CN114317638B - Method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis - Google Patents
Method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis Download PDFInfo
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Abstract
The invention discloses a method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis, which mainly comprises four steps of pretreatment, secondary pretreatment, pre-enzymolysis and enzymolysis for saccharifying poplar, and simple recovery of enzymolysis liquid for cyclic enzymolysis. The invention mainly pretreats poplar by sodium hydroxide, and uses laccase to pretreat the poplar subjected to alkali treatment for the second time. The xylanase and the surfactant are utilized to pre-enzymolysis the poplar pretreated in two steps. And finally, performing enzymolysis saccharification on the pre-hydrolyzed poplar by utilizing cellulase and a surfactant. Wherein, the laccase, the xylanase after the pre-enzymolysis, the cellulase after the enzymolysis and the surfactant in the filtrate after the secondary pretreatment are subjected to suction filtration and recycling so as to achieve the aim of reducing the cost.
Description
Technical Field
The invention relates to the field of bioconversion, in particular to a method for saccharifying poplar by step pretreatment and multienzyme synergistic surfactant cyclic enzymolysis.
The background technology is as follows:
lignocellulosic biomass exists in large quantities in nature, being the most abundant, cheapest renewable resource on earth, and meeting the requirements of sustainable development. However, since cellulose, hemicellulose and lignin contained in lignocellulose are entangled with each other, a compact three-dimensional network space structure is formed, and enzymolysis efficiency is low. Lignin forms an amorphous, nonlinear and heterogeneous stereo polymeric structure, hemicellulose penetrates between cellulose and lignin, limiting accessibility of the lignocellulose degradation catalytic factors. Thus, during the conversion of lignocellulose, a pretreatment must be performed before enzymolysis to break down its dense structure, making the enzyme more accessible to the substrate. Acid treatment can dissolve 80-90% of hemicellulose in the feedstock, but lignin is not removed effectively. The barrier effect of lignin and obvious ineffective adsorption to cellulase cause the problems of high cellulase consumption, high subsequent enzymolysis saccharification cost and the like. Therefore, lignin removal, reduction of cellulase enzymatic resistance and improvement of cellulase utilization are critical to sustainable development.
The alkali treatment can cause cellulose swelling, increase the specific surface area of the raw material, reduce the polymerization degree and crystallinity of the raw material, improve the degradability of cellulose and hemicellulose, reduce the ineffective combination of enzyme on lignin in the enzyme hydrolysis process, and improve the conversion rate. However, alkali treatment only removes part of lignin, and laccase can further remove lignin without producing inhibitors, which is a green lignin removal method. Xylanase is an enzyme for specifically degrading xylan, the xylan is a main component of hemicellulose and is wrapped on the surface of cellulose, and the accessibility of cellulose by cellulose can be improved by removing the xylan. In the enzymolysis process, the adsorption and desorption of the enzyme on the substrate are simultaneous, and the surfactant can be competitively adsorbed on lignin with the enzyme, so that the ineffective adsorption of lignin is reduced.
Although the addition of surfactants and the synergistic action of various enzymes can increase the enzymatic hydrolysis yield, the problem of high enzyme costs remains unsolved. At present, the problem of excessive cost exists in lignocellulose enzymolysis saccharification, which is mainly caused by the high enzyme consumption. Therefore, reducing the amount of enzyme used is a key to advance the industrialization of cellulosic ethanol. In the degradation process of lignocellulose, enzyme is firstly adsorbed on a substrate, then enzymolysis is carried out, desorption is carried out after the enzymolysis is finished, and then the enzyme is adsorbed on a new substrate again, and the next round of hydrolysis is carried out. The addition of the surfactant enables the enzyme to be competitively adsorbed on the lignin, reduces the adsorption of the lignin on the enzyme, and promotes the desorption and the recycling of the enzyme. By utilizing the characteristic, after the enzymolysis reaction is finished, the enzyme is recovered for recycling, so that the enzyme consumption and the use cost can be reduced.
The invention comprises the following steps:
the invention aims to provide a method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis. The method has high enzymolysis efficiency, can recycle enzyme, has low cost and has good application prospect.
