CN115896185B - Method for producing ethanol by composite additive lifting synchronous saccharification and fermentation and application thereof - Google Patents
Method for producing ethanol by composite additive lifting synchronous saccharification and fermentation and application thereof Download PDFInfo
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- CN115896185B CN115896185B CN202211547582.6A CN202211547582A CN115896185B CN 115896185 B CN115896185 B CN 115896185B CN 202211547582 A CN202211547582 A CN 202211547582A CN 115896185 B CN115896185 B CN 115896185B
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Abstract
The invention discloses a method for producing ethanol by lifting and synchronous saccharification and fermentation of a composite additive and application thereof, comprising the following steps: s1) adding NaOH and ethanol water solution into poplar raw materials in a reaction kettle, pretreating at a certain temperature for a certain time, and carrying out solid-liquid separation to obtain pretreated poplar; s2) adding deionized water, nutrient salt, adjusting pH and sterilizing to the pretreated poplar to obtain a sterilized poplar mixture; s3) adding a composite additive, cellulase and yeast to perform synchronous saccharification and fermentation. According to the method for producing the ethanol by improving the synchronous saccharification and fermentation of the poplar by adopting the composite additive which contains specific components and has specific compound mass ratio, the dosage of cellulase can be obviously reduced, the synchronous saccharification and fermentation time of the poplar (fermentation time is not more than 24 h) can be greatly shortened, the ethanol yield of the synchronous saccharification and fermentation can be obviously improved to 92.70%, the technical support is provided for preparing the liquid fuel ethanol by the poplar, and the method has a good application prospect.
Description
Technical Field
The invention belongs to the technical field of biomass conversion and utilization, and particularly relates to a method for producing ethanol by composite additive lifting synchronous saccharification and fermentation and application thereof.
Background
Along with the continuous occurrence of problems such as petroleum resource shortage, environmental deterioration, and greenhouse effect aggravation, the wood fiber fuel ethanol is used as a clean and environment-friendly renewable energy source, and the fermentation technology of the wood fiber fuel ethanol is gradually valued and actively supported by more and more countries. The method for preparing liquid fuel from lignocellulose biomass is to pretreat cellulose in the raw material, hydrolyze the cellulose into monosaccharides such as glucose through enzymolysis, and then ferment the monosaccharides to produce fuel ethanol.
The poplar has the characteristics of fast growth, high economic efficiency, high nutrition circulation, high drought resistance and disease and pest resistance, wide distribution in various parts of the world, rich yield, high sustainable utilization and the like. The cellulose content of poplar is relatively higher than other lignocellulose biomasses (bagasse, straw, wheat, etc.), and is a main biorefinery raw material for biofuel production and high-value products. However, because the cellulose in the poplar is tightly connected with hemicellulose and lignin, the structure is stable, and the cellulose is not easy to damage, when biomass resources are converted, the cellulose needs to be pretreated, and then enzymolysis and fermentation are carried out on the pretreated poplar.
There are a great deal of literature currently demonstrating that additives are used to enhance the ability of cellulases to promote hydrolysis of lignocellulosic biomass, but most are used only for enzymolysis, and few additives are used for simultaneous saccharification and fermentation to produce ethanol. Chinese patent application CN109457000a discloses that the enzymolysis time can be shortened to a certain extent by adding tween 80, the enzyme dosage can be reduced, and the glucose yield can be improved. Other researches show that some proteins and the like can reduce the dosage of the cellulase, but the problems of higher use cost and poor synergistic effect exist. Although the enzymolysis efficiency can be improved to a certain extent by the different treatment methods, the higher enzymolysis efficiency is usually achieved by using higher cellulase, and the subsequent simultaneous saccharification and fermentation to produce ethanol has the defects of overlong fermentation time and lower ethanol production efficiency.
Disclosure of Invention
Aiming at the defects of the prior art, the primary aim of the invention is to provide a method for producing ethanol by simultaneous saccharification and fermentation of poplar by using a composite additive, which can obviously reduce the dosage of cellulase, greatly shorten the simultaneous saccharification and fermentation time of poplar (the fermentation time is not more than 24 h), and obviously improve the ethanol production efficiency of simultaneous saccharification and fermentation (the ethanol yield of simultaneous saccharification and fermentation liquid is 92.7%).
