Bacillus aryabhattai, preparation containing bacillus aryabhattai and application of bacillus aryabhattai

Abstract

The invention relates to bacillus aryabhattai, a preparation containing the bacillus aryabhattai and application of the bacillus aryabhattai. The bacillus aryabhattai provided by the invention is bacillus aryabhattai LY-2, the bacillus aryabhattai is preserved in the China Center for Type Culture Collection (CCTCC) on February 27, 2023, and the preservation number is CCTCC NO: M 2023202. The bacillus aryabhattai bred by the invention is a multifunctional new strain with the capabilities of removing nitrogen and phosphorus and reducing ferric iron, 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 argentea, preparations containing the bacterium, and their applications. Background Technology

[0002] Iron is the fourth most abundant element in the Earth's crust, with an average content of approximately 5.6%, and it is highly abundant in soils and sediments. The reduction of iron in soils and sediments is driven by microorganisms. Under anaerobic conditions, iron-reducing microorganisms oxidize the matrix (electron donor) within their bodies and utilize external Fe(III) as an electron acceptor at the end of the respiratory chain, facilitating electron transfer and creating a transmembrane proton concentration potential gradient. This gradient is then converted into energy required for their metabolism, thereby reducing Fe(III) to Fe(II). Therefore, the microbial reduction of Fe(III) plays a crucial role in environmental remediation.

[0003] CN202310418750.X discloses a fermentation preparation of *Priscilla argentea* YZ-151, its preparation method, and its application. The preparation method includes: activating *Priscilla argentea* YZ-151; preparing a fermentation seed liquid from the activated *Priscilla argentea* YZ-151; inoculating the fermentation seed liquid into a fermentation medium to start fermentation; adjusting the fermentation pH to 4.0–9.0, fermentation temperature to 25℃–40℃, inoculum size to 1%–10%, fermentation volume to 30mL / 250mL–200mL / 250mL, fermentation speed to 110r / min–200r / min, and fermentation time to 1d–6d to obtain the *Priscilla argentea* YZ-151 fermentation preparation. This *Priscilla argentea* YZ-151 fermentation preparation has good control effects on black spot disease and gray mold of *Eleutherococcus senticosus*.

[0004] CN202310802913.4 discloses *Priestia aryabhattai* H2 and its applications. This strain not only has strong extracellular polysaccharide production capabilities but also produces indoleacetic acid (IAA) and siderophores, and possesses phosphate-dissolving capabilities. It can tolerate a salt concentration of 7% and can grow normally without inhibition between pH 5.0 and 9.0. Applying strain H2 to saline soils not only promotes the stability of soil aggregates but also further stabilizes the water-stable structure of soil aggregates and increases soil porosity; simultaneously, strain H2 also promotes plant growth.

[0005] CN202210706553.3 discloses a strain of Bacillus and its applications, relating to the field of plant rhizosphere growth-promoting bacteria technology. This Bacillus aureus, in addition to fixing atmospheric nitrogen, also possesses 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] The existing technology discloses that *Priestia aryabhattai* possesses multiple functions, including nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA secretion, plant growth promotion, extracellular polysaccharide production, and control of black spot and gray mold diseases. It shows promise in agricultural biological control, farmland ecological environment restoration, plant growth promotion, and fermentation. However, *Priestia aryabhattai* strains with iron reduction and nitrogen and phosphorus removal capabilities have not yet been discovered. Therefore, breeding new multifunctional strains will help expand the application areas of this type of bacteria, making them more suitable for the management of complex environmental systems. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of *Bacillus argentea*, preparations containing this bacterium, and its applications. The *Bacillus argentea* strain selected in this invention is a novel, multifunctional strain capable of denitrification, phosphorus removal, and reduction of ferric iron, showing promising application prospects in environmental pollution control.

[0008] The first aspect of this invention provides a strain of Priestia aryabhattai LY-2, which was deposited at the China Center for Type Culture Collection on February 27, 2023, with accession number CCTCC NO: M 2023202.

[0009] The main morphological characteristics of Priestia aryabhattai LY-2 provided by this invention are as follows: using LB medium, the colonies are white, round, moist, opaque, and have irregular edges.

[0010] The 16S rDNA gene sequencing analysis results of Priestia aryabhattai LY-2 provided by this invention are shown in the sequence listing.

[0011] A second aspect of the present invention provides a formulation comprising Priestia aryabhattai LY-2. Further, the formulation may also include at least one of inorganic nutrients, organic nitrogen, preservatives, etc.

