Bacillus, preparation containing bacillus and application

Abstract

The invention relates to a bacillus strain, a preparation containing the bacillus strain and application of the bacillus strain, the bacillus strain is bacillus sp. LY-3, the bacillus strain is preserved in China Center for Type Culture Collection on February 27, 2023, and the preservation number is CCTCC NO: M 2023203. The bacillus bred by the invention is a multifunctional new strain with the capabilities of reducing ferric iron and degrading aniline at low temperature, and has a good application prospect in environmental pollution treatment.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and pollution control technology, specifically to a strain of Bacillus and preparations containing the strain and their applications. Background Technology

[0002] Bacillus is a type of aerobic and facultative anaerobic bacteria that is widely found in nature. Currently discovered strains have the ability to decompose organic matter, remove nitrogen, and remove phosphorus, and have broad application prospects in wastewater treatment, ecological restoration, and promoting plant growth.

[0003] CN202011512248.8 discloses a strain of denitrifying Bacillus subtilis and its application in strain detoxification. This strain is salt-tolerant and osmotically tolerant, and possesses denitrification capabilities. It can also be used in conjunction with nitrifying bacteria to synergistically reduce the ammonia nitrogen concentration in wastewater containing toxic pollutants from pharmaceutical, pesticide, pharmaceutical, papermaking, and petroleum refining industries. It can serve as an antidote for nitrifying bacteria's resistance to inhibitors. That is, when nitrifying bacteria cannot achieve their nitrification function due to the toxicity of substances in the wastewater, the addition of this strain restores the function of the nitrifying bacteria.

[0004] CN201811399378.8 discloses a strain of Bacillus cereus DIF1, its microbial agent and its application. The Bacillus cereus DIF1 has a strong dissimilatory reduction function of Fe(III) and can efficiently reduce soluble Fe(III) and solid Fe(III). At the same time, this Bacillus cereus DIF1 has a strong ability to reduce hexavalent chromium and generate electricity.

[0005] CN202210706553.3 discloses a strain of Bacillus and its application. This Bacillus is Bacillus argentis, which, in addition to fixing atmospheric nitrogen, also has the functions of dissolving organic and inorganic phosphorus, solubilizing potassium, and secreting IAA. Its application in promoting the growth of thin-shelled pecans is of great significance for enriching the resource bank of growth-promoting bacteria strains for thin-shelled pecans, developing microbial fertilizers for thin-shelled pecans, and reducing the use of chemical fertilizers and promoting plant growth in agricultural and forestry production.

[0006] CN202011354207.0 discloses an efficient aniline-degrading bacterium and its application. The strain is Bacillus albus. Compared with other aniline-degrading bacteria, this bacterium can tolerate higher aniline concentrations. Under the condition of an initial aniline concentration of 5 g / L, the removal rate of aniline can reach 100% within 48 hours, and the degradation effect is very significant.

[0007] Existing Bacillus strains possess various functions, including salt tolerance, denitrification, detoxification, iron reduction, aniline degradation, and plant growth promotion. They show promising applications in agricultural biological control, farmland ecological environment restoration, and plant growth promotion. However, the number of strains currently selected is relatively small, and their performance is relatively limited. Therefore, selecting new strains with superior performance or multiple functions will help expand the application areas of these microorganisms, making them more suitable for pollution control in complex environmental systems. Summary of the Invention

[0008] The purpose of this invention is to provide a strain of Bacillus, preparations containing this bacterium, and its applications. The Bacillus strain selected in this invention is a novel multifunctional strain that possesses both the ability to reduce ferric iron and the ability to degrade aniline at low temperatures, showing promising application prospects in environmental pollution control.

[0009] The first aspect of this invention provides a strain of Bacillus sp. LY-3, which was deposited at the China Center for Type Culture Collection on February 27, 2023, with accession number CCTCC NO: M 2023203.

[0010] The main morphological characteristics of Bacillus sp. LY-3 provided by this invention are as follows: using LB medium, the colonies are white, round, moist, opaque, and have irregular edges.

[0011] The sequencing analysis results of the 16S rDNA gene of Bacillus sp. LY-3 provided by this invention are shown in the sequence listing.

