CN115820750A - Method for producing ethanol by poplar enzymolysis and fermentation and application thereof - Google Patents
Method for producing ethanol by poplar enzymolysis and fermentation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for producing ethanol by poplar enzymolysis and fermentation, which comprises the steps of pretreating poplar by adopting a metal chloride and ethanol aqueous solution, and then synchronously saccharifying and fermenting the pretreated poplar to produce ethanol; the metal chloride is aluminum chloride and/or chromium chloride. After poplar is pretreated by the method, synchronous saccharification and fermentation are carried out, and when the substrate concentration of the poplar is 30%, the yield of glucose produced by enzymolysis of the poplar can reach 62.28g/L; when the concentration of the poplar substrate is 32%, the yield of glucose produced by enzymolysis of poplar can reach 64.85g/L. The method can also improve the reaction activity of the cellulase under the condition of effectively removing the lignin, effectively improve the efficiency of the poplar enzymolysis and fermentation and improve the yield of the ethanol.
Description
Technical Field
The invention relates to the technical field of ethanol preparation, in particular to a method for producing ethanol by poplar enzymolysis and fermentation and application thereof.
Background
The problems of increasing exhaustion of fossil resources and environmental pollution are the strong driving force for developing the conversion of lignocellulose biomass resources. Lignocellulosic biomass, a promising abundant resource, has been developed for the production of sustainable alternatives (biofuels, chemicals and materials) from its major constituents, providing a sustainable avenue to alleviate long-term energy safety requirements.
The poplar resources are rich, the poplar resources are important raw materials for producing biological energy sources and biomass chemicals, and the biological energy sources and the chemicals prepared by biotransformation of poplar have great potential. But because the cellulose, hemicellulose and lignin in the poplar are tightly connected together, the structure is stable and is not easily damaged by the cellulase. In general, in the process of producing bioethanol, it is first necessary to release glucose from lignocellulose by hydrolysis of cellulose with cellulase enzymes, and then to obtain bioethanol by fermentation of monosaccharides (glucose) with yeast. However, the recalcitrant structure of biomass still limits the efficiency of cellulose conversion to glucose.
Therefore, the biomass resource conversion needs to be pretreated when the biomass resource conversion is carried out. The pretreatment methods used at present include acid pretreatment, alkaline pretreatment, organic solvent pretreatment and ionic liquid pretreatment.
The organic solvent method is concerned by people because the organic solvent method can fully dissolve lignin to generate solid materials with stronger reaction activity, and the solvent is easy to recycle. Addition of an acidic or basic catalyst to the organic solvent can further cleave heterocyclic ether linkages between the polysaccharides, releasing monosaccharides, resulting in a digestible cellulose-rich substrate. However, high concentrations of mineral acids degrade xylose in lignin to produce furfural and other fermentation plants, while destroying a portion of the cellulose, which is detrimental to the whole-component biorefinery of the raw material (wood).
Therefore, a method capable of effectively improving the efficiency of poplar enzymatic fermentation is needed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for producing ethanol by poplar enzymolysis and fermentation and application thereof.
The first purpose of the invention is to provide a method for producing ethanol by poplar enzymolysis and fermentation.
The second purpose of the invention is to provide the application of the method in improving the efficiency of producing ethanol by poplar enzymolysis.
In order to achieve the purpose, the invention is realized by the following scheme:
a method for producing ethanol by poplar enzymolysis and fermentation comprises the following steps:
s1, poplar pretreatment: mixing the crushed poplar, the metal chloride and the ethanol water solution to obtain a mixed solution, reacting for 10-35 min at 160-200 ℃, carrying out solid-liquid separation and collecting filter residues;
the mass volume ratio of the crushed poplar to the ethanol water solution is 1g: 8-15 mL; the volume concentration of the ethanol water solution is 30-80%; the final concentration of the metal chloride in the mixed solution is 0.01-0.03 mol/L; the metal chloride is aluminum chloride and/or chromium chloride;
s2, mixing the filter residue obtained in the step S1, cellulase, yeast activating solution and nutrient solution to obtain fermentation liquor, and performing saccharification and fermentation on the fermentation liquor to obtain ethanol;
the nutrient solution is prepared from 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, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate and water; the temperature of the saccharification and fermentation is 30-38 ℃; the mass volume ratio of the filter residue obtained in the step S1 to the fermentation liquor is 10-32 g:100mL; the dosage ratio of the filter residue obtained in the step S1 to the cellulase is 1g:10 to 30FPU; the concentration of the yeast in the fermentation liquor is 1-5 g/L.
Preferably, the crushed poplar in the step S1 is 20-60 mesh poplar.
