CN110283870A - A kind of method of double bacterial strains mixed solid fermentation corn stover - Google Patents

Abstract

The invention discloses a kind of method of double bacterial strains mixed solid fermentation corn stover, the methods are as follows: (1) configures LB plate/fluid nutrient medium and YPD plate/fluid nutrient medium；(2) bacillus amyloliquefaciens and saccharomycete are inoculated in respectively on LB plate and YPD plate, are put into culture carton upside down culture, obtain bacterium colony；(3) bacterium colony of bacillus amyloliquefaciens and saccharomycete is respectively connected in LB liquid medium and YPD fluid nutrient medium, is put into shaking table and continues to cultivate；(4) corn stover solid medium is configured；(5) bacillus amyloliquefaciens are linked into corn stover solid medium, again in bacillus amyloliquefaciens after effect 1~3 day: saccharomycete, continuation fermented maize stalk are added into culture medium for ratio of the number of saccharomycete than=1:1；(6) ethyl alcohol in corn stover solid medium is collected in concentration.This method can reduce environmental pollution for the degradation of corn stover, while remaining substrate can be made into protein rich animal feeding-stuff.

Description

A kind of method for two-strain mixed solid-state fermentation of corn stover

technical field

The invention belongs to the field of fermentation engineering, and in particular relates to a method and application of a two-strain mixed solid-state fermentation corn stover.

Background technique

Today's human society is mainly faced with a variety of problems, such as population expansion, energy crisis, resource shortage, and environmental pollution, among which environmental problems are particularly serious. my country is a big agricultural country, producing hundreds of millions of tons of straw waste every year. Crop straw is the general term for crop stems and leaves that remain after the ripe fruit is picked. Straw is the place where crops carry out photosynthesis and product accumulation, and straw contains a variety of organic matter and mineral elements. Therefore, as a by-product of crops, straw is also an important renewable biological resource with multiple functions. Common types of important crop straws in my country include: rice, wheat, corn, beans, potatoes, rapeseed, sugarcane, cotton, and tobacco. As a major agricultural country, my country's crop straw is not only huge in output, but also very rich in variety. The three types of crop straw that account for the most include rice, corn, and wheat. According to statistics, the total annual output of crop straw in my country is more than 600 million tons, accounting for more than 20% of the total global straw output. Corn stalks are the most productive of all crop stalks. Every year, my country can obtain more than 200 million tons of corn stalks while obtaining a bumper grain harvest.

The resource reuse of corn stover is related to many social, economic and environmental issues. In the era of extensive economy in the long past, the commonly used methods of treating corn stalks in rural areas of our country are often disposal, uncontrolled burning and returning of decomposed stalks to the field. This rude and disorderly treatment method, although a number of national and local policies have been introduced to control the field burning of crops, but because there is no more effective solution, the effect is not obvious. In today's agriculture, there is still a lot of waste of resources, and it also brings serious ecological problems such as air pollution and land destruction. With the adjustment and optimization of my country's agricultural structure, the annual amount of corn stover waste gas is still rising. Therefore, improving the reuse efficiency of corn stalks and innovating and developing ways to reuse resources is an important way for the sustainable development of agricultural resources in my country.

The dry matter contained in the dried corn stalk is divided into inorganic matter of mineral elements, nitrogen-containing organic matter and nitrogen-free organic matter composed of crude protein according to the type. Among them, the weight of the latter accounts for more than 80% of the dry weight of corn stalks, and is mainly divided into cellulose, hemicellulose, xylan uronic acid and lignin and other components. The main component of the cell wall of corn stover is cellulose, which is an organic macromolecular polysaccharide composed of 8,000 to 12,000 glucose monomers connected by glycosidic bond condensation. Because glucose is tightly arranged and surrounded by hemicellulose and lignin, cellulose is insoluble in common organic solvents and difficult to undergo hydrolysis. Because the main vitamins, hemicellulose and lignin in corn stover are difficult to degrade and occupy a very high proportion, the natural degradation rate is very slow in the natural environment. Therefore, the degradation of corn stover in industrial production is aimed at accelerating the degradation rate and improving the conversion efficiency.

