CN110734942A - Method for improving enzymolysis saccharification effect by pretreating xylose residues - Google Patents

Method for improving enzymolysis saccharification effect by pretreating xylose residues Download PDF

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CN110734942A
CN110734942A CN201911017304.8A CN201911017304A CN110734942A CN 110734942 A CN110734942 A CN 110734942A CN 201911017304 A CN201911017304 A CN 201911017304A CN 110734942 A CN110734942 A CN 110734942A
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xylose residue
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王朝宇
迟雪文
毕艳红
卢艺惠
赵祥杰
李明依
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Huaiyin Institute of Technology
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Abstract

The invention discloses a method for improving enzymolysis saccharification effect by pretreating types of xylose residues, which comprises the following steps of weighing the xylose residues, adding a Deep Eutectic Solvent (DES) for pretreatment, carrying out solid-liquid separation after the reaction is finished, adding a KOH solution into the separated solid for pretreatment, carrying out solid-liquid separation after the reaction is finished, respectively collecting solid and liquid parts, washing the solid part to be neutral to obtain the pretreated xylose residues, carrying out enzymolysis saccharification, and determining the glucose concentration in an enzymolysis solution.

Description

Method for improving enzymolysis saccharification effect by pretreating xylose residues
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for improving enzymolysis saccharification effect by pretreating kinds of xylose residues.
Background
Meanwhile, the problems of ecological damage, environmental pollution and the like caused in the development and utilization process of fossil energy are gradually highlighted, the global climate is greatly influenced by the large emission of greenhouse gases, and the ecological safety and the sustainable development of the human society are seriously challenged due to the frequent occurrence of various natural disasters.
The annual output of Chinese wood fiber biomass resources is over 7.5 hundred million tons, and the Chinese wood fiber biomass resources are main raw materials in the industrial fields of chemical industry, papermaking, textile and the like, but the utilization rate of the Chinese wood fiber biomass resources is not high, so that if the wood fiber biomass resources can be subjected to processing pretreatment to produce biomass fuel or bulk chemicals with high added values, the global energy crisis can be relieved to the extent of , and the pressure of fossil energy is relieved.
The xylose residue is a product obtained by extracting xylose from corncobs through dilute acid treatment, and more than 500 million tons of xylose residue is generated in China every year, wherein most of the xylose residue is combusted to generate heat, so that environmental problems and waste of cellulose materials are caused.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and provides a method for improving enzymolysis saccharification effect by types of xylose residues through pretreatment.
In order to achieve the purpose, the method for improving the enzymolysis saccharification effect by pretreating kinds of xylose residues comprises the following steps:
(1) weighing the xylose residues, adding DES (Deep Eutectic Solvent, DES) for pretreatment, carrying out solid-liquid separation after the reaction is finished, and respectively collecting solid and liquid parts;
(2) adding KOH solution into the solid separated in the step (1) for pretreatment, carrying out solid-liquid separation on the solid after the reaction is finished, and respectively collecting solid and liquid parts;
(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated xylose residue, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.
The xylose residue used in the invention is purchased from Futaste Co.Ltd or other commercial companies, or according to a literature diluted acid treatment method, the resource utilization and research of Zhao Zheng Chi slag [ D ] Zheng university, 2016 ] and the effect is .
Wherein the DES consists of choline chloride: lactic acid was added in a molar ratio of 1: 1-5. Preferably, the DES consists of choline chloride: lactic acid was added in a molar ratio of 1: 2, and preparing the product.
the DES is prepared by adding choline chloride and lactic acid into a reaction flask, magnetically stirring at 60-80 deg.C until the mixture becomes colorless clear liquid, transferring to a vacuum drying oven, and adding P2O5Fully drying the mixture for later use.
Wherein, the DES pretreatment conditions of the xylose residues are as follows: the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.
Wherein the mass fraction of the KOH solution is 5-30%.
Wherein, the pretreatment conditions of the KOH solution are as follows: the treatment time is 30-180min, the treatment solid-liquid ratio is 1:6-1:18g/mL, and the treatment temperature is 50-100 ℃.
And (3) carrying out solid-liquid separation on the xylose residue after KOH pretreatment, respectively collecting solid and liquid parts, washing the solid residue to be neutral by using tap water, and then carrying out enzymolysis saccharification.
, adding citric acid buffer solution to adjust pH, adding sodium azide solution, adding cellulase, and measuring glucose concentration in the enzymatic hydrolysate by HPLC after reaction.
