阅读说明：本技术 用NaOH和BSA辅助芬顿预处理提高竹材酶解糖化效率的方法 (Method for improving enzymolysis saccharification efficiency of bamboo wood by using NaOH and BSA (bovine serum albumin) to assist Fenton pretreatment ) 是由 骆莲新 王绪崇 王婷婷 张胜 朱秋晓 于 2021-09-17 设计创作，主要内容包括：本发明公开了用NaOH和BSA辅助芬顿预处理提高竹材酶解糖化效率的方法,以竹材为原料,经盘磨后先进行NaOH预浸渍,然后进行芬顿预处理,最后进行纤维素酶解,在酶解过程中使用BSA牛血清蛋白对纤维素酶的酶解工艺进行优化。本发明方法将竹材先通过NaOH预浸渍再利用芬顿预处理,酶解处理时间可缩短至12小时,降低了预处理能耗,且去除了竹材中的大部分木质素,减少了对纤维素酶酶解的抑制,可使纤维素酶的酶解效率获得很大的提高,最高达到99.95％。(The invention discloses a method for improving enzymolysis saccharification efficiency of bamboo wood by using NaOH and BSA (bovine serum albumin) for assisting Fenton pretreatment. According to the method, the bamboo is pre-impregnated by NaOH and then subjected to Fenton pretreatment, the enzymolysis treatment time can be shortened to 12 hours, the pretreatment energy consumption is reduced, most of lignin in the bamboo is removed, the inhibition on the enzymolysis of cellulase is reduced, and the enzymolysis efficiency of the cellulase can be greatly improved to 99.95% at most.)

1. The method for improving the bamboo enzymatic saccharification efficiency is characterized in that a bamboo raw material subjected to disc grinding is subjected to NaOH preimpregnation and Fenton pretreatment, and then BSA is added for enzymatic saccharification by cellulase.

2. The method for improving the bamboo enzymatic saccharification efficiency of the claim 1, characterized in that NaOH in the NaOH preimpregnation is solid, and the addition amount of NaOH is 5% -15% of the oven-dried bamboo quality; the balance of water, and the solid-to-liquid ratio of the oven-dried bamboo weight to the NaOH solution is 1:20 g/ml.

3. The method for improving the bamboo enzymatic saccharification efficiency of claim 1, characterized in that the NaOH is preimpregnated at 95 ℃ for 0.5-1 hour.

4. The method for improving the bamboo enzymatic saccharification efficiency of claim 1, characterized in that the Fenton pretreatment treatment solution is FeSO₄Solution, H₂O₂And (4) preparing a solution, wherein the solid-to-liquid ratio of the addition amount of oven-dried bamboo to the addition amount of the treatment solution is 1:15 g/ml.

5. The method for improving bamboo enzymatic saccharification efficiency of claim 4, characterized in that the treatment liquid is FeSO₄The concentration is 16mM/L, the addition amount is 5-10 mL/g, H₂O₂The concentration is 30 wt%, 5-10 mL/g, and the balance is water.

6. The method for improving the bamboo enzymatic saccharification efficiency of claim 1, characterized in that the Fenton pretreatment temperature is 40-60 ℃, the pH value is 2.0-4.0, the treatment time is 5-10h, and the rotating speed of a shaker is 150 r/min.

7. The method for improving the enzymolysis and saccharification efficiency of the bamboo wood according to claim 1, wherein the dosage of the cellulase in the enzymolysis and saccharification of the cellulase is 15-20 FPU/g, the concentration of an enzymolysis substrate is 5-10 wt%, and the dosage of BSA (bovine serum albumin) relative to oven-dried bamboo wood is 15-20 mg/g.

8. The method for improving the bamboo enzymatic saccharification efficiency of claim 1, wherein the temperature of the cellulase enzymatic saccharification is 50-60 ℃, the pH is 4.5-5.0, the rotation speed of the water bath shaker is 150r/min, and the enzymatic hydrolysis time is 24 hours.

Technical Field

The invention relates to the field of biomass clean energy, in particular to a method for improving bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment.

