic salt promote the degradation of hemicellulose in the lignocellulose in an acidic environment, so that the hydrolysis efficiency of the pretreated lignocellulose is obviously improved, and the lignocellulose is better especially under high solid concentration. After the pretreated lignocellulose is subjected to enzymolysis at high solid concentration, the glucose yield (enzymolysis rate) is up to 91%. The yield of glucose after the hydrolysis of the lignocellulose pretreated by the dilute acid is about 73 percent, namely after the pH of the inorganic salt solution is adjusted by the dilute acid, the conversion rate of the pretreated substrate is improved by 50 to 80 percent (the specific value is related to the pH value) compared with the glucose after the enzymolysis before the adjustment.

Detailed Description

In the following examples, the lignocellulose was corn cob.

The filtration is carried out by reduced pressure filtration.

The pretreatment method of the invention has no special requirements on the types of the lignocellulose, and can be implemented by adopting the lignocellulose raw materials which are familiar to the technical personnel in the field and have higher economical efficiency, such as crop straws, gardening wastes and the like. In an embodiment of the invention, the lignocellulose is corncob purchased from Diwu farm.

In the invention, when the lignocellulose raw material is pretreated, the lignocellulose raw material is crushed, and the preferable particle size of the lignocellulose raw material is 0.18-0.85 mm. The crushing method is not particularly limited, and the crushing technical scheme known to the skilled person can be adopted, and the corn cob is crushed in the crusher of the type Kehongfa 9 FC-K23.

In the invention, the inorganic salt solutions with different concentrations have different pH values, the pH value of the solution is adjusted to the target pH value by using acid of the same acid radical, and the preferable pH value of the inorganic salt solution is less than 2.5, and the more preferable pH value is less than 2. The smaller the pH value, the better the separation of the components of the pretreatment, but the higher the water consumption in the washing process after the pretreatment. The chemicals such as inorganic salts in this example were analytically pure and were from Tianjin optochemical reagents. The pH of the solution is measured by a thunder magnetic PHS-3E type pH meter.

The invention immerses the crushed lignocellulose into inorganic salt solution with specific pH value for heating pretreatment, and the ratio of the mass of the lignocellulose particles to the volume of the inorganic salt solution after the pH value is adjusted is 1 g: (4-10) ml, only needing to ensure that the lignocellulose raw material is fully wetted.

In the invention, the crushed lignocellulose is immersed in an inorganic salt solution with a specific pH value for heating pretreatment, and the reaction equipment needs to be corrosion-resistant reaction equipment, and is selected to be a screw bottle in the embodiment. The optimal pretreatment temperature is 100-160 ℃, the optimal heating time is 0.5-3 h, and the generation of furan, furfural and other enzymolysis inhibitors can be caused by overhigh reaction temperature and overlong heating time.

In the invention, after the lignocellulose particles are immersed in the inorganic salt solution with specific pH value for heating pretreatment, the solid part needs to be washed, the washing times are not limited, and the solid part is washed until the solution is neutral. And (3) heating and drying the solid after washing, wherein the preferable drying temperature is 50-80 ℃, and the drying time is not particularly limited, so that the solid material is obtained.

The pretreated lignocellulose obtained by the pretreatment method is subjected to enzymolysis reaction, and the lignocellulose enzymolysis method comprises the following steps:

mixing the pretreated lignocellulose with cellulase and a buffer solution, and carrying out enzymolysis.

The method mixes the pretreated lignocellulose with the cellulase and a buffer solution to obtain a mixed material, does not specially limit the source and the variety of the cellulase, and adopts a commercially available composite cellulase product well known by technical personnel, such as cellulase GC220 produced by Genencor (Jenenaceae) and the like.

In the present invention, the ratio of the mass of the pretreated lignocellulose to the volume of the buffer solution is preferably 1 g: (1-100) mL, more preferably 1 g: (3-50) mL, most preferably 1 g: (5-20) mL, in this example 1 g: 10 ml. The buffer solution is citric acid/sodium citrate buffer solution, and the preferable pH value of the buffer solution is 4.0-5.5.

In the present invention, the ratio of the mass of the pretreated lignocellulose to the volume of the cellulase is preferably 1 g: (0.1-0.5) ml.

After the mixed material is obtained, the pretreated lignocellulose is subjected to enzymolysis, the enzymolysis equipment is not particularly limited, and a water bath shaking table with the model of SW22 of Jumbo company of Germany is adopted in the embodiment. The rotation speed of the shaking table is preferably 100rpm to 200rpm, and the rotation speed of the shaking table is preferably 120rpm or 150rpm in the embodiment. The enzymolysis temperature is preferably 40-55 ℃. The preferred enzymolysis time is 24h-96 h. The concentration of glucose in the solution was measured and the glucose yield (i.e., the rate of enzymatic hydrolysis) was calculated. The glucose concentration was measured by high performance liquid chromatography, in this example, a High Performance Liquid Chromatography (HPLC) system comprising a Series III analysis pump, a Laballiance workstation, an HPX-87H column from Bio-Rad, USA, and a differential refractometer from Girald instruments, Inc.

The glucose yield calculation method of the invention is as follows:

glucose yield (enzymolysis rate) ═ glucose production (g) × 0.9 × 100% ÷ mass of cellulose (g) in pretreated lignocellulose

Glucose yield (g) 0.9 x 100% ÷ mass of cellulose in comparative lignocellulose (g)

The method for determining the quality of the cellulose in the pretreated lignocellulose/comparative lignocellulose refers to a National Renewable Energy Laboratory (NREL) method (Zhanghong, Zhengrong, Chengyun, Huanghe and NREL method for determining the content of components of a lignocellulose raw material [ J ]. analysis laboratory, 2010,29(11): 15-18.).

The technical scheme of the invention is further explained by combining specific examples.