Preferably, the cellulase is added in an amount of 3-40 FPU/g-substrate (i.e. pretreated xylose residue), the solid loading is 2-20 wt.%, and the enzymatic hydrolysis is carried out at pH 4.8 and temperature 50 ℃ for 2-96 h. If the enzymatic hydrolysis reaction volume is 20mL, 3g of pretreated xylose residue (dry basis) is added at a solids loading of 15 wt.%, and the total reaction volume is 20mL × 15% solids loading (solids content) is 3g dry weight by weight.
According to IUPAC standard, the definition of Filter paper enzyme activity (FPA) is 1g of solid enzyme (or 1mL of liquid enzyme), and the enzyme dosage required for generating 1 mu mol of glucose from a Filter paper substrate per hour at 50 ℃ and pH 4.8 is defined as enzyme activity units expressed by U/g (or U/mL).
After the enzymatic saccharification process is finished, quantitative enzymatic hydrolysate is taken to be subjected to High Performance Liquid Chromatography (HPLC) detection, and the content of reducing sugar in the enzymatic hydrolysate is measured, wherein the detection conditions of HPLC are shown in Table 1.
TABLE 1 conditions of liquid chromatography detection
Figure BDA0002246103170000031
The liquid (the black liquid collected in the steps (1) and (2)) generated in the method for improving the enzymolysis saccharification effect by pretreating the xylose residue is applied to the preparation of potassium fertilizer, lignin-based fertilizer and compound fertilizer.
According to the method, the DES and KOH pretreatment is adopted to remove lignin and hemicellulose wrapped on the surface of the cellulose, so that the highly ordered and tightly arranged regions of the cellulose are damaged, pores are generated on the surface of the cellulose, more cellulose is exposed, the porosity and porosity are increased, the accessibility of the cellulose by the cellulase is improved, and the enzymolysis saccharification efficiency is promoted.
Has the advantages that: compared with the prior art, the invention has the following advantages:
according to the invention, the DES and KOH are adopted to pretreat the xylose residues, so that the wrapping of lignin and hemicellulose on cellulose is damaged, the lignin is removed, the hemicellulose is degraded, the crystal structure of the cellulose is changed, the accessibility and the porosity of the cellulose are improved, the contact of cellulase and a substrate is promoted, and further the cellulose hydrolysis yield and the reducing sugar yield are improved.
The method adopts eutectic solvents DES and KOH solution for pretreatment, has mild reaction conditions, reduces the damage to instruments and equipment, and is beneficial to industrial production and application.
Drawings
FIG. 1 is a Scanning Electron Microscope (SEM) image of xylose residue before and after pretreatment;
FIG. 2 is a Fourier Infrared Spectroscopy (FTIR) of xylose residue before and after pretreatment;
FIG. 3 is an X-ray diffraction pattern (XRD) before and after pretreatment of xylose residue;
wherein a is a xylose residue raw material; b is the xylose residue after 10 wt.% KOH pretreatment in example 2; c is the xylose residue of example 4 pretreated with 16 wt.% KOH;
observation of SEM, FTIR and XRD patterns shows that compared with the untreated xylose residue raw material, the crystallinity of the pretreated xylose residue is increased, the KOH dosage is increased, the degradation of the xylose residue is more severe, the porosity and the porosity are increased, so that the accessibility of the cellulase to the cellulose is increased, the enzymolysis saccharification efficiency is improved, but the cost is consumed due to the excessive KOH dosage, the over-hydrolysis is caused due to the excessive KOH dosage, and the 16 wt.% KOH pretreatment effect is the best.
Detailed Description
The invention is further illustrated in connection with the following examples and the accompanying drawings.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the embodiment, the xylose residues are the solid residue part of the corncobs after diluted acid treatment, and the xylose residues used in the embodiment are all calculated by absolute dry materials; DES is prepared by adding choline chloride and lactic acid into a reaction flask, magnetically stirring at 80 deg.C until the mixture turns into colorless clear liquid, transferring into a vacuum drying oven, and adding P2O5Fully drying the mixture for later use.
Example 1
Weighing 30.0 +/-0.1 g of xylose residues, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (molar ratio of 1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 7%, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished; and (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 3g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 51.8g/L, and the obtained glucose yield is 39.8%.
Example 2
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 10%, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 3g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 86.8g/L, and the obtained glucose yield is 41.5%.
Example 3
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of 13% KOH solution into the solid, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 3g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 125.8g/L, and the obtained glucose yield is 74.5%.
Example 4
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 16% into the solid, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 3g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 128.8g/L, and the obtained glucose yield is 76.9%.
Example 5
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 16% into the solid, carrying out pretreatment at 90 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 3g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 137.9g/L, and the obtained glucose yield is 76.3%.
