Bacillus cereus and application thereof

Abstract

The invention relates to a bacillus cereus strain and application thereof, the bacillus cereus strain is bacillus cereus W1, the bacillus cereus W1 is preserved in China Center for Type Culture Collection on July 21, 2022, and the preservation number is CCTCC NO: M 20221147. The bacillus cereus W1 can grow and ferment in a non-strict anaerobic environment to produce butyric acid at high yield, and has the advantages of high oxygen resistance, high butyric acid yield and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Bacillus cereus and application thereof in producing butyric acid by fermentation. Background Art

[0002] Butyric acid is a four-carbon short-chain fatty acid widely used in the chemical, plastic, textile fiber, food and beverage, and pharmaceutical industries. In the chemical industry, butyric acid is mainly used in the production of cellulose acetate butyrate plastics, which are used in the manufacture of textile fibers. By introducing butyryl groups into cellulose acetate polymers, the polymers are more flexible and have better resistance to light, low temperatures, and organic solvents. Butyric acid can also be added directly to fibers as an additive to improve the fiber's resistance to heat and light. In addition, butyric acid is also used in the pharmaceutical industry, and many derivatives of butyric acid are used to make vasoconstrictor drugs, anesthetics, and antioxidants.

[0003] In recent years, butyric acid has attracted widespread attention as a raw material for the production of biofuel butanol, which is considered to be a new generation of liquid fuel after ethanol, with higher energy density and combustion value than ethanol. Another potential application of butyric acid is in the production of biodegradable plastics: mixed carboxylic acids rich in butyric acid can be bioconverted into polyhydroxybutyrate (PHB). Polyhydroxybutyrate is the main component of polyhydroxyalkanoates (PHAs), which are the raw materials for the production of biodegradable plastics.

[0004] At present, the industrial production of butyric acid is mainly based on the n-butyraldehyde oxidation method based on fossil resources such as petroleum, coal and natural gas: that is, n-butyraldehyde is used as the raw material, oxygen or air is used as the oxidant, and a continuous reaction is carried out under the action of a catalyst, and butyric acid is obtained by distillation. The n-butyraldehyde oxidation method has become the mainstream method for the commercial production of butyric acid due to its advantages such as single and easy-to-obtain raw materials, simple and easy-to-control process, and high product yield. However, the limited and unsustainable nature of fossil resources and the environmental pollution caused by their exploitation and use have forced people to seek sustainable production methods that are in harmony with nature.

[0005] Microbial fermentation to produce butyric acid is a highly promising alternative technology. Although the current production cost of the fermentation method is relatively high, with the continuous rise in crude oil prices, the development and maturity of biomass chemical industry, and the increasing demand for organic natural products in the fields of food additives, pharmaceuticals and preservatives, the fermentation method to produce butyric acid will play a more important role in the butyric acid supply market. Under anaerobic conditions, many genera of microorganisms can produce butyric acid, including Clostridium, Butyrivibrio, Butyribacterium, Sarcina, Eubacterium, Fusobacterium and Megasphera, but the fermentation performance needs to be improved.

[0006] The factors that restrict the development and commercialization of butyric acid production by microbial fermentation are mainly: (1) The final concentration of butyric acid is low. In the fermentation system, the growth of butyric acid-producing bacteria will be inhibited by the fermentation end products (butyric acid, acetic acid), so the final concentration of butyric acid in the fermentation broth is low; (2) The separation cost is high. The low final concentration of the product in the butyric acid fermentation broth and the presence of by-products increase the difficulty of separating and purifying butyric acid; (3) The price of fermentation raw materials is relatively high. At present, the most commonly used raw materials for butyric acid fermentation are glucose, corn, etc. The prices of these raw materials are relatively high, which makes the production cost high.

[0007] The document "Screening of Bacillus cereus and its hydrogen production performance" (Wang Haiyan et al. Journal of Beijing University of Technology, October 2014, Vol. 40, No. 10) isolated pure hydrogen-producing bacteria XN12 from activated sludge in a sewage plant. The experimental results showed that the maximum hydrogen production of the bacteria under neutral conditions was 1.6 mol per mole of glucose, and the test results of the liquid phase product of the reaction showed that the bacteria was a butyrate fermentation bacteria that produced hydrogen through the pyruvate metabolic pathway. However, this strain was selected for the purpose of hydrogen production, and its performance in the production of butyrate by fermentation needs to be improved. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention aims to provide a strain of Bacillus cereus and its application. The strain can grow and ferment to produce butyric acid in a non-strict anaerobic environment, and has the advantages of strong oxygen tolerance and high butyric acid production.