At present, the widely used and relatively mature practice is to first use physical or chemical methods to pretreat large-scale corn stalks, and then use biological methods for efficient and specific transformation. The physical method is to directly destroy the binding force of cellulose and lignin, so that the intact straw can be transformed into particles with small particle size, increase the contact surface area of subsequent biological reactions, and promote the reaction speed and efficiency of microorganisms or biological enzymes and straw. At present, the physical treatment methods that can be widely used in industrial production in practical applications include mechanical pulverization, high temperature pyrolysis, steam explosion and radiation. The immediate purpose of the chemical approach was to increase the solubility of the three natural macromolecular structural complexes in the cell wall of corn stover, making it easier for subsequent enzymatic digestion. However, relying only on these two methods, the obtained product is complex, and the content of the target product is low, and further separation and purification are required in the follow-up, which increases the complexity of the process and causes further loss of the target product. At the same time, the commonly used strong acids, strong bases and organic solvents will cause serious environmental pollution, which runs counter to the original intention of reusing corn stalks and reducing environmental pollution.

The biodegradation method mainly uses the biological activity of cellulose-degrading bacteria to process and degrade corn stalks. This method has high efficiency, low cost and environmental protection. The efficiency of the biodegradation method is not only related to the reaction environment, but also mainly depends on the choice of microorganisms. Microorganisms with cellulose degrading ability that have been found in nature include: bacteria, fungi and actinomycetes. Bacterial microorganisms with stronger adaptability are mainly used, and strains of the genus Bacillus are widely used, which can The formation of spores with high temperature resistance and strong viability makes it very low in the reaction environment. Among the currently practical microbial degradation methods of corn stalks, single-strain method is mostly used, that is, a well-known strain is mainly introduced and involved in the degradation of corn stalks. However, the obvious disadvantage of single-strain degradation is the slow degradation rate and low transformation rate, so dual or multi-strain biodegradation methods can be introduced, which can show advantages at multiple levels.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the present invention provides a method and application of a two-strain mixed solid-state fermentation of corn stalks. Through the single factor experimental design, the fermentation process of corn stalk was optimized, and the optimal fermentation conditions for degrading corn stalk were screened out. effect of reaction conditions. It can reduce environmental pollution for the degradation of corn stalks, and at the same time, the residual substrate can be made into protein-rich livestock feed.

The present invention is achieved through the following technical solutions:

A method for mixed solid-state fermentation of corn stover with two strains, comprising the following steps:

Step 1) configure common LB plate, LB liquid medium and YPD plate and YPD liquid medium containing glucose substrate, and sterilize the medium;

Step 2) inoculate Bacillus amyloliquefaciens on the LB plate, inoculate the yeast on the YPD plate, put it into an incubator for upside-down culture, and obtain colonies;

Step 3) inserting the colony of Bacillus amyloliquefaciens into the LB liquid medium, inserting the yeast colony into the YPD liquid medium, and putting it into a shaker to continue culturing;

Step 4) configure the corn stover solid medium, and sterilize the medium;

Step 5) inserting Bacillus amyloliquefaciens into the corn stalk solid medium for 1 to 3 days; and then culturing the corn stalk solid medium at the ratio of Bacillus amyloliquefaciens: yeast number ratio=1:1 Add yeast to the base to continue fermenting corn stalks;

Step 6) Concentrate and collect the ethanol in the fermented corn stover solid medium by means of distillation and condensation, and the remaining solid residue can be used as biological bacterial fertilizer.

Preferably, the concentration of the glucose substrate in the YPD liquid medium in step 3) is 20 g/L.

Preferably, the configuration process of the corn stalk solid culture medium in step 4) is as follows: the corn stalk is dried in the sun and then pulverized to 100 meshes with a pulverizer, and is configured with a ratio of corn stalk:sterile water mass ratio=1:6 to form Viscous solid, then autoclaved.

Preferably, the Bacillus amyloliquefaciens in step 5) is inserted into the corn stover solid medium for 2 days.

Application of a method for mixed solid-state fermentation of corn stalks with two strains in degrading corn stalks.

The beneficial effects of the present invention are as follows:

(1) The present invention selects yeast and Bacillus amyloliquefaciens as starting strains to degrade corn stalks and cultivate a large amount of bacterial proteins, and studies the degradation reactions of single and double strains simultaneously and sequentially fermented to corn stalks. The single factor experimental design was used to optimize the fermentation process of corn stalks, and the optimal degradation conditions were screened out.

