Spherical lysinibacillus, composition comprising same, preparation and use

Abstract

The application provides a spherical lysinibacillus, a composition containing the same, preparation and application, the spherical lysinibacillus (Lysinibacillus sphaericus) MnO-1 belongs to the lysinibacillus genus (Lysinibacillus), has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2023, and the preservation number is CGMCC No. 27228. Compared with the prior art, the bacteria screened have higher degradation efficiency of carbamazepine, strong manganese oxidation capacity, and good treatment effect on decabromodiphenyl ether and Cr(VI), and can tolerate various adverse environments; the composition preparation method is simple, green and pollution-free; the composition can efficiently treat carbamazepine, decabromodiphenyl ether, Cr(VI) and Mn(II), and is conducive to large-scale application.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, and more particularly to a spherical lysine-containing Bacillus, compositions comprising it, their preparation and application. Background Technology

[0002] With economic development and technological advancements, the ecological environment, including water bodies and soil, has suffered increasingly severe damage, accompanied by serious environmental pollution. In recent years, various organic pharmaceuticals have been frequently detected in wastewater, attracting widespread attention due to their high concentration and stable properties, leading to their long-term presence in the environment. Studies summarizing organic pharmaceutical pollution worldwide have found that carbamazepine, triclosan, anti-inflammatory drugs, and sulfamethoxazole are commonly detected in surface water. Carbamazepine concentrations in drinking water are high, reaching 600 ng / L, while other drugs are only 1 / 10 or even lower. In wastewater treatment plants, influent carbamazepine concentrations can reach 20 µg / L, exceeding the no-effect concentration of 13.8 µg / L. Because urban wastewater treatment plants are not equipped with facilities to treat complex organic pharmaceuticals, large quantities of these drugs are discharged into natural water bodies. According to incomplete statistics, the global annual average consumption of carbamazepine is 1014 tons, and it is widely distributed, having been detected in industrial and agricultural wastewater.

[0003] Due to the dismantling of electronic waste, the soil environment of dismantling sites is increasingly polluted by a combination of brominated flame retardants and heavy metals. Polybrominated diphenyl ethers (PBDES), widely used brominated flame retardants, remain in the natural environment for extended periods due to their slow degradation and long half-life, causing ecological pollution. Common commercial PBDES flame retardants include pentabromodiphenyl ether (PBDES), octabromodiphenyl ether (OCD), and decabromodiphenyl ether (DBD), with decabromodiphenyl ether having the largest global usage and the highest proportion of total PBDES in soil near electronic waste dismantling sites. Statistics show that my country uses approximately 20-40 million kilograms of commercial decabromodiphenyl ether annually. Decabromodiphenyl ether is widely distributed, mainly found at depths of 2-15 cm in soil, with a concentration reaching 670 µg / kg in 2018. In sediments, decabromodiphenyl ether accounts for 36%-93.7% of the total PBDES concentration, significantly higher than other PBDES homologues by one to two orders of magnitude. In addition to organic pollution, heavy metal pollution is also a significant concern near electronic waste dismantling sites.

[0004] Carbamazepine's environmental hazards have been confirmed, affecting the normal growth of microorganisms and aquatic organisms. Due to its persistent nature, its concentration in the environment increases over time, potentially posing a greater threat to the ecological environment and human health in the future. Decabromodiphenyl ether (DBD) enters the human body through multiple pathways, exhibits strong bioaccumulation, and possesses endocrine-disrupting effects, neurotoxicity, hepatotoxicity, reproductive toxicity, carcinogenicity, and genotoxicity. Cr typically exists in Cr(VI) and Cr(III) forms, with Cr(VI) being far more toxic than Cr(III) and exhibiting extremely high mobility. DBD interacts with coexisting heavy metal ions, resulting in synergistic or cumulative toxicological effects on the environment and organisms. While organic matter in the environment does not readily produce acute toxic effects through concentration levels, long-term exposure to organic pollutants affects biological metabolic activities, leading to irreversible and persistent damage.

[0005] Currently, various methods for removing organic matter have been proposed, mainly categorized into biological methods, physical methods, chemical methods, and combined treatment methods. Among these, biological methods generally suffer from low removal efficiency and long treatment cycles; physical methods also have problems such as high costs and secondary pollution; chemical treatment is effective in degrading organic matter, but may produce toxic byproducts and has relatively high operating costs.

