Bacillus subtilis zymophyte liquid, microbial composite nano-bactericide and preparation method of microbial composite nano-bactericide

Abstract

The invention relates to the technical field of oilfield chemistry, in particular to bacillus subtilis zymophyte liquid, a microbial composite nano bactericide and a preparation method thereof, and the preparation method of the bacillus subtilis zymophyte liquid comprises the following steps: culturing a bacillus subtilis strain in a culture medium to obtain the bacillus subtilis zymophyte liquid; wherein the culture medium is a fermentation culture medium or a hydrocarbon degradation culture medium. Fermentation bacteria liquid of the bacillus subtilis strain contains lipopeptide biosurfactants, can play roles of dispersing agents, stabilizing agents, blocking agents, reducing agents and the like in metal nanometer synthesis, can inhibit excessive growth of nanometer particles and prevent the nanometer particles from being gathered / condensed in colloid synthesis, and has the capacity of inducing tiny changes of the nanometer particles. The microbial composite nano bactericide is prepared on the basis of the zymophyte liquid, AgNPs in the microbial composite nano bactericide is small in particle size and good in dispersity and stability, and harmful florae such as sulfate reducing bacteria in the oil field environment can be well inhibited.

Description

Technical Field

[0001] This invention relates to the field of oilfield chemical technology, specifically to a Bacillus subtilis fermentation broth, its preparation method and application, and a microbial composite nano-bactericide and its preparation method. Background Technology

[0002] Nanoparticles are generally defined as small particles with a core-shell structure ranging from 1 nm to 100 nm. Key factors for nanoparticles in biological fluids are biocompatibility and stability; therefore, the surface of nanoparticles can be modified with different functional groups. Over the past decade, AgNPs (nano silver) have become a widely used alternative to chlorine-free bactericides due to their drug resistance properties, finding applications across various industries.

[0003] Nanoparticles are typically synthesized using two strategies: top-down and bottom-up approaches. In the top-down approach, van der Waals forces between large particles are physically broken down through high-energy processes, gradually decomposing bulk materials into nanoscale materials, primarily used in the microelectronics industry. Bottom-up metal nanoparticle synthesis methods refer to assembling ions or basic units into nanoscale metal molecular structures. This can be further divided into chemical synthesis and green synthesis. Both methods include three main components: a metal ion source, a reducing agent, and a capping agent. Utilizing microbial metabolites such as biosurfactants as capping agents can regulate the surface chemical functions, morphology, and size distribution of nanoparticles. Furthermore, they can prevent nanoparticle aggregation and enhance reduction kinetics by forming complex structures with metal ions in the precursor salt. Simultaneously, bacterial extracts act as both reducing agents and stabilizers in nanoparticle synthesis. Therefore, using novel eco-friendly formulations to replace chemical agents to reduce risks, and employing green and environmentally friendly biological methods to achieve non-toxic nanoparticle synthesis, holds broad development potential. However, there is a lack of microbial agents specifically designed for the preparation of nanoparticle bactericides, and there is an urgent need to find microbial agents that can be used specifically for sterilization and bacteriostasis in oilfield pipelines. Summary of the Invention

[0004] This invention provides a Bacillus subtilis fermentation broth, a microbial composite nano-bactericide, and a method for preparing the same, overcoming the shortcomings of the prior art. It can effectively solve the need for morphology and size control in the preparation of existing nanoparticles, and provides a bactericide that inhibits harmful bacteria such as sulfate-reducing bacteria in the oil reservoir environment.

[0005] One of the technical solutions of this invention is achieved through the following measures: a method for preparing Bacillus subtilis fermentation broth, comprising the following steps: Bacillus subtilis strains were cultured in a culture medium to obtain Bacillus subtilis fermentation broth; The culture medium is either a fermentation medium or a hydrocarbon degradation medium.

[0006] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: The fermentation medium consists of: liquid paraffin 1 g / L to 10 g / L, yeast extract 0.1 g / L to 1 g / L, NaNO3 5 g / L to 20 g / L, KH2PO4 0.1 g / L to 0.5 g / L, Na2HPO4 0.5 g / L to 2 g / L, and MgSO4·7H2O. 0.1 g / L to 0.5 g / L, trace element solution 5 mL / L to 20 mL / L; wherein, the trace element solution is prepared by nitrogen triacetic acid, diethylenetriaminepentaacetic acid, MgSO4·7H2O, MnSO4·2H2O, NaCl, FeSO4·7H2O, CoSO4·7H2O, CaCl2·2H2O, ZnSO4·7H2O, CuSO4·5H2O, KAl(SO4)2·12H2O, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, Na2SeO3·5H2O and water.

[0007] The composition of the above trace element solution is as follows: Nitrogen triacetic acid 1.000 g / L, diethylenetriaminepentaacetic acid 0.600 g / L, MgSO4·7H2O 3.500 g / L, MnSO4·2H2O 0.580 g / L, NaCl 1.000 g / L, FeSO4·7H2O 0.180 g / L, CoSO4·7H2O 0.180 g / L, CaCl2·2H2O 0.130 g / L, ZnSO4·7H2O 0.180 g / L, CuSO4·5H2O 0.011 g / L, KAl(SO4)2·12H2O 0.023 g / L, H3BO3 0.012 g / L, Na2MoO4·2H2O 0.010 g / L, NiCl2·6H2O 0.026 g / L, Na2SeO3·5H2O 0.330 g / L, the remainder is water.

[0008] The culture temperature was 35°C to 37°C, and the pH tolerance range of the Bacillus subtilis strain was 7.0 to 7.5.

[0009] The second technical solution of the present invention is achieved through the following measures: a method for preparing Bacillus subtilis fermentation broth.

[0010] The third technical solution of the present invention is achieved through the following measures: the application of Bacillus subtilis fermentation broth in the preparation of bactericides.

