CN113122587B - Method for promoting poplar to produce ethanol by hydrothermal combination with NaOH aqueous solution pretreatment - Google Patents
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Abstract
The invention provides a method for promoting poplar to produce ethanol by hydrothermal combination with NaOH aqueous solution pretreatment. The method comprises the steps of firstly crushing poplar wood, reacting the crushed poplar wood with water at 180-200 ℃ for 5-30 min, mixing the separated filter residue with NaOH aqueous solution, reacting at 80-120 ℃ for 30-120 min, separating to obtain pretreated residue, and then adding cellulase, yeast activating solution and nutrient solution for diastatic fermentation. The method adopts a mode of combining hydrothermal and NaOH aqueous solution two-stage pretreatment, reduces the influence of lignin on the enzymolysis and fermentation processes, and can effectively promote the production of ethanol by poplar on the premise of reducing the alkali concentration, and the ethanol yield reaches 50.91 percent.
Description
Technical Field
The invention belongs to the technical field of ethanol preparation. More particularly, relates to a method for promoting poplar wood to produce ethanol by pretreatment through hydrothermal combination of NaOH aqueous solution.
Background
The conversion and utilization of lignocellulose can be divided into 3 stages of raw material pretreatment, enzyme hydrolysis and sugar fermentation, and the main technical bottlenecks are that the pretreatment technology is not mature enough and the activity of cellulase is low, so that the production cost is overhigh. Through the pretreatment of the raw materials, the crystalline structure of cellulose can be damaged, the polymerization degree of lignin is reduced, the porosity of the lignocellulose material is improved, and the contact area between enzyme and a substrate is increased, so that the enzymolysis efficiency is improved, and the purposes of saving time and reducing cost are achieved. The pretreatment method for effectively promoting the lignocellulose to produce the ethanol meets the following 4 conditions: the method is beneficial to improving the yield of the sugar or promoting the generation of monosaccharide in the subsequent hydrolysis reaction; secondly, the decomposition of monosaccharide in the reaction system is avoided; avoiding generating harmful by-products; fourthly, the economic benefit is high.
The existing commonly used pretreatment technologies include acid treatment, alkali treatment, acid catalyzed organic solvent treatment, oxidation treatment, etc. (Zhang, flourishing zang, Liu Yang, Hexiu Lian, Wen hong. lignocellulose pretreatment method research progress [ J ]. Hubei agricultural science, 2012,51(07):1306 1309.), for example Mohammad Saber Bay, etc. discloses a method for pretreating poplar wood with alkali, but the pretreatment method uses a higher sodium hydroxide concentration (8% by mass), which causes severe corrosion to equipment, and the final yield is only about 43% (Mohammad Saber Bay, Keikhos Karimi, Mohsen Nasr Efahany, Rajeev Kumar. structural modification of and plant wood alkali pretreatment process product J.J. [ 25 ].
Therefore, the method for pretreating poplar to produce ethanol effectively under the condition of low-concentration alkali is quite necessary for the technical field of ethanol preparation.
Disclosure of Invention
Aiming at the defects of the poplar pretreatment method, the invention provides a method for promoting poplar to produce ethanol by pretreatment by combining hydrothermal with a NaOH aqueous solution.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a method for promoting poplar to produce ethanol by pretreating in combination with a hydrothermal NaOH aqueous solution, which comprises the following steps:
s1, first-stage pretreatment: crushing poplar, reacting with water at 180-200 ℃ for 5-30 min, and separating to obtain filter residue;
s2, two-stage pretreatment: mixing the filter residue obtained in the step S1 with a NaOH aqueous solution, reacting at 80-120 ℃ for 30-120 min, and separating to obtain a pretreatment residue;
s3, saccharification and fermentation: and (4) adding cellulase, yeast activating solution and nutrient solution into the pretreated residues obtained in the step (S2) to obtain fermentation liquor, and performing saccharification and fermentation.
