Method for producing ethanol by poplar enzymolysis fermentation and application thereof

Abstract

The application discloses a method for producing ethanol by poplar enzymolysis fermentation, and adopts metal chloride and ethanol aqueous solution to pretreat poplar firstly, and then carries out simultaneous saccharification and fermentation to produce ethanol; the metal chloride is aluminum chloride and / or chromium chloride. After the poplar is pretreated by the method, the simultaneous saccharification and fermentation is carried out, when the substrate concentration of the poplar is 30%, the yield of glucose produced by poplar enzymolysis can reach 62.28 g / L; when the substrate concentration of the poplar is 32%, the yield of glucose produced by poplar enzymolysis can reach 64.85 g / L. The method can effectively remove lignin, improve the reaction activity of cellulase, effectively improve the efficiency of poplar enzymolysis fermentation, and improve the yield of ethanol.

Description

Technical Field

[0001] This invention relates to the field of ethanol preparation technology, specifically to a method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood and its application. Background Technology

[0002] The increasing depletion of fossil resources and environmental pollution are powerful drivers for the development of lignocellulosic biomass resource conversion. As a promising and abundant resource, lignocellulosic biomass has been developed to produce sustainable alternatives (biofuels, chemicals, and materials) from its main components, providing a sustainable path to alleviate long-term energy security needs.

[0003] Poplar wood is abundant and an important raw material for the production of bioenergy and biomass chemicals. Bioconversion of poplar wood to produce bioenergy and chemicals holds great potential. However, due to the tightly bound structure of cellulose, hemicellulose, and lignin in poplar wood, it is structurally stable and not easily destroyed by cellulase. Generally, the production of bioethanol first requires cellulase to hydrolyze cellulose to release glucose from the lignocellulose, followed by yeast fermentation of the monosaccharide (glucose) to obtain bioethanol. However, the recalcitrant structure of biomass still limits the efficiency of cellulose conversion into glucose.

[0004] Therefore, pretreatment is necessary for biomass resource conversion. Currently used pretreatment methods include acid pretreatment, alkaline pretreatment, organic solvent pretreatment, and ionic liquid pretreatment.

[0005] Organic solvent refining has attracted attention because it can fully dissolve lignin, producing highly reactive solid materials, and the solvents are easily recyclable. Adding acidic or basic catalysts to organic solvents can further cleave heterocyclic ether bonds between polysaccharides, releasing monosaccharides and generating digestible, cellulose-rich substrates. However, high concentrations of inorganic acids can degrade xylose in lignin to produce furfural and other fermentable plant matter, while also damaging some cellulose, which is detrimental to the complete biorefining of the raw material (wood).

[0006] Therefore, there is an urgent need for a method that can effectively improve the efficiency of poplar enzymatic hydrolysis and fermentation. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned shortcomings of the prior art and to provide a method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood and its application.

[0008] The primary objective of this invention is to provide a method for producing ethanol through enzymatic hydrolysis and fermentation of poplar wood.

[0009] A second objective of this invention is to provide the application of the above-described method in improving the efficiency of poplar enzymatic hydrolysis for ethanol production.

[0010] To achieve the above objectives, the present invention is implemented through the following solution:

[0011] A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood includes the following steps:

[0012] S1. Poplar wood pretreatment: Mix crushed poplar wood, metal chloride and ethanol aqueous solution to obtain a mixture, react at 160-200℃ for 10-35 min, and collect the filter residue after solid-liquid separation;

[0013] The mass-to-volume ratio of the pulverized poplar wood to the ethanol aqueous solution is 1 g: 8-15 mL; the volume concentration of the ethanol aqueous solution is 30%-80%; the final concentration of the metal chloride in the mixture is 0.01-0.03 mol / L; the metal chloride is aluminum chloride and / or chromium chloride.

[0014] S2. Mix the filter residue, cellulase, yeast activation solution and nutrient solution obtained in step S1 to obtain fermentation broth, and carry out saccharification and fermentation of fermentation broth to obtain ethanol;

[0015] The nutrient solution consists of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water in a mass-to-volume ratio of 1–3 g: 0.5–1.5 g: 0.5–1.5 g: 0.1–0.5 g: 1 L; the saccharification and fermentation temperature is 30–38 °C; the mass-to-volume ratio of the filter residue obtained in step S1 to the fermentation broth is 10–32 g: 100 mL; the ratio of the filter residue obtained in step S1 to cellulase is 1 g: 10–30 FPU; and the concentration of yeast in the fermentation broth is 1–5 g / L.

