Bacillus tequilensis sys76 and use thereof

Abstract

The application relates to the field of biotechnology, and particularly relates to a bacillus tequilensis Sys76 and application thereof; the classification name of the bacillus tequilensis Sys76 is bacillus tequilensis (Bacillus tequilensis) Bacillus tequilensis ), numbered as Sys76, which has been preserved in the China Center for Type Culture Collection on May 15, 2023, and the preservation number is CCTCC NO: M 2023746; the characteristics of the bacillus tequilensis Sys76 are utilized, a large amount of biological surfactant can be formed in the expansion culture stage, the biological surfactant can promote the dissolution of phthalate by forming micelles, so that the solubility of phthalate is improved, meanwhile, the aspergillus tubigensis Sys60 used for degrading phthalate is matched, so that phthalate can be better degraded, and thus the degradation efficiency of phthalate can be improved by the way of biodegradation.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a Bacillus tekirulatus Sys76 and its applications. Background Technology

[0002] Phthalic acid esters (PAEs) are widely used plasticizers in consumer products. The European Environmental Association has defined PAEs as endocrine disruptors (EDCs), and many PAEs have been detected in human body fluids, posing certain risks to reproductive and neurodevelopment. Among PAEs, DEHP is the most widely used; its chemical formula is C6H2O. 24 H 38 O4 has a molecular weight of 390.56 g / mol. DEHP is currently widely distributed in the environment of daily human life and has been detected in water, air, soil, and sludge, posing a significant hazard to human health.

[0003] Currently, there are three main ways to degrade PAEs: chemical degradation, physical degradation, and biological degradation. However, since DEHP is insoluble in water (its solubility in water at 25°C is only 0.27 mg / L), the degradation in solution can only occur at the water-DEHP boundary, resulting in low degradation efficiency. Therefore, there is an urgent need for a surfactant to promote the solubility of DEHP in aqueous solution and achieve efficient degradation.

[0004] Since biosurfactants can effectively increase the distribution of polycyclic aromatic hydrocarbons (PAHs) from petroleum mixtures to the aqueous phase, microbially produced surfactants have been successfully used in environmental applications, such as the remediation of pollution caused by heavy metals or petroleum hydrocarbons. However, there are currently no applications of biosurfactants in the DEHP dissolution process. Therefore, how to develop a new biosurfactant to achieve efficient DEHP degradation through biodegradation is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] This application provides a Bacillus tergentii Sys76 and its application to solve the technical problem that it is difficult to achieve efficient degradation of DEHP through biodegradation in the prior art.

[0006] In a first aspect, this application provides a *Bacillus tekirae* Sys76, which is classified as *Bacillus tekirae* (…). Bacillus tequilensis The accession number (Sys76) was deposited at the China Center for Type Culture Collection (CCTCC) on May 15, 2023, with accession number CCTCC NO: M 2023746.

[0007] Optionally, the 16S rRNA gene sequence of the Bacillus tekirae Sys76 includes the nucleotide molecule shown in SEQ ID NO. 1.

[0008] Optionally, the Bacillus tekirae Sys76 is obtained by screening and separating the fermentation products of chicken manure compost with added phthalates.

[0009] Secondly, this application provides a microbial agent, which includes Bacillus tekiria Sys76 as described in the first aspect.

[0010] Thirdly, this application provides a method for preparing the microbial inoculant described in the second aspect, the method comprising: Bacillus tekirulae Sys76 was activated using LB liquid medium, and then high-density fermentation was carried out using Sys76 liquid high-density fermentation medium to obtain Sys76 bacterial culture.

[0011] Fourthly, this application provides a microbial fertilizer, which includes Bacillus tekirae Sys76 and Aspergillus tabineum Sys60 as described in the first aspect.

[0012] Optionally, the effective viable bacteria concentration of the microbial fertilizer is ≥5.4×10⁻⁶. 11 CFU / mL.