Technical Field

With the development of economic society, the demand of energy is gradually increased, the problem of energy shortage around the world is increasingly highlighted, the national environment protection is more and more strict, and the search for clean energy capable of replacing fossil fuel is reluctant. Therefore, the majority of researchers put the research focus on renewable biomass energy, and the production of fuel ethanol by using biomass materials is environment-friendly and can relieve the energy shortage problem. Biomass resources are a huge and renewable energy resource on earth, lignocellulose is composed of cellulose, hemicellulose and the like, and the lignocellulose is a high-molecular carbohydrate compound which can be decomposed into monosaccharides like starch, so that the lignocellulose can be hydrolyzed into fermentable sugars for preparing fuel ethanol.

At present, the utilization of wood fiber is mainly limited to daily living goods, such as paper making, industrial bamboo and the like, the utilization efficiency is not high, and the fiber substances in the residual waste after the consumption of the daily living goods and the agricultural waste can be treated by a reasonable means to obtain the fermentable sugar product. The amount of wood fiber produced by earth plants under photosynthesis can reach billions of tons, and the energy contained in the wood fiber is far beyond the global energy consumption, so that the production of fuel ethanol by utilizing plant fiber raw materials has very important significance.

The bamboo wood is a grass raw material, the utilization rate in the field of pulping and papermaking is not high at present, but the bamboo wood contains rich cellulose and hemicellulose, the growth cycle of the bamboo wood is shorter than that of wood, the distribution is wide, and the annual yield is high, so that the problem of tension of global energy and wood resources can be effectively relieved by widely using the bamboo wood in the future; the current pretreatment methods mainly comprise physical methods, chemical methods, biological methods, physical and chemical combination methods and the like, wherein the chemical methods are most applied; the Fenton pretreatment has good effects on removing lignin and preventing the re-adsorption of the lignin, and the enzymolysis effect of the pure Fenton treatment is extremely poor and almost has no effect, so that the use of a chemical auxiliary agent for improving the enzymolysis effect of the Fenton pretreatment has very important significance.

Disclosure of Invention

In order to solve the problems, the invention provides a method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment, and the technical scheme adopted by the method is as follows:

the method for improving the enzymolysis saccharification efficiency of the bamboo wood by using NaOH and BSA (bovine serum albumin) for assisting Fenton pretreatment comprises the steps of carrying out NaOH preimpregnation and Fenton pretreatment on a bamboo wood raw material subjected to disc grinding, and then adding BSA (bovine serum albumin) for carrying out enzymolysis saccharification of the bamboo wood.

NaOH in the NaOH preimpregnation is solid, and the addition amount of the NaOH is 5% -15% of the mass of the oven-dried bamboo wood; the balance of water, and the solid-to-liquid ratio of the oven-dried bamboo weight to the NaOH solution is 1:20 g/ml.

The NaOH is preimpregnated, the impregnation temperature is 95 ℃, and the impregnation time is 0.5-1 hour.

The Fenton pretreatment treatment solution is prepared from FeSO₄Solution, H₂O₂And (4) preparing a solution, wherein the solid-to-liquid ratio of the addition amount of oven-dried bamboo to the addition amount of the treatment solution is 1:15 g/ml.

The treatment liquid is FeSO₄The concentration is 16mM/L, the addition amount is 5-10 mL/g, H₂O₂The concentration is 30 wt%, 5-10 mL/g, and the balance is water.

The temperature of the Fenton pretreatment is 40-60 ℃, the pH value is 2.0-4.0, the treatment time is 5-10h, and the rotating speed of the shaking table is 150 r/min.

The dosage of the cellulase in the enzymatic hydrolysis and saccharification of the cellulase is 15-20 FPU/g, the concentration of an enzymatic hydrolysis substrate is 5-10 wt%, and the dosage of BSA relative to oven-dried bamboo wood is 15-20 mg/g.

The temperature of enzymolysis and saccharification of the cellulase is 50-60 ℃, the pH is 4.5-5.0, the rotating speed of a water bath shaker is 150r/min, and the enzymolysis time is 24 hours.