[0011] The fourth technical solution of the present invention is achieved through the following measures: the application of Bacillus subtilis fermentation broth in the preparation of nanoparticle materials.

[0012] The fifth technical solution of the present invention is achieved through the following measures: a method for preparing a microbial composite nano-bactericide, comprising the following steps: Step 1: Mix the required amounts of silver nitrate solution and sodium borohydride solution to obtain the metal precursor solution; Step 2: Mix the metal precursor solution with the Bacillus subtilis fermentation broth and then culture to obtain a microbial composite nano-bactericide.

[0013] The following are further optimizations and / or improvements to the fifth technical solution of the invention described above: In step one above, the molar concentration of silver nitrate solution is 0.01 mol / L to 0.05 mol / L, and the molar concentration of sodium borohydride solution is 0.0011 mol / L to 0.021 mol / L; the volume ratio of silver nitrate solution to sodium borohydride solution is 100:0.9 to 1.1.

[0014] In step two above, the culture temperature is 40℃ to 80℃, and the culture time is 1h to 3h.

[0015] In step two above, the volume ratio of the metal precursor solution to the Bacillus subtilis fermentation broth is 1:0.9 to 1.1.

[0016] The sixth technical solution of the present invention is achieved through the following measures: a microbial composite nano-bactericide prepared by a method for preparing a microbial composite nano-bactericide.

[0017] This invention provides a fermentation broth of Bacillus subtilis strain W-2, which contains lipopeptide biosurfactants that can act as dispersants, stabilizers, blockers, and reducing agents in metal nanoparticle synthesis. It can inhibit the excessive growth of nanoparticles and prevent their aggregation / coagulation during colloidal synthesis, and has the ability to induce subtle changes in nanoparticles. Based on this fermentation broth, a microbial composite nano-bactericide is prepared. The AgNPs in this microbial composite nano-bactericide have small particle size, good dispersibility and stability, and can effectively inhibit harmful bacteria such as sulfate-reducing bacteria in oilfield environments. Attached Figure Description

[0018] Appendix Figure 1 The image shows the infrared spectrum of the metabolites of Bacillus subtilis strain W-2 in Example 13 of this invention.

[0019] Appendix Figure 2This is a chromatogram of the metabolites of Bacillus subtilis strain W-2 in Example 13 of the present invention.

[0020] Appendix Figure 3 The mass spectrum of the metabolic products of Bacillus subtilis strain W-2 in Example 13 of this invention (at 13.48 min).

[0021] Appendix Figure 4 The images show the effects of the microbial composite nano-bactericides prepared in Examples 14 to 18 of this invention.

[0022] Appendix Figure 5 This is a graph showing the laser particle size analyzer results of silver nanoparticles in Example 19 of the present invention.

[0023] Appendix Figure 6 This is a zeta potential diagram of silver nanoparticles detected by a laser particle size analyzer in Example 20 of the present invention.

[0024] Appendix Figure 7 This is a SEM image of the silver nanoparticles in Example 21 of the present invention.

[0025] Appendix Figure 8 This is an EDS diagram of the microbial composite nano-bactericide in Example 21 of the present invention.

[0026] Appendix Figure 9 This is a TEM image of the silver nanoparticles in embodiment 21 of the present invention.

[0027] Appendix Figure 10 This is an X-ray diffraction pattern of the silver nanoparticles in Example 22 of the present invention.

[0028] Appendix Figure 11 The image shows the infrared spectrum of the supernatant of the fermentation broth of the microbial composite nano-bactericide and Bacillus subtilis W-2 in Example 23 of this invention.

[0029] Appendix Figure 12 This is a graph showing the inhibitory effect of the microbial composite nano-bactericide in Example 24 of the present invention on sulfate-reducing bacteria at different concentrations.

[0030] Appendix Figure 13 This is a diagram showing the species composition distribution of the microbial community at the phylum level after the microbial composite nano-bactericide in Example 25 inhibits bacterial growth. Detailed Implementation

[0031] This invention is not limited to the following embodiments, and specific implementation methods can be determined according to the technical solution of this invention and the actual situation. Unless otherwise specified, all chemical reagents and chemical products mentioned in this invention are well-known and commonly used chemical reagents and chemical products in the prior art; unless otherwise specified, all percentages in this invention are mass percentages; room temperature in this invention generally refers to a temperature between 15°C and 25°C, and is generally defined as 25°C.

[0032] The present invention will be further described below with reference to embodiments: Example 1: The preparation method of the Bacillus subtilis fermentation broth includes the following steps: Bacillus subtilis strains were cultured in a culture medium to obtain Bacillus subtilis fermentation broth; The culture medium is either a fermentation medium or a hydrocarbon degradation medium.

[0033] In this invention, Bacillus subtilis strain W-2 was isolated from oilfield water samples. This strain was deposited on November 18, 2014, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 9985, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, China. Scanning electron microscopy observation of the Bacillus subtilis strain in this invention showed that the bacterial cells were rod-shaped, and the spores were elliptical.

[0034] Example 2: As an optimization of the above example, the fermentation medium composition includes: liquid paraffin 1 g / L to 10 g / L, yeast extract 0.1 g / L to 1 g / L, NaNO3 5 g / L to 20 g / L, KH2PO4 0.1 g / L to 0.5 g / L, Na2HPO4 0.5 g / L to 2 g / L, MgSO4·7H2O 0.1 g / L to 0.5 g / L, trace element solution 5 mL / L to 20 mL / L; wherein, the trace element solution is prepared by nitrogen triacetic acid, diethylenetriaminepentaacetic acid, MgSO4·7H2O, MnSO4·2H2O, NaCl, FeSO4·7H2O, CoSO4·7H2O, CaCl2·2H2O, ZnSO4·7H2O, CuSO4·5H2O, KAl(SO4)2·12H2O, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, Na2SeO3·5H2O and water. Preferably, the fermentation medium consists of: 5 g / L liquid paraffin, 0.5 g / L yeast extract, 10 g / L NaNO3, 0.35 g / L KH2PO4, 1 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, and 10 mL g / L trace element solution.