[0013] Fifthly, this application provides a method for preparing the microbial fertilizer described in the fourth aspect, the method comprising: The Sys76 bacterial solution obtained in the third aspect and the Aspergillus tabingii Sys60 spores were inoculated into a soil remediation microbial fertilizer culture medium and cultured to obtain microbial fertilizer.

[0014] Sixthly, this application provides an application of microbial fertilizer, in which the microbial fertilizer described in the fourth aspect is used in the degradation of phthalates.

[0015] Optionally, the microbial fertilizer is suitable for a pH of 5.0 to 9.0 during the degradation process.

[0016] The technical solutions provided in this application have the following advantages compared with the prior art: This application provides a strain of Bacillus tekirae Sys76, which is highly active and isolated from chicken manure compost fermentation products. Utilizing the characteristics of Bacillus tekirae, it can form a large amount of biosurfactant during the scale-up culture stage. This biosurfactant can promote the dissolution of phthalates by forming micelles, thereby increasing the solubility of phthalates. Simultaneously, these micelles also possess hydrophobic regions with chemically incorporated components. Therefore, they can be combined with Aspergillus tabineum Sys60, which is used for phthalate degradation. The biomolecules formed by Aspergillus tabineum Sys60, combined with the biosurfactant, allow for better degradation of phthalates. This improves the degradation efficiency of phthalates through biodegradation, indicating that this strain has good application prospects in soil pollution remediation. Attached Figure Description

[0017] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 A schematic flowchart illustrating the method for preparing microbial inoculants provided in the embodiments of this application; Figure 2 A schematic flowchart illustrating the method for preparing microbial fertilizer provided in this application embodiment; Figure 3 This is a schematic diagram of the phylogenetic tree of strain Sys76 provided in the embodiments of this application; Figure 4 This is a schematic diagram of the oil draining ring experiment results of the fermentation supernatant of strain Sys76 provided in the embodiments of this application on a mixture of waste engine oil and DEHP; Figure 5 The effect of biosurfactants on the DEHP emulsification index in the supernatant of strain Sys76 provided in the embodiments of this application ( E 24 A schematic diagram of the measurement results; Figure 6 A schematic diagram illustrating the identification results of the biosurfactant produced by strain Sys76 provided in the embodiments of this application; Figure 7 A schematic diagram illustrating the effect of different strains on the degradation of DEHP by strain Sys60, as provided in the embodiments of this application. Figure 8 A schematic diagram showing the results of measuring the DEHP degradation efficiency of strains Sys76 and Sys60 at different time points, as provided in the embodiments of this application. Figure 9 A schematic diagram showing the results of measuring the DEHP degradation efficiency of strains Sys76 and Sys60 at different initial pH values, as provided in the embodiments of this application. Figure 10 This diagram illustrates the effect of the compound microbial fertilizer prepared by strains Sys76 and Sys60 provided in the embodiments of this application on the remediation of DEHP-contaminated soil. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this application can be purchased from the market or prepared by existing methods.

[0022] The creative thinking behind this application is: Currently, there are several main methods for degrading DEHP, such as oxidative degradation via sodium persulfate and persulfate, photodegradation via UV chemical films, pulsed radiation decomposition, and electron beam radiation decomposition. Another method is biodegradation; many strains with DEHP-degrading capabilities have been reported, such as Burkholderia, Acinetobacter, Pseudomonas, Bacillus, Aspergillus, and Penicillium. However, because DEHP is insoluble in water (its solubility in water at 25°C is only 0.27 mg / L), degradation in solution can only occur at the water-DEHP critical surface, resulting in low degradation efficiency. Therefore, a surfactant is needed to promote the solubility of DEHP in aqueous solutions to achieve efficient degradation.