Compared with the prior art, the invention has the beneficial results that:

through NaOH-assisted Fenton pretreatment, the pretreatment energy consumption is reduced in the pretreatment process, lignin in the pretreated bamboo wood is removed to a great extent, the structure of part of residual lignin is also destroyed, the compact structure of the bamboo wood is destroyed, cellulase is easier to contact with cellulose, the enzymolysis reaction is easier to be carried out, the reaction time is reduced, the content of the cellulose after the pretreatment reaction is 63.55%, the content of hemicellulose is 3.25%, and the content of lignin is 10.57%, and 63.52g of fermentable sugar can be obtained per hundred grams of bamboo wood after enzymolysis, which is about 15g higher than that of fermentable sugar obtained per hundred grams of bamboo wood in the current mainstream, and the cost is saved and the economic benefit of enterprises is improved by improving the enzymolysis efficiency of the cellulose. The BSA can be combined with the residual lignin of the pretreated bamboo wood, so that the combination sites of the lignin and the cellulase are reduced, the adsorption of the lignin to the cellulase can be effectively reduced, and the positive significance is realized for improving the enzymolysis efficiency.

Compared with other pretreatment modes, the Fenton pretreatment has lower temperature, saves energy, reduces cost and maximizes economic benefit. The cellulose content of the pretreated bamboo is improved, the lignin content and the hemicellulose content are reduced, the enzymolysis efficiency of the bamboo can be improved to a great extent by combining with cellulase for enzymolysis, the enzymolysis efficiency of the cellulose can reach 99.59 percent and almost completely hydrolyzed, and the problem of fossil energy shortage can be effectively relieved by preparing fuel ethanol from fermentable sugar.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

The process of the present invention is described in further detail below with reference to examples. Example 1 as a control example, no NaOH was added.

Example 1

The method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment comprises the following operation steps:

disc grinding: and (3) grinding the bamboo wood by using a high-concentration continuous pulping machine, wherein the distances between three sections of disc mills are 0.2mm, 0.15mm and 0.1mm in sequence, and the ground bamboo wood is reserved.

Pre-soaking with NaOH:

taking 80g of oven-dried bamboo, adding only water without adding NaOH, wherein the addition amount of the water is that the solid-to-liquid ratio of the oven-dried bamboo to the water is 1:20g/ml, the preimpregnation temperature is 95 ℃, the preimpregnation time is 0.5 hour, and the reaction is more fully performed by stirring every 10 minutes.

Fenton pretreatment:

taking bamboo pre-impregnated with NaOH, and carrying out Fenton pretreatment. The chemical addition amount of the treatment solution is 16mM/L FeSO₄5mL/g, 30 wt% H₂O₂5mL/g, the balance of water, and the solid-to-liquid ratio of the addition amount of oven dried bamboo to the treatment liquid is 1:15g/ml, Fenton pretreatment conditions are that the temperature is 40 ℃, the pH is 2.0, the treatment time is 5h, the rotating speed of a shaking table is 150r/min, and the solution is stirred to react more fully in the process.

Enzymolysis and saccharification of cellulase:

carrying out enzymolysis on a raw material prepared by preimpregnation with NaOH and Fenton pretreatment by using cellulase, wherein the dosage of the cellulase is 15FPU/g, the concentration of an enzymolysis substrate is 5 wt%, the dosage of BSA relative to oven-dried bamboo wood is 15mg/g, the temperature is 50 ℃, the pH value is 4.8, the rotating speed of a shaking table is 150r/min, and the enzymolysis time is 24 h. Obtaining the enzymolysis liquid.

Enzymolysis efficiency:

and (3) taking the enzymolysis liquid to carry out concentration detection by using a high performance liquid chromatography, and calculating to obtain the cellulose enzymolysis efficiency of 4.01%.

Example 2

The method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment comprises the following operation steps:

disc grinding: and (3) grinding the bamboo wood by using a high-concentration continuous pulping machine, wherein the distances between three sections of disc mills are 0.2mm, 0.15mm and 0.1mm in sequence, and the ground bamboo wood is reserved.