Preferably, the mass-to-volume ratio of the pulverized poplar and the ethanol aqueous solution in the step S1 is 1g:10mL.
Preferably, the metal chloride in step S1 is chromium chloride.
More preferably, the final concentration of the chromium chloride in the mixed solution is 0.025mol/L.
Preferably, the ethanol aqueous solution has a volume concentration of 60%.
Preferably, the solid-liquid separation in step S1 is vacuum filtration separation and/or centrifugal separation.
More preferably, the solid-liquid separation is vacuum filtration separation.
Preferably, the mass-to-volume ratio of the filter residue obtained in the step S1 to the fermentation broth in the step S2 is 30 to 32g:100mL.
In a system containing not less than 30g of filter residue in 100mL of fermentation liquor, goldChromium chloride (CrCl) is selected as the chloride 3 ) (ii) a In a system containing less than 30g of filter residue in 100mL of fermentation liquor, the metal chloride is selected from aluminum chloride (AlCl) 3 )。
Preferably, the usage ratio of the filter residue obtained in the step S1 in the step S2 to the cellulase is 1g:15FPU.
Preferably, the yeast activation solution in step S2 is a saccharomyces cerevisiae activation solution.
Preferably, the mass-to-volume ratio of the filter residue obtained in the step S1 to the nutrient solution in the step S2 is 1 to 3.2g:9 to 10mL.
More preferably, the mass-to-volume ratio of the filter residue obtained in the step S1 to the nutrient solution in the step S2 is 3.0 to 3.2g:9 to 10mL.
Preferably, the nutrient solution in step S2 is prepared from a mixture of 2g:1g:1g:0.3g:1L of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate and water.
Preferably, the temperature of the saccharification and fermentation in the step S2 is 30-38 ℃, the rotating speed is 100-200 rpm, and the saccharification and fermentation time is 48-144 h.
More preferably, the temperature of the saccharification and fermentation is 34 ℃, the rotating speed is 130rpm, and the saccharification and fermentation time is 72h.
The invention also requests to protect the application of any one of the methods in improving the efficiency of ethanol production by poplar enzymolysis.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a method for producing ethanol by poplar enzymolysis and fermentation, which comprises the steps of pretreating poplar by adopting a metal chloride and ethanol aqueous solution, and then synchronously saccharifying and fermenting the pretreated poplar to produce ethanol; the metal chloride is aluminum chloride and/or chromium chloride. When poplar is treated by the method disclosed by the invention, the yield of glucose produced by enzymolysis of the poplar can reach 62.28g/L when the substrate concentration of the poplar is 30%; when the concentration of the poplar substrate is 32%, the yield of glucose produced by enzymolysis of poplar can reach 64.85g/L. The method can also improve the reaction activity of the cellulase under the condition of effectively removing the lignin, effectively improve the efficiency of the poplar enzymolysis and fermentation and improve the yield of the ethanol.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Some of the starting materials in the following examples:
poplar: fertilizer from a factory is obtained by air drying treatment; cellulose content 45.56%, hemicellulose content 14.98%, lignin 22.86%.
Example 1 method for producing ethanol by poplar enzymolysis and fermentation
1. Pretreating poplar: and (3) crushing the poplar wood powder to 20-60 meshes to obtain the crushed poplar. Mixing pulverized poplar 15g and 60% ethanol water solution 150mL in volume concentration in a closed reactor equipped with a thermocouple to obtain a mixed solution, and adding chromium chloride (CrCl) 3 ) To a final concentration of 0.025mol/L.
And (3) after the reaction is carried out for 10min in a closed way at the temperature of 200 ℃, immediately reducing the temperature to 25-35 ℃ by using condensed water, carrying out vacuum filtration to obtain filtrate and filter residue, collecting the filter residue and drying the filter residue for later use.
2. And (3) producing ethanol by enzymolysis and fermentation: taking the completely dried filter residue obtained in the step S1 with the mass of 30g and 95mL of nutrient solution, placing the filter residue and the nutrient solution in a 250mL conical flask, and adding sulfuric acid (H) 2 SO 4 ) Adjusting pH to 4.8, placing the conical flask with filter residue and nutrient solution into autoclave, and sterilizing at 121 deg.C for 20min.
Dissolving 2g of glucose, 2g of peptone and 1g of yeast extract in 100mL of deionized water, then adding 6.6g of saccharomyces cerevisiae powder, shaking for 10min at 36 ℃ and 150rpm on a shaking table, and then shaking for 1h at 34 ℃ and 150rpm to obtain the saccharomyces cerevisiae activation solution.
