CN111534556B - Method for preparing high-concentration monosaccharide solution by using poplar enzyme method - Google Patents
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Abstract
The invention relates to a method for preparing high-concentration monosaccharide solution by using a poplar enzyme method, which comprises the steps of mixing poplar with acetic acid-hydrogen peroxide solution for pretreatment reaction; carrying out solid-liquid separation on the pretreatment liquid after the pretreatment reaction and poplar, mixing pretreated poplar filter residues with alkali liquor, and carrying out alkali pretreatment; performing solid-liquid separation on the treatment liquid after alkali pretreatment and poplar alkali treatment filter residues, and washing the alkali treatment filter residues to be neutral to obtain a pretreatment raw material; and mixing the pretreated raw material, sodium citrate buffer solution and cellulase for enzymolysis. The method adopts two-step pretreatment to remove a large amount of lignin and acetyl groups in poplar, improves the content of glucan in the poplar and the enzyme hydrolysis efficiency, and realizes the preparation of high-concentration monosaccharide solution by using the poplar. The method has the characteristics of high monosaccharide concentration of the enzymolysis liquid, high enzymolysis efficiency, recoverable reagent, simple operation and the like, and is suitable for preparing biological energy and high-value chemicals by biotransformation of poplar.
Description
Technical Field
The invention belongs to the technical field of biological degradation and conversion of forestry biomass, and particularly relates to a method for preparing a high-concentration monosaccharide solution by poplar enzymolysis.
Background
In the biological conversion process of biomass, high-concentration sugar liquid can be obtained by enzymolysis with high substrate concentration, and the fermentation for preparing biological energy and chemicals can reduce the cost of subsequent fermentation and purification, thereby having important significance for high-value utilization of biomass.
The poplar has the advantages of high growth speed, short growth period, strong environmental adaptability and wide planting area, and is an important raw material for producing bioenergy and biomass chemicals.
The poplar has the advantages of high degradation resistance barrier and high lignin content, and the problems of low enzymolysis yield, large enzyme dosage and the like exist in the enzymolysis process, so that the preparation of high-concentration monosaccharide enzymolysis liquid by adopting high-concentration poplar in the enzymolysis process is very difficult. For example, when Tian D et al pretreat poplar in 2017,Biotechnol Biofuels.10:157 in a two-step process and then add up to 32mg of cellulase enzyme per gram of dry matter, the glucose yield of poplar is only 70%. Zhang et al report (Bioresource Technology,2009,100 (23): 5890-5897) reported that when a special stirring device was used, the enzymatic substrate concentration of pretreated poplar was 20%, the cellulose conversion after enzymatic hydrolysis was less than 85%, and the glucose concentration was 158mg/mL at the maximum. Chandra et al (Bioresource Technology,2016, 199:135-141) performed enzymatic hydrolysis on pretreated poplar at 10% substrate concentration, and the enzymatic hydrolysis yield of poplar was only 80% at an added amount of cellulase of 20mg protein/g substrate, wherein the glucose concentration was less than 61mg/mL, the xylose concentration was less than 3mg/mL, and the monosaccharide concentration was less than 64mg/mL. Hu et al (Bioresource Technology,2015, 186:149-153) pretreated poplar by steam explosion for 20% substrate concentration enzymolysis, wherein the amount of the compound cellulase added is up to 60mg protein/g substrate, however, the glucose concentration of the enzymolysis liquid of the poplar is lower than 142mg/mL (glucose yield is lower than 85%), the xylose concentration is lower than 19mg/mL, and the total monosaccharide yield is lower than 164mg/mL. Therefore, the problems of low enzymolysis yield, low monosaccharide concentration of enzymolysis liquid and the like generally exist in the poplar treated by the traditional pretreatment mode in the enzyme hydrolysis process.
Disclosure of Invention
The invention aims to provide a method for preparing a high-concentration monosaccharide solution by using poplar. According to the invention, the acetic acid-hydrogen peroxide and sodium hydroxide are sequentially treated on poplar, so that the enzymolysis yield of poplar and the monosaccharide concentration in enzymolysis liquid can be greatly improved; the monosaccharide concentration of the prepared poplar enzymolysis liquid reaches more than 250mg/mL.