[0023] Biosurfactants typically promote the solubilization of hydrophobic chemicals by forming molecular aggregates (called micelles), which contain hydrophobic regions incorporated into the chemical. Biosurfactants can also dissolve pure solid polycyclic aromatic hydrocarbons (PAHs), such as phenanthrene, primarily because they increase their bioavailability and biodegradability; although the PAHs bound to micelles are not usable on their own and must be transferred from the micelles to cells for assimilation. However, there is currently no research on the role of biosurfactants in the dissolution of DEHP. Therefore, developing a novel biosurfactant to achieve efficient DEHP degradation via biodegradation is a pressing technical problem that needs to be solved.

[0024] like Figure 1 As shown, this application provides a *Bacillus tekirae* Sys76, which is classified as *Bacillus tekirae* (…). Bacillus tequilensis The accession number (Sys76) was deposited at the China Center for Type Culture Collection (CCTCC) on May 15, 2023, with accession number CCTCC NO: M 2023746.

[0025] In some alternative embodiments, the 16S rRNA gene sequence of the Bacillus tekirae Sys76 includes the nucleotide molecule shown in SEQ ID NO. 1.

[0026] In this embodiment, controlling the 16S rRNA gene sequence of a specific Bacillus tekirae Sys76 can be achieved by using the 16S rRNA gene sequence in conjunction with a phylogenetic tree to clearly define the taxonomic name of this Bacillus tekirae (Bacillus tekirae). Bacillus tequilensis ).

[0027] In some alternative embodiments, the Bacillus tekirae Sys76 is obtained by screening and separating chicken manure compost fermentation products with added phthalates.

[0028] In this embodiment, the specific source of Bacillus tekirae Sys76 is controlled. Since chicken manure compost fermentation products contain a large number of bacteria, phthalate esters are used to selectively screen for specific Bacillus tekirae Sys76, thereby improving the accuracy of the screening.

[0029] Based on a general inventive concept, embodiments of this application provide a microbial agent, the microbial agent comprising Bacillus tekirae Sys76.

[0030] This microbial agent is based on the aforementioned Bacillus tekirulae Sys76. Specific information about Bacillus tekirulae Sys76 can be found in the above embodiments. Since this microbial agent adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0031] like Figure 1 As shown, based on a general inventive concept, this application provides a method for preparing the aforementioned microbial inoculant, the method comprising: S1. Bacillus tergentii Sys76 was activated using LB liquid medium, and then high-density fermentation was carried out using Sys76 liquid high-density fermentation medium to obtain Sys76 bacterial culture.

[0032] This method is based on the above-mentioned microbial agent. The specific composition of the microbial agent can be referred to in the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0033] Based on a general inventive concept, embodiments of this application provide a microbial fertilizer, which includes Bacillus tekirae Sys76 and Aspergillus tabineum Sys60.

[0034] This microbial fertilizer is based on the aforementioned Bacillus tekirulae Sys76. Specific information about Bacillus tekirulae Sys76 can be found in the above embodiments. Since this microbial fertilizer adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0035] In some optional embodiments, the effective viable bacteria concentration of the microbial fertilizer is ≥5.4×10⁻⁶. 11 CFU / mL.

[0036] In this embodiment, controlling the specific effective viable bacteria concentration in the microbial agent can ensure that there are enough Bacillus tekirae Sys76 and Aspergillus tabineum Sys60 in the microbial agent. This allows Bacillus tekirae Sys76 in the microbial agent to improve the solubility of phthalates, and Aspergillus tabineum Sys60 in the microbial agent to degrade phthalates, thereby achieving efficient degradation of phthalates.

[0037] like Figure 2 As shown, based on a general inventive concept, this application provides a method for preparing the microbial fertilizer, the method comprising: S1. The obtained Sys76 bacterial solution and the Aspergillus tabingii Sys60 spores were inoculated into the soil remediation microbial fertilizer culture medium and cultured to obtain microbial fertilizer.

[0038] This method is for the preparation of the above-mentioned microbial fertilizer. The specific composition of the microbial fertilizer can be referred to the above embodiments. Since this method adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be repeated here.

