CN117025713A - Method for preparing ethanol by composite bacterial system - Google Patents

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

A method for preparing ethanol with composite bacteria

Technical field

The present invention relates to the field of fermentation technology, and in particular to a method for preparing ethanol from a composite bacterial system.

Background technique

Bioethanol is one of many bioenergy types that has lower greenhouse gas emission properties and is the primary renewable fuel today. In the early days, it was usually produced by fermentation of sugar-based raw materials such as corn starch and cane sugar. The production of fuels from sugar-based feedstocks poses a threat to the food supply due to competition with food crops. This has resulted in a transition from first to second generation biofuels. Waste lignocellulosic biomass from food crops can serve as a potential resource for bioethanol production. Since abundant lignocellulosic biomass is renewable, such as wheat straw, rice straw, sugarcane bagasse, corn cobs and cotton straw, it can be used to produce bioethanol. According to current FAOStat 2022 estimates, all crops generate 3.71 × ¹⁰ kg of nutrients per year in waste, and agricultural waste production is expected to increase by more than 50% by 2050, with the burning of biomass resulting in the production of nearly 3.67 × 10 ¹³ g of CO2 equivalent emissions, which, according to calculations, could produce nearly 2.46 × 10 ¹⁰ liters of bioethanol per year.

The residue after harvesting the edible parts of crops is rich in lignocellulose and can play a large role in bioethanol production. Trees are also a potential source of energy, and they continue to grow after being cut to the ground, which is the biggest advantage of bioethanol production. The amount of wood residue produced in forested areas around the world is enormous. The biomass potential types are estimated at 33-39 exajoules/year for energy crops, 13-15 exajoules/year for wood residues, 13-30 exajoules/year for harvest residues, and 5-19 exajoules/year for firewood /Year. The production of bioethanol has increased worldwide over the past few years as awareness and concern for the environment have increased. According to the Renewable Fuels Association (RFA, USA), global ethanol production exceeded 7.57 × ^{10 liters} in 2009, with the major producing countries, namely the United States and Brazil, accounting for 86% of the total share. Currently, most bioethanol production uses sugarcane juice and corn starch as substrates. Currently the largest bioethanol producer in the world is the United States.

However, in current biological ethanol production methods, enzyme production and saccharification efficiency are generally low, which is not conducive to ultimately increasing the yield of ethanol.

Therefore, in order to address the above shortcomings, it is necessary to provide a new method for producing ethanol by bacterial strains.

Contents of the invention

The technical problems to be solved by the present invention are that the efficiency of enzyme production and saccharification in the ethanol production process of the existing composite bacteria is low, the pretreatment cost is high, and the pollution is heavy. In view of the defects in the existing technology, a method for preparing ethanol by the composite bacteria is provided. method.

In order to solve the above technical problems, the present invention provides a method for preparing ethanol by a composite bacterial system, which method includes the following steps:

(1) Perform shaker fermentation on a composite bacterial strain composed of at least 4 strains of Bacillus spp. SPY1, SPY2, BB1, BB2, BW1, BW2 or CW to produce enzymes to obtain an enzyme liquid;

(2) Adjust the pH value of the enzyme liquid obtained in step (1) to acidity, add it to the sugar-producing culture medium, and hydrolyze and saccharify the substrate to obtain a sugar liquid;

(3) Ferment Saccharomyces cerevisiae using the sugar solution obtained in step (2) as a substrate to obtain ethanol.

SPY1 belongs to Bacterium S154, accession number: MZ461741.1; SPY2 is Lysinibacillus macroides XZMYA-3, accession number: MF170826.1; BB1 is Bacillus siamensis KCTC 13613 ^T , Registration number: AJVF01000043; BB2 is Bacillus thuringiensis BPR162, registration number: KU161299.1; BW1 is Bacillus sp.CZGRY5, registration number: KJ184854.1; BW2 is Bacillus pumilus MGB05, registration number: KP298708 .2; CW belongs to Bacillus sp.CO-3, registration number: MG371987.1.

In the present invention, a lignocellulose-degrading composite bacterial strain is constructed by screening 7 strains of cellulose-degrading bacteria, and at the same time, the preparation of ethanol is achieved through the processes of fermentation and saccharification. When the substrate in the present invention is enzymatically hydrolyzed, compared with the use of single bacteria and the composite bacterial system constructed using all 7 strains of bacteria, it can produce higher cellulase enzymatic activity. After treatment, the molecular structure changes, resulting in the production of ethanol during the production of ethanol. The efficiency of enzymes and saccharification reaches a high level.

