CN113914125A - Method for separating cellulose from bagasse and carrying out enzymolysis - Google Patents
Method for separating cellulose from bagasse and carrying out enzymolysis Download PDFInfo
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- CN113914125A CN113914125A CN202111207232.0A CN202111207232A CN113914125A CN 113914125 A CN113914125 A CN 113914125A CN 202111207232 A CN202111207232 A CN 202111207232A CN 113914125 A CN113914125 A CN 113914125A
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- 241000609240 Ambelania acida Species 0.000 title claims abstract description 67
- 239000010905 bagasse Substances 0.000 title claims abstract description 67
- 229920002678 cellulose Polymers 0.000 title claims abstract description 54
- 239000001913 cellulose Substances 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 54
- 239000000243 solution Substances 0.000 claims abstract description 61
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 38
- 239000008103 glucose Substances 0.000 claims abstract description 38
- 108010059892 Cellulase Proteins 0.000 claims abstract description 27
- 229940106157 cellulase Drugs 0.000 claims abstract description 27
- 238000011282 treatment Methods 0.000 claims abstract description 24
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims abstract description 16
- 238000004321 preservation Methods 0.000 claims abstract description 14
- 238000002156 mixing Methods 0.000 claims abstract description 9
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 90
- 239000007864 aqueous solution Substances 0.000 claims description 18
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 240000000111 Saccharum officinarum Species 0.000 claims description 4
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 229920005610 lignin Polymers 0.000 abstract description 37
- 229920002488 Hemicellulose Polymers 0.000 abstract description 34
- 239000002994 raw material Substances 0.000 abstract description 18
- 230000002349 favourable effect Effects 0.000 abstract description 3
- 238000006243 chemical reaction Methods 0.000 description 30
- 239000007788 liquid Substances 0.000 description 17
- 239000007974 sodium acetate buffer Substances 0.000 description 17
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical group [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 17
- 230000007071 enzymatic hydrolysis Effects 0.000 description 16
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 16
- 230000014759 maintenance of location Effects 0.000 description 16
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 15
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 15
- 230000002779 inactivation Effects 0.000 description 15
- 239000007791 liquid phase Substances 0.000 description 15
- 239000000203 mixture Substances 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 230000035484 reaction time Effects 0.000 description 14
- 238000001816 cooling Methods 0.000 description 13
- 238000010438 heat treatment Methods 0.000 description 13
- 238000003828 vacuum filtration Methods 0.000 description 13
- 239000002028 Biomass Substances 0.000 description 10
- 244000269722 Thea sinensis Species 0.000 description 9
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 9
- 229930182490 saponin Natural products 0.000 description 9
- 150000007949 saponins Chemical class 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 7
- 229920000053 polysorbate 80 Polymers 0.000 description 7
- 230000000694 effects Effects 0.000 description 6
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 3
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 229920002522 Wood fibre Polymers 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- 239000002025 wood fiber Substances 0.000 description 2
- 229910021556 Chromium(III) chloride Inorganic materials 0.000 description 1
- 229910021577 Iron(II) chloride Inorganic materials 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- VSCWAEJMTAWNJL-UHFFFAOYSA-K aluminium chloride Substances Cl[Al](Cl)Cl VSCWAEJMTAWNJL-UHFFFAOYSA-K 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000011636 chromium(III) chloride Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000002608 ionic liquid Substances 0.000 description 1
- 239000002029 lignocellulosic biomass Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
Classifications
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
- D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- D—TEXTILES; PAPER
- D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
- D21B—FIBROUS RAW MATERIALS OR THEIR MECHANICAL TREATMENT
- D21B1/00—Fibrous raw materials or their mechanical treatment
- D21B1/02—Pretreatment of the raw materials by chemical or physical means
- D21B1/021—Pretreatment of the raw materials by chemical or physical means by chemical means
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Mechanical Engineering (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for separating cellulose from bagasse and carrying out enzymolysis, wherein the operation of separating the cellulose comprises the steps of uniformly mixing the bagasse, an ethanol water solution and copper chloride, carrying out heat preservation treatment at 130-180 ℃, and separating to obtain a pretreatment solution and the cellulose. Adding buffer solution and cellulase into cellulose for enzymolysis to obtain glucose solution. The method has mild conditions for separating the cellulose, can effectively remove most of hemicellulose and part of lignin in the bagasse, retains the cellulose, and can obtain high-quality cellulose. The prepared cellulose has low content of hemicellulose and lignin, reduces the influence of the hemicellulose and the lignin on enzymolysis, preliminarily improves the enzymolysis efficiency, and is more favorable for being used as a raw material for enzymolysis of cellulase.