(2) The present invention first uses Bacillus amyloliquefaciens to act on the corn stalk, and then treats it with yeast, so as to achieve a good effect of degrading the corn stalk, and can cultivate a large number of bacterial cells, which can be used for the production of animal protein feed. Bacillus amyloliquefaciens can synthesize enzymes such as cellulase that degrade corn stalks, and has the same function as yeast to ferment alcohol. Yeast is rich in protein, fat, sugar, B vitamins, enzymes (coenzymes), ribonucleic acid, sterols and other nutrients. Bacillus amyloliquefaciens is an animal intestinal probiotic, which can improve the balance of animal intestinal flora. Enhance animal immunity, promote animal growth and development, and improve animal production performance.

(3) The present invention uses Bacillus amyloliquefaciens to first produce cellulase to degrade corn stalks to form reducing sugars, and then the bacteria use reducing sugars to carry out the next step of metabolism. At this time, yeast is added to promote the growth of two kinds of bacteria, so that the number of bacteria increases , the amount of enzymes also increases, forming a virtuous cycle in which enzymes degrade corn stalks to produce reducing sugars, and bacteria use reducing sugars to grow and then produce enzymes.

Description of drawings

Fig. 1 is the maximum alcohol concentration of different strains of fermentation;

Figure 2 shows the measured absorbance values of standard gradient glucose.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and embodiments.

The Bacillus amyloliquefaciens and Saccharomyces cerevisiae strains used in the examples of the present invention are general-purpose strains and cells purchased in the laboratory. The deposit number of Bacillus amyloliquefaciens is CGMCC No.1.7463. The deposit number of Saccharomyces cerevisiae is CGMCC No. 2.1638.

Example 1

A method for the mixed solid-state fermentation of corn stalks with two bacterial strains, the specific steps are as follows:

1. Preparation of activation medium

(1) Configure ordinary LB liquid medium: each 100mL medium contains 1g peptone, 1g sodium chloride and 0.5g yeast powder, and 2g agar powder should be added to the solid (plate) medium, and sterile water should be added to the volume. Then put it into an autoclave at 121°C for 20 minutes for sterilization;

(2) YPD liquid medium with a glucose concentration of 20g/L is prepared. Each 100mL medium contains 2g peptone, 2g glucose and 2g yeast powder. In the solid (plate) medium, 2g agar powder should be added, and sterile water should be added. Put it into an autoclave and sterilize it at 115°C for 25min.

2. Strain expansion and adaptation

(1) The Bacillus amyloliquefaciens and yeast strains stored in the -80°C refrigerator were streaked and inoculated on the LB and YPD plate media with inoculation loops, and were placed in an incubator at 28 ± 2°C for inverted culture for 2 days;

(2) Two days later, the colony of Bacillus amyloliquefaciens was inserted into the LB liquid medium, and the yeast colony was inserted into the YPD liquid medium, and the seed culture process used a conical flask, put it into a shaker at 180 rpm, 28±2° C. Incubate for 2 days.

3. Configuration of corn stover medium

After drying the corn stalks, pulverize them to about 100 mesh with a pulverizer, configure them into viscous solids in the ratio of corn stalks:sterile water mass ratio=1:6, and sterilize them at high temperature at 115°C for 25 minutes.

4. Fermentation on solid medium

The OD values of Bacillus amyloliquefaciens and yeast cultured for two days were measured to obtain the OD values of Bacillus amyloliquefaciens and yeast. According to the ratio of Bacillus amyloliquefaciens: yeast number ratio = 1:1, the Bacillus amyloliquefaciens culture solution and yeast solution were respectively added to the corn stalk medium, and the Bacillus amyloliquefaciens were treated for 1 day, 2 days, 3 days. After a few days, add the yeast solution for a week.

The culture medium is configured in the following manner for bacterial solid fermentation: Medium 1 is fermentation by Bacillus amyloliquefaciens alone, Medium No. 2 is fermentation by yeast alone, Medium No. 3 is fermentation by Bacillus amyloliquefaciens and yeast at the same time, No. 4 medium was treated with Bacillus amyloliquefaciens for 1 day and then connected to yeast for fermentation, No. 5 medium was treated with Bacillus amyloliquefaciens for 2 days and then connected to yeast for fermentation, and No. 6 medium was treated with Bacillus amyloliquefaciens for 3 days and then connected Yeast fermentation.

