Method for improving moso bamboo cellulase hydrolysis conversion efficiency through microwave-assisted deep eutectic solvent pretreatment

Abstract

The invention discloses a method for improving moso bamboo cellulase hydrolysis conversion efficiency through microwave-assisted deep eutectic solvent pretreatment, and belongs to the technical field of biomass recycling. The method comprises the following steps: mixing arginine and choline to prepare an alkaline eutectic solvent, then pretreating moso bamboos by using a microwave-assisted eutectic solvent, carrying out solid-liquid separation on the mixture, carrying out enzymolysis on the solid by using cellulase, adjusting the pH value of the filtrate by using hydrochloric acid, and centrifuging to obtain a lignin solid; and carrying out rotary evaporation on the centrifuged liquid to obtain the regenerated eutectic solvent for cyclic utilization. According to the method, the moso bamboo components can be efficiently separated under the conditions of low temperature and short time, the cellulose enzymolysis saccharification capacity is improved, and effective utilization of all the moso bamboo components is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of biomass resource utilization, and particularly relates to a method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted low eutectic solvent pretreatment. Background Art

[0002] The development of green and renewable lignocellulosic resources to replace fossil resources that pollute the environment has important social and economic significance. Among them, bamboo is widely used as a fast-growing and abundant lignocellulosic resource. Bamboo is rich in three components: cellulose, hemicellulose and lignin. Cellulose and hemicellulose can be converted into monosaccharides through enzymatic hydrolysis to prepare biofuels or various chemicals, while lignin can be used to produce functional materials. However, the stubbornness of the bamboo cell wall structure makes it difficult to directly carry out bioconversion, so pretreatment technology is needed to destroy the dense structure of bamboo. Pretreatment technology can effectively destroy the connecting bonds between cellulose and hemicellulose and lignin, fully expose the enzyme binding sites of cellulose, and improve the efficiency of enzymatic hydrolysis. However, traditional pretreatment technologies have the disadvantages of corroding equipment, polluting the environment, and high operating costs. Moreover, a single pretreatment method is difficult to achieve high-value utilization of all components of bamboo. Therefore, it is necessary to find a combined and efficient green pretreatment technology.

[0003] A low eutectic solvent is a solvent with low toxicity, simple preparation and recyclability. It can effectively deconstruct the cell wall structure of bamboo and separate the components through hydrogen bond interactions, which is conducive to the subsequent high-value utilization of bamboo. However, low eutectic solvents often use corrosive compounds such as formic acid and oxalic acid, which do not really meet the green and safety requirements of the pretreatment process. Therefore, a low eutectic solvent composed of natural compounds can be designed and developed to achieve efficient separation of bamboo components.

[0004] At present, natural amino acid-based deep eutectic solvents have been proven to break the interaction network between the hemicellulose-lignin structure and cellulose, destroy the highly ordered crystalline structure of cellulose and selectively dissolve lignin. At the same time, the polar side chain of alkaline arginine can give the deep eutectic solvent a stronger hydrogen bond snatching ability, making it more suitable for constituting a pretreatment solvent for bamboo. Compared with acidic deep eutectic solvents, alkaline deep eutectic solvent pretreatment can show stronger lignin and hemicellulose removal ability at low temperatures. In addition, in order to overcome the disadvantage of long single treatment time of low eutectic solvents, microwave technology can be combined to promote the ionization characteristics of low eutectic solvents, achieve internal stress damage and improve the heat transfer efficiency of pretreatment, so as to achieve the purpose of quickly and efficiently destroying the dense structure of bamboo in a shorter time. In short, in order to cleanly separate bamboo components and improve the subsequent bioconversion efficiency of bamboo, a technology of pretreatment of bamboo with alkaline arginine deep eutectic solvent and microwave is improved. Summary of the invention

[0005] The technical problem solved by the invention is to provide a method for pretreating bamboo with microwave-assisted low eutectic solvent and improving its enzymatic hydrolysis efficiency. The method is simple and safe and meets the requirements of green and sustainable development.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, comprising the following steps:

[0008] 1) Preparation of a low eutectic solvent: mixing the choline solution and the arginine solution, shaking, and rotary evaporating to obtain a clear and uniform alkaline low eutectic solvent;

[0009] 2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: bamboo and deep eutectic solvent were mixed evenly, and then pretreated by microwave;

[0010] 3) After the reaction is completed, the mixture is cooled, an acetone aqueous solution is added and stirred evenly, and then solid-liquid separation is performed, the solid residue is soaked in a sodium hydroxide solution, and then washed with deionized water until neutral, and the solid residue is air-dried for standby use;

[0011] 4) Enzymatic hydrolysis: hydrolyzing and saccharifying the solid residue with cellulase;

[0012] 5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is adjusted with hydrochloric acid solution, and lignin solid is obtained after centrifugation; the liquid after centrifugation is rotary evaporated and then recovered to obtain regenerated low eutectic solvent for recycling.