[0016] Preferably, the poplar wood to be crushed in step S1 is 20-60 mesh poplar wood.

[0017] Preferably, the mass-to-volume ratio of the pulverized poplar wood and the ethanol aqueous solution in step S1 is 1g:10mL.

[0018] Preferably, the metal chloride in step S1 is chromium chloride.

[0019] More preferably, the final concentration of chromium chloride in the mixture is 0.025 mol / L.

[0020] Preferably, the volume concentration of the ethanol aqueous solution is 60%.

[0021] Preferably, the solid-liquid separation in step S1 is vacuum filtration separation and / or centrifugal separation.

[0022] More preferably, the solid-liquid separation is vacuum filtration separation.

[0023] Preferably, the mass-to-volume ratio of the filter residue and fermentation broth obtained in step S1 in step S2 is 30-32 g: 100 mL.

[0024] In a system where 100 mL of fermentation broth contains no less than 30 g of filter residue, chromium chloride (CrCl3) is selected as the metal chloride; in a system where 100 mL of fermentation broth contains less than 30 g of filter residue, aluminum chloride (AlCl3) is selected as the metal chloride.

[0025] Preferably, the ratio of filter residue obtained in step S1 to cellulase in step S2 is 1g:15FPU.

[0026] Preferably, the yeast activation solution in step S2 is a brewer's yeast activation solution.

[0027] Preferably, the mass-to-volume ratio of the filter residue and nutrient solution obtained in step S1 in step S2 is 1-3.2 g: 9-10 mL.

[0028] More preferably, the mass-to-volume ratio of the filter residue and nutrient solution obtained in step S1 in step S2 is 3.0-3.2 g: 9-10 mL.

[0029] Preferably, the nutrient solution in step S2 is composed of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate and water in a mass-to-volume ratio of 2g:1g:1g:0.3g:1L.

[0030] Preferably, the saccharification and fermentation temperature in step S2 is 30-38°C, the rotation speed is 100-200 rpm, and the saccharification and fermentation time is 48-144 h.

[0031] More preferably, the saccharification and fermentation temperature is 34°C, the rotation speed is 130 rpm, and the saccharification and fermentation time is 72 h.

[0032] The present invention also claims protection for the application of any of the above-described methods in improving the efficiency of poplar enzymatic hydrolysis for ethanol production.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] This invention provides a method for producing ethanol from poplar wood through enzymatic fermentation. The method involves pretreating poplar wood with a metal chloride and an aqueous ethanol solution, followed by simultaneous saccharification and fermentation to produce ethanol. The metal chloride is aluminum chloride and / or chromium chloride. Using this method, the glucose yield from poplar wood enzymatic fermentation can reach 62.28 g / L when the substrate concentration is 30%, and 64.85 g / L when the substrate concentration is 32%. This method effectively removes lignin while increasing the reactivity of cellulase, thus improving the efficiency of poplar wood enzymatic fermentation and increasing ethanol yield. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0036] Some of the raw materials used in the following examples:

[0037] Poplar wood: obtained from factory fertilizer and air-dried; cellulose content 45.56%, hemicellulose content 14.98%, lignin 22.86%.

[0038] Example 1: A method for producing ethanol by enzymatic fermentation of poplar wood

[0039] 1. Poplar wood pretreatment: Poplar wood is crushed to 20-60 mesh to obtain crushed poplar wood. 15g of crushed poplar wood and 150mL of 60% ethanol aqueous solution are placed in a closed reactor equipped with a thermocouple and mixed to obtain a mixture. Chromium chloride (CrCl3) is added to the mixture until the final concentration is 0.025mol / L.

[0040] After reacting in a sealed environment at 200℃ for 10 minutes, the temperature was immediately lowered to 25-35℃ with cooling water, and vacuum filtration was performed to obtain filtrate and filter residue. The filter residue was collected, dried, and set aside for later use.

[0041] 2. Enzymatic fermentation to produce ethanol: Take 30g of the filter residue obtained in step S1 and 95mL of nutrient solution, place them in a 250mL Erlenmeyer flask, adjust the pH to 4.8 with sulfuric acid (H2SO4), and then put the Erlenmeyer flask containing the filter residue and nutrient solution into an autoclave and sterilize at 121℃ for 20min.

