CN114891844B - Method for efficiently preprocessing corncob by using ultrasonic-assisted ternary eutectic solvent - Google Patents
Method for efficiently preprocessing corncob by using ultrasonic-assisted ternary eutectic solvent Download PDFInfo
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- CN114891844B CN114891844B CN202210704749.9A CN202210704749A CN114891844B CN 114891844 B CN114891844 B CN 114891844B CN 202210704749 A CN202210704749 A CN 202210704749A CN 114891844 B CN114891844 B CN 114891844B
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- 239000002904 solvent Substances 0.000 title claims abstract description 66
- 230000005496 eutectics Effects 0.000 title claims abstract description 65
- 238000000034 method Methods 0.000 title claims abstract description 10
- 238000007781 pre-processing Methods 0.000 title claims abstract description 7
- 229920005610 lignin Polymers 0.000 claims abstract description 39
- 229920002488 Hemicellulose Polymers 0.000 claims abstract description 34
- 239000001913 cellulose Substances 0.000 claims abstract description 32
- 229920002678 cellulose Polymers 0.000 claims abstract description 32
- 229960001231 choline Drugs 0.000 claims abstract description 20
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 230000007062 hydrolysis Effects 0.000 claims abstract description 11
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 11
- 238000010438 heat treatment Methods 0.000 claims abstract description 7
- JXEKSKVJJYKJFQ-UHFFFAOYSA-N Cl.CC(O)C(O)=O.OCC(O)CO Chemical compound Cl.CC(O)C(O)=O.OCC(O)CO JXEKSKVJJYKJFQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 27
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000000203 mixture Substances 0.000 claims description 19
- 239000007787 solid Substances 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 17
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 16
- 238000011084 recovery Methods 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 239000001257 hydrogen Substances 0.000 claims description 14
- 229910052739 hydrogen Inorganic materials 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 13
- 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 description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 238000012360 testing method Methods 0.000 claims description 12
- 240000008042 Zea mays Species 0.000 claims description 11
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 11
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 11
- 235000005822 corn Nutrition 0.000 claims description 11
- 239000000706 filtrate Substances 0.000 claims description 11
- 238000000967 suction filtration Methods 0.000 claims description 11
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 claims description 8
- 235000019743 Choline chloride Nutrition 0.000 claims description 8
- 229960003178 choline chloride Drugs 0.000 claims description 8
- 239000004310 lactic acid Substances 0.000 claims description 8
- 235000014655 lactic acid Nutrition 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000002604 ultrasonography Methods 0.000 claims description 8
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 7
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 6
- 238000003756 stirring Methods 0.000 claims description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 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 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 238000004364 calculation method Methods 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims description 2
- 238000003760 magnetic stirring Methods 0.000 claims description 2
- 238000002844 melting Methods 0.000 claims description 2
- 230000008018 melting Effects 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000004064 recycling Methods 0.000 claims description 2
- 238000002390 rotary evaporation Methods 0.000 claims description 2
- 238000002791 soaking Methods 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 238000005303 weighing Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 claims 3
- 238000001816 cooling Methods 0.000 claims 1
- 239000002028 Biomass Substances 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 7
- -1 chloride-glycerol-citric acid Chemical compound 0.000 abstract description 6
- 230000007071 enzymatic hydrolysis Effects 0.000 abstract description 5
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 abstract description 5
- 230000001965 increasing effect Effects 0.000 abstract description 4
- XKIXAJFXHBRNCO-UHFFFAOYSA-N Cl.NC(N)=O.OCC(O)CO Chemical compound Cl.NC(N)=O.OCC(O)CO XKIXAJFXHBRNCO-UHFFFAOYSA-N 0.000 abstract description 3
- GSEMSXAZDCIEIF-UHFFFAOYSA-N OCCO.OCC(CO)O.Cl Chemical compound OCCO.OCC(CO)O.Cl GSEMSXAZDCIEIF-UHFFFAOYSA-N 0.000 abstract description 3
- 230000002349 favourable effect Effects 0.000 abstract 1
- 239000002994 raw material Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 13
- 108010059892 Cellulase Proteins 0.000 description 12
- 229940106157 cellulase Drugs 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 6