[0012] A second aspect of the present invention provides a formulation comprising Bacillus sp. LY-3. Further, the formulation may also include at least one of inorganic nutrients, organic nitrogen, preservatives, etc.

[0013] A third aspect of the invention provides the use of the aforementioned Bacillus sp. LY-3 or a preparation containing the bacterium in iron reduction. This strain or preparation containing the bacterium can reduce ferric iron to ferrous iron under anaerobic conditions.

[0014] In applications of iron reduction, the concentration of ferric iron in the system is generally 1-10 mM, preferably 1-5 mM.

[0015] In applications involving iron reduction, at least one of lactic acid / lactate, citric acid / citate, etc., is used as an electron donor. Furthermore, sodium lactate is preferred as the lactate, and sodium citrate is preferred as the citrate.

[0016] In the application of iron reduction, the reaction temperature is 15-25℃, preferably 15-20℃, and the pH is 6-9.

[0017] A fourth aspect of this invention provides the application of Bacillus sp. LY-3 or a formulation containing this bacterium in the degradation of aniline. This bacterium or a formulation containing this bacterium can degrade aniline under aerobic conditions, and when the aniline concentration is below 600 mg / L, preferably 30-300 mg / L, the aniline removal rate is greater than 75%. The aniline can be aniline from industrial wastewater, or aniline from polluted surface water and groundwater.

[0018] In the system for degrading aniline, the degradation conditions are: temperature of 15-25℃, preferably 15-20℃, dissolved oxygen of 0.5-2.5 mg / L, and pH of 6-9.

[0019] The fifth aspect of the present invention provides a method for culturing the Bacillus sp. LY-3, using LB medium and culturing under aerobic conditions to the stationary phase.

[0020] The culture conditions described above are: pH 6-9, temperature 10-25℃, dissolved oxygen 0.5-2.5 mg / L, and culture time 18-72 h.

[0021] In the above cultivation methods, the bacterial culture that has reached the stable phase can be directly used as an inoculum in iron reduction systems or aniline degradation systems.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The Bacillus LY-3 strain selected in this invention possesses both aerobic degradation of aniline and anaerobic reduction of ferric iron. Under anaerobic conditions, it can degrade Fe... 3+ Reduced to Fe 2+ It has excellent iron reduction ability; under aerobic conditions, it can grow with aniline as the sole carbon and nitrogen source, achieving efficient degradation of aniline.

[0024] (2) Bacillus LY-3 has better performance in degrading aniline at low temperatures, and its degradation effect at 20℃ is better than that at 30℃.

[0025] (3) Bacillus LY-3 and its preparations are green and environmentally friendly, have diverse functions and excellent performance, and provide microbial resources for pollution remediation, industrial wastewater treatment and other aspects, with broad application prospects.

[0026] Instructions for the Preservation of Biological Materials

[0027] The Bacillus sp. LY-3 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC); accession number: CCTCC NO: M 2023203; deposit date: February 27, 2023; deposit address: Wuhan University, Wuhan, China. Attached Figure Description

[0028] Figure 1 This is a colony morphology diagram of the strain LY-3 of this invention on a plate culture medium. Detailed Implementation

[0029] The isolation, screening, culture, and application of the strains of the present invention will be further described in detail below with reference to specific embodiments. These embodiments are carried out 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.

[0030] 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.

[0031] In this invention, the aniline content was determined using the method specified in "Determination of Aniline Compounds in Water - N-(1-Naphthyl)ethylenediamine azo Spectrophotometric Method" (GB / T 11889-1989). Fe 2+ The total iron content was determined by the o-phenanthroline spectrophotometric method.

[0032] Example 1: Isolation and screening of Bacillus LY-3

[0033] Strain LY-3 was isolated and screened from soil samples using the dilution plate separation method. The samples were obtained in November 2020 from soil contaminated with petroleum at an oil depot in Shanghai.