The method sequentially comprises the following steps:
(1) Alkali treatment of wood: mixing the wood chips crushed to 80 meshes and dried by sodium hydroxide, washing with water to neutrality, and drying;
(2) Laccase secondary pretreatment: suspending the sawdust again by using buffer solution, adding hydrogen peroxide and laccase for secondary treatment, and carrying out suction filtration to recover filtrate and filter residue for later use;
(3) Pre-enzymolysis of xylanase: re-suspending the filter residue obtained in the step (2) by using a buffer solution, adding xylanase and a surfactant for pre-enzymolysis, and carrying out suction filtration to recover a filtrate, wherein the filter residue is for later use;
(4) Enzymatic saccharification of cellulase: and (3) re-suspending the filter residue obtained in the step (3) by using a buffer solution, adding cellulase and a surfactant for enzymolysis saccharification, and carrying out suction filtration to collect filtrate, thus obtaining a saccharification product.
The wood chips are cleaned and crushed by a crusher, and are put into a 60 ℃ oven after being sieved by a 80-mesh sieve, and are baked to constant weight; and washing the wood chips subjected to alkali treatment by water until the pH is neutral, and drying at 60 ℃ until the wood chips are absolute dried. The method comprises the steps of carrying out a first treatment on the surface of the
In the method, the concentration of sodium hydroxide used in the step (1) is 1% -4%, preferably: the sodium hydroxide is treated at 115-125 ℃ for 60-150 min.
The enzyme solution used in the steps (2), (3) and (4) is filtered and recycled, and preferably can be recycled for 5-8 times.
In the method, the concentration of the hydrogen peroxide used in the step (2) is 8% -20%, and the dosage of laccase used is 10U/g-100U/g of wood dust.
The method comprises the steps of pretreating hydrogen peroxide and laccase at 37-60 ℃ for 6-24 h.
In the method, the xylanase used in the step (3) is 10U/g-150U/g of wood chips, the surfactant used is one or two of polyethylene glycol and tween-80, and the dosage is 0.01 g/g-0.1 g/g of wood chips.
The xylanase and the surfactant are subjected to pre-enzymolysis at 37-60 ℃ for 0.5-6 h.
According to the method, the dosage of the cellulase used in the step (4) is 4U/g-60U/g of wood chips, the surfactant used is one or two of polyethylene glycol and tween-80, and the dosage of the polyethylene glycol is 0.01 g/g-0.1 g/g of wood chips.
The method comprises the steps of carrying out enzymolysis on cellulase and surfactant at 37-60 ℃ for 24-84 h.
According to the method, the buffer solution is sodium acetate buffer solution, the pH value of the buffer solution is 4.8, and the ratio of wood chips or filter residues to the buffer solution is 1:5-1:20 (g: mL).
The filtrate recovered by the invention is pumped and filtered by a vacuum pump, and is stored at the temperature of minus 20 ℃ so as to be convenient for reuse.
Compared with the prior art, the invention has the following advantages:
(1) Compared with single pretreatment, the two-step pretreatment further removes lignin, uses xylanase to perform pre-enzymolysis, removes part of hemicellulose, increases the contact area of cellulose and cellulose, and reduces ineffective adsorption of the lignin to the cellulose by adding a surfactant, thereby further improving enzymolysis efficiency.
(2) The enzymes and the surfactant in the pretreatment and enzymolysis processes can be recycled, the operation is simple, and the enzymolysis cost is reduced.
The specific embodiment is as follows:
the following examples may help to better understand the present invention. Furthermore, the description of the embodiments is merely illustrative of the invention and should not be taken as limiting the invention as detailed in the claims.
Example 1: pretreatment of wood
(1) Cleaning poplar, crushing by using a crusher, sieving by using a 80-mesh sieve, putting wood chips into a 60 ℃ oven, and drying to constant weight;
(2) Taking 50g of wood chips, fully and uniformly mixing the wood chips with 4% sodium hydroxide according to a feed liquid ratio of 1:10 (g: mL), and placing the wood chips at 121 ℃ for 90min;
(3) Washing the poplar wood chips to be neutral by using pure water, and then placing the poplar wood chips in a 60 ℃ oven to be dried to constant weight for later use.
Example 2: secondary pretreatment of hydrogen peroxide and laccase
(1) The wood chips treated in example 1 were thoroughly mixed with sodium acetate buffer at a feed liquid ratio of 1:10 (g: mL);
(2) Adding 50mL of 10% hydrogen peroxide and 50U/g of laccase in the wood chips, and pre-treating for 12 hours at 50 ℃;
(3) And filtering the secondarily pretreated wood chips by using a vacuum pump to obtain filtrate and filter residues, wherein the filtrate is recovered, refrigerated and stored, so that the secondary pretreated wood chips are convenient to reuse, and the filter residues are used.