The first-stage pretreatment temperature of poplar fermentation is set to be 180-200 ℃ specifically, because the compact structure of poplar cannot be damaged effectively when the temperature is too low, and the requirement on equipment is severer when the temperature is too high, so that the method has no universality. In addition, the time for the first stage of pretreatment is specifically set to be 5-30 min, because under the method, complete pretreatment cannot be realized due to too short time, and the intensity of pretreatment is not obviously influenced due to too long time, so that the pretreatment process can be efficiently finished by setting the time length of 5-30 min.
After the pretreatment by the high-temperature hot water in the first stage, the compact structure of the poplar is damaged, but a large amount of lignin is still remained, and the subsequent fermentation efficiency is seriously influenced.
Preferably, the concentration of the NaOH aqueous solution in step S2 is 1.0-1.5% (w/v).
The invention adopts low-concentration sodium hydroxide, has less corrosion to equipment, and can still effectively promote the production of ethanol by poplar.
Further preferably, the ratio of the oven-dried mass of the poplar wood, the volume of water and the volume of the aqueous solution of NaOH is 1 g: 8-15 mL: 8-15 mL.
Most preferably, the ratio of the oven dry mass of the poplar, the volume of water and the volume of the aqueous solution of NaOH is 1 g: 10mL of: 10mL, see example 1.
More preferably, the ratio of the dosage of the cellulase in the step S3 to the absolute dry mass of the pretreatment residue in the step S2 is 10 to 30 FPU: 1g of the total weight of the composition.
When the cellulase is too low, the fermentation process of poplar is not facilitated, so that the fermentation is incomplete; and too high cellulase brings too high cost and is not suitable for industrial application.
Most preferably, the ratio of the dosage of the cellulase in the step S3 to the oven dry mass of the pretreatment residue in the step S2 is 15 FPU: 1g, see example 1.
Further preferably, the concentration of the yeast in the fermentation liquid in the step S3 is 1-5 g/L.
Most preferably, the yeast is saccharomyces cerevisiae.
Further preferably, the saccharification and fermentation in the step S3 is carried out at the temperature of 30-38 ℃ for 48-144 h at the rotation speed of 100-200 rpm.
Most preferably, the temperature of the saccharification fermentation in step S3 is 34 ℃, the time is 72h, and the rotation speed is 130rpm, see example 1.
Further preferably, the nutrient solution in step S3 includes a mixture of 1-3 g by mass:0.5-1.5 g: 0.5-1.5 g: 0.1-0.5 g: 1L of yeast extract, NH4Cl、KH2PO4、MgSO4And water.
Most preferably, the nutrient solution of step S3 includes a mass-to-volume ratio of 2 g: 1 g: 1 g: 0.3 g: 1L of yeast extract, NH4Cl、KH2PO4、MgSO4And water.
Further preferably, the ratio of the volume of the nutrient solution in the step S3 to the absolute dry mass of the filter residue in the step S1 is 9-10 mL: 1g of the total weight of the composition.
Preferably, the separation in steps S1 and S2 is vacuum filtration or centrifugation.
Preferably, in the step S1, the wood powder of poplar is ground to 20-60 mesh.
The method adopts a mode of combining hydrothermal and NaOH aqueous solution two-stage pretreatment, reduces the influence of lignin on the enzymolysis and fermentation processes, and can effectively promote the poplar to produce the ethanol on the premise of reducing the alkali concentration, so that the application of the method in the aspect of promoting the poplar to produce the ethanol is also in the claimed range.
The invention has the following beneficial effects:
the method adopts a mode of combining hydrothermal and NaOH aqueous solution two-stage pretreatment, effectively destroys the recalcitrant structure of the lignocellulose, improves the accessibility of the cellulose to enzyme in the subsequent enzymolysis process, improves the enzymolysis conversion rate, and effectively promotes the poplar to produce the ethanol on the premise of reducing the alkali concentration, wherein the ethanol yield reaches 50.91%.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
The poplar adopted by the invention is from factory waste, and is obtained by air drying treatment, and the component content is measured as follows: cellulose 45.56%, hemicellulose 14.98%, lignin 22.86%; the cellulase of the invention is purchased from novitin; saccharomyces cerevisiae was purchased from Angel Yeast, Inc.