Preferably, the bacterial strain described in step (1) is a composite bacterial strain composed of SPY1, BBl, BB2 and BW2.

In the present invention, the composite bacterial strain composed of the above four bacterial strains can be used to achieve a higher level of enzyme production efficiency and saccharification efficiency.

Preferably, the shaking speed of the shaking table fermentation in step (1) is 120 to 180 rpm, for example, it can be 120 rpm, 140 rpm, 160 rpm or 180 rpm, etc.; the temperature of the shaking table fermentation is 25 to 50°C, for example, it can be 25°C. , 30℃, 35℃, 40℃, 45℃ or 50℃, etc.; the inoculum amount of the bacterial strain is 5% to 10% (v/v), for example, it can be 5%, 6%, 7%, 8%, 9 % or 10% etc.

Preferably, the shaker rotation speed of the shaker fermentation described in step (1) is 165 rpm, the temperature is 34°C, and the inoculum amount of the bacterial strain is 7.5%.

Preferably, the pH value adjusted to be acidic in step (2) is 4.8.

Preferably, the composition of the sugar-producing culture 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, calcium carbonate. 0.02g/L.

Preferably, the shaking speed of the hydrolysis and saccharification process in step (2) is 125 to 175 rpm, for example, it can be 125 rpm, 130 rpm, 135 rpm, 140 rpm, 145 rpm, 150 rpm, 155 rpm, 160 rpm, 165 rpm, 170 rpm or 175 rpm, etc.; the temperature is 50～60℃, for example, it can be 50℃, 52℃, 55℃, 56℃, 57℃, 58℃, 59℃ or 60℃, etc.; the pH value is 5.5～7.5, for example, it can be 5.5, 6, 6.5, 7 Or 7.5 etc.

Preferably, the shaking speed of the hydrolysis and saccharification process in step (2) is 162 rpm, the temperature is 56.3°C, and the pH value is 6.09.

In the present invention, the substrate is straw, specifically corn straw, etc.

Preferably, the fermentation temperature in step (3) is 20-40°C, for example, it can be 20°C, 25°C, 30°C, 35°C or 40°C, etc.; the time is 30-50h, for example, it can be 30h, 35h , 40h, 45h or 50h, etc.; the pH value is 6 to 8, for example, it can be 6, 6.5, 7, 7.5 or 8, etc.

Preferably, the fermentation temperature in step (3) is 30°C, the time is 48 hours, and the pH value is 7.2.

Implementing the present invention has the following beneficial effects:

(1) The composite bacterial strain provided by the present invention produces enzymes. The optimal shaker speed is 165 rpm, the optimal temperature is 34°C, and the optimal inoculum amount is 7.5%.

(2) When the present invention uses corn straw as the substrate, the optimal sugar production conditions are shaker speed of 162.21 rpm, temperature of 56.27°C, and pH=6.09. Under these conditions, the sugar production rate can reach up to 24.34%.

(3) For the composite bacterial strain provided by the invention, when corn straw is used as the fermentation substrate, the optimal temperature is 30°C, 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 microbial pretreatment and preparation of bioethanol, has high ethanol production efficiency, and is suitable for further industrial production.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not All examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without any creative work fall within the scope of protection of the present invention.

Example 1

This embodiment provides a method for preparing ethanol from a composite bacterial system

(1) Directly mix SPY1, BB1, BB2, and BW2 to construct a composite bacterial strain, and perform shaker fermentation. The shaker speed is 165 rpm, the temperature is 34°C, and the inoculum amount of the bacterial strain is 7.5% to obtain the enzyme liquid after fermentation. Among them, the filter paper enzyme activity (FPA) enzyme activity is 6.85U/mL;

(2) Centrifuge the enzyme solution obtained in step (1) for 5 minutes at 8000 rpm to obtain the crude enzyme solution, adjust the pH to 4.8, and add 100 mL to the sugar-producing culture medium (with corn straw as substrate, 0.5 g of straw powder /L, ammonium sulfate 0.05g/L, potassium dihydrogen sulfate 0.02g/L, magnesium sulfate 0.01g/L, calcium carbonate 0.02g/L) to carry out the hydrolysis and saccharification process. The sugar production conditions are shaker speed 162rpm and temperature 56.3 ℃, pH is 6.09, and a sugar liquid is obtained with a sugar production rate of 24.34%, in which the sugar production rate is the amount of reducing sugar produced per unit mass of straw powder;

(3) Ferment the sugar liquid obtained in step (2) with Saccharomyces cerevisiae. The fermentation temperature is 30°C, the fermentation time is 48h, and the fermentation pH value is 7.2 to obtain ethanol. The yield is 6.78%, where the yield is unit The amount of ethanol produced from the mass of straw.