[0009] The present invention provides a strain of Bacillus cereus, which is Bacillus cereus W1, which has been deposited in the China Center for Type Culture Collection on July 21, 2022, with the deposit number: CCTCC NO: M 20221147.

[0010] The Bacillus cereus W1 provided by the present invention has a rod-shaped cell morphology under a microscope (such as the attached Figure 1 ), 4-7 μm long, a Gram-positive bacterium.

[0011] The Bacillus cereus W1 provided by the invention belongs to facultative anaerobic bacteria and can grow under non-strict anaerobic conditions. The pH suitable for its growth is 4.5-9.0 and the temperature is 20-40°C.

[0012] The Bacillus cereus W1 provided by the present invention is weakly positive for arginine dihydrolase, positive for pyruvate VP reaction, positive for gelatin hydrolysis reaction, weakly positive for starch hydrolysis reaction, and has physiological and biochemical characteristics as shown in Table 1 and Table 2.

[0013] The Bacillus cereus W1 provided by the present invention can utilize any one or a combination of glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol, sorbitol and the like.

[0014] Table 1 Enzyme activity and carbon source oxidation

[0015]

[0016]

[0017] Table 2 Acid production using carbon sources

[0018]

[0019]

[0020] *In the table, +: positive reaction; -: negative reaction, w: weak positive.

[0021] The 16S rRNA gene sequencing analysis results of Bacillus cereus W1 provided by the present invention are shown in the sequence table.

[0022] The present invention also provides the use of the Bacillus cereus W1 in producing butyric acid by fermentation.

[0023] In the application of the present invention, the specific process of fermenting butyric acid with Bacillus cereus W1 is as follows: (1) inoculating the strain into a seed culture medium and culturing to obtain a seed liquid; (2) transferring the seed liquid to a fermentation culture medium and fermenting to produce butyric acid under a non-strict anaerobic environment.

[0024] In the application of the present invention, the strain is inoculated into a seed culture medium and cultured at 28-42°C for 12-48 hours to obtain a seed solution, preferably cultured under a non-strict anaerobic environment. The seed culture medium is a culture medium conventionally used in the art, preferably RCM culture medium.

[0025] In the application of the present invention, the inoculation amount of the seed liquid is 2%-20% of the volume of the fermentation medium.

[0026] In the application of the present invention, anaerobic environment refers to deoxygenation during the whole culture process, and non-strict anaerobic environment refers to deoxygenation 24 hours before fermentation, preferably deoxygenation in the first 2 hours, and then no deoxygenation until the end of fermentation. The deoxygenation can be achieved by passing N2, adding a deoxygenating agent, etc.

[0027] In the application of the present invention, the fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, trace elements and vitamins. The carbon source can be at least one of glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol, sorbitol, etc., preferably glucose, and the concentration is 20-100g / L, preferably 30-80g / L. The nitrogen source can be at least one of ammonium acetate, ammonium chloride and ammonium sulfate, preferably ammonium acetate, and the concentration is 0.1-10g / L, preferably 0.5-3.0g / L. The inorganic salt can be at least one of sodium bicarbonate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, calcium chloride, cysteine ​​hydrochloride, etc., preferably potassium dihydrogen phosphate, and the concentration is 0.1-1.0g / L, preferably 0.2-0.6g / L. The trace elements may be at least one of ferric chloride, zinc chloride, copper chloride, manganese sulfate, boric acid, sodium molybdate, etc., with a concentration of 0.0001-0.05 g / L. The vitamins may be at least one of vitamin B1, vitamin B2, vitamin B6, vitamin H, folic acid, niacin, calcium pantothenate, lipoic acid, para-aminobenzoic acid, etc., with a concentration of 0.00001-0.001 g / L.

[0028] In the application of the present invention, the fermentation conditions are: temperature of 28-42°C, preferably 34-38°C; natural pH in the initial stage, pH controlled to 4.0-9.0 after the logarithmic phase, preferably 5.0-7.0; fermentation using a fermenter with stirring, stirring speed of 10-100r / min; fermentation time of 72-120h.