[0035] Example 3: As an optimization of the above example, the composition of the trace element solution is as follows: 1.000 g / L of nitrogen triacetic acid, 0.600 g / L of diethylenetriaminepentaacetic acid, 3.500 g / L of MgSO4·7H2O, 0.580 g / L of MnSO4·2H2O, 1.000 g / L of NaCl, 0.180 g / L of FeSO4·7H2O, 0.180 g / L of CoSO4·7H2O, 0.130 g / L of CaCl2·2H2O, 0.180 g / L of ZnSO4·7H2O, 0.011 g / L of CuSO4·5H2O, 0.023 g / L of KAl(SO4)2·12H2O, 0.012 g / L of H3BO3, and Na2MoO4·2H2O. 0.010 g / L, NiCl2·6H2O 0.026 g / L, Na2SeO3·5H2O 0.330 g / L, the remainder is water.

[0036] Example 4: As an optimization of the above example, the culture temperature is 35°C to 37°C, and the pH tolerance range of the Bacillus subtilis strain is 7.0 to 7.5. Specifically, the culture is carried out in a shaker at a speed of 150 rpm to 300 rpm, preferably 180 rpm; preferably, the culture temperature is 35°C, and the pH tolerance value of the Bacillus subtilis strain is 7.0.

[0037] Example 5: Bacillus subtilis fermentation broth prepared by the method described above.

[0038] In this invention, the viable cell count (OD600) of the Bacillus subtilis fermentation broth is 0.8 to 1.0.

[0039] Example 6: Application of the Bacillus subtilis fermentation broth in the preparation of bactericides.

[0040] Example 7: Application of the Bacillus subtilis fermentation broth in the preparation of nanoparticle materials.

[0041] Example 8: The preparation method of this microbial composite nano-bactericide includes the following steps: Step 1: Mix the required amounts of silver nitrate solution and sodium borohydride solution to obtain the metal precursor solution; Step 2: Mix the metal precursor solution with the Bacillus subtilis fermentation broth and then culture to obtain a microbial composite nano-bactericide.

[0042] Example 9: As an optimization of the above example, in step one, the molar concentration of silver nitrate solution is 0.01 mol / L to 0.05 mol / L, and the molar concentration of sodium borohydride solution is 0.0011 mol / L to 0.021 mol / L; the volume ratio of silver nitrate solution to sodium borohydride solution is 100:0.9 to 1.1.

[0043] Preferably, the molar concentration of silver nitrate is 0.03 mol / L to 0.05 mol / L, and the molar concentration of sodium borohydride solution is 0.01 mol / L to 0.02 mol / L. More preferably, the molar concentration of silver nitrate is 0.04 mol / L, and the molar concentration of sodium borohydride solution is 0.02 mol / L. This invention does not have any particular limitation on the source of silver nitrate and sodium borohydride; conventional silver nitrate and sodium borohydride reagents in the art can be used. This invention does not have any particular limitation on the preparation of the silver nitrate and sodium borohydride solutions; conventional solution preparation methods in the art can be used.

[0044] Example 10: As an optimization of the above example, in step two, the culture temperature is 40°C to 80°C and the culture time is 1h to 3h.

[0045] Example 11: As an optimization of the above example, in step two, the volume ratio of the metal precursor solution to the Bacillus subtilis fermentation broth is 1:0.9 to 1.1.

[0046] This invention prepares a microbial composite nano-bacterial agent using the fermentation broth of Bacillus subtilis W-2 and a metal precursor solution, yielding a crude microbial composite nano-bacterial agent. The crude agent is then purified as needed to obtain the final microbial composite nano-bacterial agent. The purification method involves filtering the crude agent through a 0.22 μm vacuum filter membrane to remove bacterial interference, followed by centrifugation at 8000 rpm for 5 minutes. After discarding the supernatant, an appropriate amount of deionized water is added, followed by shaking, centrifugation, and then discarding the supernatant. This process is repeated three times to obtain a relatively pure microbial composite nano-bacterial agent. The purified composite nano-bacterial agent can be further dried to obtain a solid microbial composite nano-bacterial agent.

[0047] The microbial composite nano-bactericide prepared by the method of the present invention is a liquid formulation. This microbial composite nano-bactericide can also be a solid formulation. The solid formulation can be obtained by drying the liquid formulation of the microbial composite nano-bactericide; the drying method is vacuum freeze-drying or low-temperature drying; wherein, the preferred low-temperature drying temperature is 70°C. The solid formulation of the microbial composite nano-bactericide can be dispersed in water and directly applied for the sterilization and bacteriostatic effects on sulfate-reducing bacteria in oil fields.

[0048] The preparation method of the microbial composite nano-bactericide of the present invention must be carried out in a sterile environment. The addition of silver nitrate and sodium borohydride bacterial solution to the metal precursor solution must be strictly performed in a sterile laminar flow hood to prevent contamination. Furthermore, the metal precursor solution must not contain chloride ions; otherwise, the reducing power of the fermentation broth will be insufficient to displace the elemental metal particles.

[0049] In this invention, the molar concentrations of silver nitrate and sodium borohydride solutions in the metal precursor solution are beneficial for the rational formulation of the prepared nano-silver, resulting in good dispersibility and stability of the metal nanoparticles in the prepared microbial composite nano-bactericide. Simultaneously, the molar ratio of silver nitrate to sodium borohydride solutions in the metal precursor solution helps to obtain metal nanoparticles with smaller particle sizes. Furthermore, the mixing ratio of the metal precursor solution to the fermentation broth also contributes to obtaining metal nanoparticles with smaller particle sizes. The particle size of the metal nanoparticles in the microbial composite nano-bactericide prepared by this invention is 20 nm to 100 nm, more preferably 20 nm to 40 nm. Under optimal preparation conditions, the particle size of the metal nanoparticles is 26 nm. These metal nanoparticles exist in the fermentation broth in a fluid form. The metal nanoparticles have the advantages of uniform shape and small particle size, and possess greater surface energy, which is beneficial for damaging the bacterial membrane, leading to cell death and providing superior antibacterial and bactericidal effects.