Activation liquid of saccharomyces cerevisiae: weighing 2g of glucose, 2g of peptone and 1g of yeast extract, dissolving in 100mL of deionized water, inoculating 6.6g of brewing yeast powder, and activating on a shaking table in two steps: oscillating at the rotation speed of 150rpm for 10min at the temperature of 36 ℃; ② shaking at the rotating speed of 150rpm for 1h at the temperature of 34 ℃.
Nutrient solution: comprises the following components in a mass-volume ratio of 2 g: 1 g: 1 g: 0.3 g: 1L of yeast extract, NH4Cl、KH2PO4、MgSO4And water.
The method utilizes the high performance liquid chromatography to analyze and obtain the ethanol concentration in the fermentation liquor, and then calculates the ethanol yield according to the following formula:
wherein Y represents ethanol yield (%); c represents the ethanol concentration (g/L) in the fermentation liquor; v represents the volume of fermentation broth (L); m represents the mass (g) of cellulose in the pulverized poplar.
Example 1 method for promoting poplar to produce ethanol by hydrothermal combination with NaOH aqueous solution pretreatment
First, experiment method
S1, first-stage pretreatment: crushing the dried poplar powder to 20-60 meshes, placing 15g of the poplar powder in a closed reactor provided with a thermocouple, carrying out closed reaction with 150mL of water at 200 ℃ for 20min, immediately cooling the reaction to room temperature with condensed water, centrifuging to obtain filtrate and filter residue, and drying the filter residue for later use;
s2, second-stage pretreatment: mixing the filter residue obtained in the step S1 with 150mL of 1.0% (w/v) NaOH aqueous solution, reacting at 120 ℃ for 60min, immediately cooling the reaction to room temperature by using condensed water, and carrying out vacuum filtration to obtain pretreated residue;
s3, saccharification and fermentation: the pretreatment residue obtained in step S2 and having a dry mass of 10g, and 95mL of a nutrient solution were taken, placed in a 250mL Erlenmeyer flask, and washed with H2SO4Adjusting pH to 4.8, sterilizing at 121 deg.C for 20min, and sterilizing in ultra-clean condition150FPU cellulase and 5mL of saccharomyces cerevisiae activation solution are added on a table, and the mixture is shaken at the temperature of 34 ℃ and the rpm on a shaking table and is saccharified and fermented for 72 hours.
Second, experimental results
The ethanol yield was calculated to be 50.91%.
Embodiment 2 method for promoting poplar to produce ethanol by pretreating with hydrothermal combination NaOH aqueous solution
First, experiment method
S1, a first stage of pretreatment: crushing the dried poplar powder to 20-60 meshes, placing 15g of the poplar powder in a closed reactor provided with a thermocouple, carrying out closed reaction on the poplar powder and 120mL of water at 200 ℃ for 5min, immediately cooling the reaction to room temperature by using condensed water, centrifuging to obtain filtrate and filter residue, and drying the filter residue for later use;
s2, second-stage pretreatment: mixing the filter residue obtained in the step S1 with 120mL of 1.5% (w/v) NaOH aqueous solution, reacting at 80 ℃ for 120min, immediately cooling the reaction to room temperature by using condensed water, and carrying out vacuum filtration to obtain pretreated residue;
s3, saccharification and fermentation: taking the absolute dry residue obtained in the step S2 and 90mL of nutrient solution, placing the absolute dry residue and the 90mL of nutrient solution in a 250mL conical flask, and using H2SO4Adjusting pH to 4.8, sterilizing at 121 deg.C for 20min, adding 300FPU cellulase and 5mL Saccharomyces cerevisiae activation solution on a superclean bench, shaking at 30 deg.C and 100rpm on a shaking table, and performing diastatic fermentation for 144 h.
Second, experimental results
The ethanol yield was calculated to be 50.69%.