[0039] Based on a general inventive concept, this application provides an application of microbial fertilizer, which is used in the degradation of phthalates.

[0040] This application is based on the above-mentioned microbial fertilizer. The specific composition of the microbial fertilizer can be referred to in the above embodiments. Since the microbial fertilizer adopts some or all of the technical solutions of the above embodiments, it has at least all the beneficial effects brought about by the technical solutions of the above embodiments, which will not be elaborated here.

[0041] In some optional embodiments, the microbial fertilizer is suitable for a pH of 5.0 to 9.0 during the degradation process.

[0042] In this embodiment of the application, the specific pH level for degradation is controlled. Within this pH range, Bacillus tertii Sys76 in the microbial fertilizer can improve the solubility of phthalates, and Aspergillus tabineus Sys60 in the microbial agent can further degrade the phthalates, thereby achieving efficient degradation of phthalates.

[0043] The present application is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the application. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. If there is no corresponding national standard, then general international standards, conventional conditions, or conditions recommended by the manufacturer are followed.

[0044] Example 1 Involving culture medium and reagent components: LB medium: 5.0 g yeast extract, 10.0 g tryptone, 10.0 g NaCl, add deionized water to 1.0 L, pH 7.0-7.2, 121℃, 20 min.

[0045] PDA medium: 200 g potatoes, cut into small pieces and boiled until soft, filtered through eight layers of gauze, add 20 g glucose and 15-20 g agar, add distilled water to a final volume of 1 L, natural pH. Sterilize at 115 ℃ for 20 min.

[0046] Congo red medium: CMC-Na 10.0 g, K2HPO4 1.0 g, MgSO4 0.1 g, FeSO4 0.1 g, MnSO4 5×10-4 g, peptone 10.0 g, yeast extract 10.0 g, Congo red 0.2 g, pH 7.0-7.2.

[0047] GYE medium: 10 g glucose, 5 g yeast extract, 0.6 g KH₂PO₄, 0.5 g MgSO₄·7H₂O, 0.4 g K₂HPO₄, 0.25 g CuSO₄·5H₂O, 0.05 g FeSO₄·7H₂O, 0.05 g MnSO₄, 0.001 g ZnSO₄·7H₂O, 1000 ml distilled water. Sterilize at 115 ℃ for 20 min.

[0048] Sys76 high-density fermentation medium: glucose 5.0g, peanut powder 10.0g, peptone 5.0g, K2HPO4 2.7g, KH2PO4 0.7g, MgSO4 2.0g, MnSO4 0.2g, FeSO4 0.2g, CaCl2 0.02g, 1000mL distilled water, sterilized at 121 ℃ for 30min.

[0049] Culture medium for preparing soil remediation microbial fertilizer: 30 parts sawdust, 15 parts wheat bran, 5 parts soybean cake powder, 0.03 parts K2HPO4, 0.02 parts KH2PO4, 0.001 parts FeSO4, 0.05 parts MgSO4·7H2O, 0.05 parts CaCO3, 0.01 parts MnSO4·H2O, and 50 parts water.

[0050] 1. Bacterial screening and identification of emulsified DEHP: (1) Sample collection: samples were taken from the fermentation products of chicken manure compost with 500 mg / kg DEHP.

[0051] (2) Initial screening: Take 10g of chicken manure compost product and 90mL of sterile water, stir at 180rpm for 30min, take 1mL of sample for serial dilution, and dilute by 10 times. -7 10 -8 0 -9 The sample was spread on Congo red medium and incubated at 60℃ for 48 h to obtain a pure culture of heat-resistant cellulose-degrading strain.

