Paenibacillus parafacii and application thereof in selenium oxidation

Abstract

This invention discloses a strain of *Bacillus paramycoides* and its application in selenium oxidation, belonging to the field of agricultural microbiology. A strain of *Bacillus paramycoides* H1 was isolated from the soil of a selenium-nutrition experimental field at Huazhong Agricultural University and deposited at the China Center for Type Culture Collection (CCTCC) on June 27, 2023, with accession number CCTCCNO: M 20231107. Experimental verification showed that this strain can simultaneously oxidize elemental selenium and organic selenium. This invention utilizes this strain to convert organic and elemental selenium into water-soluble selenium that promotes plant absorption, laying a scientific foundation for the preparation of safe and low-cost plant selenium-enriching agents and selenium oxidants. This is of great significance for the research and agricultural cultivation of selenium conversion model strains.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a strain of Bacillus paramicularis and its application in selenium oxidation. Background Technology

[0002] Selenium (Se) is located in Group VI of the periodic table, and its common valence state in nature is Se. 2- Se 0 Se 4+ Se 6+ Se 2- Selenium mainly exists in the form of organic selenium, and is mostly found in living organisms, such as selenoamino acids and selenium-binding proteins; H₂Se is the main form of gaseous selenium and also belongs to the selenium family. 2- Se 4+ Se 6+ It is the main form in which inorganic selenium exists, most commonly as two oxygen-containing anions, SeO3. 2- and SeO4 2- Selenium is also the main form of selenium absorption by plants. Most of the selenium in selenium-containing agricultural soils exists in the form of organic selenium, accounting for 56-81% of the total soil selenium, of which SeMet accounts for about 60% of organic selenium.

[0003] Currently, there are various methods for cultivating selenium-enriched plants, such as directly applying selenium fertilizer to the plants. However, most selenium fertilizers are inorganic selenium, which is highly toxic. Excessive intake of inorganic selenium can be life-threatening for organisms, thus posing certain safety risks. In contrast, introducing microorganisms capable of oxidizing organic or elemental selenium into selenium-containing agricultural soils converts organic and elemental selenium into inorganic selenium that is easily absorbed by plants. This method not only effectively utilizes the selenium in the soil without the need for additional selenium fertilizer but also eliminates safety concerns, making it a safe and low-cost method for cultivating selenium-enriched plants.

[0004] There are few reports on selenium oxidation to date. The earliest report was published in 1981, which found that Bacillus megaterium could oxidize elemental selenium. Subsequent studies have also reported relatively few selenium-oxidizing microorganisms, with the total number of related strains not exceeding 10.

[0005] Bacillus paramycoides H1 is a newly screened selenium-oxidizing microorganism in our laboratory. There have been no previous reports on Bacillus paramycoides being able to oxidize elemental selenium and organic selenium. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of Bacillus paramicularis and its application in selenium oxidation, in order to solve the problems existing in the prior art. This strain can oxidize both elemental selenium and organic selenium.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a strain of Bacillus paramycoides H1, with accession number CCTCC NO: M 20231107, deposited on June 27, 2023, at the China Center for Type Culture Collection, Wuhan University, Wuhan, China.

[0009] The present invention also provides the application of the aforementioned *Bacillus paramicularis*, including any one of the following (1)-(3):

[0010] (1) Application in selenium oxidation;

[0011] (2) Application in the preparation of selenium oxidants;

[0012] (3) Application in the preparation of selenium-enriched bacterial agents.

[0013] Preferably, the selenium oxidation includes the oxidation of elemental selenium and / or organic selenium.

[0014] The present invention also provides a selenium oxidant containing the aforementioned Bacillus parasiticus or its fermentation broth.

[0015] The present invention also provides a selenium-enriched bacterial agent containing the aforementioned Bacillus parasiticus or its fermentation broth.