And (3) adding 450FPU (fermented liquid) cellulase and 5mL of saccharomyces cerevisiae activation solution (3.33 g/L) on a superclean bench after sterilization to obtain fermentation liquid, oscillating the fermentation liquid on a shaker at 34 ℃ and 130rpm, performing saccharification and fermentation for 72h, and detecting the concentration of ethanol obtained by fermentation.
Embodiment 2 method for producing ethanol by poplar enzymolysis and fermentation
1. Pretreating poplar: and (3) crushing the poplar wood powder to 20-60 meshes to obtain the crushed poplar. Mixing pulverized poplar 15g and 30% ethanol aqueous solution 225mL in a closed reactor equipped with a thermocouple to obtain a mixed solution, and adding chromium chloride (CrCl) 3 ) To a final concentration of 0.025mol/L.
And (3) after the reaction is carried out for 20min in a closed manner at 190 ℃, immediately cooling to 25-35 ℃ by using condensed water, carrying out vacuum filtration to obtain filtrate and filter residue, collecting the filter residue and drying for later use.
2. And (3) producing ethanol by enzymolysis and fermentation: taking the filter residue obtained in the step S1 with the absolute dry mass of 32g and 95mL of nutrient solution, placing the filter residue and the nutrient solution in a 250mL conical flask, and adding sulfuric acid (H) 2 SO 4 ) Adjusting pH to 4.8, placing the conical flask with filter residue and nutrient solution into autoclave, and sterilizing at 121 deg.C for 20min.
Dissolving 2g of glucose, 2g of peptone and 1g of yeast extract in 100mL of deionized water, then adding 6.6g of saccharomyces cerevisiae powder, shaking for 10min at 36 ℃ and 150rpm on a shaking table, and then shaking for 1h at 34 ℃ and 150rpm to obtain the saccharomyces cerevisiae activation solution.
And (3) adding 320FPU (fermented product unit) cellulase and 5mL of saccharomyces cerevisiae activation solution (3.33 g/L) on a superclean bench after sterilization to obtain fermentation liquor, oscillating the fermentation liquor on a shaker at 34 ℃ and 130rpm, performing saccharification and fermentation for 72h, and detecting the concentration of ethanol obtained by fermentation.
Embodiment 3 method for producing ethanol by poplar enzymolysis and fermentation
1. Pretreating poplar: the poplar wood powder is crushed into 20-60 meshes to obtain the crushed poplar wood. Mixing pulverized poplar 15g and 80 vol% ethanol aqueous solution 120mL in a closed reactor equipped with a thermocouple to obtain a mixed solution, and adding chromium chloride (CrCl) 3 ) To a final concentration of 0.01mol/L.
And (3) after the reaction is carried out for 35min in a closed manner at the temperature of 200 ℃, immediately cooling to 25-35 ℃ by using condensed water, carrying out centrifugal filtration to obtain filtrate and filter residue, collecting the filter residue and drying for later use.
2. And (3) producing ethanol by enzymolysis and fermentation: taking the filter residue obtained in the step S1 with the absolute dry mass of 10g and 95mL of nutrient solution, placing the filter residue and the nutrient solution in a 250mL conical flask, and adding sulfuric acid (H) 2 SO 4 ) Adjusting pH to 4.8, placing the conical flask with filter residue and nutrient solution into autoclave, and sterilizing at 121 deg.C for 20min.
Dissolving 2g of glucose, 2g of peptone and 1g of yeast extract in 100mL of deionized water, then adding 6.6g of saccharomyces cerevisiae powder, shaking for 10min at 36 ℃ and 150rpm on a shaking table, and then shaking for 1h at 34 ℃ and 150rpm to obtain the saccharomyces cerevisiae activation solution.
After sterilization, 150FPU cellulase and 5mL of saccharomyces cerevisiae activation solution (3.33 g/L) are added on a superclean bench to obtain fermentation liquor, the fermentation liquor is shaken on a shaking table at 34 ℃ and 130rpm, and is subjected to saccharification and fermentation for 72 hours, and the concentration of the ethanol obtained by fermentation is detected.
Embodiment 4 method for producing ethanol by poplar enzymolysis and fermentation
1. Pretreating poplar: and (3) crushing the poplar wood powder to 20-60 meshes to obtain the crushed poplar. Mixing pulverized poplar 15g and 60% ethanol water solution 150mL in a closed reactor with thermocouple to obtain a mixed solution, and adding chromium chloride (CrCl) 3 ) To a final concentration of 0.03mol/L.
And (3) after the reaction is carried out for 10min in a closed way at the temperature of 200 ℃, immediately cooling to 25-35 ℃ by using condensed water, carrying out centrifugal filtration to obtain filtrate and filter residue, collecting the filter residue and drying for later use.