- 239000000370 acceptor Substances 0.000 description 5
- 238000004380 ashing Methods 0.000 description 5
- 238000001035 drying Methods 0.000 description 5
- 238000001704 evaporation Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 238000001914 filtration Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 239000005457 ice water Substances 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 239000007974 sodium acetate buffer Substances 0.000 description 5
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 5
- 230000001954 sterilising effect Effects 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 238000002203 pretreatment Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000002029 lignocellulosic biomass Substances 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- HJJPJSXJAXAIPN-UHFFFAOYSA-N arecoline Chemical compound COC(=O)C1=CCCN(C)C1 HJJPJSXJAXAIPN-UHFFFAOYSA-N 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 229940044631 ferric chloride hexahydrate Drugs 0.000 description 2
- 239000002608 ionic liquid Substances 0.000 description 2
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- NQCBIMOYRRMVNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrochloride Chemical compound Cl.OCC(O)CO NQCBIMOYRRMVNA-UHFFFAOYSA-N 0.000 description 2
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 239000003225 biodiesel Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 229940093956 potassium carbonate Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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
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Abstract
The invention discloses a method for efficiently preprocessing corncob by using an ultrasonic-assisted ternary eutectic solvent, and belongs to the field of biomass comprehensive utilization. The novel ternary eutectic solvent choline chloride-glycerin-lactic acid; choline chloride-glycerol-citric acid; choline chloride-glycerol-urea; choline chloride-glycerol-ethylene glycol; choline chloride-glycerin-potassium carbonate are respectively and uniformly mixed with corncob, and are put into a multi-frequency combined ultrasonic transmitter to act for a period of time, and are put into an oil bath for heating after being taken out, and after the reaction is completed, the corncob is subjected to component determination and cellulose hydrolysis; the novel ultrasonic-assisted ternary eutectic solvent system constructed by the invention is simple to operate, green, low in cost and good in pretreatment effect, lignin and hemicellulose can be obviously removed, the enzymatic hydrolysis saccharification rate of cellulose is increased, and the novel ultrasonic-assisted ternary eutectic solvent system has a wide application prospect, and is favorable for realizing the purposes of separating all components of biomass raw materials and fully utilizing all the components.
Description
Technical Field
The invention belongs to the field of comprehensive utilization of biomass, and particularly relates to a method for efficiently preprocessing corncobs by using an ultrasonic-assisted ternary eutectic solvent.
Background
Along with the continuous exhaustion of fossil resources and the continuous increase of environmental pollution pressure, the development of novel renewable green energy sources becomes an important subject with global strategic significance. Corncob is a byproduct of corn production and processing, and the yield is huge. It is often used as feed, fuel or waste and is not fully utilized. Therefore, the development and comprehensive utilization of agricultural and sideline products such as corncobs can improve economic benefit and social benefit. Lignocellulosic biomass such as corncob consists mainly of cellulose, hemicellulose and lignin. In the biomass conversion process, cellulose and hemicellulose can be converted into monosaccharides such as glucose and xylose through enzymatic hydrolysis, and various platform compounds can be prepared through microbial conversion and chemical catalytic conversion reaction. However, due to the complex structure of lignocellulose, there are a large number of covalent and hydrogen bonds between cellulose and lignin and hemicellulose both intramolecular and intermolecular, forming a dense structure that is difficult to decompose and utilize, which is a major reason that limits the efficient utilization of lignocellulosic biomass.
Many methods can pretreat lignocellulosic biomass to remove lignin and hemicellulose entrapped in the cellulose, break down dense three-dimensional structures, fully exposing the cellulose, thereby further increasing the binding sites and accessibility of the cellulase enzymes to increase enzymatic hydrolysis efficiency. The traditional pretreatment method comprises the following steps: physical and chemical treatment modes such as dilute acid, dilute alkali, steam explosion and the like have a plurality of unresolved defects such as high cost, environmental pollution, high equipment requirement and the like.