[0013] The molar ratio of arginine to choline in the low eutectic solvent of step 1) is 1:1-2.

[0014] The microwave pretreatment temperature in step 2) is 90-130°C.

[0015] In the step 2), the mass ratio of bamboo to alkaline low eutectic solvent is 1:20.

[0016] In the step 2), the microwave auxiliary power is 600 W, the heating time is 10 min, and the holding time is 5 min.

[0017] The cellulase in step 4) is Celic Tec2 cellulase.

[0018] The amount of cellulase added in step 4) is 20 FPU / g glucan.

[0019] The enzymolysis time in step 4) is 72 hours.

[0020] The enzymatic hydrolysis temperature in step 4) is 50°C.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The arginine-based low eutectic solvent prepared by the present invention is simple, green and safe, has the ability to separate the components of bamboo and promote their enzymatic saccharification, and meets the requirements of green and sustainable development.

[0023] (2) The present invention uses microwave technology to assist the pretreatment of bamboo with a choline-arginine low eutectic solvent, thereby retaining cellulose more completely and removing up to 94.4% and 75.3% of lignin and xylan.

[0024] (3) The glucose yield of bamboo pretreated by low eutectic solvent-microwave after enzymatic hydrolysis is increased from 6.2% to 69.5%-91.8%, and the xylose yield is increased from 8.9% to 56.7%-76.8%.

[0025] (4) The present invention uses a low eutectic solvent-microwave pretreatment to separate high-quality bamboo lignin. The low pretreatment temperature of this method can avoid the condensation reaction of lignin. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a graph showing the cellulose recovery rate, hemicellulose and lignin removal rate of bamboo samples after pretreatment with a choline-arginine eutectic solvent with a molar ratio of 1:1 and microwave combination;

[0027] Figure 2 is a graph showing the cellulose recovery rate, hemicellulose and lignin removal rate of bamboo samples after pretreatment with a choline-arginine eutectic solvent with a molar ratio of 2:1 and microwave combination;

[0028] Figure 3 The yields of glucose and xylose from bamboo samples pretreated with a choline-arginine eutectic solvent and microwave combined with 72h cellulase hydrolysis of the original bamboo, where A is the original bamboo, and B-F are samples treated at 90℃, 100℃, 110℃, 120℃ and 130℃, respectively.

[0029] Figure 4 Glucose and xylose yields of bamboo samples pretreated with a choline-arginine eutectic solvent in a molar ratio of 2:1 and microwave and the original bamboo after 72h cellulase hydrolysis, where A is the original bamboo, and G~K are the samples treated at 90℃, 100℃, 110℃, 120℃ and 130℃, respectively. DETAILED DESCRIPTION

[0030] The present invention is further illustrated below in conjunction with specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0031] Example 1

[0032] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0033] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 1:1 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0034] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 90 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0035] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0036] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0037] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate was slowly adjusted to 2 using a 1 M hydrochloric acid solution. The precipitated lignin sample was obtained after centrifugation and washing. The solid was subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively. The sugar content was determined by high performance liquid chromatography, and the lignin content was determined by muffle furnace ignition weighing method and spectrophotometry. Finally, the liquid after centrifugation was subjected to rotary evaporation to remove acetone and water. The regenerated low eutectic solvent was recovered and recycled.

[0038] The results showed that the cellulose recovery rate was 93.0%, the hemicellulose removal rate was 29.6%, the lignin removal rate was 65.5%, the glucose yield at 72 h was 69.5%, and the xylose yield at 72 h was 56.7%.

[0039] Example 2

[0040] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0041] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 1:1 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0042] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 100 °C with a power of 600 W for 10 min and maintained for 5 min;

[0043] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0044] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0045] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0046] The results showed that the cellulose recovery rate was 94.5%, the hemicellulose removal rate was 34.7%, the lignin removal rate was 68.8%, the glucose yield at 72 h was 75.7%, and the xylose yield at 72 h was 59.8%.