Description
Technical Field
The invention relates to the technical field of efficient conversion and utilization of biomass, in particular to a method for separating cellulose from bagasse by copper chloride catalytic ethanol pretreatment, and further relates to a method for hydrolyzing cellulose into glucose.
Background
With the reduction of fossil energy, environmental pollution and the enhancement of environmental awareness, people are forced to find a new clean energy. Biomass has attracted attention as one of them. Among abundant biomass resources, wood fiber biomass is various and easy to obtain. The method for preparing the liquid fuel by using the wood fiber biomass has important significance for not only resource utilization of wastes, but also energy safety in China, and is concerned by people. However, because the cellulose in the lignin biomass is tightly connected with the hemicellulose and the lignin, the structure is stable, which causes difficulty in effectively separating the cellulose in the lignin biomass and influences further utilization of the cellulose. Therefore, when biomass resources are converted, pretreatment is required to be carried out, the compact structure of the biomass resources is damaged, lignin in the biomass resources is removed, hemicellulose and cellulose are hydrolyzed into monosaccharides such as xylose and glucose in the enzymolysis process, and ethanol is produced through fermentation.
The current common pretreatment methods include acid pretreatment, alkali pretreatment, organic solvent pretreatment and ionic liquid pretreatment. Among them, the pretreatment with an organic solvent based on an ethanol solution is preferred because of its advantages such as recyclability, low toxicity and the possibility of generating a pretreatment residue which is easy to be subjected to enzymatic hydrolysis. Generally, the conditions provided by ethanol-based organic solvent pretreatment are not enough to degrade lignin and hemicellulose in the lignocellulosic biomass, and an acidic or alkaline catalyst is added to remove most of the hemicellulose and lignin, break the original compact structure, improve the accessibility of cellulase and facilitate the subsequent utilization of cellulose.
The inventors disclose in CN109457000A that bagasse starting materials are mixed with a certain amount of metal salt solution (MgCl)2Aqueous solution, FeCl2Aqueous solution, ZnCl2Aqueous solution, CuCl2Aqueous solution, AlCl3Aqueous solution, CrCl3Aqueous solution and FeCl3Any one of the water solutions) is placed in a closed reactor, and the pretreatment is carried out for 5-15 min at the temperature of 150-170 ℃, so that part of lignin and hemicellulose is removed. Studies have shown that the lignin and hemicellulose removal effect of such treatments is general and needs to be further improved.
Disclosure of Invention
The object of the present invention is to overcome at least one of the drawbacks of the prior art and to provide a process for the separation of cellulose from sugar cane bagasse and for enzymatic hydrolysis.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a process for separating cellulose from sugar cane bagasse, comprising the steps of: uniformly mixing bagasse, an ethanol water solution and copper chloride, carrying out heat preservation treatment at 130-180 ℃, and separating to obtain a pretreatment solution and pretreated bagasse, wherein the pretreated bagasse is cellulose obtained by separation.
In some examples, the mixing ratio of bagasse, aqueous ethanol is 1 g: 8-15 mL, and the bagasse is calculated according to absolute dry mass.
In some examples, the concentration of the aqueous ethanol solution is 40-75% (v/v).
In some examples, the concentration of the aqueous ethanol solution is 60% (v/v).
In some examples, the final concentration of copper chloride is 0.005-0.1 mol/L.
In some examples, the final concentration of copper chloride is 0.01 to 0.05 mol/L.
In some examples, the final concentration of copper chloride is 0.025 to 0.05 mol/L.
In some examples, the incubation time is 5-40 min.
In some examples, the incubation time is 10-20 min.
