CN117025713B - Method for preparing ethanol by composite bacterial system - Google Patents
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 26
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- 108090000790 Enzymes Proteins 0.000 claims abstract description 37
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- 238000004519 manufacturing process Methods 0.000 claims abstract description 23
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 11
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- 101710151560 Speedy protein A Proteins 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 5
- 230000002378 acidificating effect Effects 0.000 claims abstract description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims abstract description 4
- 230000001105 regulatory effect Effects 0.000 claims abstract description 4
- 239000010902 straw Substances 0.000 claims description 13
- 238000011081 inoculation Methods 0.000 claims description 8
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- ILRLTAZWFOQHRT-UHFFFAOYSA-N potassium;sulfuric acid Chemical compound [K].OS(O)(=O)=O ILRLTAZWFOQHRT-UHFFFAOYSA-N 0.000 claims description 3
- 241000194103 Bacillus pumilus Species 0.000 claims description 2
- 241000505551 Bacillus siamensis KCTC 13613 Species 0.000 claims description 2
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- 239000002609 medium Substances 0.000 claims description 2
- 241000894006 Bacteria Species 0.000 abstract description 10
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- 239000000243 solution Substances 0.000 description 16
- 230000000694 effects Effects 0.000 description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 6
- 244000005700 microbiome Species 0.000 description 6
- 229910052760 oxygen Inorganic materials 0.000 description 6
- 239000001301 oxygen Substances 0.000 description 6
- 108010059892 Cellulase Proteins 0.000 description 5
- 240000008042 Zea mays Species 0.000 description 5
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 5
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 5
- 229940106157 cellulase Drugs 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 235000005822 corn Nutrition 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000006911 enzymatic reaction Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
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- 241000609240 Ambelania acida Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 241001671705 Bacillus sp. CZGRY5 Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 241001468261 Lysinibacillus macroides Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
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- 239000002154 agricultural waste Substances 0.000 description 1
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- 229960004793 sucrose Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P39/00—Processes involving microorganisms of different genera in the same process, simultaneously
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
- C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
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- C12R2001/00—Microorganisms ; Processes using microorganisms
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- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
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Abstract
The invention relates to the technical field of fermentation, in particular to a method for preparing ethanol by a composite bacterial system, which comprises the following steps: (1) Performing shaking table fermentation on a composite bacterial system consisting of at least 4 strains in SPY1, SPY2, BB1, BB2, BW1, BW2 or CW bacillus to produce enzyme to obtain an enzyme solution; (2) Regulating the pH value of the enzyme solution obtained in the step (1) to be acidic, adding the enzyme solution into a sugar production culture medium, and carrying out hydrolysis saccharification on a substrate to obtain a sugar solution; (3) And (3) fermenting the saccharomyces cerevisiae by taking the sugar solution obtained in the step (2) as a substrate to obtain the ethanol. In the invention, a lignocellulose degrading composite bacterial system is constructed by screening 7 cellulose degrading bacteria, and meanwhile, the preparation of ethanol is realized through the fermentation and saccharification processes.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to a method for preparing ethanol by a composite bacterial system.
Background
Bioethanol is one of many bioenergy types, with less greenhouse gas emissions, and is the primary renewable fuel today. Early, it was typically produced by fermentation of sugar-based materials such as corn starch and cane sugar. The production of fuels from sugar-based raw materials poses a threat to the supply of food due to competition with food crops. This results in a transition from the first generation biofuel to the second generation biofuel. Waste lignocellulosic biomass from food crops can be used as a potential resource for bioethanol production. Since the rich lignocellulosic biomass is renewable, such as wheat straw, rice straw, bagasse, corn cobs, and cotton straw, it can be used to produce bioethanol. According to the estimation of FAOStat 2022, all crops produce 3.71×10 wastes each year 10 Kg of nutrients, it is expected that the agricultural waste yield will increase by more than 50% by 2050, and biomass combustion will result in approximately 3.67 x 10 13 Gram equivalent carbon dioxide emissions, which, according to calculations, can produce approximately 2.46 x 10 per year 10 Ascending bioethanol.