Based on the current technical problems of single pretreatment method, poor pretreatment effect, high pretreatment cost and the like in pretreatment of lignocellulose biomass, the finding of a method for efficiently pretreating biomass in a combined way has important significance. The eutectic solvent is developed and utilized as a novel ionic liquid consisting of a hydrogen bond acceptor and a hydrogen bond donor, and has the greatest advantages of simple, cheap and green preparation, and great adjustability due to a plurality of alternative hydrogen bond donors and hydrogen bond acceptors, for example, the pretreatment of lignocellulose biomass by the eutectic solvent consisting of betaine and amino acid in Chinese patent CN110258157B enhances the enzymatic hydrolysis efficiency, and the rapid decomposition of lignocellulose biomass by the eutectic solvent of organic carboxylic acid and polyalcohol is invented by Chinese patent CN 114108350A. Glycerol is a cheap natural compound and a main byproduct produced by biodiesel industry can be used as a hydrogen bond donor to synthesize a eutectic solvent, however, according to the Chinese patent CN113603899A, the pretreatment of the choline chloride-glycerol eutectic solvent has extremely poor lignin removal effect, so that a novel ternary eutectic solvent of a double hydrogen bond acceptor (donor) is synthesized based on the binary eutectic solvent, the internal hydrogen bond network structure is changed, and the corncob is pretreated, thereby achieving the purposes of improving quality and enhancing efficiency of poor solvent. Ultrasonic is used as a physical processing mode, and lignocellulose is often pretreated by being combined with other pretreatment methods in recent years, so that the aim of synergistically enhancing pretreatment effects is fulfilled. Therefore, the pretreatment of the corncob by combining the ultrasonic treatment and the ternary eutectic solvent has great significance in green and efficient removal of lignin and hemicellulose.
Disclosure of Invention
The invention aims to invent a method for efficiently preprocessing corncob by using an ultrasonic-assisted ternary eutectic solvent, which has the characteristics of simplicity in operation, low cost, high efficiency and greenness, and the removal rate of lignin and hemicellulose in the preprocessed corncob is remarkably improved, and the high glucose yield can be obtained after enzyme hydrolysis.
In order to achieve the above purpose, the invention provides a method for preprocessing corncob by using a three-frequency ultrasonic auxiliary ternary eutectic solvent, which comprises the following steps:
(1) Synthesis of ternary eutectic solvent:
Mixing a hydrogen bond donor and a hydrogen bond acceptor according to a certain molar ratio, placing the mixture into a magnetic stirring water bath kettle, heating and stirring at 80 ℃ and 500rmp, melting the mixture to be converted into a uniform and transparent solution, namely the prepared ternary eutectic solvent, and placing the ternary eutectic solvent into a dryer for standby.
(2) Ultrasound-assisted ternary eutectic solvent pretreatment of corncob: the corncob powder and the ternary eutectic solvent are placed in a glass test tube with a plug according to a certain mass ratio, are uniformly mixed, are placed in ultrasonic reactors with different frequencies, are treated for a certain time, and are placed in an oil bath for heating.
(3) Immediately after the reaction was completed, the vessel was taken out and cooled to room temperature. Preparing a mixed solution of acetone and water in a volume ratio of 1:1, washing the pretreated sample into a conical flask, soaking and stirring overnight, and then carrying out suction filtration on the sample by using a vacuum suction filtration device to obtain solid residues and filtrate. After the filtrate is subjected to rotary evaporation to remove the detergent, the ternary eutectic solvent can be recovered so as to realize recycling.
(4) Weighing a certain amount of solid residues, namely pretreated corncob samples, and measuring the contents of cellulose, hemicellulose and lignin in corncob components by adopting a national renewable energy laboratory standard analysis program, wherein the specific steps are as follows: uniformly stirring corncob and 72% concentrated sulfuric acid, preserving heat for 1h at 30 ℃, washing the corncob and 72% concentrated sulfuric acid into a conical flask with 84mL distilled water, preserving heat for 45min at 121 ℃, carrying out suction filtration after the reaction is finished, measuring the contents of glucose and xylose in filtrate by using a high performance liquid chromatograph to calculate the contents of cellulose and hemicellulose, measuring the content of lignin by weight analysis of residues, and finally obtaining the recovery rate of cellulose and the removal rate of lignin and hemicellulose by calculation; and simultaneously carrying out enzymatic hydrolysis on the treated sample.