2. And (3) producing ethanol by enzymolysis and fermentation: taking the filter residue obtained in the step S1 with the absolute dry mass of 20g and 95mL of nutrient solution, placing the filter residue and the nutrient solution in a 250mL conical flask, and adding sulfuric acid (H) 2 SO 4 ) Adjusting pH to 4.8, placing the conical flask with filter residue and nutrient solution into autoclave, and sterilizing at 121 deg.C for 20min.
Dissolving 2g of glucose, 2g of peptone and 1g of yeast extract in 100mL of deionized water, then adding 6.6g of saccharomyces cerevisiae powder, shaking for 10min at 36 ℃ and 150rpm on a shaking table, and then shaking for 1h at 34 ℃ and 150rpm to obtain the saccharomyces cerevisiae activation solution.
And (3) adding 300FPU (fermented soy protein) cellulase and 5mL of saccharomyces cerevisiae activation solution (3.33 g/L) on a superclean bench after sterilization to obtain fermentation liquor, oscillating the fermentation liquor on a shaker at 34 ℃ and 130rpm, performing saccharification and fermentation for 72h, and detecting the concentration of ethanol obtained by fermentation.
Example 5 method for producing ethanol by poplar enzymolysis and fermentation
1. Pretreating poplar: and (3) crushing the poplar wood powder to 20-60 meshes to obtain the crushed poplar. Mixing pulverized poplar 15g and 30% ethanol aqueous solution 225mL in volume concentration in a closed reactor equipped with a thermocouple to obtain a mixed solution, and adding aluminum chloride (AlCl) into the mixed solution 3 ) To a final concentration of 0.025mol/L.
And (3) after the reaction is carried out for 20min in a closed manner at 190 ℃, immediately cooling to 25-35 ℃ by using condensed water, carrying out vacuum filtration to obtain filtrate and filter residue, collecting the filter residue and drying for later use.
2. And (3) producing ethanol by enzymolysis and fermentation: taking the filter residue obtained in the step S1 with the absolute dry mass of 32g and 95mL of nutrient solution, placing the filter residue and the nutrient solution in a 250mL conical flask, and adding sulfuric acid (H) 2 SO 4 ) Adjusting pH to 4.8, placing the conical flask with filter residue and nutrient solution into autoclave, and sterilizing at 121 deg.C for 20min.
Dissolving 2g of glucose, 2g of peptone and 1g of yeast extract in 100mL of deionized water, then adding 6.6g of saccharomyces cerevisiae powder, shaking for 10min at 36 ℃ and 150rpm on a shaking table, and then shaking for 1h at 34 ℃ and 150rpm to obtain the saccharomyces cerevisiae activation solution.
And (3) adding 320FPU (fermented product unit) cellulase and 5mL of saccharomyces cerevisiae activation solution (3.33 g/L) on a superclean bench after sterilization to obtain fermentation liquor, oscillating the fermentation liquor on a shaker at 34 ℃ and 130rpm, performing saccharification and fermentation for 72h, and detecting the concentration of ethanol obtained by fermentation.
Example 6 method for producing ethanol by poplar enzymolysis and fermentation
Example 6 differs from example 2 in that: chromium chloride (CrCl) in the pretreatment process of poplar 3 ) Replacement by aluminium chloride (AlCl) 3 ) And the rest steps are the same.
Example 7 method for producing ethanol by poplar enzymolysis and fermentation
Example 7 same as example 4The difference lies in that: chromium chloride (CrCl) in the pretreatment process of poplar 3 ) Replacement by aluminium chloride (AlCl) 3 ) The rest steps are the same.
Embodiment 8 method for producing ethanol by poplar enzymolysis and fermentation
Example 8 differs from example 3 in that: the reaction temperature in the poplar pretreatment process is 190 ℃, and the rest steps are the same.
Example 9 method for producing ethanol by poplar enzymolysis and fermentation
Example 9 differs from example 3 in that: the reaction temperature in the poplar pretreatment process is 180 ℃, and the rest steps are the same.
Example 10 method for producing ethanol by poplar enzymolysis and fermentation
Example 10 differs from example 3 in that: the reaction temperature in the poplar pretreatment process is 160 ℃, and the rest steps are the same.
Comparative example 1 method for producing ethanol by poplar enzymolysis and fermentation
Comparative example 1 differs from example 3 in that: and replacing the ethanol solution in the poplar pretreatment process with water, and carrying out the same steps.
Comparative example 2 method for producing ethanol by poplar enzymolysis and fermentation
Comparative example 2 differs from example 8 in that: and replacing the ethanol solution in the poplar pretreatment process with water, and carrying out the same steps.