In some examples, the temperature of the incubation is 160-180 ℃.
In some examples, the temperature of the heat preservation is 160-180 ℃, and the heat preservation time is 10-20 min.
In a second aspect of the present invention, there is provided:
a method for improving sugar production efficiency of bagasse enzymolysis comprises the following steps:
s1) preparing the pretreated bagasse by the method of the first aspect of the invention;
s2) adding a buffer solution and cellulase into the pretreated bagasse for enzymolysis to obtain sugar solution.
In some examples, an enzymatic hydrolysis promoter is further added during the enzymatic hydrolysis, and the enzymatic hydrolysis promoter is one of tween 80, BSA or tea saponin.
In some examples, the cellulase is used in an amount of 10 to 20 FPU/g oven-dried mass of pretreated bagasse.
In some examples, the tween 80 is used in an amount of 0 to 100mg/g of oven-dried mass of the pretreated bagasse.
In some examples, the BSA is used in an amount of 0-60 mg/g absolute dry mass of the pretreated bagasse.
In some examples, the tea saponin is used in an amount of 0-60 mg/g oven-dried mass of pretreated bagasse.
In some examples, the buffer is an acetic acid-sodium acetate buffer solution.
In some examples, the mass to volume ratio of the pretreated bagasse to the buffer is 1 g: (40-100) mL.
In some examples, the acetic acid-sodium acetate buffer solution has a concentration of 0.03 to 0.06 mol/L.
The invention has the beneficial effects that:
the method for separating the cellulose from the bagasse has mild reaction conditions, can effectively remove most of hemicellulose and part of lignin in the bagasse, retains the cellulose, and can obtain high-quality cellulose.
According to the method for separating the cellulose from the bagasse, disclosed by the invention, the prepared cellulose is low in hemicellulose and lignin content, the influence of the hemicellulose and the lignin on enzymolysis is reduced, the enzymolysis efficiency is preliminarily improved, and the cellulose is more favorable for being used as a raw material for enzymolysis of cellulase.
According to some embodiments of the invention, the sugar production efficiency and the enzymolysis rate can be further improved by adding the additive with a specific composition, and the enzymolysis cost can be effectively reduced.
Detailed Description
A process for separating cellulose from sugar cane bagasse, comprising the steps of: uniformly mixing bagasse, an ethanol water solution and copper chloride, carrying out heat preservation treatment at 130-180 ℃, and separating to obtain a pretreatment solution and pretreated bagasse, wherein the pretreated bagasse is cellulose obtained by separation.
In some examples, the mixing ratio of bagasse, aqueous ethanol is 1 g: 8-15 mL, and the bagasse is calculated according to absolute dry mass. The proportion can be adjusted to a certain extent according to the concentration of the ethanol water solution and the subsequent hydrolysis effect.
The concentration of the ethanol aqueous solution can be adjusted correspondingly according to the application condition. In some examples, the concentration of the aqueous ethanol solution is 40-75% (v/v).
In some examples, the concentration of the ethanol aqueous solution is 40-60% (v/v), preferably 40-50% (v/v). Experimental data show that the concentration of the ethanol water solution is in the range, hemicellulose and lignin can be well removed, and cellulose is not damaged.
In some examples, the final concentration of copper chloride is 0.005-0.1 mol/L. At this concentration, it has a certain promoting effect.
In some examples, the final concentration of copper chloride is 0.01 to 0.05 mol/L. Experimental data show that the compound has a better promoting effect at the concentration.
In some examples, the final concentration of copper chloride is 0.025 to 0.05 mol/L. This concentration had the best promoting effect under the experimental conditions.
The heat preservation time can be adjusted to a certain extent according to the enzymolysis effect. In some examples, the incubation time is 5-40 min.
In some examples, the incubation time is 10-20 min. Too short a time, weak effect of promoting enzymolysis, too long a time, not beneficial to subsequent enzymolysis, may be related to the cellulose structure being destroyed at high temperature.
In some examples, the final concentration of copper chloride is 0.005-0.1 mol/L.
In some examples, the final concentration of copper chloride is 0.01 to 0.05 mol/L.
In some examples, the final concentration of copper chloride is 0.025 to 0.05 mol/L.