It is a further object of the present invention to provide the use of the above method for facilitating simultaneous saccharification and fermentation.
The above object of the present invention is achieved by the following technical solutions:
the invention provides a method for producing ethanol by improving poplar through simultaneous saccharification and fermentation by a composite additive, which comprises the following steps:
s1) adding NaOH and ethanol water solution into poplar raw materials in a reaction kettle, pretreating at a certain temperature for a certain time, and carrying out solid-liquid separation to obtain pretreated poplar;
s2) adding deionized water, nutrient salt, adjusting pH and sterilizing to the pretreated poplar to obtain a sterilized poplar mixture;
s3) adding a composite additive, cellulase and yeast into the sterilized poplar mixture to perform synchronous saccharification and fermentation.
In some embodiments, in step S1), the concentration of the aqueous ethanol solution is 50-70% (v/v), and the absolute dry mass of poplar to volume ratio of the aqueous ethanol solution is 1 g: (8-12) mL; the concentration of NaOH is 1.0-1.5%; the pretreatment reaction has the following technological conditions: reacting for 15-25 min at 200-210 ℃, wherein the solid-liquid separation adopts vacuum suction filtration or centrifugal separation.
In some embodiments, in step S2), the ratio of the oven dry mass of the pretreated poplar to deionized water is (3-8): 100.
in some embodiments, in step S2), the nutrient salt comprises a yeast extract, NH 4 Cl、KH 2 PO 4 、MgSO 4 ·7H 2 O, the mass ratio of which is 2:1:1:0.3; the addition amount of the nutrient salt is 4.0-4.5% g/L Water and its preparation method The method comprises the steps of carrying out a first treatment on the surface of the The pH is regulated to 4.6-5.0; the sterilization is carried out in an autoclave at 121 ℃ for 15-25 min.
In some embodiments, in step S3), the composite additive comprises bovine serum albumin, compounded with one or more of PEG 8000, tween 80, tea saponin, calcium lignosulfonate.
In some embodiments, in step S3), the complex additive is selected from the group consisting of a formulation of 25 mg/g PEG 8000 with 75 mg/g bovine serum albumin BSA; the compound additive is selected from the compound of 150 mg/g Tween 80 and 75 mg/g bovine serum albumin BSA; the compound additive is selected from the compound of 150 mg/g tea saponin and 75 mg/g bovine serum albumin BSA; the composite additive is selected from the compound of 25 mg/g calcium lignosulfonate and 75 mg/g bovine serum albumin BSA.
In some embodiments, in step S3), the yeast is a yeast activation solution in an amount of 40 to 60 mL/L Water and its preparation method The method comprises the steps of carrying out a first treatment on the surface of the The process conditions of the synchronous saccharification and fermentation are as follows: fermenting at 32-36 deg.c and rotation speed of 120-140 rpm for not longer than 24 hr.
In some embodiments, in step S3), the method for preparing the yeast activation solution comprises: 1 to 3 of g glucose is weighed and dissolved in 80 to 120 mL deionized water, 6.4 to 6.8 g saccharomyces cerevisiae, 1 to 3 g peptone and 0.5 to 1.5 g yeast extract are added, and the mixture is firstly activated for 8 to 12 minutes at 34 to 38 ℃ on a shaking table at 150 to 170 rpm and then activated for 50 to 70 minutes at 32 to 36 ℃ on the shaking table at 150 to 170 rpm.
In some embodiments, in step S3), the cellulase is used in an amount of 3 to 15FPU/g Insulating intervention treatment of poplar 。
In addition, the invention also provides application of the method in promoting synchronous saccharification and fermentation.
Compared with the prior art, the invention has the following beneficial effects:
according to the method for producing the ethanol by improving the synchronous saccharification and fermentation of the poplar by adopting the composite additive which contains specific components and has specific compound mass ratio, the dosage of cellulase can be obviously reduced, the synchronous saccharification and fermentation time of the poplar (fermentation time is not more than 24 h) can be greatly shortened, the ethanol yield of the synchronous saccharification and fermentation can be obviously improved to 92.70%, the technical support is provided for preparing the liquid fuel ethanol by the poplar, and the method has a good application prospect.