[0050] The present invention provides a simple method for preparing a microbial composite nano-bactericide, which, compared with previous technologies, has advantages such as significant bactericidal and bacteriostatic effects, wide availability of raw materials, and low cost. The bactericidal and bacteriostatic effects of the microbial composite nano-bactericide of the present invention are positively linearly correlated with the concentration of the microbial composite nano-bactericide. The metal nanoparticles in the microbial composite nano-bactericide prepared by the method of the present invention have the advantages of uniform shape and small particle size, and possess greater surface energy, which is beneficial for damaging bacterial membranes, leading to cell death and providing superior bactericidal and bacteriostatic effects.

[0051] This invention provides the application of the microbial composite nano-bactericide described above in the sterilization and bacteriostasis of oilfields. In this invention, the fermentation broth of the microbial composite nano-bactericide contains surfactant components, mainly surfactantin, which can act as a reducing agent, dispersant, and stabilizer. The metal nanoparticles in this microbial composite nano-bactericide have small particle sizes and good dispersibility, enabling them to adsorb well onto the surface of the bacteria, increasing cell membrane permeability, leading to cell membrane damage and cell death. Results show that the microbial composite nano-bactericide provided by this invention can effectively inhibit sulfate-reducing bacteria and other harmful bacterial communities in oilfield produced fluids, exhibiting good sterilization and bacteriostasis effects.

[0052] Example 12: Preparation of Bacillus subtilis (W-2) fermentation broth.

[0053] Bacillus subtilis W-2 was inoculated onto a fermentation medium and cultured on a shaker to obtain the fermentation broth of Bacillus subtilis W-2.

[0054] The fermentation medium consisted of the following raw materials (g / L): 5 g / L liquid paraffin, 0.5 g / L yeast extract, 10 g / L NaNO3, 0.35 g / L KH2PO4, 1 g / L Na2HPO4, 0.2 g / L MgSO4·7H2O, and 10 mL trace element solution; the pH was adjusted to 7.0 using 0.1 mol / L NaOH solution and 0.1 mol / L HCl solution.

[0055] The temperature for incubation on the shaker was 35℃; the incubation time was 3 days; and the shaking speed was 180 rpm.

[0056] After the shaker culture was completed, the fermentation broth of Bacillus subtilis W-2 was obtained, with an OD of 600 The values ​​range from 0.8 to 1.

[0057] Example 13: Isolation, purification, and identification of biosurfactants in the metabolites of Bacillus subtilis W-2 fermentation broth, as detailed below: ① Surface tension measurement of fermentation broth: A platinum sheet was heated to red-hot using the outer flame of an alcohol lamp. After cooling to room temperature, the surface tension meter was zeroed using deionized water to approximately 72.0 mN / m. The platinum sheet was then slowly brought into contact with the surface of the liquid to be tested (the Bacillus subtilis W-2 fermentation broth prepared in Example 12). The surface tension of the liquid pulled the platinum sheet downwards. When the test value stabilized, the surface tension of the sample was recorded as 30.8 mN / m. Compared to the initial surface tension of 72.0 mN / m in the surface tension meter, the surface tension decreased significantly, indicating that the Bacillus subtilis W-2 fermentation broth contains surfactant components.

[0058] ② Separation and purification of biosurfactants: 10 L of Bacillus subtilis W-2 fermentation broth was centrifuged at 8000 rpm for 10 min at room temperature. The precipitate was removed, and the remaining liquid was the supernatant. The supernatant was then acidified to pH 2 with 6 mol / L hydrochloric acid, and the acidified solution was refrigerated at 4℃ for 12 h. The acidified supernatant was then removed by centrifugation. An equal volume of chloroform-methanol (2:1) was added to the precipitate to extract the crude biosurfactant. The extract was filtered using a vacuum pump, filter paper, and Buchner funnel. The resulting brownish-yellow liquid was concentrated using a rotary evaporator at 40℃. The concentrate was then placed in a 40℃ oven until the solvent completely evaporated, yielding a yellowish-brown viscous substance, which was the biosurfactant sample. Infrared spectroscopy analysis was performed on the sample. Figure 1 As shown, the results indicate the presence of typical cyclic lactam bonds and nitrogen-hydrogen bonds in amino acids, suggesting that the metabolites of Bacillus subtilis W-2 are lipopeptide biosurfactants.

[0059] ③ Chromatographic mass spectrometry analysis of biosurfactants: Chromatographic conditions: Mobile phase: 0.1% acetic acid aqueous solution + acetonitrile; Flow rate: 0.2 ml / min; Column temperature: 35℃; Injection volume: 5 μL; Column: WatersAcquity UPLC C18 1.7um 2.1×100mm.

[0060] Mass spectrometry conditions: Ion source: ESI(+); Capillary voltage: 3.0KV; Cone voltage: 40V; Desolventization temperature: 300℃; Ion source: 150℃; Nebulizer gas: 650L / h; Acquisition mode: Ms1 Scan.

[0061] Liquid chromatography, such as Figure 2 As shown, there are elution peaks at 8 time intervals, with the mass spectrum showing a local retention time of 13.48 min ( Figure 3 The results show that the main peaks are 995.07, 1008.04, and 1022.06, corresponding to the peak values ​​of the surfactantin standard, indicating that the sample mainly contains surfactantin. Additionally, a small peak at 325.14 is observed, presumably representing a small peptide chain that did not participate in cyclization. Therefore, it can be concluded that the metabolites of Bacillus subtilis W-2 are lipopeptide biosurfactants containing surfactantin.