[0029] In the application of the present invention, the fermentation can be any one of batch fermentation, batch fed fermentation, continuous fermentation, in situ extraction fermentation and gas in situ extraction fermentation.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] (1) The present invention selects and breeds a strain of Bacillus cereus W1, which has strong oxygen tolerance, reduces the deoxygenation process of traditional anaerobic fermentation, does not need to introduce N2 throughout the process, avoids the risks of poor bacterial growth and fermentation failure caused by incomplete deoxygenation, and reduces operating energy consumption.

[0032] (2) Bacillus cereus W1 also has strong acid resistance. Under the same substrate concentration conditions, it has an excellent butyrate fermentation level. The products are mainly butyrate and acetic acid, of which butyrate accounts for no less than 70% and acetic acid accounts for no less than 20%.

[0033] (3) Bacillus cereus W1 can simultaneously utilize pentoses and hexoses as fermentation substrates, thus expanding the range of choices for fermentation substrates.

[0034] Description of biological material deposit

[0035] The Bacillus cereus W1 provided by the present invention is deposited in the China Center for Type Culture Collection; deposit number: CCTCC NO:M 20221147; deposit date: July 21, 2022; deposit address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a photo of the Bacillus cereus of the present invention magnified 1000 times under a microscope;

[0037] Figure 2 It is the variation trend of glucose concentration and product concentration in the fermentation broth of the present invention over time. DETAILED DESCRIPTION

[0038] The strain of the present invention and its effects are further described in detail below in conjunction with specific examples. The examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

[0039] The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0040] In the present invention, the butyric acid concentration, acetic acid concentration and substrate concentration in the fermentation broth are all detected by high performance liquid chromatography. Liquid chromatography analysis conditions: instrument: Agilent 1260; chromatographic column: BIO-RAD HPX-87H Ion Exclusion Column; detector: differential refractive index detector (RID); mobile phase: 0.005 mol / L H2SO4 aqueous solution; flow rate: 0.7 mL / min; column temperature: 65°C; RID temperature: 40°C; injection volume: 5 μL.

[0041] Example 1 Screening of Bacillus cereus W1

[0042] In March 2022, anaerobic sludge was sampled from a domestic sewage treatment plant in Dalian. After crushing, it was filtered with 3 layers of gauze, treated at 80°C for 1h, and 2-bromoethane sulfonate sodium (BES) was added as a methanogen inhibitor. It was mixed with corn straw powder and glucose and placed in a sterile triangular flask. An appropriate amount of sterile water was added to enrich the cultured bacteria. After 1 week of cultivation, 1.0mL of culture was placed in a test tube with 9.0mL of liquid culture medium, and cultured at 35°C for 72h. The same conditions were repeated 3 times. The culture solution was centrifuged and the supernatant was filtered and then detected by liquid chromatography. The butyric acid concentration can reach 2.8g / L. Continue to take the test tube culture solution and spread it on the solid culture medium plate, culture at 35°C for 72h, and pick a single colony for fermentation test. A total of 40 single colonies were selected for fermentation culture, of which 14 cultures produced butyric acid, and the butyric acid concentration in the 6th single colony culture was the highest. The bacteria was named W1 and the strain was identified.

[0043] The liquid culture medium formula screened is as follows: glucose 50g / L, potassium dihydrogen phosphate 0.3g / L, sodium bicarbonate 4.0g / L, ammonium acetate 1.0g / L, sodium chloride 0.6g / L, magnesium chloride 0.8g / L, calcium chloride 0.1g / L, cysteine ​​hydrochloride 0.1g / L, ferric chloride 0.01g / L, manganese chloride 0.001g / L, zinc chloride 0.001g / L, pH adjusted to 7.0±0.1, sterilized at 115°C for 30min. The solid culture medium is a liquid culture medium with 2.0% agar powder added.

[0044] Example 2 Identification of strain W1

[0045] The pure strain W1 was sent to the China Center for Type Culture Collection for strain identification. The physiological and biochemical characteristics are shown in Tables 1 and 2. The 16S rRNA gene sequencing results are shown in the sequence table. After sequence alignment, the BLAST result showed that it was closest to Bacillus cereus, so it was identified as Bacillus cereus W1.