[0006] However, to date, there is limited research on the degradation effects of single microorganisms on multiple organic compounds. Therefore, it is imperative to screen for a manganese-oxidizing bacterium that can degrade carbamazepine, efficiently degrade decabromodiphenyl ether, and simultaneously remove the heavy metal Cr, which could fill the gaps in the research on microbial treatment of organic pollutants and heavy metal pollution. Summary of the Invention

[0007] The main objective of this invention is to provide a spherical lysine-containing Bacillus, its composition, its preparation, and its application, aiming to solve the problems of existing technologies where single microorganisms cannot degrade multiple organic substances and cannot remove heavy metals.

[0008] To achieve the above objectives, the present invention provides a spherical lysine-containing Bacillus, wherein the spherical lysine-containing Bacillus (… Lysinibacillus sphaericus MnO-1 belongs to the genus *Lysinic Bacillus*. Lysinibacillus The sample has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2023, with accession number CGMCC No. 27228.

[0009] The present invention also provides an application of the spherical lysine-containing Bacillus as described above for the oxidation of manganese.

[0010] The present invention also provides a manganese oxide / spherical lysine-containing Bacillus composition, comprising amorphous manganese oxide and spherical lysine-containing Bacillus as described above; wherein the amorphous manganese oxide is mixed with the spherical lysine-containing Bacillus in an aggregated particulate form.

[0011] The present invention also provides a method for preparing the composition as described above, comprising: inoculating each 100 mL of manganese oxidizing bacterial ion medium with 1-2% of the above-described spherical lysine-containing Bacillus, and culturing it with shaking at 25-35°C for 5-10 days to obtain the manganese oxide / spherical lysine-containing Bacillus composition.

[0012] The manganese-oxidizing bacteria ion culture medium contains 0.1~4 mmol / L Mn. 2+ .

[0013] Furthermore, the manganese-oxidizing bacterial ionic culture medium specifically comprises 1 g / L yeast extract, 1 g / L acid-hydrolyzed casein, 1 g / L glucose, 0.222 g / L CaCl2, 0.396 g / L MgSO4, 0.0006 g / L FeCl3, 1 mL / L trace element solution, and 0.1~4 mmol / L MnSO4·H2O.

[0014] Furthermore, the trace element solution comprises 10 mg / L CuSO4·5H2O, 44 mg / L ZnSO4·7H2O, 20 mg / L CoCl2·6H2O, and 13 mg / L Na2MoO4·2H2O.

[0015] The present invention also provides the application of the spherical lysine-containing Bacillus or the composition described above in the treatment of organic wastewater; wherein the organic wastewater includes one or both of carbamazepine and decabromodiphenyl ether.

[0016] The present invention also provides the application of the spherical lysine-containing Bacillus as described above or the composition as described above in the treatment of heavy metal wastewater; wherein the heavy metal wastewater includes one or both of Cr(VI) and Mn(II).

[0017] The beneficial effects achieved by this invention are as follows: The *Bacillus spheroidans* strain obtained in this invention exhibits higher degradation efficiency for carbamazepine than most single microorganisms, demonstrates greater environmental adaptability, and possesses strong manganese oxidation capabilities, making it highly valuable for research. Furthermore, its degradation efficiency for high concentrations of decabromodiphenyl ether exceeds 50%. It also exhibits strong Cr(VI) removal capabilities and can tolerate various adverse environments.

[0018] The method for preparing the manganese oxide / spherical lysine-containing Bacillus composition provided by this invention has the advantages of low cost, simple operation, and being green and pollution-free.

[0019] The manganese oxide / spherical lysine-containing Bacillus composition provided by this invention has a high efficiency in degrading carbamazepine and decabromodiphenyl ether, as well as removing Cr(VI) and Mn(II), which is beneficial for large-scale application in the field of degrading organic matter and removing heavy metals. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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 the structures shown in these drawings without creative effort.