The residue of the edible part obtained by harvesting crops contains rich lignocellulose and can play a great role in bioethanol production. Trees are also potential sources of energy, and they continue to grow after being cut into the ground, which is the greatest advantage of bioethanol production. The amount of wood residues produced in forest areas around the world is enormous. It is estimated that the biomass potential type of the energy crops is estimated to be 33-39 Ai Jiao/year, the wood residues 13-15 Ai Jiao/year, the harvest residues 13-30 Ai Jiao/year, and the firewood 5-19 Ai Jiao/year. With the increasing awareness and concern over the environment, worldwide bioethanol production has increased over the past few years. According to renewable fuel Association (RFA, USA) data, 2009 global ethanol production exceeded 7.7X10 10 Ascending mainly and generatingThe country of origin, us and brazil, represents 86% of the total share. Currently, bioethanol is mostly produced using sugarcane juice and corn starch as substrates. The largest bioethanol producing country in the world is currently the united states.
However, in the current biological method for producing ethanol, the enzyme production and saccharification efficiency are generally low, which is unfavorable for finally improving the yield of ethanol.
Accordingly, in order to solve the above-mentioned drawbacks, a new method for producing ethanol by using a bacterial strain has been desired.
Disclosure of Invention
The invention aims to solve the technical problems that the existing composite bacteria system has low enzyme production and saccharification efficiency in the ethanol production process, high pretreatment cost and heavy pollution, and provides a method for preparing ethanol by the composite bacteria system aiming at the defects in the prior art.
In order to solve the technical problems, the invention provides a method for preparing ethanol by a composite bacterial system, which comprises the following steps:
(1) Performing shaking table fermentation on a composite bacterial system consisting of at least 4 strains in SPY1, SPY2, BB1, BB2, BW1, BW2 or CW bacillus to produce enzyme to obtain an enzyme solution;
(2) Regulating the pH value of the enzyme solution obtained in the step (1) to be acidic, adding the enzyme solution into a sugar production culture medium, and carrying out hydrolysis saccharification on a substrate to obtain a sugar solution;
(3) And (3) fermenting the saccharomyces cerevisiae by taking the sugar solution obtained in the step (2) as a substrate to obtain the ethanol.
SPY1 genus bacillus S154, accession no: MZ461741.1; SPY2 is Bacillus tenuius, lysinibacillus macroides XZMYA-3, accession number: MF170826.1; BB1 is Siamese bacillus Bacillus siamensis KCTC 13613 T Accession number: AJVF01000043; BB2 is Bacillus thuringiensis Bacillus thuringiensis BPR162, accession number: KU161299.1; BW1 Bacillus sp.CZGRY5 accession: KJ184854.1; BW2 is Bacillus pumilus Bacillus pumilus MGB05, accession number: KP298708.2; bacillus sp.CO-3, accession No.: MG371987.1.
In the invention, a lignocellulose degrading composite bacterial system is constructed by screening 7 cellulose degrading bacteria, and meanwhile, the preparation of ethanol is realized through the fermentation and saccharification processes. When the substrate is subjected to enzymolysis, compared with a composite bacterial system constructed by single bacteria and all 7 bacteria, the substrate can generate higher enzyme activity of cellulase, and the treated molecular structure changes, so that the enzyme production and saccharification efficiency in the ethanol production process reaches higher level.
Preferably, the strain in the step (1) is a composite strain composed of SPY1, BB2 and BW 2.
In the invention, the compound bacterial system composed of the 4 strains is selected, so that the enzyme production efficiency and saccharification efficiency can reach higher level.
Preferably, the rotation speed of the shaking table in the shaking table fermentation in the step (1) is 120-180 rpm, for example, 120rpm, 140rpm, 160rpm or 180rpm, etc.; the temperature of the shaking table fermentation is 25-50deg.C, such as 25deg.C, 30deg.C, 35deg.C, 40deg.C, 45deg.C or 50deg.C; the inoculation amount of the bacterial system is 5% to 10% (v/v), and may be, for example, 5%, 6%, 7%, 8%, 9% or 10%.