[0052] (3) Secondary screening: The obtained thermostable cellulose-degrading strains were inoculated into 5 mL of LB liquid medium and cultured at 28 °C for 48 h. After centrifugation at 6000 rpm / min for 5 min, the fermentation supernatant was collected. Using a 90 mm culture dish, 15 mL of distilled water and 20 μL of DEHP (Sudan Red I staining) were added. After the mixture dispersed, 20 μL of fermentation supernatant was added, and the diameter of the dispersed ring was measured. The strain with the largest dispersed ring diameter, Sys76, was selected.

[0053] (4) Identification of DEHP-emulsified strains: PCR amplification was performed using universal 16S rRNA primers 27F and 1492R. The PCR reaction system consisted of 50 μL: 1 μL each of upstream and downstream primers 27F and 1492R, 1 μL DNA template, 1 μL Easy Taq DNA Polymerase (5 U / μL), 5 μL 10× Easy Taq buffer, 4 μL dNTPs (2.5 mM), and 37 μL ddH2O. The PCR amplification program was as follows: 95℃ for 10 min; 94℃ for 30 s, 61℃ for 45 s, 72℃ for 1 min and 45 s, for 30 cycles; 72℃ for 10 min. The PCR amplification product was ligated into the pMD18-T plasmid, transformants were selected, and after PCR positive clone detection, they were sent to Wuhan Qingke Biotechnology Co., Ltd. for sequence analysis. The obtained sequence is shown in SEQ ID NO.1. The sequencing results were compared online using BLAST on NCBI, and a phylogenetic tree was constructed using MEGA X software. The results are shown below. Figure 3 As shown, the screened strain Sys76 was determined to belong to Bacillus tekirae in taxonomy (Bacillus tekirae). Bacillus tequilensis ).

[0054] like Figure 4 As shown, when the fermentation supernatant of strain Sys76 was added, the diameter of the spreading ring reached 71.8 mm, indicating that the fermentation product of strain Sys76 can effectively emulsify DEHP.

[0055] Example 2 Based on the Sys76 strain obtained in Example 1, further operations were performed: 2. Preparation of microbial agents A single colony was picked from the plate and transferred to a 5 mL LB PA bottle. The culture was activated by incubation at 37°C and 200 rpm for 24 h. The activated strain was then inoculated at a 2% inoculum into 1 L of Sys76 high-density fermentation medium and incubated at 37°C and 200 rpm for 48 h. The fermentation broth was then serially diluted and plated. The current biomass was recorded as 5.46 × 10⁻⁶. 11 CFU / mL.

[0056] Example 3 Based on the bacterial agent obtained in Example 2, further operations were performed: 3. Preparation, identification, and emulsification effect evaluation of surfactants (1) Preparation and identification of biosurfactants of strain Sys76: The high-density fermentation broth was centrifuged at 6000 rpm / min for 5 min, and 200 μL of supernatant was taken and filtered through a 0.22 μm filter membrane to prepare liquid chromatography-mass spectrometry samples. The intermediate products were then identified by HPLC-ESI-QTOF-MS.

[0057] (2) DEHP emulsification index (E 24 Determination: The obtained pure culture strain was inoculated into 5 mL of LB liquid medium and cultured at 37℃ for 24 h. After centrifugation at 6000 rpm / min for 5 min, the supernatant was collected. 5 mL of sterile water and 5 mL of 1% SDS solution were used as controls. 5 mL of DEHP was added to the liquid. After shaking for 5 min and standing for 24 h, the foaming height was measured and compared with the sterile water control. The DEHP emulsification index was calculated, and the strain with the highest emulsification index, Sys76, was selected.

[0058]

[0059] like Figure 5 As shown, a total of 5 biosurfactant substances were identified, including C 13 Surfactants, C 14 Surfactants, C 15 Surfactants, C 14 Iridin, C 15 Iridin. These substances together promote the emulsification of DEHP.

[0060] like Figure 6 As shown, after adding the supernatant of strain Sys76, the emulsification index of DEHP reached 53.6%, which was comparable to that of 1% SDS solution, indicating that the fermentation product of strain Sys76 has a significant emulsifying effect.