[0021] Figure 1 This is a SEM image of the manganese oxide / spherical lysine-containing Bacillus composition of manganese-oxidizing bacteria obtained in Example 2 of the present invention; Figure 2 The images shown are TEM images of the manganese oxide / spherical lysine-containing Bacillus composition of manganese oxidizing bacteria prepared in Example 2 of the present invention; wherein, (a) is a TEM image (scale bar, 200 nm), and (b) is a TEM image (scale bar, 500 nm). Figure 3 This is a phylogenetic tree of the *Bacillus spheroidans* isolated in Example 1 of this invention; Figure 4 The image shows the XRD pattern of the manganese oxide / spherical lysine-containing Bacillus composition of manganese-oxidizing bacteria obtained in Example 2 of this invention. Figure 5 The image shows an HRTEM image of the manganese oxide / spherical lysine-containing Bacillus composition of manganese-oxidizing bacteria obtained in Example 2 of this invention. Figure 6 XPS image of the manganese oxide / spherical lysine-containing Bacillus composition of manganese-oxidizing bacteria obtained in Example 2 of this invention; Figure 7 This is a graph showing the manganese oxidation capacity of *Bacillus spheroidans* in Example 3 of the present invention. Figure 8 This is a growth curve of *Bacillus spheroidans* in Example 3 of the present invention; Figure 9 This is a graph showing the pH changes during the growth of *Bacillus spheroidae* in Example 3 of this invention. Figure 10 This is a comparison chart of carbamazepine degradation rates in Example 4 of the present invention; Figure 11 This is a comparison chart of the degradation rates of decabromodiphenyl ether in Example 5 of the present invention; Figure 12 This is a comparison chart of the removal efficiency of Cr(VI) in Example 6 of the present invention.

[0022] The realization of the objective, functional characteristics and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. It should also be understood that the terminology used in the embodiments of this invention is for describing specific implementations and not for limiting the scope of protection of this invention.

[0025] Unless otherwise defined, all technical and scientific terms used in this invention are consistent with the prior art known to those skilled in the art and the description of this invention. This invention can also be implemented using any prior art methods, devices, and materials similar to or equivalent to those described, used, or made of materials in the embodiments of this invention. It should be understood by those skilled in the art that, as an explanation of this application, without affecting the actual understanding of the technical solutions of this application, "TEM" can represent transmission electron microscopy, "XRD" can represent X-ray diffractometer, "HRTEM" can represent high-resolution transmission electron microscopy, "XPS" can represent X-ray photoelectron spectroscopy, "2θ / degree" can represent diffraction angle, "Intensity (au)" can represent intensity, "Binding energy (ev)" can represent binding energy, and "satellite" can represent satellite peak.

[0026] When numerical ranges are given in the examples, it should be understood that, unless otherwise stated in the invention, both endpoints of each range and any value between the two endpoints may be used. Test methods in the following examples that do not specify specific conditions are generally performed under conventional conditions or as recommended by the respective manufacturers. Unless otherwise specified, all materials or reagents required in the following examples are commercially available.

[0027] To address the limitations of existing technologies that rely on a single microorganism to degrade multiple organic compounds and remove heavy metals, this invention provides a spherical lysine-containing Bacillus. Lysinibacillus sphaericus MnO-1 belongs to the genus *Lysinic Bacillus*. Lysinibacillus The sample has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2023, with accession number CGMCC No. 27228.

[0028] Specifically, the supernatant of a soil sample from a mine can be collected after settling and added to LB liquid medium containing MnSO4·H2O. After one week of constant temperature and shaking culture in LB liquid medium, the sample is inoculated into solid medium containing MnSO4·H2O, and the colonies growing on the medium are used for initial screening. Well-grown colonies are inoculated into LB liquid medium containing MnSO4·H2O and cultured at a constant temperature. Samples are taken at the beginning of the culture and after 7 days of culture, and Mn is determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+ Removal rate. The culture media of strains with high removal rates were further screened using LBB indicator. Those that showed a blue color on the LBB indicator were separated and purified. The culture media of the color-changing strains were then further diluted and spread onto solid medium containing MnSO4·H2O. After incubation at a constant temperature, the obtained strains were mixed with 50% glycerol at a 1:1 ratio and stored at -80℃.

[0029] Finally, the *Bacillus spheroidae* obtained through screening in this invention was deposited on April 27, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, with the accession number CGMCC No. 27228.

[0030] The selected *Bacillus spheroidans* exhibits higher degradation efficiency for carbamazepine than most single microorganisms, demonstrates greater environmental adaptability, and possesses strong manganese oxidation capabilities, making it a valuable research resource. Furthermore, it achieves a degradation efficiency of over 50% for high concentrations of decabromodiphenyl ether. It also demonstrates strong Cr(VI) removal capabilities and can tolerate various adverse environments.

[0031] The present invention also provides an application of the above-mentioned spherical lysine-containing Bacillus for the oxidation of manganese.

[0032] The present invention also provides a manganese oxide / spherical lysine-containing Bacillus composition, comprising amorphous manganese oxide and spherical lysine-containing Bacillus as described above; wherein the amorphous manganese oxide is mixed with the spherical lysine-containing Bacillus in an aggregated particulate form.

