A strain of Bacillus cereus and its application

Abstract

This invention relates to a strain of Bacillus cereus and its applications, specifically Bacillus cereus (… Bacillus cereus Bacillus cereus (W1) was deposited at the China Center for Type Culture Collection on July 21, 2022, with accession number CCTCC NO: M 20221147. Bacillus cereus W1 can grow and ferment high-yield butyric acid under non-strict anaerobic conditions, and has the advantages of strong oxygen tolerance and high butyric acid yield.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus cereus and its application in the fermentation of butyric acid. Background Technology

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

[0003] In recent years, butyric acid has attracted widespread attention as a feedstock for the production of butanol, a biofuel considered a next-generation liquid fuel following ethanol, which boasts higher energy density and calorific value compared to ethanol. Another potential application of butyric acid lies in the production of biodegradable plastics: butyric acid-rich mixed carboxylic acids can be bioconverted into polyhydroxybutyrate (PHB). PHB is a major component of polyhydroxyalkanoates (PHAs), which are raw materials for the production of biodegradable plastics.

[0004] Currently, the industrial production of butyric acid mainly relies on the butyraldehyde oxidation method, which is based on fossil resources such as petroleum, coal, and natural gas. This method uses butyraldehyde as a raw material, oxygen or air as an oxidant, and a continuous reaction under the action of a catalyst, followed by distillation to obtain butyric acid. Due to its advantages such as readily available and simple raw materials, easy process control, and high product yield, the butyraldehyde oxidation method has become the mainstream method for the commercial production of butyric acid. However, the finite and unsustainable nature of fossil resources and the environmental pollution caused by their extraction and use are forcing people to seek sustainable production methods that can harmonize with nature.

[0005] Microbial fermentation for butyric acid production is a promising alternative technology. Although the production cost of fermentation is currently relatively high, with the continuous rise in crude oil prices, the maturation of biomass chemical industry, and the increasing demand for organic and natural products in the food additives, pharmaceuticals, and preservatives sectors, fermentation for butyric acid production 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 their fermentation performance needs improvement.

[0006] The main factors restricting the development and commercialization of butyric acid production by microbial fermentation are: (1) Low final concentration of butyric acid. In the fermentation system, the growth of butyric acid-producing bacteria is inhibited by the final fermentation products (butyric acid and acetic acid), resulting in a low final concentration of butyric acid in the fermentation broth; (2) High separation cost. The low final concentration of products in the butyric acid fermentation broth and the presence of byproducts increase the difficulty of separating and purifying butyric acid; (3) High price of fermentation raw materials. Currently, the most commonly used raw materials for butyric acid fermentation are glucose, corn, etc., which are relatively expensive, resulting in high production costs.

[0007] The literature "Screening and Hydrogen Production Performance of Bacillus cereus" (Wang Haiyan et al., Journal of Beijing University of Technology, October 2014, Vol. 40, No. 10) isolated a pure hydrogen-producing strain XN12 from activated sludge in a wastewater treatment plant. Experimental results showed that, based on one mole of glucose, the strain achieved a maximum hydrogen production of 1.6 mol under neutral conditions. Analysis of the liquid-phase products indicated that the strain is a butyric acid fermenting bacterium, producing hydrogen through pyruvate metabolism. However, this strain was screened specifically for hydrogen production, and its performance in dedicated butyric acid fermentation needs further improvement. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a strain of Bacillus cereus and its applications. This strain can grow and ferment to produce butyric acid in a non-strict anaerobic environment, and has advantages such as strong oxygen tolerance and high butyric acid yield.

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

[0010] The Bacillus cereus W1 provided by this invention exhibits a rod-shaped cell morphology under a microscope (as shown in the attached image). Figure 1 ), 4-7 μm in length, are Gram-positive bacteria.

[0011] The Bacillus cereus W1 provided by this invention is a facultative anaerobic bacterium that can grow under non-strictly anaerobic conditions. The optimal growth pH is 4.5-9.0 and the optimal temperature is 20-40℃.

