阅读说明：本技术 一种甘蔗渣预处理改善酶解糖化效果的方法 (Method for improving enzymolysis saccharification effect by pretreating bagasse ) 是由 王朝宇 迟雪文 毕艳红 张睿 杨荣玲 刁怡涵 于 2019-10-24 设计创作，主要内容包括：本发明公开了一种甘蔗渣预处理改善酶解糖化效果的方法,包括如下步骤：称取甘蔗渣,加入低共熔溶剂(Deep Eutectic Solvent,DES)进行预处理,反应结束后对其进行固液分离；将分离的固体中加入KOH溶液进行预处理,反应结束后对其进行固液分离,分别收集固体和液体部分；将固体部分洗涤至中性即得到预处理后的甘蔗渣,进行酶解糖化,测定酶解液中的葡萄糖浓度。本发明甘蔗渣清洁高效预处理的方法可以改善纤维素酶水解效率低的问题,从而减少纤维素酶的用量,提高还原糖的转化效率,为甘蔗渣的综合利用提供一定的理论依据。(The invention discloses a method for improving enzymatic saccharification effect by bagasse pretreatment, which comprises the following steps of weighing bagasse, adding a Deep Eutectic Solvent (DES) for pretreatment, carrying out solid-liquid separation after the reaction is finished, adding a KOH solution into the separated solid for pretreatment, carrying out solid-liquid separation after the reaction is finished, respectively collecting solid and liquid parts, washing the solid part to be neutral to obtain the pretreated bagasse, carrying out enzymatic saccharification, and determining the glucose concentration in an enzymatic hydrolysate.)

1, method for improving enzymolysis saccharification effect by bagasse pretreatment, which is characterized by comprising the following steps:

(1) weighing bagasse, adding DES (data encryption standard) for pretreatment, performing solid-liquid separation after the reaction is finished, and respectively collecting solid and liquid parts;

(2) adding KOH solution into the solid separated in the step (1) for pretreatment, carrying out solid-liquid separation on the solid after the reaction is finished, and respectively collecting the solid part and the liquid part;

(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated bagasse, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.

2. The method for improving enzymolysis saccharification effect by bagasse pretreatment according to claim 1, wherein the DES is prepared by mixing choline chloride: lactic acid is preferably used in a molar ratio of 1: 1-5.

3. The method for improving the enzymolysis saccharification effect by bagasse pretreatment according to claim 1, wherein the conditions of the DES pretreatment of bagasse are as follows: the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.

4. The method for improving enzymatic saccharification effect by bagasse pretreatment according to claim 1, wherein the mass fraction of the KOH solution is 5-40 wt.%.

5. The method for improving enzymatic saccharification effect by bagasse pretreatment according to claim 1, wherein the pretreatment conditions of the KOH solution are as follows: the treatment time is 0.5-5h, the treatment solid-liquid ratio is 1:6-1:15g/mL, and the treatment temperature is 50-100 ℃.

6. The method for improving enzymatic saccharification effect through bagasse pretreatment according to claim 1, wherein the enzymatic saccharification is implemented by adding citric acid buffer solution to the pretreated bagasse to adjust the pH value, adding sodium azide solution, then adding cellulase, and measuring the glucose concentration in the enzymatic hydrolysate by using HPLC after reaction.

7. The method for improving enzymatic saccharification effect by bagasse pretreatment according to claim 6, wherein the cellulase is added in an amount of 3-30 FPU/g-substrate, the solid loading is 2-20 wt.%, and the enzymatic hydrolysis is performed at pH 4.8 and temperature of 50 ℃ for 2-96 h.

Use of liquid produced in the method for improving enzymatic saccharification effect by bagasse pretreatment as described in claim 1 for the production of potash fertilizer, lignin-based fertilizer and compound fertilizer.

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for improving enzymolysis saccharification effect by pretreating bagasse.