[0062] As demonstrated by the tests in this embodiment, Bacillus subtilis strain W-2 can secrete surfactant components, the fermentation broth of Bacillus subtilis W-2 can reduce surface tension, and the fermentation broth contains surfactantin.

[0063] Example 14: Preparation of microbial composite nano-bactericide, the specific process is as follows: The first step is to prepare the required amount of silver nitrate and sodium borohydride metal precursor solution; In the second step, under magnetic stirring, the metal precursor solution was added to the fermentation broth of Bacillus subtilis W-2 prepared in Example 12 at a volume ratio of 1:1 and mixed evenly. The mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 was placed in a constant temperature incubator and cultured for a certain period of time to obtain the crude product of the microbial composite nano-bactericide, which contains silver nanoparticles (AgNPs). The third step involves filtering the crude microbial composite nano-bactericide using a 0.22μm vacuum filter membrane to remove bacterial interference. Then, the sample is centrifuged at 8000 rpm for 5 minutes. After discarding the supernatant, an appropriate amount of deionized water is added, followed by shaking and centrifugation. The supernatant is then discarded. This process is repeated three times to obtain a relatively pure microbial composite nano-bactericide.

[0064] This embodiment investigates the concentration of AgNO3 in the metal precursor solution during the preparation of the microbial composite nano-bactericide. Five groups of experiments with different concentrations of AgNO3 were conducted, and the resulting microbial composite nano-bactericides were denoted as BS-1(1), BS-1(2), BS-1(3), BS-1(4), and BS-1(5), respectively. In this embodiment, the concentrations of AgNO3 in the metal precursor solution (range 10 mmol / L to 50 mmol / L), NaBH4 concentration (10 mmol / L), culture time (1 h), temperature (40 °C), and pH value (7) of the mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 are shown in Table 1.

[0065] Example 15: The preparation process of this microbial composite nano-bactericide is the same as in Example 14.

[0066] In this embodiment, the concentration of NaBH4 in the metal precursor solution was investigated during the preparation of the microbial composite nano-bactericide. Three groups of NaBH4 with different concentrations were used in the experiment, and the resulting microbial composite nano-bactericides were denoted as BS-2(1), BS-2(2), and BS-2(3), respectively.

[0067] In this embodiment, the concentrations of AgNO3 and NaBH4 in the metal precursor solution, the culture time, temperature, and pH value of the mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 are shown in Table 1.

[0068] Example 16: The preparation process of this microbial composite nano-bactericide is the same as in Example 14.

[0069] In this embodiment, the culture time of the mixture of metal precursor solution and Bacillus subtilis W-2 fermentation liquid was investigated in the preparation process of microbial composite nano-bactericide. Three groups of culture time were used for the experiment, and the obtained microbial composite nano-bactericides were recorded as BS-3(1), BS-3(2), and BS-3(3), respectively.

[0070] In this embodiment, the concentrations of AgNO3 and NaBH4 in the metal precursor solution, the culture time, temperature, and pH value of the mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 are shown in Table 1.

[0071] Example 17: The preparation process of this microbial composite nano-bactericide is the same as in Example 14.

[0072] In this embodiment, the culture temperature of the mixture of metal precursor solution and Bacillus subtilis W-2 fermentation liquid was investigated during the preparation of microbial composite nano-bactericide. Three sets of culture temperatures were used in the experiment, and the obtained microbial composite nano-bactericides were recorded as BS-4(1), BS-4(2), and BS-4(3), respectively.

[0073] In this embodiment, the concentrations of AgNO3 and NaBH4 in the metal precursor solution, the culture time, temperature, and pH value of the mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 are shown in Table 1.

[0074] Example 18: The preparation process of this microbial composite nano-bactericide is the same as in Example 14.

[0075] In this embodiment, the pH value of the mixture of metal precursor solution and Bacillus subtilis W-2 fermentation liquid was investigated during the preparation of microbial composite nano bactericide. Five pH values ​​were used in the experiment, and the obtained microbial composite nano bactericides were recorded as BS-5(1), BS-5(2), BS-5(3), BS-5(4), and BS-5(5).

[0076] In this embodiment, the concentrations of AgNO3 and NaBH4 in the metal precursor solution, the culture time, temperature, and pH value of the mixture of the metal precursor solution and the fermentation broth of Bacillus subtilis W-2 are shown in Table 1.

[0077] The microbial composite nano-bactericides prepared in Examples 14 to 18 are as follows: Figure 4 As shown. Figure 4 In this example, BS-1 corresponds to the microbial composite nano-bactericide prepared in Example 14, BS-2 corresponds to the microbial composite nano-bactericide prepared in Example 15, BS-3 corresponds to the microbial composite nano-bactericide prepared in Example 16, BS-4 corresponds to the microbial composite nano-bactericide prepared in Example 17, and BS-5 corresponds to the microbial composite nano-bactericide prepared in Example 18.

[0078] The particle size of the metallic silver nanoparticles in the microbial composite nano-bactericides prepared in Examples 14 to 18 was detected using a laser particle size analyzer.

[0079] Detection method: 1. First, turn on the computer, then turn on the instrument's power switch and preheat for about half an hour to stabilize the laser's output power.

[0080] 2. Prepare a clean cuvette. Clean the surface with alcohol or similar disinfectant, then let it air dry to ensure there are no foreign objects on the surface. Then, pour purified water into the cuvette until the water level is about 2 / 3 of the cuvette's height. Make sure the water level covers the laser beam at a position 5mm above the cuvette.

[0081] 3. Open the sample chamber cover, place the cuvette filled with pure water into the fixed slot of the sample chamber, then remove the baffle in front of the detector, adjust the position of the cuvette so that the light spot reflected to the detector is slightly offset upwards, ensuring that the reflected light spot does not shine on the receiving surface of the detector, and then close the cover.