[0042] Dissolve 2g glucose, 2g peptone and 1g yeast extract in 100mL deionized water, then add 6.6g brewer's yeast powder. Shake on a shaker at 36℃ and 150rpm for 10min, then at 34℃ and 150rpm for 1h to obtain brewer's yeast activated solution.

[0043] After sterilization, 450 FPU cellulase and 5 mL of Saccharomyces cerevisiae activation solution (3.33 g / L) were added to a clean bench to obtain a fermentation broth. The fermentation broth was then shaken on a shaker at 34°C and 130 rpm for saccharification and fermentation for 72 h. The concentration of ethanol obtained from the fermentation was then measured.

[0044] Example 2: A method for producing ethanol by enzymatic fermentation of poplar wood

[0045] 1. Poplar wood pretreatment: Poplar wood is crushed to 20-60 mesh to obtain crushed poplar wood. 15g of crushed poplar wood and 225mL of 30% ethanol aqueous solution are placed in a closed reactor equipped with a thermocouple and mixed to obtain a mixture. Chromium chloride (CrCl3) is added to the mixture until the final concentration is 0.025mol / L.

[0046] After reacting in a sealed environment at 190℃ for 20 minutes, the temperature was immediately lowered to 25-35℃ with cooling water, and vacuum filtration was performed to obtain filtrate and filter residue. The filter residue was collected, dried, and set aside for later use.

[0047] 2. Enzymatic fermentation to produce ethanol: Take 32g of the filter residue obtained in step S1 and 95mL of nutrient solution, place them in a 250mL Erlenmeyer flask, adjust the pH to 4.8 with sulfuric acid (H2SO4), and then put the Erlenmeyer flask containing the filter residue and nutrient solution into an autoclave and sterilize at 121℃ for 20min.

[0048] Dissolve 2g glucose, 2g peptone and 1g yeast extract in 100mL deionized water, then add 6.6g brewer's yeast powder. Shake on a shaker at 36℃ and 150rpm for 10min, then at 34℃ and 150rpm for 1h to obtain brewer's yeast activated solution.

[0049] After sterilization, 320 FPU cellulase and 5 mL of Saccharomyces cerevisiae activation solution (3.33 g / L) were added to a clean bench to obtain a fermentation broth. The fermentation broth was then shaken on a shaker at 34°C and 130 rpm for saccharification and fermentation for 72 h. The concentration of ethanol obtained from the fermentation was then measured.

[0050] Example 3: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0051] 1. Poplar wood pretreatment: Poplar wood is crushed to 20-60 mesh to obtain crushed poplar wood. 15g of crushed poplar wood and 120mL of 80% ethanol aqueous solution are placed in a closed reactor equipped with a thermocouple and mixed to obtain a mixture. Chromium chloride (CrCl3) is added to the mixture until the final concentration is 0.01mol / L.

[0052] After reacting in a sealed environment at 200℃ for 35 minutes, immediately cool the temperature to 25-35℃ with condensing water, centrifuge and filter to obtain filtrate and filter residue. Collect the filter residue, dry it, and set it aside for later use.

[0053] 2. Enzymatic fermentation to produce ethanol: Take 10g of the filter residue obtained in step S1 and 95mL of nutrient solution, place them in a 250mL Erlenmeyer flask, adjust the pH to 4.8 with sulfuric acid (H2SO4), and then put the Erlenmeyer flask containing the filter residue and nutrient solution into an autoclave and sterilize at 121℃ for 20min.

[0054] Dissolve 2g glucose, 2g peptone and 1g yeast extract in 100mL deionized water, then add 6.6g brewer's yeast powder. Shake on a shaker at 36℃ and 150rpm for 10min, then at 34℃ and 150rpm for 1h to obtain brewer's yeast activated solution.

[0055] After sterilization, 150 FPU cellulase and 5 mL of Saccharomyces cerevisiae activation solution (3.33 g / L) were added to a clean bench to obtain a fermentation broth. The fermentation broth was then shaken on a shaker at 34°C and 130 rpm for saccharification and fermentation for 72 h. The concentration of ethanol obtained from the fermentation was then measured.

[0056] Example 4: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0057] 1. Poplar wood pretreatment: Poplar wood is crushed to 20-60 mesh to obtain crushed poplar wood. 15g of crushed poplar wood and 150mL of 60% ethanol aqueous solution are placed in a closed reactor equipped with a thermocouple and mixed to obtain a mixture. Chromium chloride (CrCl3) is added to the mixture until the final concentration is 0.03mol / L.

