阅读说明：本技术 一种以秸秆水解液为原料发酵生产abe的方法 (Method for producing ABE by fermenting straw hydrolysate serving as raw material ) 是由 郜晓峰 苏亚蕊 贺洁 马芳园 王深垒 于 2019-10-23 设计创作，主要内容包括：本申请属于生物质能源制备技术领域,具体涉及一种以秸秆水解液为原料发酵生产ABE的方法。具体包括：秸秆预处理、生物酶解、制备ABE发酵培养液、发酵水解液等步骤。本申请中,在以秸秆酸水解液为原料发酵丁醇时,配合特定微生物菌株发酵处理时,通过添加谷胱甘肽后可显著影响溶剂代谢途径中相关关键酶表达量的变化,进而改变了菌株细胞膜的化学成分,提高了产丁醇能力。本申请将秸秆预处理制成水解液后直接发酵生产ABE,避开了复杂的脱毒工艺,从而有利于降低生产成本和促进ABE发酵生产的产业化；进一步通过发酵工艺的优化,尤其是通过发酵过程中谷胱甘肽的添加和利用,进一步提高了ABE的发酵产率,有助于促进ABE工业化生产。(The application belongs to the technical field of biomass energy preparation, and particularly relates to a method for producing ABE by fermenting straw hydrolysate serving as a raw material. The method specifically comprises the following steps: straw pretreatment, biological enzymolysis, ABE fermentation culture solution preparation, hydrolysate fermentation and the like. According to the application, when the straw acid hydrolysate is used as the raw material for fermenting the butanol and is matched with a specific microbial strain for fermentation treatment, the change of related key enzyme expression quantity in a solvent metabolic pathway can be obviously influenced by adding the glutathione, so that the chemical components of cell membranes of the strain are changed, and the butanol production capacity is improved. According to the method, the ABE is directly fermented and produced after the straws are pretreated to prepare the hydrolysate, so that a complex detoxification process is avoided, and the production cost is reduced and the industrialization of ABE fermentation production is promoted; further, the fermentation yield of the ABE is further improved by optimizing the fermentation process, particularly by adding and utilizing glutathione in the fermentation process, and the promotion of the industrial production of the ABE is facilitated.)

1. A method for producing ABE by fermenting straw hydrolysate serving as a raw material is characterized by comprising the following steps:

preparation of straw hydrolysate

When preparing the straw hydrolysate, the method comprises the steps of straw pretreatment and biological enzymolysis, and specifically comprises the following steps:

(1) pretreatment of straw

Crushing straws, mixing the crushed straws with sulfuric acid uniformly, performing high-temperature sterilization treatment, and then adjusting the pH to be =4 ~ 6;

(2) biological enzymolysis

Adding biological enzyme into the sterilized straw mixture obtained in the step (1), uniformly mixing, performing enzymolysis treatment at 40 ~ 60 ℃ for 24 ~ 48h, and centrifuging to remove solid precipitates, wherein the supernatant is the straw hydrolysate to be fermented;

the biological enzyme is a mixture of cellulase, xylanase and beta-1, 4-mannase, the specific dosage is 40 ~ 60 muL of cellulase, 40 ~ 60 muL of xylanase and 40 ~ 60 mug of beta-1, 4-mannase in every 100mL of straw hydrolysate, and the specific enzyme activity requirement is as follows:

the standard enzyme activity of cellulose is more than 1 multiplied by 10⁵u/mL; the standard enzyme activity of xylanase is more than 1 multiplied by 10⁸U/mL; the standard enzyme activity of beta-1, 4-mannase is more than 5 multiplied by 10⁴U/g；

(II) preparing ABE fermentation culture solution

Using clostridium acetobutylicum or clostridium beijerinckii as a fermentation strain to prepare seed liquid;

(III) fermentation of the hydrolysate to produce ABE

Firstly, the hydrolysate after the enzymolysis treatment in the step (I) is subjected to component blending to be used as fermentation liquor to facilitate subsequent fermentation, specifically, 1 ~ 3g of CH is added into each liter of straw hydrolysate₃COO(NH₄)、0.5~0.7g KH₂PO₄、0.3~0.5gK₂HPO₄1 ~ 2g bean powder, adjusting pH to neutral, and removing O₂Sterilizing, and cooling to room temperature;

secondly, inoculating the seed solution prepared in the step (II) into the fermentation liquor after the blending and sterilization treatment under the aseptic condition, and performing static culture at 35 ~ 40 ℃ for 18-24 h;

finally, adding glutathione into the fermentation liquor, continuing static fermentation at 35 ~ 40 ℃ for 48-72 h, wherein the addition amount of the glutathione in the fermentation liquor is 0.05-0.5 g/L.