[0082] 4. Add the sample and run the main program.

[0083] The particle size of the metallic silver nanoparticles in the microbial composite nano-bactericides prepared in Examples 14 to 18 is shown in Table 2. Table 2 shows that different conditions have varying degrees of influence on the particle size of the metallic silver nanoparticles in the microbial composite nano-bactericides. The order of influence of different conditions on particle size is: metal ion concentration > reaction time > reaction temperature > pH.

[0084] Example 19: The preparation process of this microbial composite nano-bactericide is the same as in Example 14.

[0085] Based on the influence of different conditions on the particle size of metallic silver nanoparticles in the microbial composite nano-bactericide in Examples 14 to 18, the optimized preparation conditions for the synthesis of metallic silver nanoparticles were determined to be: AgNO3 concentration of 40 mmol / L, NaBH4 concentration of 20 mmol / L, reaction time of 2 hours, temperature of 60℃, and pH of 9.

[0086] In this embodiment, under the optimized preparation conditions described above, the particle size of the metallic silver nanoparticles in the prepared microbial composite nano-bactericide is 26 nm (see...). Figure 5 Since metal ions are easily aggregated and grown after being reduced to zero-valent metals, finding a suitable stabilizer to maintain the particles at the nanoscale is crucial. The Bacillus subtilis W-2 fermentation broth used in this invention contains a large number of macromolecules such as hydroxyl and carbonyl groups, which are beneficial for the stable dispersion of silver nanoparticles in the system and for maintaining the particles at the nanoscale.

[0087] Example 20: Detection of the microbial composite nano-bactericide prepared in Example 19 using a laser particle size analyzer.

[0088] The Zeta potentials of the synthesized AgNPs were measured using a Zetasizer Nano ZSP nanoparticle potentiometer. All Zeta potentials were negative, indicating that the AgNPs containing surfactants in the fermentation broth of Bacillus subtilis W-2 are inherently negatively charged. The nanoparticles are relatively stable within the range of ±30mV to ±40mV. The detection results are as follows: Figure 6 As shown. Figure 6 The zeta potential of AgNPs is −31.3 mV. The negative charge of the nanoparticles causes the formation of a double electron layer between identical particles, resulting in electrostatic repulsion. This prevents the agglomeration of AgNPs particles and stabilizes the nanoparticles in the solution, indicating that the AgNPs colloidal system synthesized by the method of this invention has good stability.

[0089] Example 21: Characterization of Microbial Composite Nanoparticle Bactericide SEM characterization: The shape profile of the microbial composite nanoparticles was determined using a Zeiss SIGMA SEM running at 5.00 kV to 6.00 kV, and the equipped energy dispersive spectroscopy (EDS) was used to confirm the presence of silver in the microbial composite nanoparticles.

[0090] The sample preparation method for SEM and EDS analysis is as follows: A suitable amount of microbial composite nano-bactericide is dropped onto a silica plate, and the solvent is slowly evaporated at room temperature or by irradiation with an infrared lamp. The sample is then observed and analyzed under a Zeiss SIGMA SEM. In this example, AgNPs prepared from fermentation broth without the addition of Bacillus subtilis W-2 are used as a blank control (the remaining preparation conditions are the same as in Example 19).

[0091] TEM characterization: After washing away the biosurfactant coated on the surface of the microbial composite nano-bactericide with ethanol, it was dispersed with ethanol, sonicated with ultrasound for 10 min, dropped onto a carbon-coated copper grid, vacuum dried for 20 min, and then placed under a JEM-2100 TEM to observe its morphology.

[0092] Figure 7 The SEM observations are shown from left to right. Figure 7In the image, the leftmost image shows AgNPs synthesized from fermentation broth without Bacillus subtilis W-2 (blank control); the second from the left shows AgNPs synthesized mediated by Bacillus subtilis W-2; and the rightmost image shows AgNPs after removal of the surface biosurfactant. The results indicate that AgNPs synthesized directly from fermentation broth without Bacillus subtilis W-2 exhibit uneven size distribution, irregular spherical shapes, and significant aggregation. In contrast, AgNPs synthesized from fermentation broth with Bacillus subtilis W-2 show better dispersibility. Due to electrostatic repulsion between particles, the AgNP particles are uniformly dispersed in the medium without aggregation. However, when the biosurfactant coating the AgNP surface is washed with ethanol, AgNP particles aggregate again, indicating that the biosurfactant acts as a stabilizer by adsorbing onto the nanoparticle surface.

[0093] EDS elemental analysis results ( Figure 8 The spectrum showed a characteristic absorption peak of silver at 3 keV, confirming that the microbial composite nano-bactericide of this invention contains silver. AgNPs accounted for 25.82% by weight. This indicates that the reducing properties of the Bacillus subtilis W-2 fermentation broth have the potential to synthesize metallic silver nanoparticles.

[0094] Figure 9 The images from left to right show TEM observations. The left image shows AgNPs synthesized from the fermentation broth of Bacillus subtilis W-2 without the addition of Bacillus subtilis W-2 (blank control), and the right image shows AgNPs synthesized from the fermentation broth of Bacillus subtilis W-2. The AgNPs in the control group synthesized from the fermentation broth without Bacillus subtilis W-2 have a larger diameter, approximately 180 nm to 200 nm, and are irregularly ellipsoidal or angular rod-shaped. Furthermore, there are still irregular AgNPs on the surface that continue to coarsen and aggregate. This indicates that AgNPs exhibit anisotropic growth in the absence of a stabilizer. At a scale bar of 50 nm, the AgNPs synthesized from the fermentation broth of Bacillus subtilis W-2 show a certain degree of dispersion, with an average size of 5 nm to 18 nm, and spherical or ellipsoidal shapes. It can be seen that the fermentation broth of Bacillus subtilis W-2, when used as a stabilizer, can effectively prevent the aggregation and continued growth of nanoparticles.