[0046] Example 3 Application of Bacillus cereus W1

[0047] The formula of the seed culture medium (RCM) is (g / L): peptone 10g / L, beef powder 10g / L, yeast powder 3g / L, glucose 5g / L, soluble starch 1.0g / L, sodium chloride 5.0g / L, sodium acetate 3.0g / L, L-cysteine ​​hydrochloride 0.5g / L. Sterilize at 121℃ for 15min.

[0048] The fermentation medium formula is: glucose 60g / L, potassium dihydrogen phosphate 0.3g / L, sodium bicarbonate 4.0g / L, ammonium acetate 1.0g / L, sodium chloride 0.6g / L, magnesium chloride 0.8g / L, calcium chloride 0.1g / L, cysteine ​​hydrochloride 0.1g / L, trace element mother solution 10mL, vitamin mother solution 10mL, adjust pH to 7.0±0.1. Sterilize at 115℃ for 30min. The trace element composition is ferric chloride 1.35g / L, manganese chloride 0.1g / L, zinc chloride 0.1g / L, copper chloride 0.025g / L, boric acid 0.01g / L, sodium molybdate 0.024g / L. The vitamin composition includes vitamin H 2.0 mg / L, folic acid 2.0 mg / L, vitamin B6 10.0 mg / L, vitamin B1 5.0 mg / L, vitamin B2 5.0 mg / L, niacin 5.0 mg / L, calcium pantothenate 5.0 mg / L, P-aminobenzoic acid 5.0 mg / L, and lipoic acid 5.0 mg / L.

[0049] The fermentation process is as follows: (1) Bacillus cereus W1 is inoculated into the seed culture medium, and cultured at 35°C for 24 hours to obtain the seed liquid; (2) The seed liquid is transferred to the fermentation medium at 10% of the inoculation amount, and fermented to produce butyric acid in a non-strict anaerobic environment (nitrogen is introduced in the first 2 hours). The volume of the fermenter is 1.2L, the liquid volume is 0.8L, the stirring rate is 50r / min, and the temperature is 35°C. Nitrogen is introduced for 24 hours before fermentation to maintain an anaerobic environment, the initial pH is natural, and after 24 hours, 10M NaOH solution is added to adjust the pH to 6.5±0.2. After 96 hours of fermentation, it was found that the glucose was almost consumed, and the main products in the fermentation broth were butyric acid and acetic acid, of which the concentration of butyric acid was 17.72g / L, accounting for 78.06%, and the proportion of acetic acid was 21.94%. The trend of glucose and product concentrations in the fermentation broth over time is as follows Figure 2 described.

[0050] Example 4

[0051] Same as Example 3, except that sucrose was used as the carbon source in the fermentation medium. After 96 hours of fermentation, the main products in the fermentation broth were butyric acid and acetic acid, of which the concentration of butyric acid was 16.73 g / L, accounting for 77.85%, and the proportion of acetic acid was 22.15%.

[0052] Example 5

[0053] Same as Example 3, except that mannitol was used as the carbon source in the fermentation medium. After 96 hours of fermentation, the main products in the fermentation broth were butyric acid and acetic acid, of which the concentration of butyric acid was 11.36 g / L, accounting for 76.96%, and the proportion of acetic acid was 23.04%.

[0054] Example 6

[0055] Same as Example 3, except that xylose was used as the carbon source in the fermentation medium. After 96 hours of fermentation, the main products in the fermentation broth were butyric acid and acetic acid, with a butyric acid concentration of 6.62 g / L, of which butyric acid accounted for 76.87% and acetic acid accounted for 23.13%.

[0056] Example 7

[0057] Same as Example 3, except that ammonium chloride was used as the nitrogen source in the fermentation medium. After 96 hours of fermentation, the main products in the fermentation broth were butyric acid and acetic acid, with a butyric acid concentration of 17.05 g / L, of which butyric acid accounted for 73.52% and acetic acid accounted for 26.48%.

[0058] Comparative Example 1

[0059] The same as Example 3, except that the anaerobic environment was maintained throughout the fermentation process, and N2 was introduced. After 96 hours of fermentation, the main products were butyric acid and acetic acid, of which the concentration of butyric acid was 16.11 g / L, of which butyric acid accounted for 79.16% and acetic acid accounted for 20.84%.