The ternary eutectic solvent in the step (1) is choline chloride-glycerol-lactic acid; choline chloride-glycerol-citric acid; choline chloride-glycerol-urea; choline chloride-glycerol-ethylene glycol; choline chloride-glycerol-potassium carbonate; choline chloride-glycerol-ferric chloride hexahydrate, wherein the hydrogen bond acceptors are choline chloride, potassium carbonate and ferric chloride hexahydrate; wherein the hydrogen bond donor is glycerol, lactic acid, citric acid, urea and ethylene glycol.
The molar ratio of choline chloride, glycerol and lactic acid in the ternary eutectic solvent choline chloride-glycerol-lactic acid in the step (1) is 1: (0.5-2): (0.5-2).
The multi-frequency combined ultrasonic frequency in the step (2) is 20kHz,40kHz,60kHz,20+40kHz,20+60kHz,40+60kHz,20+40+60kHz, and the treatment time is 10-90 min.
The mass ratio of the corncob to the ternary eutectic solvent in the step (2) is 1: (5-30).
The heating temperature of the oil bath in the step (3) is 50-150 ℃.
And (3) heating the oil bath for 0.5-5 h.
The enzyme hydrolysis time in the step (4) is 24-72 h, and the temperature is 50 ℃.
The invention has the beneficial effects that:
(1) Compared with the traditional ionic liquid, the novel ternary eutectic solvent used in the invention has the advantages of simple preparation, low cost and green degradability, achieves the aim of green efficient pretreatment of corncob to a great extent, and meets the requirement of sustainable development of green chemistry.
(2) The invention combines physical ultrasonic pretreatment and chemical ternary eutectic solvent pretreatment, can achieve the effect of physical-chemical synergistic enhancement pretreatment, and has the lignin and hemicellulose removal rate which is 13.28 percent and 12.08 percent higher than that of the lignin and hemicellulose which only use ternary eutectic solvent after ultrasonic addition.
(3) The invention realizes better corn cob pretreatment effect, the lignin and hemicellulose removal rate is only 14.79% and 13.35% after pretreatment by using the binary eutectic solvent choline chloride-glycerol, and is increased to 87.54% and 67.00% after pretreatment by using the ternary eutectic solvent, which respectively improves 72.75% and 53.65%, and can still keep the cellulose recovery rate as high as 82.18%.
(4) The corn cob pretreated by the ternary eutectic solvent can realize the glucose yield of 76.94 percent after being hydrolyzed by the enzyme for 72 hours, which is 61.58 percent higher than the original corn cob and 46.32 percent higher than the corn cob pretreated by the binary eutectic solvent. The pretreatment method can significantly remove lignin and hemicellulose, thereby increasing the yield of cellulose hydrolysis glucose.
Drawings
FIG. 1 is a graph showing lignin, hemicellulose removal and cellulose recovery from corncobs after pretreatment with different ternary eutectic solvents. ( a-E represent different ternary eutectic solvents, wherein a choline chloride-glycerol-lactic acid; b is choline chloride-glycerol-citric acid; c is choline chloride-glycerol-urea; d is choline chloride-glycerol-ethylene glycol; e is choline chloride-glycerol-potassium carbonate )
Figure 2 is a graph of cob lignin, hemicellulose removal and cellulose recovery after pretreatment with different molar ratios of ternary eutectic solvents. ( G-K represents different molar ratios, wherein G is 1:1:1, a step of; h is 1:2:1, a step of; i is 1:1:2; j is 1:2:2; k is 1:0.5:0.5 )
FIG. 3 shows glucose yields after 72h cellulase hydrolysis of corncob with different molar ratios of ternary eutectic solvent pretreatment. ( L-K represents different molar ratios, wherein L is 0:0:0, i.e., untreated corncob of control group; g is 1:1:1, a step of; h is 1:2:1, a step of; i is 1:1:2; j is 1:2:2; k is 1:0.5:0.5 )
Detailed Description
The present invention will be described in further detail with reference to specific examples.
Example 1:
(1) Preparing a ternary eutectic solvent: choline chloride, glycerol and lactic acid were mixed according to 1:1:1 in a beaker, and stirred at a speed of 500rmp at 80 ℃ until a clear solution is formed.
(2) 10G of ternary eutectic solvent and 1g of corncob are added into a test tube and uniformly mixed, so that the ternary eutectic solvent and the corncob are fully contacted.