[0016] The present invention also provides a method for culturing the aforementioned Bacillus paramyophyte, comprising the step of inoculating Bacillus paramyophyte into LB liquid medium for fermentation culture to obtain fermentation broth.

[0017] The present invention discloses the following technical effects:

[0018] (1) This invention obtained a strain of Bacillus paramycoides H1 through screening and isolation. Experiments verified that this strain has the ability to oxidize organoselenium (SeMet) to Se. 4+ This ability is the first of its kind discovered in the genus Bacillus. In selenium-rich soils, organic selenium typically constitutes the largest proportion, exceeding 50%. Using Bacillus paramycoides H1, organic selenium can be oxidized into Se2+, a form more easily absorbed by plants. 4+ .

[0019] (2) The present invention, Bacillus paramycoides H1, can oxidize water-insoluble elemental selenium to water-soluble Se. 4+ It can promote the absorption of selenium by plants and also provides a new member for the scarce selenium-oxidizing microorganisms.

[0020] (3) The strain of this invention can oxidize both elemental selenium and organic selenium at the same time, which is of great significance for the scientific research and agricultural cultivation of selenium conversion model strains.

[0021] (4) The present invention provides a safe and low-cost plant selenium-enriched bacterial agent. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Image A shows the culture plate of strain H1 and the Gram-stained electron micrograph (B).

[0024] Figure 2 This is a schematic diagram of the bacterial growth curve, where CK represents the growth curve of the strain without selenium; 100μM SeMet indicates that SeMet was added to the culture medium at a concentration of 100μM; 0.1g / L Se 0 This indicates that Se has been added to the culture medium. 0 And make its concentration 0.1 g / L;

[0025] Figure 3 For strain H1, Se 0 Oxidized to Se 4+ Subsequently, the supernatant detected Se. 4+ The concentration of 0.1 g / L Se changes over time; 0 The culture medium uninoculated with strain H1 serves as a control; 0.1 g / L Se 0 H1 indicates that the culture medium was inoculated with strain H1;

[0026] Figure 4 For strain H1, SeMet is oxidized to Se. 4+ Subsequently, the Se detected in the supernatant 4+ The concentration changes over time; where 100 μM SeMet represents the culture medium without inoculation of strain H1, serving as a control; 100 μM SeMet H1 represents the culture medium inoculated with strain H1;

[0027] Figure 5 Strain H1 oxidizes SeMet in soil containing 0.04 g / kg SeMet to Se. 4+ Subsequently, the supernatant detected Se. 4+The concentration of SeMet changes over time; where 0.04 g / kg SeMet represents selenium-containing soil that was not inoculated with strain H1, serving as a control; and 0.04 g / kg SeMet H1 represents selenium-containing soil inoculated with strain H1. Detailed Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] Example 1: Isolation, screening, identification, and preservation of strains

[0034] ① Sample collection

[0035] Soil samples were collected from the selenium nutrition experimental field of Huazhong Agricultural University in May 2021.

[0036] ② Strain isolation and screening

[0037] Take 10g of soil into an Erlenmeyer flask, add 90mL of single-distilled water, and shake on a constant-temperature shaker at 180rpm and 30℃ for 30min. Then take 1mL of water sample for 10... -1 10 -2 10 -3 10 -4 10 -5 Gradient dilution, take 10 -2 10 -3 10 -4 10 -5 100 μL of serially diluted solution was spread onto 0.58 mM Na₂SeO₃ LB solid medium and incubated at 30 °C for 3 days. Strains whose colonies did not turn red were selected and preliminarily identified as strains that do not reduce Na₂SeO₃, preparing for later work on selenium-oxidizing strains. The screened strains were stored in screw-cap tubes at a 1:1 ratio of glycerol to bacterial culture and incubated at -80 °C.