Detailed Description
The invention will be further described with reference to the following specific embodiments, but the examples are not intended to limit the invention in any way. Raw materials reagents used in the examples of the present invention are conventionally purchased raw materials reagents unless otherwise specified.
Part of the starting materials and methods in the following examples:
the poplar raw material is air-dried, thread-rolled and crushed, the grain diameter is less than 1 mm, the component content is 43.08% of cellulose, 13.91% of hemicellulose and 23.65% of lignin.
The preparation method of the yeast activating solution comprises the following steps: 2 g glucose, 2 g peptone and 1 g yeast extract were weighed and dissolved in 100 mL deionized water, 6.6 g yeast powder was added, activated at 160 rpm at 36℃for 10 min on a shaking table, and then activated at 34℃for 60 min on a 160 rpm shaking table.
The ethanol concentration in the synchronous saccharification fermentation liquid is directly measured by adopting a high performance liquid chromatography method: samples of 0.5 mL were taken for measurement after 6 h, 12 h and fermentation endpoint.
The method for testing the ethanol yield in the synchronous saccharification fermentation liquid comprises the following steps:
example 1
A method for producing ethanol by simultaneous saccharification and fermentation of poplar with a composite additive comprises the following steps:
s1) adding 1.2% NaOH into poplar raw materials in a reaction kettle, wherein the absolute mass volume ratio is 1 g:10 Adding 60% (v/v) ethanol water solution into the mL, reacting for 20 min at 205 ℃ for pretreatment, and centrifuging to obtain pretreated poplar;
s2) adding deionized water and nutrient salt into the pretreated poplar, adjusting the pH to 4.8, and placing the poplar into an autoclave for sterilization at 121 ℃ for 20 min to obtain a sterilized poplar mixture; wherein, the mass ratio of the absolute dry mass of the pretreated poplar to the deionized water is 5:100, the addition amount of the nutrient salt is 4.3 g/L Deionized water Yeast extract and NH in nutrient salt 4 Cl、KH 2 PO 4 、MgSO 4 ·7H 2 The mass ratio of O is 2:1:1:0.3;
s3) adding a composite additive and 15FPU/g into the 5 g (absolute dry mass) sterilized poplar mixture Insulating intervention treatment of poplar Cellulase of (A), 50 mL/L Deionized water The yeast activation liquid of (2) is subjected to synchronous saccharification and fermentation on a shaking table at 34 ℃ and 120 rpm; wherein, the composite additive comprises the following two components: 75 mg/g bovine serum albumin BSA and 150 mg/g tea saponin; the bovine serum albumin BSA in the step is a biological protein additive, and is required to be added in an ultra-clean workbench operation after a sample bottle is sterilized; the process conditions of the synchronous saccharification and fermentation are as follows: fermentation was carried out at 34℃and 130 rpm.
The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Example 2
The same method as in example 1 for producing ethanol by simultaneous saccharification and fermentation of poplar with the composite additive is distinguished in that the composite additive added in the simultaneous saccharification and fermentation process is a compound of 150 mg/g Tween 80 and 75 mg/g bovine serum albumin BSA.
The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Example 3
The same method as in example 1 for producing ethanol by simultaneous saccharification and fermentation of poplar with the composite additive is distinguished in that the composite additive added in the simultaneous saccharification and fermentation process is a compound of 25 mg/g PEG 8000 and 75 mg/g bovine serum albumin BSA.
The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Example 4
The same method as in example 1 for producing ethanol by simultaneous saccharification and fermentation of poplar with the composite additive is distinguished in that the composite additive added in the simultaneous saccharification and fermentation process is a compound of 25 mg/g calcium lignosulfonate and 75 mg/g bovine serum albumin BSA.