Embodiment 3 method for promoting poplar to produce ethanol by pretreating with hydrothermal combination NaOH aqueous solution
First, experiment method
S1, first-stage pretreatment: crushing the dried poplar powder to 20-60 meshes, placing 15g of the poplar powder in a closed reactor provided with a thermocouple, carrying out closed reaction with 225mL of water at 180 ℃ for 20min, immediately cooling the reaction to room temperature by using condensed water, centrifuging to obtain filtrate and filter residue, and drying the filter residue for later use;
s2, second-stage pretreatment: mixing the filter residue obtained in the step S1 with 225mL of 1.0% (w/v) NaOH aqueous solution, reacting at 120 ℃ for 60min, immediately cooling the reaction to room temperature by using condensed water, and carrying out vacuum filtration to obtain pretreated residue;
s3, saccharification and fermentation: taking the absolute dry residue obtained in the step S2 and 100mL of nutrient solution, placing the absolute dry residue and the nutrient solution in a 250mL conical flask, and using H2SO4Adjusting pH to 4.8, sterilizing at 121 deg.C for 20min, adding 100FPU cellulase and 5mL Saccharomyces cerevisiae activation solution on a superclean bench, shaking at 38 deg.C and 200rpm on a shaking table, and performing diastatic fermentation for 48 h.
Second, experimental results
The ethanol yield was calculated to be 45.57%.
Embodiment 4 method for promoting poplar to produce ethanol by using hydrothermal combination NaOH aqueous solution for pretreatment
First, experiment method
S1, first-stage pretreatment: crushing the dried poplar powder to 20-60 meshes, placing 15g of the poplar powder in a closed reactor provided with a thermocouple, carrying out closed reaction with 150mL of water at 180 ℃ for 30min, immediately cooling the reaction to room temperature with condensed water, centrifuging to obtain filtrate and filter residue, and drying the filter residue for later use;
s2, second-stage pretreatment: mixing the filter residue obtained in the step S1 with 150mL of 1.5% (w/v) NaOH aqueous solution, reacting at 120 ℃ for 60min, immediately cooling the reaction to room temperature by using condensed water, and carrying out vacuum filtration to obtain pretreated residue;
s3, saccharification and fermentation: taking the absolute dry weight of 10g of the pretreatment residue in the step S2 and 95mL of nutrient solution, placing the mixture in a 250mL conical flask, and using H2SO4Adjusting pH to 4.8, sterilizing at 121 deg.C for 20min, adding 150FPU cellulase and 5mL Saccharomyces cerevisiae activation solution on a superclean bench, shaking at 34 deg.C and 130rpm on a shaking table, and performing diastatic fermentation for 72 h.
Second, experimental results
The ethanol yield was calculated to be 46.11%.
Comparative example 1
First, experiment method
The experimental procedure of example 1 was followed, except that the temperature of the reaction in step S1 was 160 ℃.
Second, experimental results
The ethanol yield was calculated to be 33.16%.
Comparative example 2
First, experiment method
The experimental procedure of example 1 was followed, except that the temperature of the reaction in step S1 was 220 ℃.
Second, experimental results
The calculated ethanol yield was 39.11%.
Comparative example 3
First, experiment method
The experimental procedure of example 1 was followed, except that the reaction time in step S1 was 1 min.
Second, experimental results
The ethanol yield was calculated to be 37.67%.
Comparative example 4
First, experiment method
The experimental procedure of example 1 was followed, except that the reaction time in step S1 was 60 min.
Second, experimental results
The ethanol yield was calculated to be 40.22%.
Comparative example 5
First, experiment method
The experimental procedure of example 1 is followed, with the difference that the temperature of the reaction described in step S2 is 60 ℃.
Second, experimental results
The ethanol yield was calculated to be 30.75%.
Comparative example 6
First, experiment method
The experimental procedure of example 1 was followed, except that the temperature of the reaction in step S2 was 150 ℃.
Second, experimental results
The ethanol yield was calculated to be 40.24%.
Comparative example 7
First, experiment method
The experimental procedure of example 1 was followed, except that the reaction time in step S2 was 20 min.
Second, experimental results
The ethanol yield was calculated to be 32.44%.