2. The method for producing ABE by fermenting with straw hydrolysate as raw material as claimed in claim 1, wherein the step of (1) straw pre-treatment comprises pulverizing dried straw, sieving with 40 mesh sieve, mixing with 0.75 vol.% sulfuric acid at a mass ratio of 1: 6 ~ 12, and treating at 121 deg.C for 60 min.

3. The method for producing ABE by fermentation with straw hydrolysate as raw material as claimed in claim 1, wherein in the step (1) of straw pretreatment, the straw is any one or mixture of several of wheat straw, rice straw and corn straw.

4. The method for producing ABE by fermentation of straw hydrolysate as claimed in claim 1, wherein the specific dosage in the step (2) of biological enzymolysis is as follows: in every 100mL of straw hydrolysate, 50 muL of cellulase, 50 muL of xylanase, and 50 mug of beta-1, 4-mannanase are added.

5. The method for producing ABE by fermentation of straw hydrolysate as claimed in claim 4, wherein the ABE is treated at 55 ℃ for 24 ~ 48h and then centrifuged at 3000r/min for 5min to remove solid substances.

6. The method for producing ABE by fermentation of straw hydrolysate as claimed in claim 1, wherein in step (II), the formulation of the seed culture medium and the specific preparation method of the seed liquid are as follows:

cooking 4-6 wt% of corn flour uniformly mixed with water for 30min, supplementing evaporated water, and removing O₂Then sterilizing to prepare a seed culture medium;

adding 5-10% of strain spore liquid into a seed culture medium cooled to normal temperature, and performing static culture at 35 ~ 40 ℃ for 18-36 h.

7. The method for producing ABE by fermentation of straw hydrolysate as claimed in claim 1, wherein in the step (III), the hydrolysate is prepared by the following specific formula: adding the following components in each liter of straw hydrolysate: 2g CH₃COO(NH₄)、0.6gKH₂PO₄、0.4g K₂HPO₄1.5g of bean flour, and adjusting the pH value to 7.0.

8. The application of glutathione in fermentation production of ABE is characterized in that when ABE is produced by fermentation of clostridium acetobutylicum or clostridium beijerinckii, glutathione is added into fermentation liquor to adjust the expression quantity of ptb, pta, adhE, ctfAB CoA and bdhB genes.

Technical Field

The application belongs to the technical field of biomass energy preparation, and particularly relates to a method for producing ABE by fermenting straw hydrolysate serving as a raw material.

Background

In the process of developing and utilizing biomass energy technology, acetone, ethanol and butanol (ABE) fermentation is a mature fermentation technology and is the second most important fermentation process in the world after ethanol fermentation. With the rapid development of global economy, energy problems become an important limiting factor in economic development, and the search for potential energy sources capable of replacing fossil fuels is urgent.

Biofermentation is one of the most efficient methods for producing fuels, while biobutanol is one of the most promising studies for biofuels. The biobutanol is not only an important organic chemical raw material, but also a novel biofuel with great potential. However, the production of ABE from grain crops raises concerns about the influence of grain safety and environment, so scientists have a great interest in producing ABE from straw, and compared with the first generation of biofuel using grains such as corn and soybean as raw materials, the straw ABE has the greatest advantage of avoiding the 'moral risk', and once the production is industrialized, the straw ABE can solve the 'struggle for grain with people' and can also change waste into valuable.

In the actual fermentation production process, the butanol producing bacteria cannot directly utilize the main components of cellulose and hemicellulose of the straw biomass, so that the straw needs to be pretreated to a certain degree before fermentation. The main pretreatment at present comprises gas explosion, acid hydrolysis, enzyme treatment and the like. The straws can generate fermentable sugar such as glucose, xylose and the like after being pretreated, but can generate a large amount of microbial fermentation inhibitors such as salts, acids, aldehydes, phenols and the like. Therefore, complex detoxification treatment process is often needed when straw hydrolysate is used as a substrate for fermentation. The detoxification method commonly used at present comprises a physical method, a chemical method, a biological method and the like. The physical method mainly comprises rotary evaporation, activated carbon adsorption, ion exchange resin and the like, wherein the rotary evaporation is suitable for removing volatile substances such as acetic acid, furfural and the like. The activated carbon adsorption mainly depends on adsorption to remove partial lignin degradation products and other toxic substances, the ion exchange resin can remove inorganic ions and remove most furfural, acetic acid and the like in the hydrolysate, the chemical method achieves the aim of reducing toxicity by chemical precipitation, changing pH and the ionization characteristics of inhibitors, and the biological method reduces toxicity by using specific enzyme or microorganism to act on fermentation inhibitors and changing the structures of the inhibitors. However, in practical application, a single method is often difficult to meet the fermentation requirements, and various methods are generally comprehensively utilized. The detoxification methods obviously increase the production cost and limit the industrialization of producing ABE by using straws. Therefore, the method develops a production process with low cost and high operability in a targeted manner, avoids a detoxification process, and has very important technical significance for truly realizing the production of ABE by taking straws as raw materials.