Example 6
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 16% into the solid, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 1g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 5 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 20 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 47.2g/L, and the obtained glucose yield is 92.4%.
Example 7
Weighing 30.0 +/-0.1 g of xylose residue, adding 300.0 +/-0.1 g of DES (choline chloride: lactic acid (1: 2)) for pretreatment at 90 ℃ for 1h, carrying out solid-liquid separation after the reaction is finished, then adding 300.0 +/-0.1 mL of KOH solution with the mass fraction of 16% into the solid, carrying out pretreatment at 70 ℃ for 90min, and carrying out solid-liquid separation after the reaction is finished. And (4) washing the solid part to be neutral by using tap water, and carrying out an enzymolysis saccharification experiment. The enzymatic hydrolysis reaction volume was 20mL, 0.4g of pretreated xylose residue (absolute dry basis) was added at a solids loading of 2 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result shows that the glucose concentration in the enzymolysis liquid is 18.7g/L, and the obtained glucose yield is 97.1%.
Example 8
Example 8 is the same as example 4 except that: by adopting KOH solution with the mass fraction of 20 percent, the yield of the obtained glucose is 75.4 percent.
Example 9
Example 8 is the same as example 4 except that: by adopting 30 percent of KOH solution, the yield of the obtained glucose is 68.2 percent.
Example 10
Example 10 is the same as example 4 except that: DES consists of choline chloride: lactic acid was added in a molar ratio of 1:3, preparing; the DES pretreatment of the xylose residues is carried out for 6 hours, the pretreatment solid-to-liquid ratio is 1:8g/mL, and the pretreatment temperature is 60 ℃; the pretreatment conditions of the KOH solution are as follows: the treatment time is 30min, the treatment solid-to-liquid ratio is 1:6g/mL, and the treatment temperature is 100 ℃; the enzymatic hydrolysis reaction volume was 20mL, with a 20 wt.% solids loading of 4g of pretreated xylose residue (absolute dry); cellulase was added at 3 FPU/g-substrate, solids loading at 2 wt.%, and enzymatic hydrolysis at pH 4.8 and temperature 50 ℃ for 2 h.
Example 11
Example 11 is the same as example 4 except that: the DES pretreatment of the xylose residues is carried out for 0.5h, the pretreatment solid-to-liquid ratio is 1:30g/mL, and the pretreatment temperature is 130 ℃; the pretreatment conditions of the KOH solution are as follows: the treatment time is 180min, the treatment solid-to-liquid ratio is 1:18g/mL, and the treatment temperature is 50 ℃; cellulase was added at 40 FPU/g-substrate, solids loading at 20 wt.%, and enzymatic hydrolysis at pH 4.8 and temperature 50 ℃ for 96 h.
Example 12
Example 12 was prepared identically to example 4, except that: the enzymatic hydrolysis reaction volume was 20mL, 3g of untreated xylose residue raw material (absolute dry basis) was added at a solids loading of 15 wt.%, 0.05mol/L citric acid buffer solution was added to adjust the pH to 4.8, 0.2mL of sodium azide solution (0.02g/mL) was added, and then cellulase was added at a rate of 5 FPU/g-substrate at 120rpm for 72h at 50 ℃. Then, the glucose concentration in the enzymatic hydrolysate was measured by HPLC. The result showed that the glucose concentration in the enzymolysis solution was 106.7g/L, and the glucose yield was 47.8%.
Comparing examples 4 and 5, it can be seen that example 12 and examples 4 and 5 have the same conditions for enzymatic hydrolysis of the same xylose residue feedstock, but example 12 directly performs enzymatic saccharification on the untreated xylose residue feedstock, whereas examples 4 and 5 perform DES and 16 wt.% KOH pretreatment on the xylose residue prior to enzymatic hydrolysis, and the final glucose concentration in the enzymatic hydrolysate is 22.1g/L and 31.2g/L higher than example 12, and the final glucose yield in examples 4 and 5 is 29.1% and 28.5% higher than example 12.
By observing the graph 1, the graph 2 and the graph 3, the crystallinity of the pretreated xylose residue is increased, the porosity and the porosity are increased, so that the accessibility of cellulose by cellulase is increased, and the enzymolysis saccharification efficiency is improved compared with the untreated xylose residue raw material, the degradation of the xylose residue is more severe, and the series characteristics of XRD, FTIR, SEM and the like prove the action principle of the pretreatment and are favorable for the downstream enzymolysis saccharification process.
According to the invention, the DES and KOH are combined for treatment, lignin can be removed by two methods independently to improve the sugar yield after enzymolysis and saccharification, but the removal rate of the lignin is not high and the effect is not obvious when the DES is independently carried out for treating the lignin, the KOH used is larger as the effect of the KOH pretreatment alone is , the two methods are combined to have obvious synergistic effect, the DES is a green solvent, parts of lignin are removed by the DES treatment, and then the KOH treatment is added, so that the use amount of the KOH can be reduced to achieve the same effect.
Example 13
The liquid fraction collected by the solid-liquid separation in steps (1) and (2) of example 4 was used as a raw material for the preparation of a potassium fertilizer, a lignin-based fertilizer or a compound fertilizer.