[0095] Example 22: The microbial composite nano-bactericide solution prepared in Example 19 was centrifuged, the precipitate was washed with ethanol, and dried in a constant temperature oven to obtain AgNPs powder. XPERT-PRO technology was used for analysis, and the obtained images were compared with the powder diffraction standard (JCPDS) library to illustrate the crystal structure. The X-ray diffraction (XRD) curve of AgNPs powder in the microbial composite nano-bactericide synthesized in Example 19 is shown below. Figure 10 As shown, the crystal properties of the AgNPs powder synthesized in this invention are confirmed. The XRD diffraction peaks of the AgNPs at 2θ are 38.18, 44.34, 64.57, and 77.41°, corresponding to lattice planes (111), (200), (220), and (311), which are characteristic of the face-centered cubic (fcc) structure of metallic silver (JCPDS No. 04-0873). Among them, the Ag(111) diffraction peak has a larger intensity, indicating that AgNPs were successfully synthesized in this embodiment.

[0096] Example 23: The supernatant of the microbial composite nano-bactericide prepared in Example 19 and the fermentation broth of Bacillus subtilis W-2 was dried in an oven at 100°C, and the powder was used to prepare samples. The sample wavelengths in the range of 4000–4000 cm⁻¹ were recorded using a Thermo Scientific Nicolet 6700 FTIR spectrometer. −1 The spectral range (4000 cm⁻¹) is used to determine the specific components of the sample. The potassium bromide pellet method is employed. 1-2 mg of powdered sample and 200 mg of pure KBr are collected, ground uniformly, placed in a mold, and pressed into transparent sheets using a hydraulic press. The sample is then placed in an infrared spectrometer for testing. −1 up to 400 cm −1 The number of scans was 32, and the resolution was 4cm. -1 Based on the scanning results, the functional group composition involved in AgNP synthesis in the fermentation broth of Bacillus subtilis W-2 was analyzed. The infrared spectral results are as follows: Figure 11 As shown. 1116cm -1 This represents the CH bond stretching vibration of an aliphatic carbon chain; 1387 cm⁻¹ -1 This represents the CN stretching vibration, 1605cm. -1 This represents the amide bond (-CO-NH-) in the carbonyl (C=O) stretching vibration region, indicating the presence of a peptide component; 2900 cm −1 up to 3300 cm -1 The broadband amplitude is related to strong hydrogen bonding and the presence of NH and OH stretching vibrations. Figure 11 It can be seen that the spectrum of AgNPs in the microbial composite nano-bactericide ( Figure 11 The W-AgNP in the middle reached 1605 cm. -1 3371 cm -1The peak indicates that the aliphatic carbon chains and peptide components of the biosurfactants in the fermentation broth of Bacillus subtilis W-2 participated in the synthesis of AgNPs. Peptides can generate electrostatic attraction with negatively charged carboxyl groups and attract nanoparticles through free amine groups. Therefore, negatively charged lipopeptide biosurfactants can adsorb onto the surface of AgNPs, altering the surface properties of the particles and acting as end-capping agents and stabilizers. Furthermore, the imide, amine, and micellar functional groups in lipopeptide biosurfactants can also further stabilize the synthesized AgNPs.

[0097] Example 24: The microbial composite nano-bactericide prepared in Example 19 was used to conduct an inhibition test on sulfate-reducing bacteria (SRB) in a sample from an oilfield in western China.

[0098] ① Enrichment culture of sulfate-reducing bacteria in oilfield samples SRB medium: potassium dihydrogen phosphate 0.5 g / L, ammonium chloride 1.0 g / L, sodium sulfate 1.0 g / L, calcium chloride 0.05 g / L, magnesium chloride hexahydrate 2.0 g / L, yeast extract 1.0 g / L, ascorbic acid 0.1 g / L, sodium thioglycolate 0.1 g / L, ferrous sulfate heptahydrate 0.5 g / L, sodium D-lactic acid 1.1 g / L, pH 7.0 to 7.2.

[0099] Prepare SRB medium and dispense it into anaerobic culture tubes containing iron nails. Sterilize in an autoclave at 121°C for 20 minutes. Inoculate the produced fluid from the oilfield into anaerobic bottles containing SRB medium at an inoculation rate of 5%. After purging the anaerobic culture tubes with nitrogen, place them in an anaerobic bag with an oxygen indicator and incubate at 33°C. Subculture every 7 days until the fourth generation.

[0100] After adding 5% water sample, a very obvious black precipitate appeared in the culture medium starting on the third day, and a pungent H2S odor was detected upon opening, indicating the presence of SRB in the produced fluid from the oilfield. On the fourth and fifth days, the amount of black precipitate in the culture medium increased significantly. After subculturing, the growth of SRB stabilized.

[0101] ② Determination of the minimum inhibitory concentration for sulfate-reducing bacteria The powder of the microbial composite nano-bactericide prepared in Example 19 was weighed out. Sterilized SRB liquid culture medium was used as the solution. The final concentrations of the silver nano-solution were adjusted using a two-fold dilution method to 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, and 0 μg / mL, respectively. The culture media containing different concentrations of antibacterial material were dispensed into sterilized vials containing iron nails. A 5% SRB enrichment sample was injected using a syringe needle. Nitrogen gas was purged to remove oxygen from the vials, and the vials were incubated at 33°C for one week. The experimental results are as follows: Figure 12 As shown in Table 3.

[0102] Figure 12 The results showed that when the concentration of the microbial composite nano-bactericide was below 62.5 μg / mL, a large amount of black precipitate appeared in the bottle and adhered to the surface of the iron nails, growing there. When the concentration of the nano-silver solution was above 62.5 μg / mL, no black substance was produced in the solution, and the deposits on the surface of the iron nails decreased or even disappeared, indicating that the growth of SRB was inhibited.