[0058] After reacting in a sealed environment at 200℃ for 10 minutes, immediately cool the temperature to 25-35℃ with condensing water, centrifuge and filter to obtain filtrate and filter residue. Collect the filter residue, dry it, and set it aside for later use.

[0059] 2. Enzymatic fermentation to produce ethanol: Take 20g of the filter residue obtained in step S1 and 95mL of nutrient solution, place them in a 250mL Erlenmeyer flask, adjust the pH to 4.8 with sulfuric acid (H2SO4), and then put the Erlenmeyer flask containing the filter residue and nutrient solution into an autoclave and sterilize at 121℃ for 20min.

[0060] Dissolve 2g glucose, 2g peptone and 1g yeast extract in 100mL deionized water, then add 6.6g brewer's yeast powder. Shake on a shaker at 36℃ and 150rpm for 10min, then at 34℃ and 150rpm for 1h to obtain brewer's yeast activated solution.

[0061] After sterilization, 300 FPU cellulase and 5 mL of Saccharomyces cerevisiae activation solution (3.33 g / L) were added to a clean bench to obtain a fermentation broth. The fermentation broth was then shaken on a shaker at 34°C and 130 rpm for saccharification and fermentation for 72 h. The concentration of ethanol obtained from the fermentation was then measured.

[0062] Example 5: A method for producing ethanol by enzymatic fermentation of poplar wood.

[0063] 1. Poplar wood pretreatment: Poplar wood is crushed to 20-60 mesh to obtain crushed poplar wood. 15g of crushed poplar wood and 225mL of 30% ethanol aqueous solution are placed in a closed reactor equipped with a thermocouple and mixed to obtain a mixture. Aluminum chloride (AlCl3) is added to the mixture until the final concentration is 0.025mol / L.

[0064] After reacting in a sealed environment at 190℃ for 20 minutes, the temperature was immediately lowered to 25-35℃ with cooling water, and vacuum filtration was performed to obtain filtrate and filter residue. The filter residue was collected, dried, and set aside for later use.

[0065] 2. Enzymatic fermentation to produce ethanol: Take 32g of the filter residue obtained in step S1 and 95mL of nutrient solution, place them in a 250mL Erlenmeyer flask, adjust the pH to 4.8 with sulfuric acid (H2SO4), and then put the Erlenmeyer flask containing the filter residue and nutrient solution into an autoclave and sterilize at 121℃ for 20min.

[0066] Dissolve 2g glucose, 2g peptone and 1g yeast extract in 100mL deionized water, then add 6.6g brewer's yeast powder. Shake on a shaker at 36℃ and 150rpm for 10min, then at 34℃ and 150rpm for 1h to obtain brewer's yeast activated solution.

[0067] After sterilization, 320 FPU cellulase and 5 mL of Saccharomyces cerevisiae activation solution (3.33 g / L) were added to a clean bench to obtain a fermentation broth. The fermentation broth was then shaken on a shaker at 34°C and 130 rpm for saccharification and fermentation for 72 h. The concentration of ethanol obtained from the fermentation was then measured.

[0068] Example 6: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0069] The difference between Example 6 and Example 2 is that chromium chloride (CrCl3) in the poplar pretreatment process is replaced with aluminum chloride (AlCl3), while the other steps are the same.

[0070] Example 7: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0071] The difference between Example 7 and Example 4 is that chromium chloride (CrCl3) in the poplar pretreatment process is replaced with aluminum chloride (AlCl3), while the other steps are the same.

[0072] Example 8: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0073] The difference between Example 8 and Example 3 is that the reaction temperature in the poplar pretreatment process is 190°C, while the other steps are the same.

[0074] Example 9: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0075] The difference between Example 9 and Example 3 is that the reaction temperature in the poplar pretreatment process is 180℃, while the other steps are the same.

[0076] Example 10: A method for producing ethanol by enzymatic fermentation of poplar wood.

[0077] The difference between Example 10 and Example 3 is that the reaction temperature in the poplar pretreatment process is 160°C, while the other steps are the same.

[0078] Comparative Example 1: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood

[0079] The difference between Comparative Example 1 and Example 3 is that the ethanol solution in the poplar pretreatment process is replaced with water, while the other steps are the same.