[0033] The present invention also provides a method for preparing the above composition, comprising: inoculating each 100 mL of manganese oxidizing bacterial ion medium with 1-2% of the above-mentioned spherical lysine-containing Bacillus, and culturing it with shaking at 25-35°C for 5-10 days to obtain a manganese oxide / spherical lysine-containing Bacillus composition.

[0034] The ion culture medium for manganese oxidizing bacteria contains 0.1–4 mmol / L Mn. 2+ When Mn in the ion culture medium of manganese oxidizing bacteria 2+ A relatively large amount of manganese oxide minerals can be formed at concentrations of 0.1–4 mmol / L; during the preparation process, it was found that when Mn 2+ A concentration >4 mmol / L inhibits the growth of Bacillus spheroidans and prevents mineral formation.

[0035] Furthermore, the manganese-oxidizing bacterial ion medium specifically comprises 1 g / L yeast extract, 1 g / L acid-hydrolyzed casein, 1 g / L glucose, 0.222 g / L CaCl2, 0.396 g / L MgSO4, 0.0006 g / L FeCl3, 1 mL / L trace element solution, and 0.1–4 mmol / L MnSO4·H2O. This manganese-oxidizing bacterial ion medium perfectly meets the conditions for manganese oxide formation and is suitable for the growth of *Bacillus spheroidae*.

[0036] The method for preparing the manganese oxide / spherical lysine-containing Bacillus composition provided by this invention has the advantages of low cost, simple operation, and being green and pollution-free.

[0037] Furthermore, the trace element solution includes 10 mg / L CuSO4·5H2O, 44 mg / L ZnSO4·7H2O, 20 mg / L CoCl2·6H2O, and 13 mg / L Na2MoO4·2H2O.

[0038] This invention also provides the application of the above-mentioned spherical lysine-containing Bacillus or the above-mentioned composition in the treatment of organic wastewater; the organic wastewater includes one or both of carbamazepine and decabromodiphenyl ether. The manganese oxide / spherical lysine-containing Bacillus composition provided by this invention has a highly efficient degradation effect on carbamazepine and decabromodiphenyl ether, which is beneficial for large-scale application in the field of organic matter degradation.

[0039] This invention also provides an application of the above-mentioned *Bacillus spheroidans* and its composition in the treatment of heavy metal wastewater; the heavy metal wastewater includes one or both of Cr(VI) and Mn(II). The manganese oxide / *Bacillus spheroidans* composition provided by this invention has a highly efficient removal effect on Cr(VI) and Mn(II), which is beneficial for large-scale application in the field of heavy metal removal.

[0040] Specifically, both the spherical lysine-containing Bacillus and the manganese oxide / spherical lysine-containing Bacillus composition can efficiently remove carbamazepine, decabromodiphenyl ether, Cr(VI), Mn(II) and other pollutants on their own; they can also treat carbamazepine, decabromodiphenyl ether, Cr(VI), Mn(II) and other pollutants simultaneously.

[0041] To further illustrate the present invention, the following examples are provided: Example 1 Screening, isolation and purification of Bacillus spheroidae 1. Preparation of LB liquid culture medium: Weigh 5g of yeast extract, 10g of tryptone, and 10g of sodium chloride. Add 1000mL of distilled water, adjust the pH to 7, and dispense 100mL into 250mL Erlenmeyer flasks. Autoclave at 121℃ for 20min. Filter using a 0.22µm sterile filter and add 3mL of 100mmol / L MnSO4·H2O stock solution to achieve the desired Mn content. 2+ The final concentration was 3 mmol / L.

[0042] 2. Preparation of solid culture medium: Dissolve 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, and 15g of agar in 1000mL of distilled water. Autoclave at 121℃ for 20min. After sterilization, adjust the pH to 7. Filter using a 0.22µm sterile filter and add 3mL of 100mmol / L MnSO4·H2O stock solution to achieve the desired Mn content. 2+ The final concentration was 3 mmol / L. After cooling to 50°C, the plates were poured into a UV sterilization pan and allowed to condense before use.