5. Post-fermentation treatment

The ethanol in the fermentation medium is concentrated and collected by the method of distillation and condensation, and the remaining solid residue contains a large number of cells and nutrients produced by the degradation of corn stover, which can be used as biological bacterial fertilizer.

6. Result analysis

(1) Determination of alcohol content of liquid culture medium

Take 5mL samples of each bottle of fermentation broth in the ultra-clean workbench every day, and then add 5mL of YPD medium with a concentration of 20g/L. The alcohol content of the sample taken out was measured with an alcohol meter, as shown in Figure 1.

It can be seen from Figure 1 that Bacillus amyloliquefaciens alone, yeast alone and mixed bacteria can ferment the medium and produce ethanol, and the amount of alcohol produced by fermentation in the mixed state is more than that produced by single strain fermentation. .

According to the experimental process, the growth rate of Bacillus amyloliquefaciens is slow, and a large amount of ethanol is produced by fermentation when the bacterial density is high in the later stage of fermentation. The growth rate of yeast is fast, but the amount of ethanol produced by fermentation is small. By mixing the two kinds of bacteria, the advantages of the two can be combined. The bacterial growth rate is fast, the bacterial density is high, and the ethanol fermentation speed is fast, and the amount of ethanol produced by fermentation is more than that of the single bacterial species.

(2) Determination of absorbance value of medium suspension and cell number, observation of plate

In medium 1-6, take 1 g of uninoculated corn stalk medium and add 100 mL of sterile water as a reference, measure the OD value of the bacterial suspension obtained by adding 1 g of inoculated solid medium to 100 mL of sterile water, and then use Microscope and 25×16 hemocytometer were observed to calculate the number of bacteria in the suspension, and the results of different media were compared. The bacterial suspension was then added to the plate, and the growth of yeast and Bacillus amyloliquefaciens on the plate was observed after culturing for two days.

After table 1 corn stover solid medium is fermented, add water to make suspension OD value

As can be seen from Table 1, the bacterial count and OD value of the medium suspension for culturing yeast are the lowest, indicating that the yeast does not grow well in the medium, while the bacterial count of Bacillus amyloliquefaciens is significantly higher, and the OD of the suspension is significantly higher. The higher value indicates that Bacillus amyloliquefaciens can effectively utilize corn stover medium and grow well. In mixed culture, bacteria can grow by adding bacterial liquid at the same time, but because of less glycogen produced, adding mixed growth at the same time does not have as many Bacillus amyloliquefaciens alone. There is a certain amount of glucose accumulation, and yeast can be added at this time to grow well, and the number of cells is more, and a double-strain mixed fermentation of corn stalks is better than single-strain fermentation. This shows that Bacillus amyloliquefaciens alone can act on corn stalks, and after adding yeast, the two can grow synergistically, degrading corn stalks more effectively, and also producing a large number of bacteria that can be used as animal feed, improving the utilization of corn stalks Rate.

(3) Determination of enzyme activity in medium

The enzyme activity in the filtrate was determined by 3,5-dinitrosalicylic acid (DNS) method. First prepare 1mg/mL glucose stock solution, respectively take 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL, 1.0mL, 1.2mL in test tubes, and make up to 2mL with distilled water. Add 1.5mL DNS solution and shake well, take a boiling water bath for 5min, add distilled water to make up to 25mL after cooling, mix well, measure the absorbance value at 540nm, draw the standard curve with the glucose concentration as the abscissa and the absorbance value as the ordinate, as shown in the figure 2 shown.

Take 5 g of inoculated corn stalk medium, add 50 mL of sterile water, and after complete mixing, centrifuge at 5000 r/min for 30 min, take the supernatant, add ammonium sulfate to insoluble state, and precipitate overnight. The next day, the liquid was taken, centrifuged again, and the pellet was taken. 2 mL of phosphate buffer solution (KH ₂ PO ₄ : NaHPO ₄ =1:1) was added to the precipitate, and then 0.5 mL of 1% carboxymethyl cellulose sodium salt solution was added to mix well for the reaction. Take 0.5mL of sample and make up to 2mL with distilled water, add 1.5mL of DNS solution and shake well, take a boiling water bath for 5min, add distilled water to make the volume to 25mL after cooling, mix well, measure the absorbance value at 540nm, and substitute the data into the standard curve to get the sample's value. The size of the enzyme activity is shown in Table 2.