[0038] ③ Screening of selenium-oxidizing strains

[0039] The preliminarily screened non-Na₂SeO₃-reducing strains were inoculated into sterile LB medium containing 100 μM selenomethionine (SeMet) or 0.1 g / L elemental selenium. Uninoculated sterile LB medium containing 100 μM SeMet and 0.1 g / L elemental selenium served as controls. After inoculation, the medium was shaken well, and samples were taken immediately, recorded as 0 h. All samples were incubated at 30℃ and 180 rpm in a constant-temperature shaker, with samples taken every 12 h until 120 h. The samples were centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane. The Se content of the filtrate was measured using atomic fluorescence spectrometry. 4+ Concentration. If the filtrate Se 4+ The concentration increased significantly, proving that the strain has the ability to oxidize elemental selenium or organic selenium.

[0040] ④ Identification and preservation of selenium-oxidizing strains

[0041] The 16S rRNA gene of the obtained selenium-oxidizing strain, named H1, was sequenced. Blast analysis was performed in NCBI, and the 16S rRNA amplified sequence of strain H1 showed 100% homology with Bacillus paramycoides NH24A2, thus identifying it as Bacillus paramycoides.

[0042] Strain H1, with the Latin name Bacillus paramycoides H1, was deposited on June 27, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M20231107.

[0043] like Figure 1 As shown, the cells of Bacillus paramycoides H1 are rod-shaped and stain purple with Gram stain, indicating that it is a Gram-positive strain. Bacillus paramycoides H1 was cultured on LB agar at 30°C for 24 hours. This strain grows rapidly, with obvious colonies appearing within 8 hours, and the culture conditions are simple. The colonies are white, opaque, round, with raised edges and irregular margins.

[0044] ⑤ Growth curve plotting

[0045] Strain H1 strain was inoculated at a 1% inoculum into sterile LB medium containing 100 μM SeMet or 0.1 g / L elemental selenium, with LB medium without SeMet and elemental selenium serving as a control. The cultures were incubated at 30°C and 180 rpm using a constant temperature shaker. Samples were taken every 12 hours from 0 to 120 hours, and the OD values ​​of the samples were measured using a UV spectrophotometer. 600 Values, plot growth curves (see) Figure 2 ).

[0046] Example 2: Oxidation curve of elemental selenium by strain H1

[0047] The specific steps are as follows:

[0048] ① Prepare 6 bottles of 100mL sterile LB medium with a selenium concentration of 0.1g / L. Inoculate the H1 strain seed culture into 3 of the bottles at a 1% inoculation rate, leaving the other 3 bottles uninoculated as a control. Mix well after inoculation and take a sample immediately. Record this sampling time point as 0h. Take samples every 12 hours thereafter until 120 hours have elapsed.

[0049] ② After each sampling, centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane and store it in a 4℃ refrigerator.

[0050] ③ After all samples have been processed, the Se content of the supernatant was measured using an atomic fluorescence spectrometer manufactured by Beijing Jitian Instruments Co., Ltd. 4+ concentration.

[0051] ④With Se 4+ Plot the supernatant Se concentration on the Y-axis and time on the X-axis. 4+ Concentration change curve.

[0052] ⑤ For example Figure 3As shown, H1 begins to oxidize elemental selenium in 48-60 hours, and Se in 60 hours... 4+ The concentration increased significantly over time, and subsequently Se... 4+ The concentration continued to rise, and at 120 h, the Se in the supernatant... 4+ The concentration reached approximately 60 μg / L (0.76 μM), and no Se was detected in the supernatant of the control group. 4+ Concentration. As shown in Table 1 below:

[0053] Table 1

[0054]

[0055] Example 3: Oxidation curve of strain H1 on organoselenium

[0056] Specific steps:

[0057] ① Prepare 6 bottles of 100mL LB medium with a 100μM organic selenium concentration (Se concentration of 40.26μM) sterilized. Inoculate the H1 strain seed culture into 3 of the bottles at a 1% inoculation rate, leaving the other 3 bottles uninoculated as a control. Mix well after inoculation and take a sample immediately. Record this sampling time point as 0h. Take samples every 12 hours thereafter until 120 hours have elapsed.