(3) Placing a test tube filled with a sample into an ultrasonic tank of three-frequency combined ultrasonic, setting the power of a 20kHz ultrasonic transmitter to be 300W, and setting the work intermittent ratio to be 10s:5s, and ensures that the time of the ultrasound is 30min. Three sets of replicates were performed simultaneously for each set of experiments to ensure accuracy of the results.
(4) After the end of the ultrasound, all samples were placed in an oil bath and kept at 120 ℃ for 2h. After the reaction was completed, 200mL of a volume ratio of 1 was added: 1, vacuum filtering, washing with the same acetone-water mixture to colorless, drying the obtained solid part in a 40 ℃ oven, combining the filtrates, spin-evaporating to remove acetone, centrifuging, washing to obtain lignin sample, and storing in a hermetic glass vial.
(5) 0.1G of cellulase was mixed with the formulated acetic acid-sodium acetate buffer (1 l, ph=4.6) and stirred well to form a cellulase solution. 50mL of enzyme solution and 0.2g of treated corncob powder were sequentially added into a conical flask, sealed and placed in a constant temperature shaker at a set temperature of 50℃and a shaking speed of 120rpm. After hydrolysis for 72h, the reaction was stopped by placing in an ice-water bath. The sugar content was analyzed by high performance liquid chromatography using a syringe to take a volume of supernatant.
(6) The component content of corncob was determined using the national renewable energy laboratory standard analytical procedure: uniformly mixing 300mg of corncob with 3mL of 72% sulfuric acid solution, adding 84mL of distilled water after keeping the mixture in a water bath kettle at 30 ℃ for 1h, putting the mixture into a high-pressure steam sterilization kettle at 121 ℃ for 45min, and finally carrying out solid-liquid separation on the reacted solution by combining a vacuum suction filtration device with a G3 type sand core crucible funnel; wherein the solid component is washed to be neutral by distilled water, then dried, put into a muffle furnace at 550 ℃ for ashing, the lignin content is calculated, and the cellulose and hemicellulose contents of the liquid component are analyzed by a high performance liquid chromatograph. After the corn cob is pretreated by the ultrasonic-assisted ternary eutectic solvent, the contents of lignin, cellulose and hemicellulose are 6.06%,61.53% and 17.54%; the solid recovery rate, lignin removal rate, hemicellulose removal rate and cellulose recovery rate were 71.40%, 76.49%,61.22% and 83.76%, respectively. The yield of glucose hydrolyzed by the enzyme for 72 hours is 70.28 percent.
Example 2:
(1) Preparing a ternary eutectic solvent: choline chloride, glycerol and lactic acid were mixed according to 1:2:1 in a beaker, and stirred at a speed of 500rmp at 80 ℃ until a clear solution is formed.
(2) 10G of ternary eutectic solvent and 1g of corncob are added into a test tube and uniformly mixed, so that the ternary eutectic solvent and the corncob are fully contacted.
(3) Placing a test tube filled with a sample into an ultrasonic tank of three-frequency combined ultrasonic, setting the power of a 20kHz ultrasonic transmitter to be 300W, setting the work intermittent ratio to be 10 s/5 s, and ensuring the ultrasonic time to be 30min. Three sets of replicates were performed simultaneously for each set of experiments to ensure accuracy of the results.
(4) After the end of the ultrasound, all samples were placed in an oil bath and kept at 120 ℃ for 2h. After the reaction was completed, 200mL of a volume ratio of 1 was added: 1, vacuum filtering, washing with the same acetone-water mixture to colorless, drying the obtained solid part in a 40 ℃ oven, combining the filtrates, spin-evaporating to remove acetone, centrifuging, washing to obtain lignin sample, and storing in a hermetic glass vial.
(5) 0.1G of cellulase was mixed with the formulated acetic acid-sodium acetate buffer (1 l, ph=4.6) and stirred well to form a cellulase solution. 50mL of enzyme solution and 0.2g of treated corncob powder were sequentially added into a conical flask, sealed and placed in a constant temperature shaker at a set temperature of 50℃and a shaking speed of 120rpm. After hydrolysis for 72h, the reaction was stopped by placing in an ice-water bath. The sugar content was analyzed by high performance liquid chromatography using a syringe to take a volume of supernatant.