In order to achieve the above object, the present invention provides the following technical solutions;
the invention provides a method for preparing high-concentration monosaccharide solution by using a poplar enzyme method, which comprises the following steps:
(1) Mixing poplar with acetic acid-hydrogen peroxide solution, and carrying out pretreatment reaction;
(2) Carrying out solid-liquid separation on the pretreatment liquid after the pretreatment reaction and poplar, mixing pretreated poplar filter residues with alkali liquor, and carrying out alkali pretreatment;
(3) Performing solid-liquid separation on the treatment liquid after alkali pretreatment and poplar alkali treatment filter residues, and washing the alkali treatment filter residues to be neutral to obtain a pretreatment raw material;
(4) And mixing the pretreated raw material, sodium citrate buffer solution and cellulase for enzymolysis.
Preferably, the ratio of the weight of the pretreated raw material in the step (4) to the volume of the sodium citrate buffer solution is 150-250 mg:1mL; the pH of the sodium citrate buffer solution is 5.0-6.0.
Preferably, the cellulase in the step (4) is added with 20-40 FPU of enzyme activity per gram of pretreated raw material.
Preferably, the mass concentration of the alkali liquor in the step (2) is 0.1-0.5%.
Preferably, the ratio of the mass of the pretreated filter residue to the volume of the alkali liquor in the step (2) is 1g: 8-10 mL.
Preferably, the temperature of the alkali pretreatment in the step (2) is 60-80 ℃ and the time is 1-2 h.
Preferably, the volume percentage of the acetic acid hydrogen peroxide in the acetic acid-hydrogen peroxide solution in the step (1) is 60%.
Preferably, the mass ratio of the poplar to the acetic acid-hydrogen peroxide solution in the step (1) is 1g: 8-10 mL.
The high-concentration monosaccharide solution obtained by the poplar enzymolysis method is characterized in that the concentration of the high-concentration monosaccharide solution is higher than 140g/mL
According to the invention, the degradability of poplar can be improved by utilizing acetic acid-hydrogen peroxide and alkali, a large amount of lignin and acetyl groups in the poplar are removed, the glucan component of the poplar is greatly improved, and on the basis, cellulose can be further subjected to enzymolysis, so that the concentration of monosaccharide in an enzymolysis solution is improved. The embodiment shows that the mode of pretreatment and enzymatic hydrolysis of poplar provided by the invention can ensure that the monosaccharide concentration of the enzymatic hydrolysate reaches more than 250mg/mL, and can greatly improve the monosaccharide concentration in the enzymatic hydrolysate of poplar and the enzymatic hydrolysis efficiency of poplar.
Detailed Description
The invention provides a method for preparing a high-concentration monosaccharide solution by using poplar, which comprises the following steps:
(1) Mixing poplar with acetic acid-hydrogen peroxide solution, and carrying out pretreatment reaction;
(2) Carrying out solid-liquid separation on the pretreatment liquid after the pretreatment reaction and poplar, mixing pretreated poplar filter residues with alkali liquor, and carrying out alkali pretreatment;
(3) Performing solid-liquid separation on the treatment liquid after alkali pretreatment and poplar alkali treatment filter residues, and washing the alkali treatment filter residues to be neutral to obtain a pretreatment raw material;
(4) And mixing the pretreated raw material, sodium citrate buffer solution and cellulase for enzymolysis.