[0080] Comparative Example 2: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0081] The difference between Comparative Example 2 and Example 8 is that the ethanol solution in the poplar pretreatment process is replaced with water, while the other steps are the same.

[0082] Comparative Example 3: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0083] The difference between Comparative Example 3 and Example 9 is that the ethanol solution in the poplar pretreatment process is replaced with water, while the other steps are the same.

[0084] Comparative Example 4: A method for producing ethanol by enzymatic hydrolysis and fermentation of poplar wood.

[0085] The difference between Comparative Example 4 and Example 10 is that the ethanol solution in the poplar pretreatment process is replaced with water, while the other steps are the same.

[0086] Experimental example: Detection of ethanol concentration

[0087] The ethanol concentration in the fermentation broth after saccharification and fermentation for 72 h in Examples 1 to 10 and Comparative Examples 1 to 4 was determined by high performance liquid chromatography.

[0088] The ethanol concentrations in the fermentation broth after saccharification and fermentation for 72 hours in Examples 1 to 10 and Comparative Examples 1 to 4 are shown in Table 1.

[0089] Table 1. Ethanol concentration in fermentation broth after 72 hours of saccharification and fermentation in Examples 1-10 and Comparative Examples 1-4

[0090]

[0091]

[0092] As can be seen from the results shown in Table 1, by using the method described in this invention to pretreat poplar wood with metal chlorides (chromium chloride or aluminum chloride), when the concentration of poplar wood substrate is 10% to 32% (i.e., when the filter residue: fermentation liquid = 10 to 32 g: 100 mL as described in the examples), the concentration of ethanol produced by simultaneous saccharification and fermentation of poplar wood can be effectively increased, and the ethanol concentration can reach 8.76 g / L to 64.85 g / L.

[0093] Furthermore, the results shown in Table 1 clearly demonstrate that when the poplar substrate concentration is less than 30%, the simultaneous saccharification and fermentation of poplar pretreated with aluminum chloride to produce ethanol results in a higher ethanol concentration; when the poplar substrate concentration is not less than 30%, the simultaneous saccharification and fermentation of poplar pretreated with chromium chloride to produce ethanol results in a higher ethanol concentration.

[0094] When the concentration of poplar substrate is 30% (i.e., Example 1 of the present invention), the ethanol concentration can reach 62.28 g / L; when the concentration of poplar substrate is 32% (i.e., Example 2 of the present invention), the ethanol concentration can reach 64.85 g / L.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for producing ethanol by enzymatic fermentation of poplar wood, characterized in that, Includes the following steps: S1. Poplar wood pretreatment: Mix crushed poplar wood, chromium chloride and ethanol aqueous solution to obtain a mixture, react at 160-200℃ for 10-35 min, and collect the filter residue after solid-liquid separation; The mass-to-volume ratio of the pulverized poplar wood to the ethanol-water solution is 1 g: 15 mL; the volume concentration of the ethanol-water solution is 30%; and the final concentration of chromium chloride in the mixture is 0.025 mol / L. S2. Mix the filter residue, cellulase, yeast activation solution and nutrient solution obtained in step S1 to obtain fermentation broth, and carry out saccharification and fermentation of fermentation broth to obtain ethanol; The nutrient solution consists of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water in a mass-to-volume ratio of 1–3 g: 0.5–1.5 g: 0.5–1.5 g: 0.1–0.5 g: 1 L; the saccharification and fermentation temperature is 30–38 °C; the mass-to-volume ratio of the filter residue obtained in step S1 to the fermentation broth is 32 g: 100 mL; the ratio of the filter residue obtained in step S1 to cellulase is 1 g: 10 FPU; and the concentration of yeast in the fermentation broth is 3.33 g / L.

2. The method according to claim 1, characterized in that, The solid-liquid separation described in step S1 is vacuum filtration separation and / or centrifugal separation.

3. The method according to claim 1, characterized in that, The yeast activation solution mentioned in step S2 is a brewer's yeast activation solution.

4. The method according to claim 1, characterized in that, The nutrient solution described in step S2 consists of yeast extract, ammonium chloride, potassium dihydrogen phosphate, magnesium sulfate, and water in a mass-to-volume ratio of 2 g: 1 g: 1 g: 0.3 g: 1 L.

5. The application of the method according to any one of claims 1 to 4 in improving the efficiency of poplar enzymatic hydrolysis for ethanol production.

Images