The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Example 5
The procedure of example 1 was repeated except that the enzyme was used in the simultaneous saccharification and fermentation in an amount of 3 FPU/g. The ethanol concentration and the conversion rate of the ethanol in the fermentation liquid obtained by carrying out 6 h, 12 h and the fermentation end point of the synchronous saccharification and fermentation are shown in the table 1
Example 6
The procedure of example 1 was repeated except that the enzyme was used in an amount of 5FPU/g in the simultaneous saccharification and fermentation process. The ethanol concentration and the conversion rate of the ethanol in the fermentation liquid obtained by carrying out 6 h, 12 h and the fermentation end point of the synchronous saccharification and fermentation are shown in the table 1
Example 7
The procedure of example 1 was repeated except that 8 FPU/g of enzyme was used in the simultaneous saccharification and fermentation. The ethanol concentration and the conversion rate of the ethanol in the fermentation liquid obtained by carrying out 6 h, 12 h and the fermentation end point of the synchronous saccharification and fermentation are shown in the table 1
Example 8
The procedure of example 1 was repeated except that the enzyme was used in the simultaneous saccharification and fermentation in an amount of 10 FPU/g. The ethanol concentration and the conversion rate of the ethanol in the fermentation liquid obtained by carrying out 6 h, 12 h and the fermentation end point of the synchronous saccharification and fermentation are shown in the table 1
Comparative example 1
The procedure of example 1 was repeated except that no additive was added during simultaneous saccharification and fermentation, and that after 6 h, 12 h, 24h and 36 h, 0.5 mL sample was taken at an enzyme dosage of 15FPU/g, and that the ethanol concentrations in the fermentation broths were determined by high performance liquid chromatography and were 7.10, 12.24, 13.33 and 11.97 g/L, respectively. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Comparative example 2
The procedure of example 1 was repeated except that 75 mg/g bovine serum albumin BSA was used as an additive in the simultaneous saccharification and fermentation process. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Comparative example 3
The same procedure as in example 1 was followed except that 150 mg/g Tween 80 was used as an additive in the simultaneous saccharification and fermentation process. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Comparative example 4
The same procedure as in example 1 was repeated except that 150 mg/g tea saponin was used as an additive in the simultaneous saccharification and fermentation. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Comparative example 5
The procedure of example 1 was repeated except that 25 mg/gPEG 8000 was used as the additive in the simultaneous saccharification and fermentation process. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
Comparative example 6
The same procedure as in example 1 was followed except that 25 mg/g calcium lignosulfonate was used as an additive in the simultaneous saccharification and fermentation process. The ethanol concentration and conversion rate of the simultaneous saccharification and fermentation in the fermentation broth obtained by carrying out 6 h, 12 h and the fermentation end point are shown in table 1.
TABLE 1 concentration of ethanol (g/L) and conversion (%)
From the results in table 1, it can be seen that:
(1) As can be seen from comparative examples 1 to 4 and comparative examples 1 to 6, the concentration of fermented ethanol and the conversion rate thereof can be improved by adding the composite additive compounded by the specific components of the invention in the synchronous saccharification and fermentation process under the same other conditions. The addition of BSA in the composite additive can effectively improve the fermentation efficiency compared with other single additives, wherein the best effect is that the composite additive of 75 mg/g BSA and 150 mg/g tea saponin can improve the ethanol concentration to 18.82 g/L at 24h, and the conversion rate is 92.71%; compared with the poplar fermentation without the additive (13.33 g/L) at this time, the conversion rate is increased by 41.19 percent and the conversion rate is increased by 27.04 percent. Other composite additives have good effect without BSA and tea saponin, but can improve the fermentation efficiency in a short time, advance the reaction to be completed by 12 h, and greatly shorten the time required by fermentation.
(2) In addition, as is evident from comparative example 1, examples 5-8 and comparative example 1, poplar samples with the composite additive produced higher ethanol concentrations than without the additive (enzyme amount 15 FPU/g) at enzyme amounts of only 8 FPU/g. The addition of the composite additive can greatly reduce the enzyme dosage while improving the fermentation efficiency.
Therefore, the method for adding the composite additive in the synchronous saccharification and fermentation process can effectively reduce the enzyme dosage and improve the ethanol yield of poplar fermentation, and has good application prospect in the aspect of preparing liquid fuel ethanol from poplar.