[0043] 3. Screening and separation: Soil was collected from a manganese mine in Xiangtan, Hunan Province. After settling, the supernatant sludge liquid was added to LB liquid medium from step 1 at a 1% inoculum concentration. The medium was incubated for 7 days on a shaker at 30°C and 150 rpm. The resulting culture was then spread onto solid plates containing 3 mmol / L MnSO4·H2O for initial screening. After incubation at a constant temperature, well-grown colonies were selected and inoculated into LB liquid medium containing MnSO4·H2O for further purification. Samples were taken at the beginning of the incubation and after 7 days. Mn content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 2+ Removal rate. Cultures of strains with high removal rates were further screened using LBB indicator; those that showed a blue color on the LBB indicator were separated.

[0044] The LBB indicator is prepared as follows: Weigh 0.04g of LBB powder, dissolve it in 0.25mL of glacial acetic acid aqueous solution (45mmol / L), add deionized water and make up to 100mL, and store at 4℃ protected from light.

[0045] 4. Purification: The culture medium that turns blue to the LBB indicator in step 3 is diluted and spread onto the solid culture medium in step 2. It is then placed in a 30°C biochemical incubator for constant temperature culture and purification. The purified strain is then mixed with 50% glycerol at a ratio of 1:1 and stored in a -80°C refrigerator.

[0046] Example 2 Preparation, characterization, and molecular identification of manganese oxide / spherical lysine-containing Bacillus compositions. 1. Preparation of manganese oxide / spherical lysine-containing Bacillus composition (1) Preparation of manganese oxidizing bacteria ion culture medium: Based on the Leptothrix medium, a suitable culture medium for manganese-oxidizing bacteria isolated from a freshwater system, some modifications were made: 1 g / L yeast extract, 1 g / L acid-hydrolyzed casein, 1 g / L glucose, 0.222 g / L CaCl2, 0.396 g / L MgSO4, 0.0006 g / L FeCl3, 1 mL / L trace element solution (10 mg / L CuSO4·5H2O, 44 mg / L ZnSO4·7H2O, 20 mg / L CoCl2·6H2O, 13 mg / L Na2MoO4·2H2O), and 3 mmol / L MnSO4·H2O were added to 1000 mL of deionized water and sterilized in an autoclave at 121°C for 20 min. The MnSO4·H2O was filtered through a sterile filter and added to 100 mL of the medium.

[0047] Set up parallel experiments and prepare three 100mL aliquots of manganese oxidizing bacteria ion culture medium for later use.

[0048] (2) Generation of manganese oxide / spherical lysine-containing Bacillus composition Add 1% of the *Bacillus spheroides* obtained in Example 1 to 100 mL of manganese oxidizing bacteria ion culture medium prepared in step (1). Place all three conical flasks in a constant temperature shaker at 30°C and 150 rpm for 7 days. After centrifugation at 8000 rpm for 3 minutes, discard the supernatant and wash three times with deionized water. The resulting precipitate is the manganese oxide / *Bacillus spheroides* composition.

[0049] 2. Characterization analysis: The SEM image of the manganese oxide / spherical lysine-containing Bacillus composition obtained in step 1 above is shown below. Figure 1 As shown; its TEM image (scale bar, 200nm) is as follows. Figure 2 As shown in (a), its TEM image (scale bar, 500 nm) is as follows. Figure 2 As shown in (b).

[0050] according to Figure 1 , Figure 2 Observations show that the strain of *Bacillus spheroidans* is spherical in shape, with a diameter of approximately 710 nm. Mineral particles adhere to the surface of the *Bacillus spheroidans*, preliminarily indicating that the bacteria form manganese oxides through surface mineralization. Furthermore, the surface of this manganese oxide / *Bacillus spheroidans* composition is highly irregular, exhibiting aggregated granular structures. The manganese-oxidizing bacteria and the generated minerals are mixed together to form the overall manganese oxide / *Bacillus spheroidans* composition.

[0051] The manganese oxide / Bacillus spheroidae composition was analyzed by XRD, HRTEM, and XPS. The XRD, HRTEM, and XPS images are shown below. Figure 4 , Figure 5 , Figure 6 As shown. From Figure 4 and Figure 5 It is known that the manganese oxide in the manganese oxide / spherical lysine Bacillus composition is in an amorphous state, with disordered diffraction fringes, consistent with most reports on biogenic manganese oxides.

[0052] from Figure 6 XPS analysis revealed that the content of high-valence manganese (III, IV) in the biogenic manganese oxide was 42.45%, indicating that the spherical lysine-containing Bacillus has a strong oxidizing ability for Mn(II).