[0012] A third aspect of the invention provides the use of *Priestia aryabhattai* LY-2 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.

[0013] In applications of iron reduction, the concentration of ferric iron is generally 1-10 mM, preferably 1-5 mM.

[0014] In iron reduction applications, at least one of sodium lactate, sodium citrate, etc., is used as an electron donor.

[0015] In iron reduction applications, the reaction temperature is 25-35℃ and the pH is 6-9.

[0016] The fourth aspect of the present invention provides the application of the aforementioned Priestia aryabhattai LY-2 or a preparation containing the bacterium in wastewater denitrification and phosphorus removal, wherein the strain is capable of simultaneously removing ammonia nitrogen and phosphate from wastewater.

[0017] In nitrogen and phosphorus removal applications, the ammonia nitrogen concentration is 20-100 mg / L and the total phosphorus concentration is 1-15 mg / L. The removal rates of both total phosphorus and ammonia nitrogen can reach over 85%.

[0018] In nitrogen and phosphorus removal applications, a carbon source of 2-10 g / L needs to be added. The carbon source can be at least one of sodium acetate, sodium lactate, sodium citrate, sodium succinate, etc.

[0019] In nitrogen and phosphorus removal applications, the application conditions are: temperature 20-35℃, pH 6-9, and dissolved oxygen concentration 1-2.5 mg / L.

[0020] The fifth aspect of the present invention provides a method for culturing Priestia aryabhattai LY-2, using LB medium and culturing to the stationary phase under aerobic conditions.

[0021] The culture conditions described above are: pH 6-9, temperature 15-40℃, dissolved oxygen concentration 0.5-2.5 mg / L, and culture time 18-48 h.

[0022] 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 nitrogen and phosphorus removal systems.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The Bacillus LY-2 strain selected in this invention can, under anaerobic conditions, convert Fe... 3+ Reduced to Fe2+ It has excellent iron reduction ability.

[0025] (2) The Bacillus LY-2 selected in this invention can remove ammonia nitrogen and phosphate from wastewater simultaneously under aerobic conditions, and has heterotrophic nitrification and dephosphorization capabilities.

[0026] (3) The strains and their preparations provided by this invention are green and environmentally friendly, with diverse functions, and provide microbial resources for the remediation of polluted water bodies and the treatment of industrial wastewater, with broad application prospects. Attached Figure Description

[0027] Figure 1 This is a colony morphology diagram of strain 1H-2 of the present invention on LB agar plate medium.

[0028] Instructions for the Preservation of Biological Materials

[0029] The *Priestia aryabhattai* LY-2 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC); accession number: CCTCC NO: M 2023202; deposit date: February 27, 2023; deposit address: Wuhan University, Wuhan, China. Detailed Implementation

[0030] 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.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. In this invention, all percentages are weight percentages.

[0032] In this invention, total phosphorus was determined using the ammonium molybdate spectrophotometric method (GB 11893-89); ammonia nitrogen was determined using the Nessler's reagent colorimetric method (GB7479-1987). 2+ Total iron was determined using the o-phenanthroline spectrophotometric method.

[0033] Example 1: Isolation and Screening of Strains

[0034] Strain LY-2 was obtained by screening soil samples using the dilution plate separation method. The soil samples were obtained from an oil depot of a petrochemical company in Shanghai in November 2020.

[0035] (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.

[0036] (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.

[0037] (3) Transfer the purified single colony to 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-2 was obtained.

[0038] 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.

[0039] The formula for culture medium B is as follows: NaCl 10g / L, MgSO4 0.5g / L, K2HPO4 1g / L, KH2PO4 0.5g / L, NH4Cl 0.5g / L, FeSO4 0.03g / L, CaCl2 0.2g / L, aniline 0.1g / L, and diesel oil 0.5mL / L.

[0040] The purified strain LY-2 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 it showed the closest homology to *Priestiaaryabhattai*. Therefore, this strain was identified as *Priestiaaryabhattai*.

[0041] Example 2: Culture of Bacillus LY-2

[0042] Prepare LB medium: 10 g / L NaCl, 10 g / L peptone, 5 g / L yeast extract.

[0043] Pure Bacillus LY-2 was inoculated into LB medium and cultured in a shaker at 25°C, pH 7.5-8.0, under aerobic conditions for 24 hours to obtain activated bacterial solution.