Table 2 Sample absorbance value (OD value) and calculated enzymatic activity

As shown in Table 2, the presence of cellulase in the medium was confirmed because the measured results had absorbance. The absorbance value in the No. 2 medium is almost 0, indicating that the yeast alone cannot produce cellulase-acting straw, while the No. 3 medium is added to the mixed fermentation at the same time. The absorbance value is slightly, but not high, and the No. 2 Bacillus amyloliquefaciens alone The absorbance value of yeast added after 2 or 3 days of fermentation and fermentation was relatively high in several groups, indicating that Bacillus amyloliquefaciens can produce cellulase, and after a period of time, the yeast can also produce no lower than that of single culture. amount of enzyme.

According to the results obtained from the above experiments, it can be concluded that Bacillus amyloliquefaciens can produce cellulase. When Bacillus amyloliquefaciens is added to the corn stalk at the beginning, it will produce cellulase and act on cellulose to produce glucose. Then, yeast is added, and the yeast uses The glucose produced by the action of cellulase grows in large quantities, and because the mixed growth of the two bacteria has a synergistic effect, it can stimulate the growth of Bacillus amyloliquefaciens, produce more cellulase, reduce more sugar, and achieve a A good cycle of enzyme production - sugar production - bacteria production. Therefore, the sequential addition of two strains to mixed fermentation is a better method than that of single strains.

At present, the research on how to rationally utilize straw is mainly focused on the following three aspects: (1) using enzymes to degrade straw cellulose on a large scale according to traditional methods, and returning straw directly to the field to increase soil fertility; (2) utilizing Microbial fermentation, fermenting crop straws to produce protein feed to feed livestock; (3) using related microorganisms to produce straw cellulose into energy such as alcohol and methane that people need. In the present invention, yeast and Bacillus amyloliquefaciens are cultured individually and in a mixed state in culture media with different substrate concentrations, and it is determined that the mixed growth of yeast and Bacillus amyloliquefaciens has a promoting effect, and then single strain of corn stalk is mixed and mixed. Simultaneous and sequential solid fermentation of strains, it was found that corn stover treated with Bacillus amyloliquefaciens and then yeast can effectively degrade corn stover and produce a large amount of protein strains.

Claims (5)

1. a method for two-strain mixed solid-state fermentation corn stover, is characterized in that, comprises the following steps: Step 1) configure common LB plate, LB liquid medium and YPD plate and YPD liquid medium containing glucose substrate, and sterilize the medium; Step 2) inoculate Bacillus amyloliquefaciens on the LB plate, inoculate the yeast on the YPD plate, put it into an incubator for upside-down culture, and obtain colonies; Step 3) inserting the colony of Bacillus amyloliquefaciens into the LB liquid medium, inserting the yeast colony into the YPD liquid medium, and putting it into a shaker to continue culturing; Step 4) configure the corn stover solid medium, and sterilize the medium; Step 5) inserting Bacillus amyloliquefaciens into the corn stalk solid medium for 1 to 3 days; and then culturing the corn stalk solid medium at the ratio of Bacillus amyloliquefaciens: yeast number ratio=1:1 Add yeast to the base to continue fermenting corn stalks; Step 6) Concentrate and collect the ethanol in the fermented corn stover solid medium by means of distillation and condensation, and the remaining solid residue can be used as biological bacterial fertilizer. 2. the method for a kind of two-strain mixed solid-state fermentation corn stover according to claim 1, is characterized in that, the concentration of glucose substrate in the described YPD liquid medium of step 3) is 20g/L. 3. the method for a kind of two-strain mixed solid-state fermentation corn stalk according to claim 1, is characterized in that, the configuration process of the described corn stalk solid culture medium in step 4) is as follows: after the corn stalk is sun-dried, pulverize with a pulverizer To 100 mesh, it is prepared into a viscous solid in the ratio of the mass ratio of corn stalk: sterile water = 1:6, and then high temperature sterilization. 4 . The method for a dual-strain mixed solid-state fermentation of corn stover according to claim 1 , wherein the time when the Bacillus amyloliquefaciens in step 5) is connected to the corn stover solid medium for action is 2 days. 5 . 5. The application of the method for the mixed solid-state fermentation of corn stover with two strains of claim 1 in degrading corn stover.