According to the invention, poplar is mixed with acetic acid-hydrogen peroxide solution for pretreatment reaction. In the invention, the mass ratio of poplar to acetic acid-hydrogen peroxide solution is preferably 1g: 8-10 mL; the volume percentage of the acetic acid hydrogen peroxide in the acetic acid-hydrogen peroxide solution is preferably 60%. The acetic acid-hydrogen peroxide treatment can remove a large amount of lignin in the poplar, improve the degradability of the poplar and realize the pretreatment of the poplar. The source of the acetic acid-hydrogen peroxide solution is not particularly limited, and the acetic acid-hydrogen peroxide solution can be prepared by self. When a self-preparation mode is adopted, the preparation of the acetic acid-hydrogen peroxide solution is preferably as follows: acetic acid with the mass fraction of 99% and hydrogen peroxide with the mass fraction of 30% are mixed in equal volume, the mixed solution is diluted by water, and sulfuric acid is added for catalysis to prepare the acetic acid-hydrogen peroxide solution. Sulfuric acid can catalyze acetic acid and hydrogen peroxide to generate peracetic acid, so that the solution becomes a more oxidizing mixed solution. The acetic acid-hydrogen peroxide pretreatment of poplar can remove a large amount of lignin components in the poplar and greatly retain the polysaccharide composition of the poplar.
After the poplar is pretreated by acetic acid-hydrogen peroxide, the pretreatment liquid after the pretreatment reaction is subjected to solid-liquid separation with the poplar, and the pretreated poplar filter residues are mixed with alkali liquor for alkali pretreatment. In the invention, the mass ratio of the pretreated filter residue to the alkali liquor is preferably 1g: 8-10 mL; the mass concentration of the alkali liquor is preferably 0.1-0.5%, more preferably 0.2-0.4%; the method can avoid insufficient removal of a large amount of acetyl groups in the enzymolysis process with the concentration of the alkali liquor being too low, can avoid the condition that the removal of a large amount of lignin and polysaccharide components caused by excessive alkali liquor is unfavorable for hydrolysis, and can effectively reduce the loss of the polysaccharide components. The alkali liquor can remove acetyl in the alkali liquor, so that the enzymolysis efficiency of poplar can be improved. The invention is not particularly limited in the kind of solute in the alkali liquor, and preferably comprises one or more of sodium hydroxide, potassium hydroxide and calcium hydroxide; the alkali pretreatment is preferably carried out by cooling to room temperature, wherein the temperature of the alkali pretreatment is preferably 60-80 ℃, more preferably 60-75 ℃, and in the embodiment of the invention, the temperature of the alkali pretreatment is specifically 60 ℃; the time is preferably 1 to 2 hours. The alkali pretreatment is preferably carried out in a sealed reaction kettle, the lining of the reaction kettle is preferably made of polytetrafluoroethylene, the lining of the reaction kettle is made of polytetrafluoroethylene and does not contain metal or other reducing materials, and the reaction kettle is corrosion-resistant and not easy to cause explosion. The proper alkali pretreatment temperature and time ensure that the acetyl groups of the poplar are removed in the alkali treatment process, and simultaneously a large amount of polysaccharide components and a small amount of necessary lignin components are reserved, so that the residual acetyl groups in the poplar can be greatly removed after the alkali pretreatment of the poplar is pretreated by the acetic acid-hydrogen peroxide, and the accessibility of cellulose to the poplar is improved, so that the hydrolysis of the poplar is promoted.
After the alkali treatment is finished, the treatment liquid after the alkali pretreatment and the poplar alkali treatment filter residue are subjected to solid-liquid separation, and the alkali treatment filter residue is washed to be neutral to obtain a pretreatment raw material. The invention washes the filter residue until the filter residue is neutral, so that the residual alkali liquor in the raw materials can be removed, and the interference of the alkali liquor on subsequent hydrolysis is reduced.
After the pretreated raw material is obtained, the pretreated raw material, a sodium citrate buffer solution and cellulase are mixed for enzymolysis. In the invention, the volume ratio of the mass of the pretreatment raw materials to the sodium citrate buffer solution is 150-250 mg:1mL, more preferably 200 to 250mg:1mL; the pH of the sodium citrate buffer is 5-6. The dosage of the cellulase is preferably 20-40 FPU of cellulase added to each gram of filter residue. In the present invention, the temperature of the enzymolysis is preferably 50 ℃; the time of the enzymolysis is preferably 168 hours. The source of the cellulase is not particularly limited, and the cellulase is a conventional cellulase in the art. After the raw materials are washed to be neutral, cellulase is added, and the sodium citrate buffer solution can buffer the release of organic acid in the hydrolysis process, so that the stability of the hydrolysis pH at the optimal activity of the enzyme is ensured.