[0044] 2% of the activated bacterial solution was inoculated into LB medium for large-scale culture. The culture conditions were: temperature 25℃, pH 7.0-7.5, 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.

[0045] Example 3: Application of Bacillus LY-2 in Iron Reduction

[0046] 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 111 mg / L), pH 7.

[0047] (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 25°C and pH 7.0-8.5 for 5 days. The Fe content was then measured. 2+ The content was 61.6 mg / L, Fe 3+ The reduction rate was 55.5%.

[0048] (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 25°C and pH 7.0-8.5 for 5 days. The Fe content was measured. 2+ The content was 67.4 mg / L, Fe 3 + The reduction rate was 60.7%.

[0049] (3) The bacterial culture from Example 2 was added to yeast extract at a concentration of 50 mg / L to obtain the formulation. This was then added to the culture medium at a volume inoculum of 5%, using 2 mL / L sodium lactate as the electron donor. The mixture was treated anaerobicly at 25°C and pH 7.0-8.5 for 5 days. The Fe content was then measured. 2+ The content was 75.8 mg / L, Fe 3+ The reduction rate was 68.2%.

[0050] Example 4: Application of Bacillus LY-2 in denitrification and phosphorus removal

[0051] The wastewater from a company's biochemical treatment unit has an ammonia nitrogen concentration of 54 mg / L and a total phosphorus concentration of 7.8 mg / L.

[0052] (1) Take the bacterial culture cultured in Example 2 and add it to the wastewater at a volume inoculum of 1%, along with 4 g / L of sodium acetate. Treat for 24 h at 25°C, pH 7.8-9, and dissolved oxygen concentration of 1-2.5 mg / L. The effluent was found to contain 0.3 mg / L ammonia nitrogen and 0.5 mg / L total phosphorus.

[0053] (2) Take the bacterial culture cultured in Example 2 and add it to the wastewater at a volume inoculum of 1%, along with 4 g / L of sodium citrate. Treat for 24 h at 25°C, pH 7.8-9, and dissolved oxygen concentration of 1-2.5 mg / L. The effluent was found to contain 0.5 mg / L ammonia nitrogen and 0.8 mg / L total phosphorus.

Claims

1. A strain of Bacillus argentea ( Priestia aryabhattai LY-2, this strain was deposited at the China Center for Type Culture Collection on February 27, 2023, with accession number CCTCC NO: M 2023202.

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

3. A method comprising the *Bacillus argentea* as described in claim 1 or 2 (… Priestia aryabhattai ) LY-2 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 argentea as described in claims 1-2 ( Priestia aryabhattai Application of LY-2 or preparations containing this bacterium in iron reduction.

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

7. The application according to claim 5 or 6, characterized in that: In iron reduction applications, at least one of sodium lactate and sodium citrate is used as an electron donor.

8. The application according to claim 5 or 6, characterized in that: In iron reduction applications, the reaction temperature is 25-35℃ and the pH is 6-9.

9. A Bacillus argentea as described in claims 1-2 ( Priestia aryabhattai Application of LY-2 or preparations containing this bacterium in wastewater denitrification and phosphorus removal.

10. The application according to claim 9, characterized in that: This strain can simultaneously remove ammonia nitrogen and phosphate from wastewater with ammonia nitrogen concentration of 20-100 mg / L and total phosphorus concentration of 1-15 mg / L. The removal rates of both total phosphorus and ammonia nitrogen can reach over 85%.

11. The application according to claim 9 or 10, characterized in that: In nitrogen and phosphorus removal applications, a carbon source of 2-10 g / L needs to be added. The carbon source is at least one of sodium acetate, sodium lactate, sodium citrate, and sodium succinate.

12. The application according to claim 9 or 10, characterized in that: The application conditions are: temperature 20-35℃, pH 6-9, and dissolved oxygen concentration 1-2.5 mg / L.

13. A Bacillus argentea as described in claims 1-2 ( Priestia aryabhattai The LY-2 culture method involves using LB medium and culturing under aerobic conditions until the stationary phase.

14. The cultivation method according to claim 13, characterized in that: The culture conditions are: pH 6-9, temperature 15-40℃, dissolved oxygen concentration 0.5-2.5 mg / L, and culture time 18-48 h.

15. The cultivation method according to claim 13, characterized in that: The bacterial culture that has reached the stable phase can be directly used as inoculum in iron reduction systems or nitrogen and phosphorus removal systems.

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