Preferably, the shaking table rotation speed of the shaking table fermentation in the step (1) is 165rpm, the temperature is 34 ℃, and the inoculation amount of the bacterial system is 7.5%.
Preferably, the pH adjusted to be acidic in step (2) is 4.8.
Preferably, the composition of the sugar-producing medium in step (2) is: straw powder 0.5g/L, ammonium sulfate 0.05g/L, potassium dihydrogen sulfate 0.02g/L, magnesium sulfate 0.01g/L and calcium carbonate 0.02g/L.
Preferably, the rotation speed of the shaking table in the hydrolysis saccharification process in the step (2) is 125-175 rpm, for example, 125rpm, 130rpm, 135rpm, 140rpm, 145rpm, 150rpm, 155rpm, 160rpm, 165rpm, 170rpm or 175rpm, etc.; the temperature is 50 to 60℃and may be, for example, 50℃52℃55℃56℃57℃58℃59℃or 60℃or the like; the pH is 5.5 to 7.5, and may be, for example, 5.5, 6, 6.5, 7 or 7.5.
Preferably, the rotation speed of the shaking table in the hydrolysis saccharification process in the step (2) is 162rpm, and the temperature is 56.3 ℃; the pH was 6.09.
In the invention, the substrate is straw, and can be corn straw and the like.
Preferably, the fermentation in step (3) is carried out at a temperature of 20 to 40 ℃, for example, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃ or the like; the time is 30 to 50 hours, for example, 30 hours, 35 hours, 40 hours, 45 hours or 50 hours, etc.; the pH is 6 to 8, and may be, for example, 6, 6.5, 7, 7.5 or 8.
Preferably, the fermentation in step (3) is carried out at a temperature of 30℃for a period of 48 hours and at a pH of 7.2.
The implementation of the invention has the following beneficial effects:
(1) The compound bacteria enzyme provided by the invention has the optimal rotation speed of a shaking table of 165rpm, the optimal temperature of 34 ℃ and the optimal inoculation amount of 7.5%.
(2) When corn straw is used as a substrate, the optimal sugar production condition is that the rotation speed of a shaking table is 162.21rpm, the temperature is 56.27 ℃, the pH=6.09, and the sugar production rate can reach 24.34% at the highest under the condition.
(3) When the composite bacterial system provided by the invention uses corn straw as a fermentation substrate, the optimal temperature is 30 ℃, the optimal fermentation time is 48 hours, the optimal pH is 7.2, and the highest ethanol yield can reach 6.78%.
The composite bacterial system provided by the invention realizes the pretreatment of microorganisms and the preparation of bioethanol, has high ethanol production efficiency, and is suitable for further industrial production.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides a method for preparing ethanol by using a composite bacterial system
(1) Directly mixing SPY1, BB2 and BW2 to construct a composite bacterial system, carrying out shaking table fermentation, wherein the rotation speed of the shaking table is 165rpm, the temperature is 34 ℃, the inoculum size of the bacterial system is 7.5%, and obtaining fermented enzyme liquid, and the enzyme activity of Filter Paper (FPA) is 6.85U/mL;
(2) Centrifuging the enzyme solution obtained in the step (1) for 5min at 8000rpm to obtain crude enzyme solution, regulating the pH to 4.8, adding 100mL into a sugar production culture medium (taking corn straw as a substrate, 0.5g/L of straw powder, 0.05g/L of ammonium sulfate, 0.02g/L of potassium dihydrogen sulfate, 0.01g/L of magnesium sulfate and 0.02g/L of calcium carbonate), and performing hydrolysis saccharification under the conditions that the rotation speed of a shaking table is 162rpm, the temperature is 56.3 ℃ and the pH is 6.09 to obtain sugar solution, wherein the sugar yield is 24.34 percent, and the sugar yield is the amount of reducing sugar produced by unit mass of straw powder;
(3) And (3) fermenting the sugar solution obtained in the step (2) by using saccharomyces cerevisiae, wherein the fermentation temperature is 30 ℃, the fermentation time is 48 hours, the fermentation pH value is 7.2, and the ethanol is obtained, the yield is 6.78%, and the yield is the amount of ethanol produced by straw per unit mass.