In some examples, the incubation time is 5-40 min.
In some examples, the incubation time is 10-20 min.
In some examples, the temperature of the incubation is 160-180 ℃.
In some examples, the temperature of the heat preservation is 160-180 ℃, and the heat preservation time is 10-20 min. Based on the existing experimental data, the optimal promotion effect is expected to be achieved under the treatment condition, and compared with other conditions, the enzymolysis efficiency is obviously improved.
In a second aspect of the present invention, there is provided:
a method for improving sugar production efficiency of bagasse enzymolysis comprises the following steps:
s1) preparing the pretreated bagasse by the method of the first aspect of the invention;
s2) adding a buffer solution and cellulase into the pretreated bagasse for enzymolysis to obtain sugar solution.
The amount of cellulase used is based primarily on cost considerations. In some examples, the cellulase is used in an amount of 10 to 20 FPU/g oven-dried mass of pretreated bagasse. With the dosage, not only can better enzymolysis efficiency be obtained, but also the cost is relatively acceptable.
In some examples, an enzymatic hydrolysis promoter is further added during the enzymatic hydrolysis, and the enzymatic hydrolysis promoter is one of tween 80, BSA or tea saponin. Experimental data show that the addition of Tween 80, BSA or tea saponin can improve the enzymolysis rate, shorten the enzymolysis time and improve the production efficiency.
In some examples, the tween 80 is used in an amount of 0 to 100mg/g of oven-dried mass of the pretreated bagasse. Preferably, the oven-dried mass of the pretreated bagasse is about 50-60 mg/g.
In some examples, the BSA is used in an amount of 0-60 mg/g absolute dry mass of the pretreated bagasse.
In some examples, the tea saponin is used in an amount of 0-60 mg/g oven-dried mass of pretreated bagasse.
The buffer mainly serves to maintain a stable pH required for the enzymatic reaction. The buffer solution has no special requirements, and only has better buffer capacity and does not influence or slightly influences the enzyme activity. The pH of the buffer may be adjusted accordingly depending on the cellulase used. In some examples, the buffer is an acetic acid-sodium acetate buffer solution.
The mixing ratio of the pretreated bagasse and the buffer solution can be adjusted to a certain extent according to actual conditions, so that the enzymolysis reaction is not influenced, and the use cost is relatively low. In some examples, the mass to volume ratio of the pretreated bagasse to the buffer is 1 g: (40-100) mL.
In some examples, the acetic acid-sodium acetate buffer solution has a concentration of 0.03 to 0.06 mol/L. At such a concentration, the buffer capacity is relatively sufficient, and the requirement of enzymolysis reaction can be met.
The technical scheme of the invention is further explained by combining the examples.
For convenience of proportion, bagasse raw materials used in the following examples are air-dried, thread-rolled and pulverized, and have particle sizes of less than 1mm, and the components mainly comprise, by mass, 40.2% of cellulose, 21.5% of hemicellulose and 25.2% of lignin; the cellulase used was a cellulase of the second generation of cellulose under Millipore.
And measuring and analyzing the glucose content in the enzymolysis liquid by using a high performance liquid chromatography.
Example 1
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) ethanol aqueous solution and 0.005mol/L ethanol aqueous solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can respectively reach 26.5 percent and 35.4 percent, and the retention rate of cellulose can reach 99.6 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample is taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 54.66%.
Example 2
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.01mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can respectively reach 50.8 percent and 45.2 percent, and the retention rate of cellulose can reach 98.9 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 69.84%.
Example 3
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 83.2 percent and 71.4 percent respectively, and the retention rate of cellulose reaches 95.0 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high performance liquid phase, and calculating to obtain the glucose yield of 89.25%.
Example 4
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.05mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 94.9 percent and 78.3 percent respectively, and the retention rate of cellulose reaches 93.1 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 86.60%.
Example 5
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 130 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 43.1 percent and 41.6 percent respectively, and the retention rate of cellulose can reach 97.3 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 55.98%.
Example 6
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 180 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 95.6 percent and 78.8 percent respectively, and the retention rate of cellulose can reach 85.8 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 83.70%.