(6) The component content of corncob was determined using the national renewable energy laboratory standard analytical procedure: uniformly mixing 300mg of corncob with 3mL of 72% sulfuric acid solution, adding 84mL of distilled water after keeping the mixture in a water bath kettle at 30 ℃ for 1h, putting the mixture into a high-pressure steam sterilization kettle at 121 ℃ for 45min, and finally carrying out solid-liquid separation on the reacted solution by combining a vacuum suction filtration device with a G3 type sand core crucible funnel; wherein the solid component is washed to be neutral by distilled water, then dried, put into a muffle furnace at 550 ℃ for ashing, the lignin content is calculated, and the cellulose and hemicellulose contents of the liquid component are analyzed by a high performance liquid chromatograph. After the corn cob is pretreated by the ultrasonic-assisted ternary eutectic solvent, the content of lignin, cellulose and hemicellulose is 8.91%,55.27% and 19.07%; the solid recovery rate, lignin removal rate, hemicellulose removal rate and cellulose recovery rate are respectively 80.37%, 61.15%,52.56% and 83.35%. The yield of glucose hydrolyzed by the enzyme for 72 hours is 59.81%.
Example 3:
(1) Preparing a ternary eutectic solvent: choline chloride, glycerol and lactic acid were mixed according to 1:1:2 in a beaker, and stirred at a speed of 500rmp at 80 ℃ until a clear solution is formed.
(2) 10G of ternary eutectic solvent and 1g of corncob are added into a test tube and uniformly mixed, so that the ternary eutectic solvent and the corncob are fully contacted.
(3) Placing a test tube filled with a sample into an ultrasonic tank of three-frequency combined ultrasonic, setting the power of a 20kHz ultrasonic transmitter to be 300W, setting the work intermittent ratio to be 10 s/5 s, and ensuring the ultrasonic time to be 30min. Three sets of replicates were performed simultaneously for each set of experiments to ensure accuracy of the results.
(4) After the end of the ultrasound, all samples were placed in an oil bath and kept at 120 ℃ for 2h. After the reaction was completed, 200mL of a volume ratio of 1 was added: 1, vacuum filtering, washing with the same acetone-water mixture to colorless, drying the obtained solid part in a 40 ℃ oven, combining the filtrates, spin-evaporating to remove acetone, centrifuging, washing to obtain lignin sample, and storing in a hermetic glass vial.
(5) 0.1G of cellulase was mixed with the formulated acetic acid-sodium acetate buffer (1 l, ph=4.6) and stirred well to form a cellulase solution. 50mL of enzyme solution and 0.2g of treated corncob powder were sequentially added into a conical flask, sealed and placed in a constant temperature shaker at a set temperature of 50℃and a shaking speed of 120rpm. After hydrolysis for 72h, the reaction was stopped by placing in an ice-water bath. The sugar content was analyzed by high performance liquid chromatography using a syringe to take a volume of supernatant.
(6) The component content of corncob was determined using the national renewable energy laboratory standard analytical procedure: uniformly mixing 300mg of corncob with 3mL of 72% sulfuric acid solution, adding 84mL of distilled water after keeping the mixture in a water bath kettle at 30 ℃ for 1h, putting the mixture into a high-pressure steam sterilization kettle at 121 ℃ for 45min, and finally carrying out solid-liquid separation on the reacted solution by combining a vacuum suction filtration device with a G3 type sand core crucible funnel; wherein the solid component is washed to be neutral by distilled water, then dried, put into a muffle furnace at 550 ℃ for ashing, the lignin content is calculated, and the cellulose and hemicellulose contents of the liquid component are analyzed by a high performance liquid chromatograph. After the corn cob is pretreated by the ultrasonic-assisted ternary eutectic solvent, the content of lignin, cellulose and hemicellulose is 4.29%,65.79% and 16.27%; the solid recovery rate, lignin removal rate, hemicellulose removal rate and cellulose recovery rate were 65.80%, 84.66%,66.87% and 81.22%, respectively. The yield of glucose hydrolyzed by the enzyme for 72 hours is 76.94 percent. The molar ratio pretreatment effect is better than other groups.
Example 4:
(1) Preparing a ternary eutectic solvent: choline chloride, glycerol and lactic acid were mixed according to 1:2:2 in a beaker, and stirred at a speed of 500rmp at 80 ℃ until a clear solution is formed.