The concentration of the high-concentration monosaccharide solution is preferably higher than 140mg/mL, more preferably 150-250 mg/mL, the subsequent high-concentration product conversion is easy, and the distillation and purification cost in the later conversion process is greatly reduced.
The following is a detailed description of a method for preparing a high concentration monosaccharide solution by means of poplar enzyme method, which is provided by the present invention, but should not be construed as limiting the scope of the present invention.
Example 1
(1) Acetic acid-hydrogen peroxide treatment
Weighing poplar with an absolute dry weight of 10g, and mixing the poplar with a feed liquid according to a feed liquid ratio of 1:10 adding an acetic acid-hydrogen peroxide solution with the volume concentration of 60% (the acetic acid-hydrogen peroxide solution is prepared by adding 100mM sulfuric acid into the diluted acetic acid-hydrogen peroxide solution system for catalysis according to the volume ratio of acetic acid with the mass concentration of 99% to hydrogen peroxide with the mass concentration of 30% being 1:1), and heating and preprocessing for 2h at the temperature of 80 ℃. After pretreatment, the reaction mixture was cooled to obtain a reaction solution.
(2) Alkali treatment
And (3) carrying out solid-liquid separation by utilizing a centrifugal machine, taking filter residues, washing the filter residues with water to be neutral, weighing 10g of absolute dry solid filter residues pretreated by acetic acid-hydrogen peroxide solution, adding sodium hydroxide solution with the mass fraction of 0.1% into a water bath at 60 ℃ according to the feed liquid ratio of 1g to 10mL, and carrying out alkali pretreatment for 1h. Cooling, performing solid-liquid separation by using a centrifugal machine, washing the alkali treatment filter residue to be neutral to obtain a pretreatment raw material for cellulose enzymolysis.
(3) Cellulase enzymolysis
15 g of pretreatment raw material is weighed into a conical flask, 20FPU/g DM (dry matter) cellulase is added, then sodium citrate buffer solution with pH equal to 5.0 is added to adjust the mass concentration of poplar in a substrate to 150mg/mL, and the ratio of the weight of the pretreatment raw material to the volume of the sodium citrate buffer solution is 150mg:1mL, shaking up and placing in a constant temperature shaking table for enzymolysis for 168 hours at 50 ℃, boiling for 10min in a boiling water bath to inactivate enzymes, and cooling to room temperature to obtain monosaccharide solution.
The method for detecting the enzymolysis rate of the monosaccharide comprises the following steps: centrifuging monosaccharide solution, detecting monosaccharide by high performance liquid chromatography on supernatant, and calculating the enzymolysis rate of the pretreated raw material monosaccharide according to the following formula:
glucose yield (%) = glucose content obtained by enzymolysis x 0.9/cellulose content in substrate x 100
Xylose yield (%) = xylose content x 0.88 obtained by enzymolysis/xylan content x 100 in substrate
The cellulose and xylan content of the substrate in the formula is determined according to the national renewable energy laboratory method;
the glucose yield in enzymolysis is calculated to be 96.6%, the xylose yield is calculated to be 98.6%, the glucose concentration is calculated to be 126.1mg/mL, the xylose concentration is calculated to be 26.3mg/mL, and the total monosaccharide concentration is calculated to be 152.4mg/mL.
Example 2
The pretreatment process and reaction conditions were the same as in example 1 except that the ratio of the weight of the pretreated feedstock to the volume of sodium citrate buffer was 250mg:1mL, the final glucose yield is 96.1%, the xylose yield is 98.1%, the glucose concentration is 209.2mg/mL, the xylose concentration is 43.7mg/mL, and the total monosaccharide concentration is 252.9mg/mL.
Example 3
The pretreatment method and reaction conditions were the same as in example 1, except that the feed liquid ratio of poplar to acetic acid-hydrogen peroxide pretreatment solution was 1g:8mL, the final enzymatic hydrolysis glucose yield was 91.7%, the xylose yield was 93.4%, the glucose concentration was 120.4mg/mL xylose concentration was 25.3mg/mL, and the total monosaccharide concentration was 145.7mg/mL.