Example 3: pre-enzymatic hydrolysis of xylanase and surfactant
(1) The poplar filter residues obtained in the example 2 are fully and evenly mixed with sodium acetate buffer solution according to the feed liquid ratio of 1:10 (g: mL);
(2) Adding xylanase of 60U/g wood chips and a nonionic surfactant Tween-80 of 0.05g/g wood chips for pre-enzymolysis for 2 hours;
(3) And filtering the wood chips subjected to pre-enzymolysis by using a vacuum pump to obtain filtrate and filter residues, wherein the filtrate is recovered, refrigerated and stored, so that the filter residues are convenient to reuse and stand by.
Example 4: enzymatic saccharification of cellulase
(1) Fully and uniformly mixing the poplar filter residues obtained in the example 3 with a sodium acetate buffer solution according to a feed liquid ratio of 1:10 (g: mL);
(2) Adding 20U/g of wood chip cellulase and 0.05g/g of wood chip nonionic surfactant polyethylene glycol for enzymolysis saccharification for 48 hours;
(3) And filtering the enzymatic hydrolysis wood chips by using a vacuum pump to obtain filter residues and filtrate, wherein the filtrate is recovered, refrigerated and stored, so that the filter residues are convenient to reuse and discarded.
Example 5: the enzymolysis saccharification effect of wood chips is compared
In order to examine the enzymolysis saccharification effect of poplar, the following treatments are designed:
treatment 1: wood chips were treated in the order of example 1, example 2, example 3 and example 4, wherein 50g of wood chips were used;
treatment 2: wood chips were treated in the order of example 2, example 3 and example 4, wherein the wood chips were changed to 50g of untreated wood chips baked to a constant weight;
treatment 3: wood chips were treated in this order as in example 1, example 3 and example 4, wherein 50g of wood chips were used;
treatment 4: wood chips were treated in the order of example 1, example 2 and example 4, wherein 50g of wood chips were used;
treatment 5: the poplar was treated in sequence as in example 3 and example 4, with the filter residue being replaced by 50g of untreated wood chips baked to constant weight;
treatment 6: the wood chips were treated in this order as in examples 2 and 4, wherein the wood chips were changed to 50g of untreated wood chips baked to a constant weight;
treatment 7: wood chips were treated in the order of example 1 and example 4, wherein 50g of wood chips were used;
treatment 8: the treatment of poplar was carried out directly as in example 4, with the filter residue being replaced by 50g of untreated wood chips baked to constant weight;
treatment 9: wood chips were treated in this order as in example 1, example 2 and example 3, wherein 50g of wood chips were used;
treatment 10: after the treatment according to example 1, hydrogen peroxide and laccase, xylanase and surfactant and cellulase were added simultaneously in the amounts of examples 2, 3 and 4, respectively, wherein 50g of wood chips were used.
The reducing sugar content of the final filtrate in treatments 1 to 10 was measured by DNS method and the results are shown in table 1 below:
TABLE 1
As can be seen from the data in the table, the final sugar yield was very low without cellulase hydrolysis (treatment 9) and without any pretreatment (treatment 8), whereas treatment 1 with four treatments, the reduced sugar content in the filtrate was highest, reaching 20.26mg/mL, i.e. 20.26% yield of reduced sugar (to substrate), which is 3.77% higher than 16.49% yield of reduced sugar with suboptimal (treatment 4), and very advantageous over the one-or two-step treatment methods.
Example 6: recycling of enzymes
Treatment 1: poplar was treated in the order of example 1, example 2, example 3 and example 4;
treatment 2: the poplar was treated 2 times in the order of example 1, example 2, example 3 and example 4, wherein the enzymes of example 2, example 3 and example 4 were the filtrate recovered from the previous time.
Treatment 3: the poplar was treated 5 times in the order of example 1, example 2, example 3 and example 4, wherein the enzymes of example 2, example 3 and example 4 were the filtrate recovered from the previous time.
Treatment 4: the poplar was treated 8 times in the order of example 1, example 2, example 3 and example 4, wherein the enzymes of example 2, example 3 and example 4 were the filtrate recovered from the previous time.