The above description of the present invention is further illustrated in detail and should not be taken as limiting the practice of the present invention. It is within the scope of the present invention for those skilled in the art to which the present invention pertains to any modification, equivalent substitution, improvement, etc. that do not depart from the spirit of the invention.
Claims (3)
1. A method for producing ethanol by improving poplar through simultaneous saccharification and fermentation by using a composite additive comprises the following steps:
s1) adding NaOH and an ethanol water solution into a poplar raw material in a reaction kettle, carrying out pretreatment at a certain temperature and for a certain time, and carrying out solid-liquid separation to obtain pretreated poplar, wherein the concentration of the ethanol water solution is 50-70% (v/v), and the volume ratio of the absolute dry mass of the poplar to the ethanol water solution is 1 g: (8-12) mL; the concentration of NaOH is 1.0-1.5%; the pretreatment reaction has the following technological conditions: reacting for 15-25 min at 200-210 ℃, wherein the solid-liquid separation adopts vacuum suction filtration or centrifugal separation;
s2) adding deionized water and nutrient salt into the pretreated poplar, adjusting pH and sterilizing to obtain a sterilized poplar mixture, wherein the mass ratio of the absolute dry mass of the pretreated poplar to the deionized water is (3-8): 100, the nutrient salt comprises yeast extract and NH 4 Cl、KH 2 PO 4 、MgSO 4 ·7H 2 O, the mass ratio of which is 2:1:1:0.3; the addition amount of the nutrient salt is 4.0-4.5% g/L Water and its preparation method The method comprises the steps of carrying out a first treatment on the surface of the The pH is regulated to 4.6-5.0;
s3) adding a composite additive, cellulase and yeast into the sterilized poplar mixture for synchronous saccharification and fermentation, wherein the dosage of the cellulase is 3-15 FPU/g Insulating intervention treatment of poplar The yeast is a yeast activating solution, and the dosage of the yeast activating solution is 40-60 mL/L Water and its preparation method The method comprises the steps of carrying out a first treatment on the surface of the The process conditions of the synchronous saccharification and fermentation are as follows: fermenting at the temperature of 32-36 ℃ and the rotating speed of 120-140 rpm for not more than 24 hours, wherein the composite additive is selected from one of the following compounds:
25 Compounding mg/g PEG 8000 with 75 mg/g bovine serum albumin BSA;
150 Compounding of mg/g Tween 80 and 75 mg/g bovine serum albumin BSA;
150 Compounding of mg/g tea saponin and 75 mg/g bovine serum albumin BSA;
25 Compounding of mg/g calcium Lignosulfonate with 75 mg/g bovine serum Albumin BSA.
2. The method according to claim 1, wherein in step S3), the method for preparing the yeast activation solution comprises: 1 to 3 of g glucose is weighed and dissolved in 80 to 120 mL deionized water, 6.4 to 6.8 g saccharomyces cerevisiae, 1 to 3 g peptone and 0.5 to 1.5 g yeast extract are added, and the mixture is firstly activated for 8 to 12 minutes at 34 to 38 ℃ on a shaking table at 150 to 170 rpm and then activated for 50 to 70 minutes at 32 to 36 ℃ on the shaking table at 150 to 170 rpm.
3. The method according to claim 1, wherein in step S3), the sterilization is performed in an autoclave at 121 ℃ for 15-25 min.
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| CN113151366A (en) * | 2021-06-04 | 2021-07-23 | 华南农业大学 | Method for improving poplar fermentation efficiency by dilute alkali and ethanol pretreatment |
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| CN107177636A (en) * | 2017-05-17 | 2017-09-19 | 吉林大学 | The method that one primary yeast high density fermentation coupling simultaneous saccharification and fermentation produces ethanol |
| CN107142297A (en) * | 2017-07-18 | 2017-09-08 | 天津科技大学 | A kind of synchronous saccharification common fermentation method |
| CN113106127A (en) * | 2021-05-27 | 2021-07-13 | 华南农业大学 | Method for improving yield of ethanol produced by synchronous saccharification and fermentation of poplar |
| CN113151366A (en) * | 2021-06-04 | 2021-07-23 | 华南农业大学 | Method for improving poplar fermentation efficiency by dilute alkali and ethanol pretreatment |
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