Example 7
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 20 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 87.0 percent and 72.8 percent respectively, and the retention rate of cellulose reaches 92.3 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 87.03%.
Example 8
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 30 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can respectively reach 88.3 percent and 76.4 percent, and the retention rate of cellulose reaches 89.3 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 20FPU of cellulase and 100mL of acetic acid-sodium acetate buffer solution having pH 4.8 were added to carry out enzymatic hydrolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 87.77%.
Example 9
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 83.2 percent and 71.4 percent respectively, and the retention rate of cellulose reaches 95.0 percent.
S2, taking 2 g (absolute dry basis) of the pretreatment residue, adding 10FPU of cellulase, 50mg/g of Tween 80 and 100mL of acetic acid-sodium acetate buffer solution with pH value of 4.8 for enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 24 hours and 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
The glucose concentration in the enzymolysis liquid is measured by using a high performance liquid phase, and the calculated glucose yield is 86.21 percent and 92.66 percent respectively.
Example 10
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 83.2 percent and 71.4 percent respectively, and the retention rate of cellulose reaches 95.0 percent.
S2, taking 2 g (absolute dry basis) of the pretreated residue, adding 10FPU of cellulase, 60mg/g of tea saponin and 100mL of acetic acid-sodium acetate buffer solution with pH value of 4.8 for enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 24 hours and 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the glucose concentration in the enzymolysis liquid by using a high performance liquid phase, and calculating to obtain the glucose yields of 88.63% and 94.07% respectively.
Example 11
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 60% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 83.2 percent and 71.4 percent respectively, and the retention rate of cellulose reaches 95.0 percent.
S2, 2 g (absolute dry basis) of the pretreated residue was taken, and 10FPU of cellulase, 60mg/g BSA and 100mL of acetic acid-sodium acetate buffer solution with pH 4.8 were added to carry out enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 24 hours and 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the glucose concentration in the enzymolysis liquid by using a high performance liquid phase, and calculating that the obtained glucose yields are 89.87% and 93.59% respectively.
Example 12
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 50% (v/v) of an aqueous ethanol solution and 0.025mol/L of an aqueous ethanol solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 87.4 percent and 67.8 percent respectively, and the retention rate of cellulose reaches 96.3 percent.
S2, taking 2 g (absolute dry basis) of the pretreated residue, adding 10FPU of cellulase, 60mg/g of tea saponin and 100mL of acetic acid-sodium acetate buffer solution with pH value of 4.8 for enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 24 hours and 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high performance liquid phase, and calculating to obtain the glucose yields of 88.58% and 91.05% respectively.
Example 13
S1, adding the bagasse raw material into a mixture according to the absolute dry mass volume ratio of 1 g: 10mL of CuCl, 40% (v/v) ethanol aqueous solution and 0.025mol/L ethanol aqueous solution2Reacting at 160 ℃ in a high-pressure reaction kettle, stopping heating when the reaction time reaches 10 minutes, immediately cooling the reaction to room temperature by using condensed water, and separating out the pretreatment residues by adopting a vacuum filtration method.
Component analysis on the pretreatment residues shows that the removal rates of hemicellulose and lignin can reach 91.5 percent and 69.8 percent respectively, and the retention rate of cellulose can reach 97.2 percent.
S2, taking 2 g (absolute dry basis) of the pretreated residue, adding 10FPU of cellulase, 60mg/g of tea saponin and 100mL of acetic acid-sodium acetate buffer solution with pH value of 4.8 for enzymolysis. The temperature is controlled at 50 ℃ in the enzymolysis process, and the rotating speed is 150 r/min. After 24 hours and 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
The glucose concentration in the enzymolysis liquid is measured by using a high performance liquid phase, and the calculated glucose yield is 86.59 percent and 91.07 percent respectively.
Comparative example 1
The control group is prepared by adding no CuCl into bagasse raw material at 160 deg.C for 10min2The pretreatment of the ethanol aqueous solution shows that the removal rate of hemicellulose and lignin is only 22.4 percent and 31.8 percent and the retention rate of cellulose reaches 99.7 percent by analyzing the components of the pretreatment residue. The cellulase enzymatic conditions were the same as in example 1 above. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high-performance liquid phase, and calculating to obtain the glucose yield of 48.62%.