The content of reducing sugar in the filtrate of the last treatment in example 4 was measured using DNS method, and recovery efficiency=final filtrate reducing sugar content/(reducing sugar content in treatment 1×number of treatments). The results are shown in Table 2 below:
TABLE 2
As can be seen from the data in the table, the final recovery efficiency of the enzyme can be 89.66% after the enzyme is recycled for 1 time (i.e. the treatment 2), and the recovery efficiency can still reach more than half after the enzyme is recycled for 4 times (i.e. the treatment 3), which shows that the enzyme recovery is significant and the cost can be greatly saved. When the recycling is performed for 7 times, the recycling efficiency is lower than 50 percent and is 48.6 percent, and the enzymolysis efficiency is lower at the moment and is not suitable for continuous recycling, so the recycling method designed by the method can have good enzymolysis effect after being reused for 4 to 6 times, and the cost can be greatly reduced.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (1)
1. A method for saccharifying lignocellulose by multi-enzyme synergistic surfactant cyclic enzymolysis, which is characterized by comprising the following steps in sequence: (1) alkali treatment of wood: mixing the crushed 80-mesh dried wood chips uniformly by using sodium hydroxide, washing with water to neutrality, and drying; (2) laccase secondary pretreatment: suspending the sawdust again by using buffer solution, adding hydrogen peroxide and laccase for secondary treatment, and carrying out suction filtration to recover filtrate and filter residues for later use; (3) pre-enzymatic hydrolysis of xylanase: re-suspending the filter residue obtained in the step (2) by using a buffer solution, adding xylanase and a surfactant for pre-enzymolysis, and carrying out suction filtration to recover a filtrate, wherein the filter residue is for later use; (4) enzymatic saccharification of cellulase: re-suspending the filter residue obtained in the step (3) by using a buffer solution, adding cellulase and a surfactant for enzymolysis saccharification, and carrying out suction filtration to collect filtrate, thus obtaining a saccharification product; filtering the enzyme solution used in the steps (2), (3) and (4), and recycling for 4-6 times;
the concentration of sodium hydroxide used in the step (1) is 1% -4%, the sodium hydroxide is treated at 115-125 ℃ for 60-150 min;
the concentration of the hydrogen peroxide used in the step (2) is 8% -20%, and the using amount of laccase is 10U/g-100U/g of wood dust;
the hydrogen peroxide and laccase are pretreated at 37-60 ℃ for 6-24 h;
the xylanase used in the step (3) is used in an amount of 10U/g to 150U/g of wood chips, the surfactant used is one or two of polyethylene glycol and tween-80, and the xylanase used is used in an amount of 0.01g/g to 0.1g/g of wood chips;
the xylanase and the surfactant are subjected to pre-enzymolysis at 37-60 ℃ for 0.5-6 h;
the dosage of the cellulase used in the step (4) is 4U/g-60U/g of wood chips, the surfactant used is one or two of polyethylene glycol and tween-80, and the dosage is 0.01 g/g-0.1 g/g of wood chips;
the cellulase and the surfactant are subjected to enzymolysis at 37-60 ℃ for 24-84 hours;
the buffer solution is sodium acetate buffer solution, the pH value of the buffer solution is 4.8, and the ratio of wood chips or filter residues to the buffer solution is 1:5-1:20 g/mL.
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| CN101255479A (en) * | 2008-04-22 | 2008-09-03 | 南京工业大学 | Pretreatment method for efficiently saccharifying lignocellulose |
| CN102191299A (en) * | 2010-03-10 | 2011-09-21 | 中国科学院青岛生物能源与过程研究所 | Method for increasing lignocellulose saccharification yield through multi-step enzymolysis |
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| US8993274B2 (en) * | 2011-08-18 | 2015-03-31 | Andritz Inc. | Enzymatic hydrolysis pretreatment of lignocellulosic materials |
| CN105671088B (en) * | 2014-11-20 | 2019-05-17 | 中国科学院大连化学物理研究所 | A kind of continuous conversion common fermentation method of the improved lignocellulosic of surfactant |
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| CN101255479A (en) * | 2008-04-22 | 2008-09-03 | 南京工业大学 | Pretreatment method for efficiently saccharifying lignocellulose |
| CN102191299A (en) * | 2010-03-10 | 2011-09-21 | 中国科学院青岛生物能源与过程研究所 | Method for increasing lignocellulose saccharification yield through multi-step enzymolysis |
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| CN107164424A (en) * | 2017-07-12 | 2017-09-15 | 天津科技大学 | It is a kind of to aoxidize the lignocellulose pretreatment method that delignification improves enzyme hydrolysis rate |
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| CN110669804A (en) * | 2019-09-25 | 2020-01-10 | 湖北大学 | Method for improving saccharification efficiency of lignocellulose |
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