[0034] (1) Take 10g of soil sample and add it to a 250mL Erlenmeyer flask, then add 100mL of culture medium A. After culturing at 28℃ and 150rpm for 5 days, take 1% of the enriched solution and transfer it to fresh culture medium A. Continue culturing under the same conditions until the OD of the bacterial solution reaches the desired level. 660 When the concentration is greater than 0.8, transfer the culture to fresh culture medium A again. Repeat the above transfer operation three times.

[0035] (2) Under aseptic conditions, transfer to solid culture medium plates (liquid medium A + 2% agar), using the plate dilution method, with a dilution concentration of 10. -4 -10 -14 Incubate until a single colony appears on the plate. Pick a single colony and streak it onto a solid culture medium plate. Repeat this process three times.

[0036] (3) Transfer the purified single colony to liquid culture medium B and incubate at 28℃ and 150 rpm until the OD of the colony is measured. 660 >0.5, a purified strain was obtained. Microscopic observation was used to determine if it was a pure strain; if not, the above steps were repeated until it was confirmed to be pure. After repeated cultivation, the dominant pure strain LY-3 was obtained.

[0037] The formula for culture medium A is as follows: peptone 5 g / L, NaCl 10 g / L, MgSO4 0.5 g / L, NH4Cl 0.5 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, FeSO4 0.03 g / L, CaCl2 0.2 g / L, aniline 0.2 g / L, and diesel oil 1.5 mL / L.

[0038] The formula for culture medium B is as follows: NaCl 10g / L, MgSO4 0.5g / L, NH4Cl 0.5g / L, K2HPO4 1g / L, KH2PO4 0.5g / L, FeSO4 0.03g / L, CaCl2 0.2g / L, aniline 0.1g / L, and diesel oil 0.5mL / L.

[0039] The purified strain LY-3 was subjected to species identification, and the 16S rDNA gene sequencing results are shown in the sequence listing. The 16S rDNA sequence was compared with a professional database based on the NCBI database, and the strain was identified as Bacillus sp.

[0040] Example 2: Culture of Bacillus LY-3

[0041] Prepare LB medium: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract.

[0042] The preserved pure Bacillus LY-3 was inoculated into LB medium and cultured in a shaker at 20℃, pH 7.5-8.0, under aerobic conditions for 24 hours to obtain activated bacterial solution.

[0043] 2% of the activated bacterial solution was inoculated into LB medium for large-scale culture. The culture conditions were: temperature 20℃, pH 7.5-9.0, dissolved oxygen 0.5-2.5 mg / L, and culture for at least 20 hours. Samples were then taken for analysis, including OD... 660 If the concentration reaches 0.8 or higher, it is considered a suitable bacterial solution for addition or use, or it can be formulated into a preparation for use.

[0044] Example 3: Application of Bacillus LY-3 in Iron Reduction

[0045] Prepare ferric citrate medium with the following formula: 0.5 g / L NH4Cl, 0.5 g / L KH2PO4, 0.1 g / L NaCl, 2 g / L NaHCO3, 2 mM ferric citrate (ferric trivalent is 113.5 mg / L), pH 7.

[0046] (1) Take the bacterial culture from Example 2 and add it to the culture medium at an inoculum volume of 5%. Use 2 mL / L sodium lactate as an electron donor and treat under anaerobic conditions at 20°C and pH 7.5-8.5 for 5 days. The Fe content was then measured. 2+ The content was 82.8 mg / L, Fe 3+ The reduction rate was 73.0%.

[0047] (2) Take the bacterial culture from Example 2 and add it to the culture medium at an inoculum volume of 5%. Use 2 g / L sodium citrate as an electron donor and treat under anaerobic conditions at 20°C and pH 7.5-8.5 for 5 days. The Fe content was measured. 2+ The content was 77.7 mg / L, Fe 3+ The reduction rate was 68.4%.

[0048] Example 4: Application of Bacillus LY-3 in the degradation of aniline

[0049] A body of water is contaminated with aniline, with an aniline concentration of 260 mg / L.

[0050] (1) Take the bacterial culture cultured in Example 2 and add it to the water at an inoculum volume of 4%. Treat the water at 20°C, pH 7.5-9.0, and dissolved oxygen of 0.5-2.5 mg / L for 5 days. The concentration of aniline in the effluent was found to be 20.7 mg / L.