Referring to the procedure of example 1, while maintaining the hydrolysis saccharification and fermentation conditions of step (2) and step (3) unchanged, the fermentation conditions in step (1) were changed to obtain the following examples 2 to 18, specifically as shown in Table 1 below, while measuring the FPA enzyme activity. The measurement method is as follows: taking a proper amount of crude enzyme solution with dilution multiple of 0.5mL in a 20mL test tube, adding 1.5mL of 0.05mol/L citric acid buffer solution with pH=5.0 and 50mg of degreasing filter paper strips, and then placing the mixture in a constant-temperature oscillator for 30min in a constant-temperature water bath with the temperature of 50 ℃; adding 3.0mL of DNS reagent, taking out after boiling water bath for 5min, rapidly cooling to room temperature, fixing volume to 10.0mL, and setting 3 repeats for each strain. The absorbance value of the treatment liquid at the wavelength of 540nm is measured by a Multiskan FC enzyme label instrument, and 4 enzyme activities are converted according to a glucose standard curve. The enzyme activity calculation formula is E=1000×S×N/T×V, wherein E is the enzyme activity of the sample, and U/mL; s is the glucose content, mg, corresponding to the average absorbance value of the sample on a standard curve; n is the dilution multiple of the crude enzyme solution; 1000 is a conversion multiple between mg and μg; t is the reaction time, min; v is the volume of crude enzyme solution participating in the reaction, mL.
TABLE 1
Referring to the procedure of example 2, while maintaining the hydrolysis saccharification and fermentation conditions of step (1) and step (3) unchanged, the conditions of fermentation in step (2) were changed to obtain the following examples 19 to 35, specifically as shown in Table 2 below, while measuring the sugar yield.
TABLE 2
| Examples | A: rotation speed/rpm of shaking table | B: temperature (temperature) | C：pH | Sugar yield/% |
| 19 | 150 | 50 | 5.5 | 9.21 |
| 20 | 125 | 55 | 5.5 | 6.10 |
| 21 | 175 | 55 | 7.5 | 6.59 |
| 22 | 175 | 60 | 6.5 | 18.81 |
| 23 | 150 | 55 | 6.5 | 19.60 |
| 24 | 150 | 60 | 7.5 | 9.43 |
| 25 | 125 | 50 | 6.5 | 10.31 |
| 26 | 125 | 55 | 7.5 | 9.01 |
| 27 | 175 | 55 | 5.5 | 22.57 |
| 28 | 150 | 55 | 6.5 | 22.78 |
| 29 | 150 | 50 | 7.5 | 15.16 |
| 30 | 150 | 60 | 5.5 | 22.32 |
| 31 | 125 | 60 | 6.5 | 7.72 |
| 32 | 150 | 55 | 6.5 | 22.02 |
| 33 | 150 | 55 | 6.5 | 21.71 |
| 34 | 175 | 50 | 6.5 | 6.41 |
| 35 | 150 | 55 | 6.5 | 23.69 |
From the experimental data of examples 1-35, it can be seen that:
the rotation speed of the shaking table is positively correlated with the dissolved oxygen content, and the dissolved oxygen content can reflect the growth of bacteria. When shaking is too slow, the content of dissolved oxygen in the fermentation system is not high enough, the substances are not uniformly mixed, and the compound bacteria system cannot fully utilize the nutrition in the environment for growth and propagation, so that the enzyme activity is low; however, when the rotation speed of the shaking table is too high, the dissolved oxygen is obviously increased, a large amount of metabolites such as organic acid are generated, the pH in the environment is reduced, and the growth and propagation of microorganisms are greatly reduced, so that the enzyme production process of the compound bacterial system is affected.