[0053] 3. Molecular identification and phylogenetic tree comparison: DNA extraction and sequencing were performed on the *Bacillus spheroidans* isolated in Example 1. This entire process was completed by Shenzhen MicroMed Technology Group Co., Ltd. The 16S rRNA results obtained from the sequencing were compared with existing 16S rRNA nucleic acid sequences using the NCBI (National Center for Biotechnology Information) BLAST program. Phylogenetic analysis and phylogenetic tree construction were performed using Mega11.0 software; the phylogenetic tree is shown below. Figure 3 .

[0054] Sequence results showed that this manganese-oxidizing bacterium was related to... Lysinibacillus sphaericus It showed the highest similarity and was identified as belonging to the genus Bacillus lysine, and named MnO-1.

[0055] Example 3 Determination of manganese oxidation capacity of Bacillus spheroidae 1. Manganese oxidation capacity of Bacillus spheroidans Select a 250 ml Erlenmeyer flask and prepare three 100 ml portions of manganese oxidizing bacteria ion culture medium according to the method described in step 1 of Example 2. Mn 2+ The final concentration was 3 mmol / L. *Bacillus spheroides* was added to manganese-oxidizing ion medium at an inoculum volume of 1%. Each conical flask was placed in a shaker at 30°C and 150 rpm for incubation. After sampling, the samples were centrifuged at 8000 rpm for 3 min, filtered through a 0.22 μm filter, and the supernatant was analyzed using inductively coupled plasma optical emission spectrometry (ICP-OES) to determine the residual Mn. 2+ concentration.

[0056] The average value of the samples from three conical flasks was used to plot the manganese oxidation capacity graph. The results are as follows: Figure 7 As shown, analysis Figure 7 The results showed that *Bacillus spheroidae* inhibited Mn within 8 hours. 2+ Oxidation removal efficiency was the highest, reaching 37.8%, and then gradually stabilized, with the final oxidation removal rate at 96.6%, indicating a high manganese oxidation capacity.

[0057] 2. Growth activity of Bacillus spheroidae. Select a 250ml Erlenmeyer flask and prepare 100ml of spherical lysine-containing Bacillus experimental culture medium according to the method of preparing manganese oxidizing bacteria ion culture medium in step 1 of Example 2, except without adding MnSO4·H2O. After sterilization, take samples at regular intervals and measure their absorbance at a wavelength of 600nm on an ultraviolet spectrophotometer.

[0058] like Figure 8 The figure shows the growth curve of this spherical lysine-containing Bacillus. The spherical lysine-containing Bacillus reaches an OD600 value of 1 at approximately 8 hours, enters a growth retardation phase at approximately 24 hours, and begins to decline after 36 hours, indicating the bacteria are entering their death phase.

[0059] like Figure 9 The figure shows the pH changes during the growth of *Bacillus spheroidae*. The pH changes during the growth of *Bacillus spheroidae* are unstable; generally, the pH increases with time, reaching 8.3 after 3 days of culture.

[0060] Example 4 Oxidative degradation experiment of carbamazepine by Bacillus spheroidae and a combination of manganese oxide / Bacillus spheroidae. Select a 250ml Erlenmeyer flask and prepare 100ml of manganese oxidizing bacteria ion culture medium according to the method described in step 1 of Example 2. After sterilization, adjust the pH to 8. Mn 2+The final concentration was 3 mmol / L. 5% of the *Bacillus lysine-containing* obtained in Example 1 was added to the manganese-oxidizing ion medium. Carbamazepine was added to a final concentration of 1 mg / L, and the mixture was cultured in a shaker at 30°C and 150 rpm, serving as the Mn-containing experimental group (…). Figure 10 (Contains Mn).

[0061] A 250ml Erlenmeyer flask was selected, and the manganese-oxidizing bacterial ion culture medium was prepared according to the method described in step 1 of Example 2, except that MnSO4·H2O was not added. 100ml of spherical lysine-containing Bacillus experimental medium was prepared, sterilized, and the pH was adjusted to 8. 5% of the spherical lysine-containing Bacillus obtained in Example 1 was added to the spherical lysine-containing Bacillus experimental medium. Carbamazepine was added to a final concentration of 1mg / L, and the mixture was cultured in a constant temperature shaker at 30℃ and 150rpm as the Mn-free experimental group. Figure 10 (Mn is absent in the middle).