[0060] Comparative Example 2

[0061] Same as Example 3, except that sodium citrate was used as the carbon source in the fermentation medium. After 96 hours of fermentation, almost no product was detected in the fermentation broth, and the carbon source was almost not consumed.

[0062] Comparative Example 3

[0063] The same as Example 3, except that: the strain of the present invention is used to ferment and produce hydrogen, and the specific process and conditions of the reference document "Screening of Bacillus cereus and its hydrogen production performance" (Journal of Beijing University of Technology, Wang Haiyan et al., October 2014, Vol. 40, No. 10) are used. After testing, the amount of hydrogen obtained is 0.63 mol per mol of glucose.

Claims

1. A strain of Bacillus cereus, characterized in that: Bacillus cereus ( Bacillus cereus )W1, was deposited in the China Center for Type Culture Collection on July 21, 2022, with the deposit number: CCTCC NO: M 20221147.

2. The Bacillus cereus according to claim 1, characterized in that: Under a microscope, the bacteria are rod-shaped, 4-7 μm long, and are Gram-positive. They are facultative anaerobic bacteria that can grow under non-strict anaerobic conditions. The suitable pH for growth is 4.5-9.0 and the temperature is 20-40°C.

3. The Bacillus cereus according to claim 1, characterized in that: The bacteria can utilize any one or a combination of glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol and sorbitol.

4. The Bacillus cereus according to any one of claims 1 to 3 ( Bacillus cereus ) Application of W1 in fermentation to produce butyric acid.

5. The use according to claim 4, characterized in that: The Bacillus cereus ( Bacillus cereus ) The specific process of W1 fermentation to produce butyric acid is as follows: (1) inoculating the strain into a seed culture medium and culturing to obtain a seed solution; (2) transferring the seed solution to a fermentation medium and fermenting to produce butyric acid under a non-strict anaerobic environment.

6. The use according to claim 4, characterized in that: The strain is inoculated into a seed culture medium, and cultured at 28-42° C. for 12-48 hours to obtain a seed solution; preferably, the culture is carried out under a non-strict anaerobic environment.

7. The use according to claim 5 or 6, characterized in that: The seed culture medium used was RCM medium.

8. The use according to claim 5 or 6, characterized in that: The inoculation amount of the seed solution is 2%-20% of the volume of the fermentation medium.

9. The use according to claim 5, characterized in that: A non-strict anaerobic environment means that oxygen is removed during the first 24 hours, preferably the first 2 hours, of the culture, and then no oxygen is removed until the end of the fermentation.

10. The use according to claim 5, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace elements and vitamins, wherein the carbon source is at least one of glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol and sorbitol, preferably glucose, and the concentration is 20-100 g / L, preferably 30-80 g / L.

11. The use according to claim 5 or 10, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, trace elements and vitamins, wherein the nitrogen source is at least one of ammonium acetate, ammonium chloride and ammonium sulfate, preferably ammonium acetate, with a concentration of 0.1-10 g / L, preferably 0.5-3.0 g / L.

12. The use according to claim 5 or 10, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, trace elements and vitamins, wherein the inorganic salt is at least one of sodium bicarbonate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, calcium chloride and cysteine ​​hydrochloride, preferably potassium dihydrogen phosphate, with a concentration of 0.1-1.0 g / L, preferably 0.2-0.6 g / L.

13. The use according to claim 5 or 10, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, trace elements and vitamins, wherein the trace element is at least one of ferric chloride, zinc chloride, copper chloride, manganese sulfate, boric acid and sodium molybdate, and the concentration is 0.0001-0.05 g / L.

14. The use according to claim 5 or 10, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, an inorganic salt, trace elements and vitamins, wherein the vitamin is at least one of vitamin B1, vitamin B2, vitamin B6, vitamin H, folic acid, nicotinic acid, calcium pantothenate, lipoic acid and p-aminobenzoic acid, and the concentration is 0.00001-0.001 g / L.

15. The use according to claim 5, characterized in that: The fermentation conditions are as follows: temperature of 28-42°C, preferably 34-38°C; natural pH in the initial stage, pH adjusted to 4.0-9.0 after the logarithmic phase, preferably 5.0-7.0; stirring speed of 10-100 r / min; fermentation time of 72-120 h.

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