The change of the oxidation-reduction environment in the fermentation liquor has great influence on cell growth and product fermentation, glutathione is often applied to the fermentation oxidation-reduction potential regulation of aerobic bacteria, and few research reports on anaerobic bacteria exist. The clostridium acetobutylicum and the clostridium beijerinckii are anaerobic bacteria and have no capacity of synthesizing glutathione. Linjiang Zhu et al (Metabolic engineering13(2011) 426-434) were overexpressed in C.acetobutylicumgshAB，The strain obtains the capacity of synthesizing glutathione, and the result shows that the damage of a solvent to the bacterial cell membrane is reduced, and the capacity of producing butanol by the strain is improved.

Disclosure of Invention

The method for producing acetone, ethanol and butanol by taking straw hydrolysate as a raw material and adding glutathione into the hydrolysate to rapidly ferment is provided, and the method can achieve the production purpose of simplifying the fermentation production process of ABE by avoiding the treatment of a detoxification process, thereby promoting the industrial development of ABE fermentation production.

The technical solution adopted in the present application is detailed as follows.

A method for producing ABE by fermenting straw hydrolysate serving as a raw material specifically comprises the following steps:

preparation of straw hydrolysate

When preparing the straw hydrolysate, the method comprises the steps of straw pretreatment, biological enzymolysis and the like, and specifically comprises the following steps:

(1) pretreatment of straw

Crushing straws, mixing the crushed straws with sulfuric acid uniformly, carrying out high-temperature sterilization (acidolysis is carried out simultaneously in the high-temperature sterilization process), and then adjusting the pH to be =4 ~ 6;

specifically, the method comprises the steps of crushing dry straws, sieving the crushed dry straws with a 40-mesh sieve, uniformly mixing dry straw powder and 0.75% sulfuric acid (in volume ratio) according to the mass ratio of 1: 6 ~ 12, treating the mixture at 121 ℃ for 60min, and adding Ca (OH)₂Adjusting the pH value to 4.8;

the straws are specifically any one or a mixture of a plurality of straws in any proportion, such as wheat straws, rice straws and corn straws;

(2) biological enzymolysis

Adding biological enzyme into the sterilized straw mixture obtained in the step (1), uniformly mixing, performing enzymolysis at 40 ~ 60 ℃ for 24 ~ 48h, and centrifuging to remove solid precipitates, wherein the supernatant is the straw hydrolysate to be fermented, and the enzyme does not need to be inactivated independently;

specifically, the solid matter is removed by treating the mixture for 24 ~ 48h at 55 ℃ and then centrifuging the mixture for 5min at 3000 r/min;

the biological enzyme is a mixture of cellulase, xylanase and beta-1, 4-mannase, the specific dosage is 40 ~ 60 muL of cellulase, 40 ~ 60 muL of xylanase and 40 ~ 60 mug of beta-mannase in every 100mL of straw hydrolysate, and the specific enzyme activity requirement is as follows:

standard enzyme activity (CMC enzyme activity) of cellulose is more than 1 x 10⁵u/mL (enzyme activity definition: 1mL enzyme solution at 50 ℃, pH4.5, 1min catalytic hydrolysis of CMC to generate 1 microgram glucose as a cellulase CMC activity unit);

the standard enzyme activity of xylanase is more than 1 multiplied by 10⁸U/mL (enzyme activity definition: 1mL enzyme solution at 50 deg.C, pH 5.0 for 1min hydrolysis of 1% xylanThe enzyme amount of 1 microgram of xylose produced by the solution is one xylanase activity unit);

the standard enzyme activity of beta-1, 4-mannase is more than 5 multiplied by 10⁴U/g (enzyme activity is defined as that the enzyme amount required for degrading and releasing 1 mu mol of reducing sugar from a beta-1, 4-mannan solution with the concentration of 3mg/mL per minute at the conditions of pH of 5.5 and temperature of 37 ℃ is one enzyme activity unit);