Example 4
The pretreatment method and reaction conditions were the same as in example 1, except that the feed liquid ratio of the alkali treatment was 1:8, the final enzymatic hydrolysis glucose yield was 91.7%, the xylose yield was 93.4%, the glucose concentration was 120.0mg/mL, the xylose concentration was 25.2mg/mL, and the total monosaccharide concentration was 147.2mg/mL.
Example 5
The pretreatment method and reaction conditions were the same as in example 1, except that the temperature of the alkali treatment was 80 ℃, the final glucose yield was 95.6%, the xylose yield was 95.0%, the glucose concentration was 130.1mg/mL, the xylose concentration was 25.3mg/mL, and the total monosaccharide concentration was 155.4mg/mL.
Example 6
The pretreatment method and reaction conditions were the same as in example 1 except that the concentration of the alkali-treated sodium hydroxide solution was 0.5%, the final glucose yield by enzymolysis was 96.6%, the xylose yield was 92.9%, the glucose concentration was 132.6mg/mL, the xylose concentration was 24.0mg/mL, and the total monosaccharide concentration was 156.6mg/mL.
Comparative example 1
The acetic acid-dioxygen hydrogen peroxide pretreatment method and the enzymatic hydrolysis conditions were the same as in example 1, except that no alkali treatment was used, the final enzymatic hydrolysis glucose yield was 76.7%, xylose yield was 80.0%, glucose concentration was 87.0mg/mL xylose concentration was 28.0mg/mL, and total monosaccharide concentration was 115.0mg/mL.
Comparative example 2
The enzymatic hydrolysis conditions were the same as in example 1 except that no acetic acid-hydrogen peroxide and alkali treatment was used, the final enzymatic hydrolysis glucose yield was 11.8%, xylose yield was 38.0%, glucose concentration was 9.0mg/mL xylose concentration was 10.0mg/mL, and total monosaccharide concentration was 19.0mg/mL.
According to the embodiment, the invention provides the poplar enzymolysis method, which can greatly improve the glucan content in the poplar enzymolysis liquid and the enzymolysis efficiency of the poplar; the monosaccharide concentration of the obtained poplar enzymolysis liquid can reach 250mg/mL.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (3)
1. A method for preparing high-concentration monosaccharide solution by using a poplar enzyme method, which is characterized by comprising the following steps:
(1) Mixing poplar with acetic acid-hydrogen peroxide solution, and carrying out pretreatment reaction;
(2) Carrying out solid-liquid separation on the pretreatment liquid after the pretreatment reaction and poplar, mixing pretreated poplar filter residues with alkali liquor, and carrying out alkali pretreatment;
(3) Performing solid-liquid separation on the treatment liquid after alkali pretreatment and poplar alkali treatment filter residues, and washing the alkali treatment filter residues to be neutral to obtain a pretreatment raw material;
(4) Mixing the pretreated raw material, sodium citrate buffer solution and cellulase for enzymolysis;
the volume ratio of the weight of the pretreated raw materials to the sodium citrate buffer solution in the step (4) is 150-250 mg:1mL; the pH value of the sodium citrate buffer solution is 5.0-6.0;
the volume percentage content of the acetic acid hydrogen peroxide in the acetic acid-hydrogen peroxide solution in the step (1) is 60%;
the mass ratio of the poplar to the acetic acid-hydrogen peroxide solution in the step (1) is 1g: 8-10 mL;
the mass concentration of the alkali liquor in the step (2) is 0.1-0.5%;
the ratio of the mass of the pretreated filter residue to the volume of the alkali liquor in the step (2) is 1g: 8-10 mL.
2. The method of claim 1, wherein the cellulase is added in an amount of 20 to 40FPU per gram of pretreated feedstock.
3. The method according to claim 1, wherein the alkaline pretreatment in step (2) is carried out at a temperature of 60 to 80 ℃ for a time of 1 to 2 hours.
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| 硫酸盐木质素对预处理杨木酶水解糖化的影响;郭天雨等;《林业工程学报》;20180927(第05期);全文 * |
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