[0012] The Bacillus cereus W1 provided by this invention is weakly positive for arginine double hydrolase, positive for pyruvate VP reaction, positive for gelatin hydrolysis reaction, and weakly positive for starch hydrolysis reaction. Its physiological and biochemical characteristics are shown in Tables 1 and 2.

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

[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, + indicates a positive reaction; - indicates a negative reaction; w indicates a weak positive reaction.

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

[0022] The present invention also provides the application of the aforementioned Bacillus cereus W1 in the fermentation of butyric acid.

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

[0024] In this invention, the strain is inoculated into a seed culture medium and statically cultured at 28-42℃ for 12-48 hours to obtain a seed solution. More preferably, the culture is carried out under a non-strictly anaerobic environment. The seed culture medium used is a conventionally used medium in the art, preferably RCM medium.

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

[0026] In this invention, an anaerobic environment refers to deoxygenation throughout the entire cultivation process, while a non-strictly anaerobic environment refers to deoxygenation for the first 24 hours of fermentation, preferably the first 2 hours, followed by no further deoxygenation until fermentation is complete. Deoxygenation can be achieved through methods such as introducing N2 or adding an oxygen scavenger.

[0027] In this invention, the fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace elements, and vitamins. The carbon source can be at least one selected from glucose, sucrose, trehalose, fructose, mannose, L-arabinose, ribose, xylose, glycerol, inositol, mannitol, sorbitol, etc., preferably glucose, with a concentration of 20-100 g / L, more preferably 30-80 g / L. The nitrogen source can be at least one selected from ammonium acetate, ammonium chloride, and ammonium sulfate, preferably ammonium acetate, with a concentration of 0.1-10 g / L, more preferably 0.5-3.0 g / L. The inorganic salt can be at least one selected from sodium bicarbonate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, calcium chloride, cysteine ​​hydrochloride, etc., preferably potassium dihydrogen phosphate, with a concentration of 0.1-1.0 g / L, more preferably 0.2-0.6 g / L. The trace elements may be at least one of the following: 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 the following: 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 this invention, the fermentation conditions are as follows: temperature is 28-42℃, preferably 34-38℃; pH is natural in the initial stage, and pH is controlled to 4.0-9.0 after the logarithmic phase, preferably 5.0-7.0; fermentation is carried out in a fermenter with stirring, and the stirring speed is 10-100 r / min; fermentation time is 72-120 h.

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

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) The present invention has selected and obtained a strain of Bacillus cereus W1, which has strong oxygen resistance, reducing the deoxygenation process of traditional anaerobic fermentation. It does not require the continuous introduction of N2, thus avoiding the risks of poor bacterial growth and fermentation failure caused by incomplete deoxygenation, and at the same time reducing operating energy consumption.

[0032] (2) Bacillus cereus W1 also has strong acid resistance and has excellent butyric acid fermentation level under the same substrate concentration conditions. The products are mainly butyric acid and acetic acid, with butyric acid accounting for no less than 70% and acetic acid accounting for no less than 20%.

[0033] (3) Bacillus cereus W1 can use pentoses, hexoses and other sugars as fermentation substrates at the same time, which expands the range of fermentation substrates.

[0034] Instructions for the Preservation of Biological Materials

[0035] The Bacillus cereus W1 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC); accession number: CCTCC NO: M 20221147; deposit date: July 21, 2022; deposit address: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province. Attached Figure Description

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

[0037] Figure 2 This shows the trend of glucose concentration and product concentration in the fermentation broth of this invention over time. Detailed Implementation

[0038] The strain and its effects of the present invention will be further described in detail below with reference to specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following embodiments.

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

[0040] In this invention, the concentrations of butyric acid, acetic acid, and substrate in the fermentation broth were all detected using high-performance liquid chromatography (HPLC). HPLC analysis conditions: Instrument: Agilent 1260; Column: BIO-RAD HPX-87H Ion Exclusion Column; Detector: Refractive index detector (RID); Mobile phase: 0.005 mol / L H₂SO₄ aqueous solution; Flow rate: 0.7 mL / min; Column temperature: 65℃; RID temperature: 40℃; Injection volume: 5 μL.