(2) 10G of ternary eutectic solvent and 1g of corncob are added into a test tube and uniformly mixed, so that the ternary eutectic solvent and the corncob are fully contacted.
(3) Placing a test tube filled with a sample into an ultrasonic tank of three-frequency combined ultrasonic, setting the power of a 20kHz ultrasonic transmitter to be 300W, setting the work intermittent ratio to be 10 s/5 s, and ensuring the ultrasonic time to be 30min. Three sets of replicates were performed simultaneously for each set of experiments to ensure accuracy of the results.
(4) After the end of the ultrasound, all samples were placed in an oil bath and kept at 120 ℃ for 2h. After the reaction was completed, 200mL of a volume ratio of 1 was added: 1, vacuum filtering, washing with the same acetone-water mixture to colorless, drying the obtained solid part in a 40 ℃ oven, combining the filtrates, spin-evaporating to remove acetone, centrifuging, washing to obtain lignin sample, and storing in a hermetic glass vial.
(5) 0.1G of cellulase was mixed with the formulated acetic acid-sodium acetate buffer (1 l, ph=4.6) and stirred well to form a cellulase solution. 50mL of enzyme solution and 0.2g of treated corncob powder were sequentially added into a conical flask, sealed and placed in a constant temperature shaker at a set temperature of 50℃and a shaking speed of 120rpm. After hydrolysis for 72h, the reaction was stopped by placing in an ice-water bath. The sugar content was analyzed by high performance liquid chromatography using a syringe to take a volume of supernatant.
(6) The component content of corncob was determined using the national renewable energy laboratory standard analytical procedure: uniformly mixing 300mg of corncob with 3mL of 72% sulfuric acid solution, adding 84mL of distilled water after keeping the mixture in a water bath kettle at 30 ℃ for 1h, putting the mixture into a high-pressure steam sterilization kettle at 121 ℃ for 45min, and finally carrying out solid-liquid separation on the reacted solution by combining a vacuum suction filtration device with a G3 type sand core crucible funnel; wherein the solid component is washed to be neutral by distilled water, then dried, put into a muffle furnace at 550 ℃ for ashing, the lignin content is calculated, and the cellulose and hemicellulose contents of the liquid component are analyzed by a high performance liquid chromatograph. After the corn cob is pretreated by the ultrasonic-assisted ternary eutectic solvent, the contents of lignin, cellulose and hemicellulose are 5.48%,64.01% and 17.64%; the solid recovery rate, lignin removal rate, hemicellulose removal rate and cellulose recovery rate are 69.32%, 79.37%,62.16% and 83.26%, respectively. The yield of glucose hydrolyzed by the enzyme for 72 hours is 72.97 percent.
Example 5:
(1) Preparing a ternary eutectic solvent: choline chloride, glycerol and lactic acid were mixed according to 1:0.5: a molar ratio of 0.5 was placed in a beaker and stirred at 80℃at a speed of 500rmp until a clear solution formed.
(2) 10G of ternary eutectic solvent and 1g of corncob are added into a test tube and uniformly mixed, so that the ternary eutectic solvent and the corncob are fully contacted.
(3) Placing a test tube filled with a sample into an ultrasonic tank of three-frequency combined ultrasonic, setting the power of a 20kHz ultrasonic transmitter to be 300W, setting the work intermittent ratio to be 10 s/5 s, and ensuring the ultrasonic time to be 30min. Three sets of replicates were performed simultaneously for each set of experiments to ensure accuracy of the results.
(4) After the end of the ultrasound, all samples were placed in an oil bath and kept at 120 ℃ for 2h. After the reaction was completed, 200mL of a volume ratio of 1 was added: 1, vacuum filtering, washing with the same acetone-water mixture to colorless, drying the obtained solid part in a 40 ℃ oven, combining the filtrates, spin-evaporating to remove acetone, centrifuging, washing to obtain lignin sample, and storing in a hermetic glass vial.
(5) 0.1G of cellulase was mixed with the formulated acetic acid-sodium acetate buffer (1 l, ph=4.6) and stirred well to form a cellulase solution. 50mL of enzyme solution and 0.2g of treated corncob powder were sequentially added into a conical flask, sealed and placed in a constant temperature shaker at a set temperature of 50℃and a shaking speed of 120rpm. After hydrolysis for 72h, the reaction was stopped by placing in an ice-water bath. The sugar content was analyzed by high performance liquid chromatography using a syringe to take a volume of supernatant.