[0047] Example 3

[0048] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0049] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 1:1 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0050] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 110 °C with a power of 600 W for 10 min and maintained for 5 min;

[0051] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0052] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0053] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0054] The results showed that the cellulose recovery rate was 97.9%, the hemicellulose removal rate was 34.3%, the lignin removal rate was 73.1%, the glucose yield at 72 h was 80.0%, and the xylose yield at 72 h was 65.3%.

[0055] Example 4

[0056] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0057] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 1:1 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0058] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 120 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0059] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0060] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0061] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute hydrochloric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace ignition weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0062] The results showed that the cellulose recovery rate was 95.0%, the hemicellulose removal rate was 35.1%, the lignin removal rate was 74.7%, the glucose yield at 72 h was 82.0%, and the xylose yield at 72 h was 65.9%.

[0063] Example 5

[0064] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0065] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 1:1 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0066] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 130 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0067] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0068] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0069] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0070] The results showed that the cellulose recovery rate was 93.4%, the hemicellulose removal rate was 44.7%, the lignin removal rate was 84.3%, the glucose yield at 72 h was 91.8%, and the xylose yield at 72 h was 71.4%.

[0071] Example 6

[0072] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0073] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 2:1 and placed in a 25°C water bath for 24 h at 150 rpm. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0074] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 90 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0075] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0076] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0077] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0078] The results showed that the cellulose recovery rate was 91.7%, the hemicellulose removal rate was 58.4%, the lignin removal rate was 86.7%, the glucose yield at 72 h was 86.0%, and the xylose yield at 72 h was 76.8%.

[0079] Example 7

[0080] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0081] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 2:1 and placed in a 25°C water bath for 24 h at 150 rpm. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0082] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 100 °C with a power of 600 W for 10 min and maintained for 5 min;

[0083] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0084] (4) Enzymatic hydrolysis: 0.5 g of the absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath with a set temperature of 50°C and an oscillation speed of 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0085] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0086] The results showed that the cellulose recovery rate was 91.2%, the hemicellulose removal rate was 62.3%, the lignin removal rate was 89.8%, the glucose yield at 72 h was 83.7%, and the xylose yield at 72 h was 75.2%.

[0087] Example 8

[0088] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0089] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 2:1 and placed in a 25°C water bath for 24 h at 150 rpm. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0090] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 110 °C with a power of 600 W for 10 min and maintained for 5 min;

[0091] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0092] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0093] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0094] The results showed that the cellulose recovery rate was 85.3%, the hemicellulose removal rate was 73.8%, the lignin removal rate was 92.6%, the glucose yield at 72 h was 79.4%, and the xylose yield at 72 h was 70.5%.

[0095] Example 9

[0096] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0097] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 2:1 and placed in a 25°C water bath for 24 h at 150 rpm. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0098] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 120 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0099] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0100] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0101] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0102] The results showed that the cellulose recovery rate was 86.7%, the hemicellulose removal rate was 75.3%, the lignin removal rate was 94.4%, the glucose yield at 72 h was 75.7%, and the xylose yield at 72 h was 68.0%.

[0103] Example 10

[0104] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0105] (1) Preparation of a low eutectic solvent: choline hydroxide solution and arginine solution were added into a conical flask at a molar ratio of 2:1 and placed in a 25°C water bath for 24 h at 150 rpm. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0106] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 130 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0107] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0108] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0109] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0110] The results showed that the cellulose recovery rate was 88.7%, the hemicellulose removal rate was 71.5%, the lignin removal rate was 92.9%, the glucose yield at 72 h was 72.0%, and the xylose yield at 72 h was 68.7%.

[0111] Embodiment 11

[0112] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0113] (1) Preparation of a low eutectic solvent: Arginine solution and lactic acid were added to a conical flask at a molar ratio of 1:5 and placed in a 25°C water bath at 150 rpm for 24 h. The mixture was rotary evaporated at 50°C to obtain a clear and uniform solution, which was then placed in a desiccator for later use.