[0051] (2) The culture temperature was changed to 25℃, and the treatment was carried out under the same conditions for 5 days. The concentration of aniline in the effluent was found to be 55.8 mg / L.

[0052] (2) The culture temperature was changed to 30℃, and the treatment was carried out under the same conditions for 5 days. The concentration of aniline in the effluent was measured to be 78.5 mg / L.

[0053] Comparative Example 1

[0054] Same as Example 3, except that Bacillus sp. 3# as described in CN116445315A, with accession number CGMCC No. 21561, was used. The bacterial culture was prepared according to the method described in Example 2, and added to the culture medium at an inoculum volume of 5%. Using 2 mL / L sodium lactate as the electron donor, the culture was treated under anaerobic conditions at 20°C and pH 7.5-8.5 for 5 days. The Fe content was detected. 2+ The content was 39.2 mg / L, Fe 3+The reduction rate was 34.5%.

[0055] Comparative Example 2

[0056] Same as Example 4, except that Bacillus sp. 3# as described in CN116445315A, with accession number CGMCC No. 21561, was used. The bacterial culture was prepared according to the method described in Example 2, and added to water at an inoculum volume of 4%. The solution was treated for 5 days at 20°C, pH 7.5-9.0, and dissolved oxygen of 0.5-2.5 mg / L. The aniline concentration in the effluent was measured to be 206.8 mg / L.

Claims

1. A strain of Bacillus, characterized in that... This strain is Bacillus ( Bacillus sp.) LY-3 was deposited at the China Center for Type Culture Collection on February 27, 2023, with accession number CCTCC NO: M 2023203.

2. The Bacillus according to claim 1, characterized in that: The main morphological characteristics of this bacterium are: on LB medium, the colonies are white, round, moist, opaque, and have irregular edges.

3. A method comprising the Bacillus subtilis of claim 1 or 2 ( Bacillus (sp.) LY-3 formulation.

4. The formulation according to claim 3, characterized in that: The formulation also includes at least one of inorganic nutrients, organic nitrogen, and preservatives.

5. A Bacillus subtilis according to claim 1 ( Bacillus sp . Application of LY-3 or preparations containing this bacterium in iron reduction.

6. The application according to claim 5, characterized in that: This strain or preparation containing this bacterium can reduce ferric iron to ferrous iron under anaerobic conditions, with the concentration of ferric iron in the system being 1-10 mM, preferably 1-5 mM.

7. The application according to claim 5 or 6, characterized in that: In the application of iron reduction, at least one of lactic acid / lactate and citric acid / citate is used as an electron donor. Further, lactate is preferably sodium lactate, and citrate is preferably sodium citrate.

8. The application according to claim 5 or 6, characterized in that: In the application of iron reduction, the reaction temperature is 15-25℃, preferably 15-20℃, and the pH is 6-9.

9. A Bacillus subtilis according to claim 1 ( Bacillus sp . The application of LY-3 or preparations containing this bacterium in the degradation of aniline.

10. The application according to claim 9, characterized in that: This bacterium or preparations containing this bacterium can degrade aniline under aerobic conditions. When the aniline concentration is below 600 mg / L, preferably 30-300 mg / L, the aniline removal rate is greater than 75%.

11. The application according to claim 9 or 10, characterized in that: In the system for degrading aniline, the degradation conditions are: temperature of 10-25℃, preferably 15-20℃, dissolved oxygen of 0.5-2.5 mg / L, and pH of 6-9.

12. A Bacillus subtilis according to claim 1 ( Bacillus sp . The cultivation method of LY-3 is characterized by... The culture was carried out in LB medium under aerobic conditions until the stationary phase.

13. The cultivation method according to claim 12, characterized in that: The culture conditions are: pH 6-9, temperature 10-25℃, dissolved oxygen 0.5-2.5 mg / L, and culture time 18-72 h.

14. The cultivation method according to claim 12, characterized in that: The bacterial culture that has reached the stable phase can be directly used as inoculum in iron reduction systems or aniline degradation systems.

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