Comparative example 2
The control group is prepared by adding FeCl 0.025mol/L into bagasse raw material at 160 deg.C for 10min3The pretreatment with the aqueous ethanol solution revealed that the removal rates of hemicellulose and lignin were 91.1% and 55.2%, the retention rate of cellulose was 91.8%, and the removal rate of lignin was low, as a result of the composition analysis of the pretreatment residue. The enzymatic conditions were identical to those described in example 1 above. After 72 hours of enzymolysis, 1mL of sample was taken and subjected to inactivation treatment.
And (3) determining the concentration of glucose in the enzymolysis liquid by using a high performance liquid phase, and calculating to obtain the glucose yield of 91.15%.
The comparison shows that:
1)CuCl2at low (0.005M) or high (0.1M), effective removal of hemicellulose and lignin is difficult, but, correspondingly, less damage to cellulose occurs.
2) At 160-180 ℃, hemicellulose and lignin can be removed more effectively, the treatment time is short, and the damage to cellulose is less.
3) 0.025-0.050 mol/L of CuCl2Can well promoteThe hemicellulose and the lignin are added, and the retention rate of the cellulose is also ideal.
4) The preferred conditions for separating cellulose from bagasse are: the heat preservation temperature is as follows: the temperature is 160-180 ℃, the heat preservation time is 10-20 min, and CuCl2The concentration of the water is 0.025-0.050 mol/L.
5) The concentration of the ethanol aqueous solution can be selected according to different applications, in order to remove hemicellulose better, the ethanol aqueous solution with the concentration of 40-50% (v/v) can be used, and for removing hemicellulose and lignin at the same time, the ethanol aqueous solution with higher concentration is better, and the ethanol aqueous solution with 50-60% (v/v) is better. Higher concentrations of aqueous ethanol may be more favorable for lignin removal.
The pretreatment method of the invention adds CuCl in the pretreatment stage2The yield of the cellulose enzyme enzymolysis glucose is improved to different degrees. Under the pretreatment condition of 160 ℃, 10min and 0.025mol/L CuCl2Under the condition, the yield of the enzymolysis glucose reaches the maximum. Adding Tween 80, tea saponin and BSA during enzymolysis stage, and allowing the glucose yield to reach the level of 20FPU enzyme load enzymolysis for 72 hours after 10FPU enzyme load enzymolysis for 24 hours. The addition of the additive can reduce the enzyme dosage, shorten the reaction time and save the enzymolysis cost.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
Claims (10)
1. A process for separating cellulose from sugar cane bagasse, comprising the steps of: uniformly mixing bagasse, an ethanol water solution and copper chloride, carrying out heat preservation treatment at 130-180 ℃, and separating to obtain a pretreatment solution and pretreated bagasse, wherein the pretreated bagasse is cellulose obtained by separation.
2. The method of claim 1, wherein: the mixing ratio of bagasse and ethanol aqueous solution is 1 g: 8-15 mL, and the bagasse is calculated according to absolute dry mass.
3. The method according to claim 1 or 2, characterized in that: the concentration of the ethanol water solution is 40-75% (v/v).
4. The method according to claim 1 or 2, characterized in that: the final concentration of the copper chloride is 0.005-0.1 mol/L.
5. The method of claim 4, wherein: the final concentration of the copper chloride is 0.025-0.05 mol/L.
6. The method according to claim 1 or 2, characterized in that: the temperature of heat preservation is 160-180 ℃.
7. The method of claim 1, wherein: the time of the heat preservation treatment is 5-40 min.
8. The method of claim 6, wherein: the heat preservation treatment time is 10-20 min.
9. The method of claim 1, wherein: the concentration of the ethanol water solution is 60% (v/v).
10. A method for improving sugar production efficiency of bagasse enzymolysis is characterized in that: the method comprises the steps of preparing the pretreated bagasse according to any one of claims 1 to 9, mixing the pretreated bagasse, a buffer solution and cellulase, and carrying out enzymolysis to obtain glucose solution.
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