Comparative example 8
First, experiment method
The experimental procedure of example 1 was followed, except that the reaction time in step S2 was 160 min.
Second, experimental results
The ethanol yield was calculated to be 38.53%.
Comparative example 9
First, experiment method
The experimental procedure of example 1 was followed, except that the concentration of the aqueous NaOH solution in step S2 was 0.5% (w/v).
Second, experimental results
The ethanol yield was calculated to be 30.09%.
Comparative example 10
First, experiment method
The experimental procedure of example 1 was followed, except that the concentration of the aqueous NaOH solution in step S2 was 3% (w/v).
Second, experimental results
The ethanol yield was calculated to be 38.41%.
Comparative example 11
First, experiment method
The experimental procedure of example 2 is followed, with the difference that the temperature of the reaction described in step S1 is 160 ℃.
Second, experimental results
The ethanol yield was calculated to be 36.63%.
Comparative example 12
First, experiment method
The experimental procedure of example 3 was followed, except that the concentration of the aqueous NaOH solution in step S2 was 0.5% (w/v).
Second, experimental results
The ethanol yield was calculated to be 28.84%.
From the embodiments 1 to 4, the method combines hydrothermal and NaOH aqueous solution to pretreat the poplar wood, so that the influence of lignin on the enzymolysis and fermentation processes can be reduced, and the production of ethanol by the poplar wood can be effectively promoted; compared with the prior art (Mohammad Saber Bay, Keikhosro Karimi, Mohsen Nasr Efahany, Rajeev Kumar. structural modification of pine and floor wood by okali pretreatment) that ethanol yield is about 43%, the ethanol yield of the invention can still be equivalent to that of the prior art (examples 3-4) even exceeds that of the prior art (examples 1-2), and the invention proves that the invention adopts a mode of combining hydrothermal and two-stage pretreatment of NaOH aqueous solution, can effectively reduce the influence on the processes of enzymolysis and fermentation, and can also effectively promote the production of ethanol by poplar under the premise of reducing alkali concentration.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.
Claims (6)
1. A method for promoting poplar to produce ethanol by hydrothermal combination with NaOH aqueous solution pretreatment is characterized by comprising the following steps:
s1, first-stage pretreatment: crushing poplar, reacting with water at 180-200 ℃ for 5-30 min, and separating to obtain filter residue;
s2, two-stage pretreatment: mixing the filter residue obtained in the step S1 with a NaOH aqueous solution, reacting at 80-120 ℃ for 30-120 min, and separating to obtain a pretreated residue;
s3, saccharification and fermentation: adding cellulase, yeast activation liquid and nutrient solution into the pretreated residues obtained in the step S2 to obtain fermentation liquor, and performing saccharification and fermentation;
wherein the ratio of the absolute dry mass of the poplar to the volume of water to the volume of the NaOH aqueous solution is 1 g: 8-15 mL: 8-15 mL;
the concentration of the NaOH aqueous solution in the step S2 is 1.0-1.5% (w/v);
the nutrient solution in the step S3 comprises the following components in a mass-volume ratio of 1-3 g: 0.5-1.5 g: 0.5-1.5 g: 0.1-0.5 g: 1L of yeast extract, NH4Cl、KH2PO4、MgSO4And water; the volume of the nutrient solution and the absolute dry mass ratio of the filter residue in the step S1 are 9-10 mL: 1g of the total weight of the composition.
2. The method as claimed in claim 1, wherein the ratio of the dosage of the cellulase in step S3 to the oven dry mass of the pretreated residue in step S2 is 10-30 FPU: 1g of the total weight of the composition.
3. The method according to claim 1, wherein the concentration of yeast in the fermentation broth in step S3 is 1-5 g/L.
4. The method of claim 1, wherein said yeast is saccharomyces cerevisiae.
5. The method of claim 1, wherein the saccharification and fermentation in step S3 is carried out at 30-38 ℃ for 48-144 h at 100-200 rpm.
6. The method of claim 1, wherein the separation in steps S1 and S2 is vacuum filtration or centrifugation.
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