(6) The component content of corncob was determined using the national renewable energy laboratory standard analytical procedure: uniformly mixing 300mg of corncob with 3mL of 72% sulfuric acid solution, adding 84mL of distilled water after keeping the mixture in a water bath kettle at 30 ℃ for 1h, putting the mixture into a high-pressure steam sterilization kettle at 121 ℃ for 45min, and finally carrying out solid-liquid separation on the reacted solution by combining a vacuum suction filtration device with a G3 type sand core crucible funnel; wherein the solid component is washed to be neutral by distilled water, then dried, put into a muffle furnace at 550 ℃ for ashing, the lignin content is calculated, and the cellulose and hemicellulose contents of the liquid component are analyzed by a high performance liquid chromatograph. After the corn cob is pretreated by the ultrasonic-assisted ternary eutectic solvent, the content of lignin, cellulose and hemicellulose is 9.95%,58.25% and 17.47%; the solid recovery rate, lignin removal rate, hemicellulose removal rate and cellulose recovery rate are respectively 76.96%, 58.33%,58.35% and 84.04%. The yield of glucose hydrolyzed by the enzyme for 72 hours is 53.74 percent.
Claims (1)
1. The method for preprocessing the corncob by using the three-frequency ultrasonic-assisted ternary eutectic solvent is characterized by comprising the following steps of:
(1) Synthesis of ternary eutectic solvent:
mixing a hydrogen bond donor and a hydrogen bond acceptor according to a certain molar ratio, placing the mixture into a magnetic stirring water bath kettle, heating and stirring at 80 ℃ and 500 rmp, melting the mixture to be converted into a uniform and transparent solution, namely the prepared ternary eutectic solvent, and placing the ternary eutectic solvent into a dryer for standby;
(2) Ultrasound-assisted ternary eutectic solvent pretreatment of corncob: corn cob powder and ternary eutectic solvent are mixed according to a certain mass ratio of 1: (5:30) placing the materials in a glass test tube with a plug, uniformly mixing, then placing the materials in ultrasonic reactors with different frequencies, treating for a certain time, and placing the materials in an oil bath with the temperature of 50-150 ℃ for heating for 0.5-5 h;
(3) Immediately taking out the container after the reaction is finished, and cooling to room temperature; preparing a mixed solution of acetone and water in a volume ratio of 1:1, washing the pretreated sample into a conical flask, soaking and stirring overnight, and then carrying out suction filtration on the sample by using a vacuum suction filtration device to obtain solid residues and filtrate; after the filtrate is subjected to rotary evaporation to remove the detergent, the ternary eutectic solvent can be recovered so as to realize recycling;
(4) Weighing a certain amount of solid residues, namely pretreated corncob samples, and measuring the contents of cellulose, hemicellulose and lignin in corncob components by adopting a national renewable energy laboratory standard analysis program, wherein the specific steps are as follows: uniformly stirring corncob and 72% concentrated sulfuric acid, preserving heat at 30 ℃ for 1 h, washing with 84 mL distilled water into a conical flask, preserving heat at 121 ℃ for 45 min, carrying out suction filtration after the reaction is finished, measuring glucose and xylose content in filtrate by using a high performance liquid chromatograph to calculate cellulose and hemicellulose content, measuring lignin content by analyzing residue weight, and finally obtaining cellulose recovery rate and lignin and hemicellulose removal rate by calculation; simultaneously carrying out enzyme hydrolysis on the treated sample at 50 ℃ for 24-72 hours;
the ternary eutectic solvent in the step (1) is choline chloride-glycerol-lactic acid, wherein the molar ratio of choline chloride, glycerol and lactic acid is 1: (0.5-2): (0.5-2);
The ultrasonic reactors with different frequencies in the step (2) are three-frequency combined ultrasonic, the three-frequency combined ultrasonic frequency is 20kHz, 40kHz, 60kHz, 20+40kHz, 20+60kHz, 40+60kHz or 20+40+60kHz, and the treatment time is 10-90 min.
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