[0062] Select a 250ml Erlenmeyer flask and prepare 100ml of blank experimental medium according to the method of manganese oxidizing bacteria ion medium preparation in step 1 of Example 2, without adding yeast extract, acid-hydrolyzed casein, or MnSO4·H2O. After sterilization, adjust the pH to 8. Add 5% of the spherical lysine-containing Bacillus obtained in Example 1 to the blank experimental medium. Add carbamazepine to a final concentration of 1mg / L, and culture in a constant temperature shaker at 30℃ and 150rpm as a blank control group. Figure 10 (Chinese blank control).

[0063] Sampling was performed at regular intervals, and the samples were pretreated after sampling to meet the conditions for carbamazepine determination. Carbamazepine was determined using ultra-high performance liquid chromatography (Acquity UPLC H-class) with a mobile phase of acetonitrile:water = 70:30, a flow rate of 0.3 mL / min, an injection volume of 10 μL, a detection wavelength of 285 nm, and a retention time of 5 min. Results of sampling at different times are shown below. Figure 10 As shown.

[0064] from Figure 10 It can be seen that the Mn-containing experimental group (manganese oxide / Bacillus spheroidae) exhibited the highest degradation efficiency, but the degradation efficiency of carbamazepine did not differ significantly among the three treatments. The Mn-containing experimental group showed faster degradation in the first 12 hours, reaching an efficiency of 26%. Between 12 and 120 hours, the degradation efficiency of this composition for carbamazepine fluctuated, but subsequently, carbamazepine was further degraded over time, eventually reaching 59%. The Bacillus spheroidae of this invention demonstrated good degradation effects on carbamazepine, which is beneficial for its application in the field of organic matter degradation in wastewater.

[0065] Example 5 Oxidative degradation experiment of decabromodiphenyl ether by Bacillus spheroidae and a manganese oxide / Bacillus spheroidae composition. Select a 250ml Erlenmeyer flask and prepare 100ml of manganese oxidizing bacteria ion culture medium according to the method for preparing manganese oxidizing bacteria ion culture medium in step 1 of Example 2, then sterilize. Mn 2+ The final concentration was 1 mmol / L. Decabromodiphenyl ether was added to a final concentration of 5 mg / L. No *Bacillus lysinus* was inoculated, and the mixture was shaken in a constant temperature shaker at 30°C and 150 rpm as the Mn blank control group. Figure 11 (Mn).

[0066] A 250ml Erlenmeyer flask was selected. Following the method for preparing the manganese-oxidizing bacterial ion culture medium in step 1 of Example 2, except that MnSO4·H2O was not added, 100ml of spherical lysine-containing Bacillus experimental medium was prepared. After sterilization, 5% of the spherical lysine-containing Bacillus obtained in Example 1 was added to the spherical lysine-containing Bacillus experimental medium. Decabromodiphenyl ether was added to a final concentration of 5mg / L, and the medium was cultured in a constant temperature shaker at 30℃ and 150rpm, serving as the bacterial experimental group (…). Figure 11 bacteria).

[0067] Select a 250ml Erlenmeyer flask and prepare 100ml of manganese oxidizing bacteria ion culture medium according to the method described in step 1 of Example 2. After sterilization, add the spherical lysine-containing Bacillus obtained in Example 1 to the manganese oxidizing bacteria ion culture medium at an inoculation rate of 5%. Mn 2+ The final concentration was 1 mmol / L. Decabromodiphenyl ether was added to a final concentration of 5 mg / L. *Bacillus lysine* was not inoculated, and the mixture was shaken in a constant temperature shaker at 30°C and 150 rpm as the Mn+ bacteria experimental group. Figure 11 Mn+ bacteria).

[0068] Example 6 Removal of heavy metal Cr(VI) by Bacillus spheroidae and a combination of manganese oxide / Bacillus spheroidae. Select a 250ml Erlenmeyer flask and prepare 100ml of manganese oxidizing bacteria ion culture medium according to the method described in step 1 of Example 2. After sterilization, add the spherical lysine-containing Bacillus obtained in Example 1 to the manganese oxidizing bacteria ion culture medium at an inoculation rate of 5%. Mn 2+ The final concentration was 3 mmol / L. Cr(VI) was added to a final concentration of 50 mg / L, and the mixture was cultured in a shaker at 30℃ and 150 rpm as the Mn+ bacteria experimental group. Figure 12 Mn+ bacteria (i.e., a combination of manganese oxide / spherical lysine-containing Bacillus).