[0041] Example 1: Screening of Bacillus cereus W1

[0042] Anaerobic sludge was sampled from a wastewater treatment plant in Dalian in March 2022. After pulverization, the sludge was filtered through three layers of gauze and treated at 80℃ for 1 hour. Sodium 2-bromoethanesulfonate (BES), a methanogen inhibitor, was added and mixed with corn stalk powder and glucose in a sterile Erlenmeyer flask. An appropriate amount of sterile water was added to enrich the bacterial cells. After one week of cultivation, 1.0 mL of the culture was transferred to a 9.0 mL liquid culture medium in a test tube and incubated statically at 35℃ for 72 hours. This process was repeated three times. The supernatant was collected after centrifugation and filtered. Liquid chromatography analysis showed a butyric acid concentration of 2.8 g / L. Further culture was plated on solid culture plates and incubated at 35℃ for 72 hours. Single colonies were then picked for fermentation testing. A total of 40 single colonies were selected for fermentation, and 14 cultures produced butyric acid. The sixth single colony showed the highest butyric acid concentration. This bacterium was named W1 and its strain was identified.

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

[0044] Example 2 Identification of strain W1

[0045] The purified strain W1 was sent to the China Center for Type Culture Collection for strain identification. Physiological and biochemical characteristics are shown in Tables 1 and 2. The 16S rRNA gene sequencing results are shown in the sequence listing. Sequence alignment and BLAST results showed the closest homology to *Bacillus cereus*, therefore it was identified as *Bacillus cereus* W1.

[0046] Example 3: Application of Bacillus cereus W1

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

[0048] The fermentation medium formula is as follows: glucose 60 g / L, potassium dihydrogen phosphate 0.3 g / L, sodium bicarbonate 4.0 g / L, ammonium acetate 1.0 g / L, sodium chloride 0.6 g / L, magnesium chloride 0.8 g / L, calcium chloride 0.1 g / L, cysteine ​​hydrochloride 0.1 g / L, trace element stock solution 10 mL, vitamin stock solution 10 mL, and pH adjusted to 7.0 ± 0.1. Sterilize at 115℃ for 30 min. The trace element composition is: ferric chloride 1.35 g / L, manganese chloride 0.1 g / L, zinc chloride 0.1 g / L, copper chloride 0.025 g / L, boric acid 0.01 g / L, and sodium molybdate 0.024 g / L. The vitamin composition is as follows: 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 Alpha-lipoic acid 5.0 mg / L.

[0049] The fermentation process was as follows: (1) Bacillus cereus W1 was inoculated into the seed culture medium and cultured statically at 35℃ for 24 hours to obtain the seed liquid; (2) the seed liquid was transferred to the fermentation culture medium at 10% of the inoculation amount and fermented to produce butyric acid under a non-strict anaerobic environment (nitrogen gas was introduced for the first 2 hours). The fermentation tank had a volume of 1.2L, a liquid volume of 0.8L, a stirring rate of 50r / min, and a temperature of 35℃. Nitrogen gas was introduced for the first 24 hours to maintain an anaerobic environment. The initial pH was natural. After 24 hours, 10M NaOH solution was added to adjust the pH to 6.5±0.2. After 96 hours of fermentation, the glucose was almost completely consumed. The main products in the fermentation liquid were butyric acid and acetic acid. The concentration of butyric acid was 17.72g / L, accounting for 78.06%, and the concentration of acetic acid was 21.94%. The trends of glucose and product concentrations in the fermentation liquid over time are as follows. Figure 2 As stated above.

[0050] Example 4

[0051] Similar to 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, with butyric acid concentration of 16.73 g / L (77.85%) and acetic acid concentration of 22.15%.

[0052] Example 5

[0053] Similar to 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, with butyric acid concentration of 11.36 g / L (76.96%) and acetic acid concentration of 23.04%.

[0054] Example 6

[0055] Similar to 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, accounting for 76.87% and acetic acid accounting for 23.13%.

[0056] Example 7

[0057] Similar to 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, accounting for 73.52% and acetic acid accounting for 26.48%.

[0058] Comparative Example 1

[0059] Similar to Example 3, except that an anaerobic environment was maintained throughout the fermentation process, using N2. After 96 hours of fermentation, the main products were butyric acid and acetic acid, with a butyric acid concentration of 16.11 g / L, accounting for 79.16% and acetic acid accounting for 20.84%.