Pre-soaking with NaOH:

taking 80g of oven-dried bamboo, adding NaOH powder according to 5% of the mass of the oven-dried bamboo, adding water to ensure that the solid-to-liquid ratio of the weight of the oven-dried bamboo to the NaOH solution is 1:20g/ml, the preimpregnation temperature is 95 ℃, the preimpregnation time is 0.5 hour, and stirring every 10min to ensure that the oven-dried bamboo reacts more fully.

Fenton pretreatment:

taking bamboo pre-impregnated with NaOH, and carrying out Fenton pretreatment. The chemical addition amount of the treatment solution is 16mM/L FeSO₄7mL/g, 30 wt% H₂O₂7mL/g, the balance of water, a solid-to-liquid ratio of the addition amount of oven-dried bamboo and the treatment solution of 1:15g/mL, Fenton pretreatment conditions of 40 ℃, pH 4.0, 7h and 150r/min of shaking table rotation speed, and stirring the solution in the process to enable the reaction to be more complete.

Enzymolysis and saccharification of cellulase:

carrying out enzymolysis on a raw material prepared by preimpregnation with NaOH and Fenton pretreatment by using cellulase, wherein the dosage of the cellulase is 15FPU/g, the concentration of an enzymolysis substrate is 7 wt%, the dosage of BSA relative to oven-dried bamboo wood is 15mg/g, the temperature is 50 ℃, the pH value is 4.5, the rotating speed of a shaking table is 150r/min, and the enzymolysis time is 24 h. Obtaining the enzymolysis liquid.

Enzymolysis efficiency:

and (3) taking the enzymolysis liquid to carry out concentration detection by using a high performance liquid chromatography, and calculating to obtain that the enzymolysis efficiency of the cellulose is 79.51%.

Example 3

The method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment comprises the following operation steps:

disc grinding: and (3) grinding the bamboo wood by using a high-concentration continuous pulping machine, wherein the distances between three sections of disc mills are 0.2mm, 0.15mm and 0.1mm in sequence, and the ground bamboo wood is reserved.

Pre-soaking with NaOH:

taking 80g of oven-dried bamboo, adding NaOH powder according to 10% of the mass of the oven-dried bamboo, adding water to ensure that the solid-to-liquid ratio of the weight of the oven-dried bamboo to the NaOH solution is 1:20g/ml, the preimpregnation temperature is 95 ℃, the preimpregnation time is 0.7 hour, and stirring every 10min to ensure that the oven-dried bamboo reacts more fully.

Fenton pretreatment:

taking bamboo pre-impregnated with NaOH, and carrying out Fenton pretreatment. The chemical addition amount of the treatment solution is 16mM/L FeSO₄7mL/g, 30 wt% H₂O₂7mL/g, the balance of water, a solid-to-liquid ratio of the addition amount of oven-dried bamboo and the treatment solution of 1:15g/mL, Fenton pretreatment conditions of 50 ℃ temperature, 2.0 pH, 10h treatment time and 150r/min table rotation speed, and the solution is stirred in the process to fully react.

Enzymolysis and saccharification of cellulase:

carrying out enzymolysis on a raw material prepared by preimpregnation with NaOH and Fenton pretreatment by using cellulase, wherein the dosage of the cellulase is 17FPU/g, the concentration of an enzymolysis substrate is 10 wt%, the dosage of BSA relative to oven-dried bamboo wood is 20mg/g, the temperature is 55 ℃, the pH is 4.8, the rotating speed of a shaking table is 150r/min, and the enzymolysis time is 24 h. Obtaining the enzymolysis liquid.

Enzymolysis efficiency:

and (3) taking the enzymolysis liquid to carry out concentration detection by using a high performance liquid chromatography, and calculating to obtain that the enzymolysis efficiency of the cellulose is 82.91%.

Example 4

The method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment comprises the following operation steps:

disc grinding: and (3) grinding the bamboo wood by using a high-concentration continuous pulping machine, wherein the distances between three sections of disc mills are 0.2mm, 0.15mm and 0.1mm in sequence, and the ground bamboo wood is reserved.