[0061] Example 4 Based on the bacterial agent obtained in Example 3, further operations were performed: 4. Effect of Sys60 on promoting DEHP degradation in solution Aspergillus tabingii Sys60 mycelial cakes were placed on PDA solid medium and incubated at 28°C for 5 days. The spores were washed with sterile water, and the number of spores was counted using a hemocytometer. The spore suspension concentration was then adjusted to 1×10⁻⁶. 8 CFU / mL.

[0062] The strain Sys76 was inoculated into 100 mL of high-density fermentation medium and cultured at 37 °C for 24 h to obtain Sys76 bacterial culture.

[0063] Sys76 bacterial suspension and Sys60 spore suspension were inoculated at a rate of 1% into GYE liquid medium containing 1000 mg / L DEHP and cultured at 28°C and 150 rpm. The DEHP content and esterase activity of the culture medium were measured daily. The culture medium was extracted with dichloromethane, and the sample was dissolved in methanol after rotary evaporation. The DEHP content was then determined by HPLC, and the degradation rate was calculated.

[0064] Sys76 bacterial suspension and Sys60 spore suspension were inoculated at a rate of 1% into GYE liquid medium containing 1000 mg / L DEHP. After culturing at 28°C and 150 rpm for 7 days under different initial pH conditions, the samples were extracted with dichloromethane, dissolved in methanol after rotary evaporation, and finally the degradation rate of DEHP was determined by high performance liquid chromatography.

[0065] like Figure 7 As shown, most of the selected and screened Bacillus strains exhibited inhibitory effects on fungi, which led to their inhibitory effect on the degradation of DEHP by strain Sys60. Meanwhile, another strain, Bacillus tekirae, had little effect on the degradation of DEHP by Sys60, indicating that only strain Sys76 showed a promoting effect.

[0066] like Figure 8 As shown, on the third day, the Sys76 strain alone showed weak DEHP degradation ability, and the Sys60 strain alone achieved a DEHP degradation rate of only 84.7%. However, the co-culture of Sys76 and Sys60 strains resulted in a DEHP degradation rate of 96.9%. With increasing culture time, the DEHP content in the culture medium continuously decreased. After 5 days of culture, the co-culture of Sys76 and Sys60 achieved a DEHP degradation rate of over 99.6%.

[0067] like Figure 9 As shown, co-culturing Sys76 and Sys60 exhibits high degradation rates (>50%) of 1000 mg / L DEHP at initial pH values ​​of 5.0–9.0, with the optimal initial pH being 9.0, under which the degradation rate can reach 100%.

[0068] Example 5 Based on Example 4, the following further steps were taken: 5. Preparation of soil remediation microbial fertilizer and its effect on DEHP degradation in soil The strain Sys76 was inoculated into 1L of high-density fermentation medium and cultured for 48h. After culturing the mycelium of Aspergillus tabingii Sys60 on a mushroom cultivation substrate for 5 days, the spore concentration was 1.7 × 10⁻⁶. 8CFU / g; The two strains were added to 1 kg of soil remediation microbial fertilizer culture medium at 2% and 2% respectively, and cultured at 28℃ for 5 days to obtain the soil remediation microbial fertilizer. The spore content of Sys60 was 8.6 × 10⁻⁶. 8 The CFU / g and Sys76 content reached 1.3×10⁻⁶. 10 CFU / g.

[0069] Soil was collected from vegetable farms, sieved, and DEHP was dissolved in acetone. The solution was then sprayed evenly onto the soil surface using a spray bottle to achieve a DEHP content of 50 mg / kg. 700g of the DEHP-containing soil was then placed in each pot. The experimental groups were as follows: (1) 15-5-20 inorganic fertilizer; (2) 1.0wt% soil remediation microbial fertilizer; (3) 5.0wt% soil remediation microbial fertilizer; (4) 10.0wt% soil remediation microbial fertilizer.