[0069] A 250ml Erlenmeyer flask was selected. Following the method for preparing the manganese-oxidizing bacterial ion culture medium in step 1 of Example 2, except that MnSO4·H2O was not added, 100ml of spherical lysine-containing Bacillus experimental medium was prepared. After sterilization, 5% of the spherical lysine-containing Bacillus obtained in Example 1 was added to the spherical lysine-containing Bacillus experimental medium. Cr(VI) was added to a final concentration of 50mg / L, and the medium was cultured in a constant temperature shaker at 30℃ and 150rpm as the bacterial experimental group (…). Figure 12 (i.e., Bacillus spheroidae).

[0070] After sampling, the samples were pretreated. The Cr(VI) content in the solution was determined by diphenylcarbazide spectrophotometry. Specifically: after centrifuging the samples at 8000 rpm for 3 min, a certain amount of supernatant was taken into a 50 ml colorimetric tube, diluted with water to the mark, and shaken well; 0.5 ml of 50% sulfuric acid solution and 50% phosphoric acid solution were added respectively, followed by 2 ml of colorimetric reagent, and shaken well. After standing for 5-10 min, the absorbance was measured at 540 nm. The removal efficiency of *Bacillus spheroides* and the manganese oxide / *Bacillus spheroides* combination for heavy metal Cr(VI) was as follows: Figure 12 As shown.

[0071] like Figure 12 It can be seen that the removal Cr(VI) efficiencies of *Bacillus spheroides* and the *Bacillus spheroides* / manganese oxide combination are similar, both achieving removal rates of over 95%. The *Bacillus spheroides* and *Bacillus spheroides* / manganese oxide combination of this invention both demonstrate excellent removal effects on the heavy metal Cr(VI), which is beneficial for application in sites with combined organic and heavy metal contamination. In summary, this invention has screened out a novel manganese-oxidizing bacterium—Bacillus spheroidae, which exhibits resistance to Mn. 2+ This bacterium possesses strong oxidizing power and has significant potential application research value; it oxidizes Mn... 2+ The resulting manganese oxide / Bacillus spheroidae composition improves the degradation efficiency of carbamazepine; the bacterium alone achieves a 56% degradation efficiency for carbamazepine. Simultaneously, the prepared manganese oxide / Bacillus spheroidae composition achieves a degradation efficiency of over 60% for decabromodiphenyl ether. MnO-1 (Bacillus spheroidae) itself also achieves a removal rate of over 95% for the heavy metal Cr(VI). This indicates that the bacterium has good environmental adaptability, can remove and degrade multiple pollutants, and can be widely applied in the field of organic-heavy metal complex pollution.

[0072] In summary, the above-described technical solutions of the present invention are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A spherical lysinibacillus sphaericus MnO-1, characterized in that, The spherical lysine-containing Bacillus MnO-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on April 27, 2023, with accession number CGMCC No. 27228.

2. The application of the spherical lysine-containing Bacillus MnO-1 as described in claim 1 in the oxidation of manganese.

3. A composition, characterized in that, The composition comprises amorphous manganese oxide and Bacillus spheroides lysine-1 as described in claim 1, wherein the amorphous manganese oxide is mixed with Bacillus spheroides lysine-1 in an aggregated particulate form.

4. A method for preparing the composition according to claim 3, characterized in that, include: Inoculate each 100 mL of manganese oxidizing bacterial ion medium with 1-2% of the *Bacillus spheroidae* MnO-1 as described in claim 1, and culture with shaking at 25-35°C for 5-10 days to obtain the composition. The manganese-oxidizing bacteria ionic culture medium comprises 1 g / L yeast extract, 1 g / L acid-hydrolyzed casein, 1 g / L glucose, 0.222 g / L CaCl2, 0.396 g / L MgSO4, 0.0006 g / L FeCl3, 1 mL / L trace element solution, and 0.1~4 mmol / L MnSO4·H2O.

5. The preparation method according to claim 4, characterized in that, The trace element solution comprises 10 mg / L CuSO4·5H2O, 44 mg / L ZnSO4·7H2O, 20 mg / L CoCl2·6H2O, and 13 mg / L Na2MoO4·2H2O.

6. The application of the *Bacillus spheroidans* MnO-1 as described in claim 1 or the composition as described in claim 3 in the treatment of organic wastewater; The organic wastewater includes one or both of carbamazepine and decabromodiphenyl ether.

7. The application of the *Bacillus spheroidans* MnO-1 as described in claim 1 or the composition as described in claim 3 in the treatment of heavy metal wastewater; The heavy metal wastewater includes one or both of Cr(VI) and Mn(II).

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