Background

The biomass energy is used as type typical representative of renewable energy, and has a relatively mature development system in developed countries such as Europe and the United states in the aspects of biological liquid fuel, biomass power generation and the like, so that if the lignocellulosic biomass raw material can be processed and pretreated to produce biomass fuel or a large amount of chemicals with high added value, the global energy crisis can be relieved to a certain extent in , and the pressure of fossil energy is relieved.

Sugarcane is a herbaceous plant with years or more of years, which is common in tropical and subtropical regions, is rich in sugar, vitamins, minerals and the like, China is the third sugarcane planting base in the world after Brazil and India, China is the third sugarcane planting base in China, the main production area of sugarcane is distributed in west, Yunnan, east, Hainan, Fujian, Sichuan and Jiangxi, and the like, the yield of sugarcane is as high as 80 metric tons/hectare, which is much higher than that of crops, so that sugarcane can be regarded as a high-efficiency storage rich in solar energy, which is important sugar crops, the produced cane sugar accounts for 80% of the total sugar content in China, which is the consuming country of sucrose, which is second only in Brazil and India, wherein sucrose is the main source of sugar, accounts for more than 92% of the total sugar content, the sugarcane has the highest weight in the sugar industry of China, bagasse is important sugar manufacturing byproducts, which is the total sugar production of cane sugar, or the sugar juice extracted vigorously by the sugar factory, the infiltration method, the cane has the highest cellulose content of cellulose, the cane juice, the cane pulp, the cellulose content of the cane pulp is about 20-30% of cellulose, the cellulose residue of bagasse is generated by-30 stalks, the bagasse is generated by-30 stalks, the bagasse is generated by the bagasse, the biomass, the biomass is used for the biomass, the biomass is used for biomass, the biomass.

Disclosure of Invention

The invention aims to solve the problems in the prior art, and provides a method for improving enzymolysis saccharification effect by pretreating bagasse.

In order to achieve the purpose, the method for improving the enzymatic saccharification effect by pretreating types of bagasse comprises the following steps:

(1) weighing bagasse, adding DES (data encryption standard) for pretreatment, performing solid-liquid separation on the bagasse after the reaction is finished, and respectively collecting solid and liquid parts;

(2) adding KOH solution into the solid separated in the step (1) for pretreatment, and carrying out solid-liquid separation after the reaction is finished;

(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated bagasse, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.

The bagasse used in the invention is purchased from Futaste Co. Ltd. (China) or other commercial companies, or the extraction method is according to the documents of Wang Yonggarden, Libuhua, Liuyu ring and the like, the latest progress of the comprehensive utilization technology of the bagasse [ J ] Chinese agricultural bulletin, 2010,26(16):370-375, and the effect is .

Wherein the DES consists of choline chloride: lactic acid was added in a molar ratio of 1: 1-5. Preferably, the DES consists of choline chloride: lactic acid was added in a molar ratio of 1:2, and preparing the product.

the DES is prepared by adding choline chloride and lactic acid into a reaction flask, magnetically stirring at 60-80 deg.C until the mixture becomes colorless clear liquid, transferring to a vacuum drying oven, and adding P₂O₅Fully drying the mixture for later use.

Wherein, the conditions of DES pretreatment of bagasse are as follows: the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.

Wherein the mass fraction of the KOH solution is 5 to 40 wt.%.

Wherein, the pretreatment conditions of the KOH solution are as follows: the treatment time is 0.5-5h, the treatment solid-liquid ratio is 1:6-1:15g/mL, and the treatment temperature is 50-100 ℃.

And (3) carrying out solid-liquid separation on the bagasse subjected to KOH pretreatment, respectively collecting solid and liquid parts, washing solid residues to be neutral by using tap water, and then carrying out enzymatic saccharification.

, adding citric acid buffer solution to adjust pH, adding sodium azide solution, adding cellulase, and measuring glucose concentration in the enzymolysis solution by HPLC after reaction.