[0070] Each group was set up with 3 replicates. After being placed at room temperature for 12 days (with soil turning over every 4 days), the samples were extracted with dichloromethane, dissolved in methanol after rotary evaporation, and finally the DEHP content in the soil was determined by high performance liquid chromatography.

[0071] Figure 10 As shown, in soils with added 1%, 5%, and 10% soil remediation microbial fertilizer, the degradation rates of DEHP reached 59.1%, 69.5%, and 90.1%, respectively.

[0072] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages: (1) The Bacillus terrestris Sys76 provided in the embodiments of this application can produce a variety of biosurfactants that can promote the dissolution of plasticizer DEHP, and this characteristic has not been described in the prior art.

[0073] (2) The microbial fertilizer provided in this application embodiment degrades DEHP by co-culturing Bacillus tekirae Sys76 and Aspergillus tabineum Sys60. Bacillus tekirae Sys76 can improve the solubility of DEHP, and combined with the DEHP degradation ability of Aspergillus tabineum Sys60, the degradation efficiency of DEHP can be improved.

[0074] (3) The microbial fertilizer provided in this application embodiment, wherein the effective viable cell concentration of Bacillus tekirii Sys76 after fermentation in a high-density fermentation medium can reach 5.4 × 10⁻⁶. 11 CFU / mL or higher.

[0075] (4) The application of the microbial fertilizer provided in the embodiments of this application has been found in actual application that it has a high degradation rate (>50%) at the initial pH of 5.0 to 9.0, and the optimal initial pH for degradation is 9.0. Under this acidity and alkalinity, the degradation rate of DEHP can reach 100%. Therefore, the microbial fertilizer can completely eliminate DEHP.

[0076] (5) The application of a microbial fertilizer provided in the embodiments of this application shows that in soils with 5% and 10% added microbial fertilizer for soil remediation, the degradation rate of DEHP can reach 69.5% and 90.1%, respectively.

[0077] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0078] In this application, unless otherwise stated, directional terms such as "upper" and "lower" specifically refer to the drawing directions in the accompanying drawings. Furthermore, in the description of this application, terms such as "comprising" and "including" mean "including but not limited to." In this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. In this document, "and / or" describes the relationship between related objects, indicating that three relationships can exist; for example, A and / or B can represent: A alone, A and B simultaneously, or B alone. A and B can be singular or plural. In this document, "at least one" means one or more, and "more than one" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or plural items. For example, "at least one of a, b, or c" or "at least one of a, b, and c" can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be a single or multiple.

[0079] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A type of Bacillus tekirae ( Bacillus tequilensis Sys76, characterized in that, The Bacillus tekirae Sys76 was deposited at the China Center for Type Culture Collection on May 15, 2023, with accession number CCTCC NO: M2023746.

2. A microbial inoculant, characterized in that, The microbial agent includes Bacillus tekirae Sys76 as described in claim 1.

3. A microbial fertilizer, characterized in that, The microbial fertilizer includes Bacillus tekirulae Sys76 and Aspergillus tabine as described in claim 1. Aspergillus tubingensis Sys60.

4. The microbial fertilizer according to claim 3, characterized in that, The effective viable bacteria concentration of the microbial fertilizer is ≥5.4×10⁻⁶. 11 CFU / mL.

5. A method for preparing the microbial fertilizer according to claim 3 or 4, characterized in that, The method includes: Bacillus tekirae Sys76 bacterial suspension and Aspergillus tabingii Sys60 spores were inoculated into soil remediation microbial fertilizer culture medium and cultured to obtain microbial fertilizer.

6. An application of a microbial fertilizer, characterized in that, The microbial fertilizer described in claim 3 or 4 is used for the degradation of DEHP.

7. The application according to claim 6, characterized in that, The microbial fertilizer is suitable for a pH of 5.0 to 9.0 during the degradation process.

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