Temperature affects the life of microorganisms mainly by affecting the mobility of the microbial cell membrane and the activity of biological macromolecules. As the temperature increases, the rate of intracellular enzymatic reactions increases, resulting in increased metabolism and growth of the cells. It was found that as the culture temperature increases, the number of viable microorganisms increases first, however, once the temperature is too high, the biologically active substance is denatured, leading to a decrease in cell function and even death. Therefore, temperature is one of the most important factors in the enzyme production process. In the process of producing sugar by enzymolysis, the temperature not only affects the enzymatic reaction, but also affects the activity of cellulase. In general, when the temperature is raised to a certain range, the enzyme activity is accelerated. Enzymatic reactions, like most chemical reactions, proceed at a faster rate as the temperature increases.
The excessive inoculation amount leads to the excessive bacterial density, which leads to insufficient nutrient substances and dissolved oxygen in the culture medium, and finally can inhibit the growth of microorganisms, thereby reducing the enzyme production capacity of the compound bacteria system; when the inoculation amount is insufficient, the microorganism grows slowly and is insufficient to achieve the highest yield of the cellulase, so that the proper inoculation amount is beneficial to the utilization of dissolved oxygen and nutrient substances in the culture medium by the composite bacterial system, thereby achieving the highest yield of the cellulase.
The initial pH change may result in failure of the cellulase and xylanase activities or may result in dissociation between the substrate and the enzyme active site, resulting in maximum enzyme activity for the enzyme-catalyzed hydrolysis reaction, which may be reduced when the pH is higher or lower than 6.0, and may be adapted to slightly different pH values relative to different strains.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for preparing ethanol by a composite bacterial system is characterized by comprising the following steps: the method comprises the following steps:
(1) Performing shaking table fermentation on a composite bacterial system consisting of SPY1, BB2 and BW2 to produce enzyme to obtain enzyme solution;
(2) Regulating the pH value of the enzyme solution obtained in the step (1) to be acidic, adding the enzyme solution into a sugar production culture medium, and carrying out hydrolysis saccharification on a substrate to obtain a sugar solution;
(3) Fermenting Saccharomyces cerevisiae with the sugar solution obtained in the step (2) as a substrate to obtain ethanol;
wherein, SPY1 genus bacillus S154, accession number:MZ461741.1; BB1 is Siamese bacillus Bacillus siamensis KCTC 13613 T Accession number: AJVF01000043; BB2 is Bacillus thuringiensis Bacillus thuringiensis BPR162, accession number: KU161299.1; BW2 is Bacillus pumilus Bacillus pumilus MGB05, accession number: KP298708.2.
2. The method according to claim 1, characterized in that: the rotation speed of the shaking table for shaking table fermentation in the step (1) is 120-180 rpm, and the temperature is 25-50 ℃; the inoculation amount of the bacterial system is 5% -10%.
3. The method according to claim 2, characterized in that: the rotation speed of the shaking table in the shaking table fermentation in the step (1) is 165rpm, the temperature is 34 ℃, and the inoculation amount of the bacterial system is 7.5%.
4. The method according to claim 1, characterized in that: the pH value of the step (2) is adjusted to be acidic, and the pH value is 4.8.
5. The method according to claim 1, characterized in that: the composition of the sugar-producing medium in the step (2) is as follows: straw powder 0.5g/L, ammonium sulfate 0.05g/L, potassium dihydrogen sulfate 0.02g/L, magnesium sulfate 0.01g/L and calcium carbonate 0.02g/L.
6. The method according to claim 1, characterized in that: the rotation speed of the shaking table in the hydrolysis saccharification process in the step (2) is 125-175 rpm, the temperature is 50-60 ℃, and the pH value is 5.5-7.5.
7. The method according to claim 6, wherein: the rotation speed of a shaking table in the hydrolysis and saccharification process in the step (2) is 162rpm, and the temperature is 56.3 ℃; the pH was 6.09.
8. The method according to claim 1, characterized in that: the fermentation temperature in the step (3) is 20-40 ℃, the time is 30-50 h, and the pH value is 6-8.
9. The method according to claim 8, wherein: the fermentation in the step (3) is carried out at a temperature of 30 ℃ for 48 hours and at a pH value of 7.2.
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