Pre-soaking with NaOH:

taking 80g of oven-dried bamboo, adding NaOH powder according to 15% of the mass of the oven-dried bamboo, adding water to enable the weight of the oven-dried bamboo to be 1:20g/ml of the solid-liquid ratio of NaOH solution, pre-soaking at the temperature of 95 ℃ for 1 hour, and stirring every 10min to enable the oven-dried bamboo to react more fully.

Fenton pretreatment:

taking bamboo pre-impregnated with NaOH, and carrying out Fenton pretreatment. The chemical addition amount of the Fenton treatment solution is 16mM/L FeSO₄10mL/g, 30 wt% H₂O₂10mL/g, the balance of water, the solid-to-liquid ratio of the addition amount of oven-dried bamboo and the treatment solution of 1:15g/mL, the Fenton pretreatment condition is that the temperature is 60 ℃, the pH value is 2.0, the treatment time is 5h, the rotating speed of a shaking table is 150r/min, and the solution is stirred in the process to enable the reaction to be more complete.

Enzymolysis of cellulase:

carrying out enzymolysis on a raw material prepared by preimpregnation with NaOH and Fenton pretreatment by using cellulase, wherein the dosage of the cellulase is 15FPU/g, the concentration of an enzymolysis substrate is 5 wt%, the dosage of BSA relative to oven-dried bamboo wood is 20mg/g, the temperature is 60 ℃, the pH is 4.5, the rotating speed of a shaking table is 150r/min, and the enzymolysis time is 24 h. Obtaining the enzymolysis liquid.

Enzymolysis efficiency:

and (3) taking the enzymolysis liquid to carry out concentration detection by using a high performance liquid chromatography, and calculating to obtain that the enzymolysis efficiency of the cellulose is 85.54%.

Example 5

The method for improving the bamboo enzymolysis saccharification efficiency by using NaOH and BSA to assist Fenton pretreatment comprises the following operation steps:

disc grinding: grinding the bamboo wood by using a high-concentration continuous pulping machine, wherein the grinding intervals of three sections of disc mills are 0.2mm, 0.15mm and 0.1mm in sequence, and the ground bamboo wood is reserved;

pre-soaking with NaOH:

taking 80g of oven-dried bamboo, adding NaOH powder according to 10% of the mass of the oven-dried bamboo, adding water to ensure that the solid-to-liquid ratio of the weight of the oven-dried bamboo to the NaOH solution is 1:20g/ml, the preimpregnation temperature is 95 ℃, the preimpregnation time is 1 hour, and stirring is carried out every 10min to ensure that the oven-dried bamboo reacts more fully.

Fenton pretreatment:

taking bamboo pre-impregnated with NaOH, and carrying out Fenton pretreatment. The chemical addition amount of the treatment solution is 16mM/L FeSO₄10mL/g, 30 wt% H₂O₂The addition amount of the bamboo powder is 10mL/g, the balance is water, the solid-to-liquid ratio of the addition amount of the oven-dried bamboo to the addition amount of the treatment liquid is 1:15g/mL, the Fenton pretreatment condition is that the temperature is 40 ℃, the pH value is 2.0, the treatment time is 5h, the rotating speed of a shaking table is 150r/min, and the solution is stirred in the process to enable the reaction to be more sufficient.

Enzymolysis of cellulase:

carrying out enzymolysis on a raw material prepared by preimpregnation with NaOH and Fenton pretreatment by using cellulase, wherein the dosage of the cellulase is 20FPU/g, the concentration of an enzymolysis substrate is 5 wt%, the dosage of BSA relative to oven-dried bamboo wood is 20mg/g, the temperature is 50 ℃, the pH value is 5.0, the rotating speed of a shaking table is 150r/min, and the enzymolysis time is 24 h. Obtaining the enzymolysis liquid.

Enzymolysis efficiency:

and (3) performing concentration detection on the enzymolysis liquid by using a high performance liquid chromatography, calculating that the enzymolysis efficiency of the cellulose is 99.95%, and calculating to obtain 63.52g of fermentable sugar per hundred grams of bamboo wood after enzymolysis.