(II) preparing ABE fermentation culture solution

Clostridium acetobutylicum (C.) (Clostridium acetobutyLicum) CICC 8012 (with a preservation number of CCTCC M2010148) and Clostridium beijerinckii (Clostridium beijerinckii: (C.))C.beijerinckii) CGMCC1.255 (platform resource number 1511C 0002100007244) is taken as an example, when the seed solution is prepared, the formula of the seed culture medium and the specific preparation method are as follows:

cooking 4-6% (by mass) of corn flour uniformly mixed with water for 30min, supplementing evaporated water, removing O₂(specifically, for example, by charging N₂By removing O₂) Then sterilizing (115 ~ 121 deg.C for 15 ~ 30 min) to obtain seed culture medium;

adding 5-10% (volume ratio) of strain spore liquid into seed culture medium cooled to normal temperature, and performing stationary culture for 4-24 h (before the culture is started, preferably performing heat shock for 30-90 s by using boiling water bath) at 35 ~ 40 deg.C (preferably 37 deg.C for constant temperature culture), thereby accelerating spore germination);

(III) fermentation of the hydrolysate to produce ABE

Firstly, the hydrolysate after the enzymolysis treatment in the step (I) is subjected to component blending to be used as fermentation liquor to facilitate subsequent fermentation, specifically, 1 ~ 3g of CH is added into each liter of straw hydrolysate₃COO(NH₄)、0.5~0.7g KH₂PO₄、0.3~0.5gK₂HPO₄1 ~ 2g of bean flour, then adjusting the pH value to be neutral, and preferably adding 2g of CH into each liter of straw hydrolysate₃COO(NH₄)、0.6g KH₂PO₄、0.4g K₂HPO₄1.5g of bean powder and Ca (OH)2 to adjust the pH value to 7.0; after the blending is finished, removing O₂(specifically, for example, by charging N₂By removing O₂) Cooling to room temperature for later use after sterilization treatment (115 ~ 121 deg.C sterilization for 15 ~ 30 min);

secondly, under the aseptic condition, inoculating the seed solution prepared in the step (II) into the fermentation liquor after the blending and sterilization treatment according to the proportion of 10 percent (volume ratio), and carrying out static culture for 4-24 h at 35 ~ 40 ℃ (preferably constant temperature culture at 37 ℃);

finally, adding glutathione into the fermentation liquor, continuing to perform stationary fermentation for 48-72 h at 35 ~ 40 ℃ (preferably constant temperature culture at 37 ℃), wherein the addition amount of the glutathione in the fermentation liquor is 0.05-0.5 g/L.

In the application, the inventor thinks that when the butanol is fermented by taking the straw acid hydrolysate as the raw material and is matched with a specific microbial strain for fermentation treatment, the change of related key enzyme expression quantity in a solvent metabolic pathway can be obviously influenced by adding the glutathione, so that the chemical components of the cell membrane of the strain are changed, and the change is probably the reason that the glutathione improves the butanol fermentation capacity of clostridium.

In general, the main technical features of the present application are as follows:

(1) in the application, the ABE is directly fermented and produced after the straws are pretreated to prepare the hydrolysate, so that a complex detoxification process is avoided, the production cost is reduced, and the industrialization of ABE fermentation production is promoted;

(2) the fermentation yield of the ABE is further improved by optimizing the fermentation process, particularly by adding and utilizing glutathione in the fermentation process, so that the industrial production of the ABE is further promoted.

In a word, the method further improves the yield of the ABE and reduces the production cost by optimizing and simplifying key process steps such as straw hydrolysis, ABE fermentation and the like, particularly optimizing the addition time and the addition amount of the glutathione, so that the method has better practical value and popularization and application significance.

Drawings

FIG. 1 shows the effect of different glutathione addition times on the production of a solvent by Clostridium beijerinckii;

FIG. 2 shows the effect of glutathione on the expression level of key enzymes of the C.acetobutylicum metabolic pathway.

Detailed Description

The present application is further explained below with reference to the drawings and examples. Before describing the specific examples, some of the materials used in the following examples will be briefly described as follows.

Biological enzyme:

cellulase, a product of the company aladin, product properties: is derived from Aspergillus niger, powder, and enzyme activity is 10000U/g;

xylanase, a product of the company aladin, product properties: the enzyme activity is more than or equal to 2500U/g, and the enzyme is expressed in aspergillus oryzae;

beta-1, 4-mannanase, product of Solardiao, product Properties: powdery, the enzyme activity is more than or equal to 60000u/g, the optimum action is pH4.5-5.0, and the action temperature is 40-65 ℃.