Referring to the steps of Example 1, keeping the hydrolysis, saccharification and fermentation conditions of steps (2) and (3) unchanged, changing the fermentation conditions in step (1), the following Examples 2-18 are obtained, as shown in Table 1 below. , while measuring FPA enzyme activity. The determination method is as follows: Take 0.5 mL of crude enzyme solution with an appropriate dilution ratio in a 20 mL test tube, add 1.5 mL of 0.05 mol/L citric acid buffer with pH=5.0, and 50 mg of defatted filter paper strips, and then place it in a constant-temperature oscillator for 50 ℃ constant temperature water bath for 30 minutes; add 3.0 mL of DNS reagent, take out the boiling water bath for 5 minutes, quickly cool to room temperature, dilute to 10.0 mL, and set 3 replicates for each strain. The absorbance value at the wavelength of 540nm was measured using a Multiskan FC microplate reader for the treatment solutions, and the activities of the four enzymes were calculated based on the glucose standard curve. The calculation formula for enzyme activity is E=1000×S×N/T×V, where E is the enzyme activity of the sample, U/mL; S is the glucose content corresponding to the average absorbance value of the sample on the standard curve, mg; N is the crude The dilution factor of the enzyme solution; 1000 is the conversion factor 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 steps of Example 2, keeping the hydrolysis, saccharification and fermentation conditions of steps (1) and (3) unchanged, changing the fermentation conditions in step (2), the following examples 19-35 are obtained, as shown in Table 2 below. , and the sugar production rate was measured at the same time.

Table 2

Example A: Shaker speed/rpm B: Temperature C:pH Sugar production rate/% 19 150 50 5.5 9.21 20 125 55 5.5 6.10 twenty one 175 55 7.5 6.59 twenty two 175 60 6.5 18.81 twenty three 150 55 6.5 19.60 twenty four 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

It can be known from the experimental data of Examples 1-35:

The shaker speed is positively correlated with the dissolved oxygen content, and the dissolved oxygen content can also reflect the growth of bacteria. When the shaking is too slow, the dissolved oxygen content in the fermentation system is not high enough, the materials are mixed unevenly, and the composite bacteria cannot fully utilize the nutrients in the environment for growth and reproduction, thus resulting in low enzyme activity; when the shaking speed is too high, At this time, the dissolved oxygen increases significantly and produces a large amount of metabolites, such as organic acids, which will reduce the pH in the environment and greatly reduce the growth and reproduction of microorganisms, thus affecting the enzyme production process of the composite bacteria.

Temperature affects the life of microorganisms mainly by affecting the fluidity of microbial cell membranes and the activity of biological macromolecules. As temperature increases, the rate of intracellular enzymatic reactions increases, resulting in increased cell metabolism and growth. Studies have found that as the culture temperature increases, the number of viable microorganisms first increases. However, once the temperature is too high, the bioactive substances will denature, leading to a decrease in cell function or even death. Therefore, temperature is one of the most important factors during enzyme production. In the process of enzymatic sugar production, temperature not only affects the enzymatic reaction, but also affects cellulase activity. Generally speaking, when the temperature is increased to a certain range, the enzyme activity will speed up. Enzyme-catalyzed reactions, like most chemical reactions, proceed at a faster rate as the temperature increases.

Excessive inoculation amount will make the bacterial density too high, resulting in insufficient nutrients and dissolved oxygen in the culture medium, which will ultimately inhibit the growth of microorganisms and reduce the enzyme-producing ability of the composite bacteria. When the inoculum amount is insufficient, the microorganisms will grow slowly and lack sufficient In order to achieve the highest yield of cellulase production, the appropriate inoculation amount is conducive to the use of dissolved oxygen and nutrients in the culture medium by the composite bacteria, thereby achieving the maximum yield of cellulase.

Changes in the initial pH value may cause the inactivation of cellulase and xylanase activities, or cause dissociation between the substrate and the active site of the enzyme, allowing the enzyme-catalyzed hydrolysis reaction to reach the maximum activity of the enzyme. When the pH When the value is above or below 6.0, the enzymatic reaction will be reduced, and different strains will adapt to slightly different pH.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

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 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.

2. The method according to claim 1, characterized in that: the strain in the step (1) is a composite strain system consisting of SPY1, BB2 and BW 2.

3. 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%.

4. A method according to claim 3, 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%.

5. 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.

6. 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.

7. 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.

8. The method according to claim 7, 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.

9. 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.

10. The method according to claim 9, 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.