[0114] (2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: 60 g of deep eutectic solvent and 3 g of bamboo were added to a microwave digestion tank, mixed evenly to ensure the uniformity of the subsequent reaction, and the microwave digestion tank was placed in a microwave digestion instrument, heated at 120 ° C with a power of 600 W for 10 min and maintained for 5 min;

[0115] (3) After the reaction is completed, the mixture is taken out and cooled, a 50% (v / v) acetone aqueous solution is added to the mixture and stirred evenly, and then the mixture is separated into solid and liquid using a vacuum filter, and the solid residue is soaked in a 1% (w / v) sodium hydroxide solution for 4 hours, and then the solid residue is washed with deionized water until the pH is neutral, and the solid residue is air-dried for standby use;

[0116] (4) Enzymatic hydrolysis: 0.5 g of absolutely dry solid residue was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzymatic hydrolysis system was 10 mL and the amount of enzyme added was 20 FPU / g of glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50° C. and the shaking speed was set to 150 rpm. After 72 h of enzymatic hydrolysis, the enzymatic hydrolyzate was analyzed for sugar content using high performance liquid chromatography;

[0117] (5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is slowly adjusted to 2 using a 1 M hydrochloric acid solution, and the precipitated lignin sample is obtained after centrifugation and washing; the solid is subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively, and the sugar content is determined by high performance liquid chromatography, and the lignin content is determined by muffle furnace burning weighing method and spectrophotometry. Finally, the liquid after centrifugation is subjected to rotary evaporation to remove acetone and water, and the regenerated low eutectic solvent is obtained after recovery for recycling.

[0118] The results showed that the cellulose recovery rate was 86.6%, the hemicellulose removal rate was 28.3%, the lignin removal rate was 42.9%, the glucose yield at 72 h was 33.8%, and the xylose yield at 72 h was 40.2%.

[0119] Therefore, the DES system prepared with arginine as the hydrogen bond donor and choline hydroxide as the hydrogen bond acceptor can significantly improve the separation and conversion rate of bamboo.

[0120] Comparative Example 1

[0121] A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, the specific steps are as follows:

[0122] (1) 0.5 g of dried bamboo sample was placed in a conical flask and 1 M citric acid buffer and tetracycline were added. The enzyme hydrolysis system was 10 mL and the enzyme addition amount was 20 FPU / g glucan. The reaction was carried out in a water bath. The reaction temperature was set to 50 ° C and the shaking speed was 150 rpm. After 72 h of enzymatic hydrolysis, the sugar content of the enzymatic hydrolyzate was analyzed by high performance liquid chromatography.

[0123] (2) Chemical composition analysis of original bamboo samples: The solids were subjected to two-step acid hydrolysis using 72% concentrated sulfuric acid and 4% dilute sulfuric acid at 30°C and 121°C, respectively. The sugar content was determined by HPLC, and the lignin content was determined by muffle furnace gravimetric method and spectrophotometry.

[0124] The results showed that the glucose yield was 6.2% after 72 h, and the xylose yield was 8.9% after 72 h.

[0125] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment, characterized in that: The following steps are involved: 1) Preparation of a low eutectic solvent: mixing the choline solution and the arginine solution, shaking, and rotary evaporating to obtain a clear and uniform alkaline low eutectic solvent; 2) Microwave-assisted deep eutectic solvent pretreatment of bamboo: bamboo and deep eutectic solvent were mixed evenly, and then pretreated by microwave; 3) After the reaction is completed, the mixture is cooled, an acetone aqueous solution is added and stirred evenly, and then solid-liquid separation is performed, the solid residue is soaked in a sodium hydroxide solution, and then washed with deionized water until neutral, and the solid residue is air-dried for standby use; 4) Enzymatic hydrolysis: hydrolyzing and saccharifying the solid residue with cellulase; 5) Recovery of lignin and low eutectic solvent: The pH of the filtrate is adjusted with hydrochloric acid solution, and lignin solid is obtained after centrifugation; the liquid after centrifugation is rotary evaporated and then recovered to obtain regenerated low eutectic solvent for recycling.

2. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The molar ratio of arginine to choline in the low eutectic solvent of step 1) is 1:1-2.

3. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The microwave pretreatment temperature in step 2) is 90-130°C.

4. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: In the step 2), the mass ratio of bamboo to alkaline low eutectic solvent is 1:

20.

5. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: In the step 2), the microwave auxiliary power is 600 W, the heating time is 10 min, and the holding time is 5 min.

6. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The cellulase in step 4) is Celic Tec2 cellulase.

7. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The amount of cellulase added in step 4) is 20 FPU / g glucan.

8. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The enzymolysis time in step 4) is 72 hours.

9. The method for improving the enzymatic hydrolysis conversion efficiency of bamboo cellulose by microwave-assisted deep eutectic solvent pretreatment according to claim 1, characterized in that: The enzymatic hydrolysis temperature in step 4) is 50°C.

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