Claims (8)

1, kinds of xylose slag pretreatment improves the enzymolysis saccharification effect method, its characteristic lies in, including the following step:
(1) weighing the xylose residues, adding DES (data encryption standard) for pretreatment, performing solid-liquid separation after the reaction is finished, and respectively collecting solid and liquid parts;
(2) adding KOH solution into the solid separated in the step (1) for pretreatment, carrying out solid-liquid separation on the solid after the reaction is finished, and respectively collecting the solid part and the liquid part;
(3) and (3) washing the solid part separated in the step (2) to be neutral to obtain pretreated xylose residue, carrying out enzymolysis saccharification, and determining the concentration of glucose in the enzymolysis liquid.
2. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the DES is prepared by mixing choline chloride: lactic acid was added in a molar ratio of 1: 1-5.
3. The method for improving enzymolysis saccharification effect of xylose residue pretreatment of claim 1, wherein the conditions of DES pretreatment of xylose residue are as follows: preferably, the pretreatment time is 0.5-6h, the pretreatment solid-to-liquid ratio is 1:8-1:30g/mL, and the pretreatment temperature is 60-130 ℃.
4. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the mass fraction of KOH solution is 5-30 wt.%.
5. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the pretreatment conditions of the KOH solution are as follows: the treatment time is 30-180min, the treatment solid-liquid ratio is 1:6-1:18g/mL, and the treatment temperature is 50-100 ℃.
6. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 1, wherein the enzymatic saccharification is that the pretreated xylose residue is added with citric acid buffer solution to adjust pH, sodium azide solution is added, then cellulase is added, and after reaction, HPLC is adopted to measure glucose concentration in the enzymatic hydrolysate.
7. The method for improving enzymatic saccharification effect of xylose residue pretreatment of claim 6, wherein said cellulase is added in an amount of 3-40 FPU/g-substrate, solid loading is 2-20 wt.%, and enzymatic hydrolysis is performed at pH 4.8 and temperature 50 ℃ for 2-96 h.
8, use of the liquid produced by the method for improving enzymolysis saccharification effect of xylose residue pretreatment as claimed in claim 1 in the preparation of potassium fertilizer, lignin-based fertilizer and compound fertilizer.
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