Preferably, the cellulase is added in an amount of 3-30 FPU/g-substrate (i.e. pretreated bagasse), with a solids loading of 2-20 wt.%, and is subjected to enzymatic hydrolysis at pH 4.8 and a temperature of 50 ℃ for 2-96 h. If the enzymatic hydrolysis reaction volume is 20mL, 1g of pretreated bagasse (dry by absolute) is added at a solids loading of 5 wt.%, and the total reaction volume is 20mL x 5% solids loading (solids content) is 1g of dry by absolute weight.

According to IUPAC standard, the definition of Filter paper enzyme activity (FPA) is 1g of solid enzyme (or 1mL of liquid enzyme), and the enzyme dosage required for generating 1 mu mol of glucose from a Filter paper substrate per hour at 50 ℃ and pH 4.8 is defined as enzyme activity units expressed by U/g (or U/mL).

After the enzymatic saccharification process is finished, quantitative enzymatic hydrolysate is taken to be subjected to High Performance Liquid Chromatography (HPLC) detection, and the content of reducing sugar in the enzymatic hydrolysate is measured, wherein the detection conditions of HPLC are shown in Table 1.

TABLE 1 conditions of liquid chromatography detection

The liquid (the black liquid collected in the steps (1) and (2)) generated in the method for improving the enzymolysis and saccharification effect by bagasse pretreatment is applied to the preparation of potassium fertilizer, lignin-based fertilizer and compound fertilizer.

According to the method, the DES and KOH pretreatment is adopted to remove lignin and hemicellulose wrapped on the surface of the cellulose, so that the highly ordered and tightly arranged regions of the cellulose are damaged, pores are generated on the surface of the cellulose, more cellulose is exposed, the porosity and porosity are increased, the accessibility of the cellulose by the cellulase is improved, and the enzymolysis saccharification efficiency is promoted.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the method comprises the steps of pretreating bagasse by DES and KOH, damaging the wrapping of lignin and hemicellulose on cellulose, removing lignin, degrading hemicellulose, changing the crystal structure of cellulose, improving the accessibility and the porosity of cellulose, promoting the contact of cellulase and a substrate, and further improving the yield of cellulase hydrolysis and the yield of reducing sugar, wherein under the pretreatment and enzymatic hydrolysis conditions of the bagasse, the optimal conditions of DES pretreatment are determined as choline chloride and lactic acid (1:2), the reaction temperature is 90 ℃, the reaction time is 1h, the solid-liquid ratio is 1:10, the optimal conditions of KOH pretreatment are that the KOH dosage is 15 wt.%, the reaction temperature is 90 ℃, the reaction time is 2h, and the solid-liquid ratio is 1:10, the research shows that under the conditions of 10% of solid content and 20 FPU/g-substrate, the highest glucose concentration is 47.6 +/-1.1 g/L, the xylose concentration is 21.6 +/-0.5 g/L, 5% of solid content is 20 FPU/g-substrate, the highest glucose yield is 87.4 +/-0.7.1 g/L, the highest xylose concentration is 21.6 +/-0.5 g/L, the highest solid content of bagasse can be found according to the theoretical requirement of waste for developing a renewable resource-rich in bagasse, and the resource can be developed according to the total resource of renewable waste.

The method adopts eutectic solvents DES and KOH solution for pretreatment, has mild reaction conditions, reduces the damage to instruments and equipment, and is beneficial to industrial production and application.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) atlas before and after bagasse pretreatment;

FIG. 2 shows Fourier Infrared Spectroscopy (FTIR) spectra before and after bagasse pretreatment;

FIG. 3 is an X-ray diffraction pattern (XRD) before and after bagasse pretreatment;

wherein a is bagasse raw material; b is bagasse after 15 wt.% KOH pretreatment in example 2;

observation of SEM, FTIR and XRD patterns shows that compared with the bagasse raw material which is not pretreated, the bagasse after pretreatment has increased crystallinity, increased porosity and porosity, and increased accessibility of cellulose by cellulase, thereby improving the efficiency of enzymatic saccharification.

Detailed Description

The invention is further illustrated in connection with the following examples and the accompanying drawings.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.