Comparative example 3 method for producing ethanol by poplar enzymolysis and fermentation
Comparative example 3 differs from example 9 in that: and replacing the ethanol solution in the poplar pretreatment process with water, and carrying out the same steps.
Comparative example 4 method for producing ethanol by poplar enzymolysis and fermentation
Comparative example 4 differs from example 10 in that: and replacing the ethanol solution in the poplar pretreatment process with water, and carrying out the same steps.
Experimental example measurement of ethanol concentration
And (3) measuring the ethanol concentration in the fermentation liquor after 72 hours of saccharification and fermentation of the fermentation liquor obtained in the example 1-the example 10 and the comparative example 1-the comparative example 4 by adopting a high performance liquid chromatography.
The ethanol concentrations in the fermentation broths after 72 hours of the saccharification and fermentation of examples 1 to 10 and comparative examples 1 to 4 are shown in table 1.
TABLE 1 ethanol concentration in fermentation broths after 72 hours of diastatic fermentation in examples 1 to 10 and comparative examples 1 to 4
As can be seen from the results shown in table 1: according to the method, the poplar is pretreated by using the metal chloride (chromium chloride or aluminum chloride), when the substrate concentration of the poplar is 10-32% (namely when the filter residue is fermentation liquor = 10-32g.
And as is apparent from the results shown in table 1: the concentration of the poplar substrate is less than 30%, the poplar is pretreated by aluminum chloride and then is subjected to synchronous diastatic fermentation to produce ethanol, and the obtained ethanol has higher concentration; when the concentration of the poplar substrate is not less than 30%, the poplar is pretreated by chromium chloride and then is subjected to synchronous saccharification and fermentation to produce ethanol, and the obtained ethanol has higher concentration.
When the concentration of the poplar substrate is 30% (namely the invention in the embodiment 1), the concentration of the ethanol can reach 62.28g/L; when the concentration of the poplar substrate is 32% (namely the invention in the embodiment 2), the concentration of the ethanol can reach 64.85g/L.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention and not to limit the scope of the present invention, and that those skilled in the art can make other variations or modifications on the basis of the above description and idea, and that all embodiments are neither necessary nor exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A method for producing ethanol by poplar enzymolysis and fermentation is characterized by comprising the following steps:
s1, poplar pretreatment: mixing the crushed poplar, the metal chloride and the ethanol water solution to obtain a mixed solution, reacting for 10-35 min at 160-200 ℃, carrying out solid-liquid separation and collecting filter residues;
the mass volume ratio of the crushed poplar to the ethanol water solution is 1g: 8-15 mL; the volume concentration of the ethanol water solution is 30-80%; the final concentration of the metal chloride in the mixed solution is 0.01-0.03 mol/L; the metal chloride is aluminum chloride and/or chromium chloride;
s2, mixing the filter residue obtained in the step S1, cellulase, yeast activating solution and nutrient solution to obtain fermentation liquor, and performing saccharification and fermentation on the fermentation liquor to obtain ethanol;
the nutrient solution is prepared from 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, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate and water; the temperature of the saccharification and fermentation is 30-38 ℃; the mass volume ratio of the filter residue obtained in the step S1 to the fermentation liquor is 10-32 g:100mL; the dosage ratio of the filter residue obtained in the step S1 to the cellulase is 1g:10 to 30FPU; the concentration of the yeast in the fermentation liquor is 1-5 g/L.
2. The method according to claim 1, wherein the mass-to-volume ratio of the poplar and the ethanol aqueous solution in step S1 is 1g:10mL.
3. The method according to claim 1, wherein the metal chloride in step S1 is chromium chloride.
4. The method according to claim 1, wherein the concentration of the ethanol aqueous solution in step S1 is 60% by volume.
5. The method according to claim 1, wherein the solid-liquid separation in step S1 is vacuum filtration separation and/or centrifugal separation.
6. The method according to claim 1, wherein the mass-to-volume ratio of the residue obtained in step S1 to the fermentation broth in step S2 is 30-32 g:100mL.
7. The method according to claim 1, wherein the ratio of the amount of the residue obtained in step S1 to the amount of the cellulase in step S2 is 1g:15FPU.
8. The method according to claim 1, wherein the yeast activating solution in step S2 is a saccharomyces cerevisiae activating solution.
9. The method according to claim 1, wherein the nutrient solution in step S2 is prepared from a mixture of 2g:1g:1g:0.3g:1L of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate and water.
10. Use of the method of any one of claims 1 to 9 for increasing the efficiency of enzymatic hydrolysis of poplar to produce ethanol.
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