[0060] Comparative Example 2

[0061] Similar to 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, indicating that the carbon source was almost completely consumed.

[0062] Comparative Example 3

[0063] Same as Example 3, except that: hydrogen production was achieved through fermentation using the strain of this invention, following the specific process and conditions described in the reference "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). The result showed that 0.63 mol of hydrogen was obtained per mole of glucose.

Claims

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

2. The Bacillus cereus according to claim 1, characterized in that: Under a microscope, the cells of this bacterium are rod-shaped, 4-7 μm long, and are Gram-positive. They are facultative anaerobic bacteria, capable of growing under non-strictly anaerobic conditions, with an optimal pH of 4.5-9.0 and a temperature of 20-40℃ for growth.

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

4. The use of Bacillus cereus W1 as described in any one of claims 1-3 in the fermentation of butyric acid.

5. The application according to claim 4, characterized in that: The specific process of Bacillus cereus W1 fermentation to produce butyric acid is as follows: (1) the strain is inoculated into a seed culture medium and cultured to obtain a seed liquid; (2) the seed liquid is transferred to a fermentation culture medium and fermented to produce butyric acid under a non-strict anaerobic environment.

6. The application according to claim 4, characterized in that: The strain was inoculated into seed culture medium and incubated statically at 28-42℃ for 12-48 hours to obtain seed liquid.

7. The application according to claim 6, characterized in that: Cultured in a non-strictly anaerobic environment.

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

9. The application according to claim 5 or 6, characterized in that: The inoculation volume of the seed culture is 2%-20% of the fermentation medium volume.

10. The application according to claim 7, characterized in that: A non-strictly anaerobic environment refers to deoxygenation 24 hours before cultivation, followed by no further deoxygenation until the end of fermentation.

11. The application according to claim 10, characterized in that: A non-strictly anaerobic environment refers to removing oxygen 2 hours before cultivation, and then not removing oxygen until the end of fermentation.

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

13. The application according to claim 12, characterized in that: The carbon source is glucose, with a concentration of 30-80 g / L.

14. The application according to claim 5 or 12, characterized in that: The fermentation medium includes carbon source, nitrogen source, inorganic salt, trace elements and vitamins. The nitrogen source is at least one of ammonium acetate, ammonium chloride and ammonium sulfate, with a concentration of 0.1-10 g / L.

15. The application according to claim 14, characterized in that: The nitrogen source is ammonium acetate, with a concentration of 0.5-3.0 g / L.

16. The application according to claim 5 or 12, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace elements, and vitamins. The inorganic salts are at least one of sodium bicarbonate, potassium dihydrogen phosphate, sodium chloride, magnesium chloride, calcium chloride, and cysteine ​​hydrochloride, with a concentration of 0.1-1.0 g / L.

17. The application according to claim 16, characterized in that: The inorganic salt is potassium dihydrogen phosphate, with a concentration of 0.2-0.6 g / L.

18. The application according to claim 5 or 12, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace elements, and vitamins. The trace elements are at least one of ferric chloride, zinc chloride, copper chloride, manganese sulfate, boric acid, and sodium molybdate, with a concentration of 0.0001-0.05 g / L.

19. The application according to claim 5 or 12, characterized in that: The fermentation medium includes a carbon source, a nitrogen source, inorganic salts, trace elements, and vitamins. The vitamins are at least one of vitamin B1, vitamin B2, vitamin B6, vitamin H, folic acid, niacin, calcium pantothenate, lipoic acid, and para-aminobenzoic acid, with a concentration of 0.00001-0.001 g / L.

20. The application according to claim 5, characterized in that: Fermentation conditions are as follows: temperature 28-42℃; initial pH at a natural level, followed by pH adjustment to 4.0-9.0 after the logarithmic phase; stirring speed 10-100 r / min; fermentation time 72-120 h.

21. The application according to claim 20, characterized in that: Fermentation conditions are: temperature 34-38℃; pH is natural in the initial stage, and pH is adjusted to 5.0-7.0 after the logarithmic phase.

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