[0058] ② After each sampling, centrifuge at 12000 rpm for 10 min, filter the supernatant through a 0.22 μm filter membrane and store it in a 4℃ refrigerator.

[0059] ③ After all samples have been processed, the Se content of the supernatant was measured using an atomic fluorescence spectrometer manufactured by Beijing Jitian Instruments Co., Ltd. 4+ concentration.

[0060] ④With Se 4+ Plot the supernatant Se concentration on the Y-axis and time on the X-axis. 4+ Concentration change curve.

[0061] ⑤ For example Figure 4 As shown, H1 begins to oxidize organic selenium within 12-24 hours, and Se... 4+ The concentration increased significantly over time, and subsequently Se... 4+ The concentration continued to rise, and at 120 h, the Se in the supernatant... 4+ The concentration reached 167.47 μg / L (2.12 μM). See Table 2 below:

[0062] Table 2

[0063]

[0064]

[0065] Example 4H1 Oxidation of organic selenium in selenium-containing soil

[0066] Specific steps:

[0067] ① Inoculate 100 μL of strain H1 from the threaded tube into a bacterial culture flask containing 10 mL of LB medium, and culture at 30°C and 180 rpm for 12 h to prepare the seed culture.

[0068] ② Prepare six 250mL Erlenmeyer flasks containing 50g of soil each. Add distilled water to each flask at a soil:water ratio of 1:2. Add SeMet to each of the six flasks to make the SeMet concentration in the flasks 0.04g / kg. Sterilize each flask at high temperature.

[0069] ③ Inoculate 2 ml of the prepared seed culture into 3 Erlenmeyer flasks containing SeMet, with the uninoculated flasks serving as a control. Shake well after inoculation, and immediately take a 2 ml sample, which is recorded as 0 h. Incubate at 30℃ and 180 rpm in a constant temperature shaker, and take samples every 24 h.

[0070] ④ Centrifuge the sample at 12000 rpm for 10 min, collect the supernatant, filter the supernatant through a 0.22 μm filter membrane, and store it in a refrigerator at 4℃.

[0071] ④ After all samples were processed, the Se content of the supernatant of each sample was measured using an atomic fluorescence spectrometer manufactured by Beijing Jitian Instruments Co., Ltd. 4+ content.

[0072] ⑥With Se 4+ Plot the supernatant Se concentration on the Y-axis and time on the X-axis. 4+ Concentration change curve.

[0073] ⑦ For example Figure 5 As shown, the selenium-containing soil supernatant inoculated with strain H1 at 24 h... 4+ The concentration increased significantly over time, and subsequently Se... 4+ The concentration continued to rise, and at 120 h, the Se in the supernatant... 4+ The concentration reached 66.92 μg / L. See Table 3 below:

[0074] Table 3

[0075]

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of *Bacillus paramyophyte* ( Bacillus paramycoides H1, characterized in that, The strain has the preservation number CCTCC NO: M 20231107.

2. The application of *Bacillus paramyophyte* as described in claim 1, characterized in that, Includes any one of the following (1)-(3): (1) Application in selenium oxidation; (2) Application in the preparation of selenium oxidants; (3) Application in the preparation of selenium-enriched bacterial agents.

3. The application as described in claim 2, characterized in that, The selenium oxidation includes the oxidation of elemental selenium and / or organic selenium.

4. A selenium oxidizing agent, characterized in that, Contains the *Bacillus paramyophyte* as described in claim 1.

5. A selenium-enriched bacterial agent, characterized in that, Contains the *Bacillus paramyophyte* as described in claim 1.

6. A method for culturing *Bacillus paramyophyte* as described in claim 1, characterized in that, The method includes the step of inoculating Bacillus paramyxoides into LB liquid medium for fermentation culture to obtain fermentation broth.

Images