[0103] As shown in Table 3, the sample with a concentration of 7.8125 μg / mL had the highest total bacterial count, at 4.08 × 10⁻⁶. 7 The copy number was 4.06 × 10⁻⁵. Specifically, when AgNPs concentrations were 0, 7.8125, 15.625, and 31.25 μg / mL, the bacterial 16S gene copy number was 4.06 × 10⁻⁵. 7 4.08×10 7 3.98×10 7 1.87×10 7 The concentration of the microbial composite nano-bacterial agent was 31.25 μg / mL, indicating that it had little impact on the growth and metabolism of bacteria in the environment, and some bacteria could still grow and reproduce normally. When the concentration of the microbial composite nano-bacterial agent exceeded 31.25 μg / mL, the bacterial 16S gene copy number decreased to 10. 6 The magnitude indicates that the growth and metabolism of some microorganisms in the culture medium are inhibited by the microbial composite nano-bactericide, leading to a decrease in bacterial count.

[0104] Example 25: The microbial composite nano-bactericide prepared in Example 19 was used to monitor the microbial community before and after antibacterial treatment.

[0105] Microbial community diversity was analyzed in SRB samples cultured for one week with different concentrations of the microbial composite nano-bactericide (0 μg / mL, 7.8125 μg / mL, 15.625 μg / mL, 31.25 μg / mL, 62.5 μg / mL, 125 μg / mL, and 250 μg / mL) using 16S rRNA gene sequencing. DNA was extracted from the cells using a DNA extraction kit. Primers 338F (5'-ACTCCTACGGGAGGCAGCA-3', primer sequence shown in SEQ ID NO:1) and 806R (5'-GGACTACHVGGGTWTCTBacillus subtilis fermentation broth T-3', primer sequence shown in SEQ ID NO:2) were used to amplify the V3-V4 region (approximately 460 bp) of the bacterial 16S rRNA gene. Finally, paired-end sequencing of the community DNA fragments was performed using the Illumina platform. Experimental results are shown below. Figure 13As shown in Table 4.

[0106] Figure 13 The distribution of species at the phylum level is mainly represented by Proteobacteria, Firmicutes, Bacteroideres, Synergistetes, Actinobacteria, Chloroflexi, Acidobacteria, Cyanobacteria, Planctomycetes, and Elusimicrobia. SRB primarily covers four bacterial phyla: Proteobateria, Firmicutes, Nitrospirae, and Thermodesulfobacteria, and one Archaea. The SRB in sample 0 are mainly composed of Proteobacteria and Firmicutes. Compared to sample 0 without the addition of the microbial composite nano-bactericide, the proportions of Proteobacteria and Synergistetes decreased with increasing concentration of the microbial composite nano-bactericide in the culture medium, indicating that AgNPs have a good inhibitory effect on these two phyla. However, for Bacteroideres, the proportion increased compared to the blank control with increasing concentration of the microbial composite nano-bactericide in the culture medium (as shown in Table 4), indicating that certain substances in the culture medium can stimulate their growth, such as Ag... + .

[0107] Taxonomically, SRB belongs to the genus *Desulfovibrio* and is generally associated with microbial corrosion. When the concentration of the microbial composite nano-bactericide increased from 0 μg / mL to 250 μg / mL, the relative abundance of *Desulfovibrio* in the sample decreased from 43.65% to 0.03%, and *Soehngenia* decreased from 25.73% to 0.46%, as shown in Table 4. This indicates that the microbial composite nano-bactericide of the present invention can effectively inhibit the growth of SRB and acid-producing bacteria in the sample.

[0108] The above embodiments and test results demonstrate that the fermentation broth of Bacillus subtilis strain W-2 of the present invention can serve as a dispersant, capping agent, reducing agent, and stabilizer for nanoparticles, preventing nanoparticle aggregation and achieving uniform dispersion. The silver nanoparticles prepared using the fermentation broth of Bacillus subtilis strain W-2 of the present invention exhibit high stability and small particle size. Simultaneously, the metal nanoparticles prepared using the fermentation broth of Bacillus subtilis strain W-2 of the present invention demonstrate a synergistic effect in catalytic metal reduction and sterilization. The microbial-based nano-bactericide prepared by reacting the fermentation broth of Bacillus subtilis strain W-2 with metal ions, when applied in oilfield environments, can inhibit harmful bacteria such as sulfate-reducing bacteria while reducing environmental pollution and secondary damage to pipelines.

[0109] The present invention has the following beneficial technical effects: (1) The fermentation broth of Bacillus subtilis strain W-2 provided by this invention can be used to prepare bactericides to inhibit harmful bacteria such as sulfate-reducing bacteria in oil reservoirs. Bacillus subtilis strain W-2 provided by this invention has broad application prospects in the field of corrosion protection for oil and gas pipelines.

[0110] (2) The fermentation broth of Bacillus subtilis strain W-2 described in this invention can be used as a dispersant, capping agent, reducing agent, and stabilizer for nanoparticles, preventing nanoparticle aggregation and achieving uniform dispersion of nanoparticles for the preparation of nanoparticles. Nanoparticles prepared using the fermentation broth of Bacillus subtilis strain W-2 of this invention exhibit high stability, good dispersibility, and small particle size. Metal nanoparticles prepared using the fermentation broth of Bacillus subtilis strain W-2 of this invention can achieve a synergistic effect in the catalytic reduction of metals.

[0111] (3) The fermentation broth of Bacillus subtilis strain W-2 of the present invention is mixed with silver nitrate and sodium borohydride and cultured to prepare a microbial composite nano bactericide; the microbial composite nano bactericide can be applied to sterilize and inhibit bacteria in oilfield pipelines to prevent pipeline corrosion.

[0112] In summary, this invention provides a fermentation broth of Bacillus subtilis strain W-2, and a microbial composite nano-bactericide is prepared based on this fermentation broth. The AgNPs in this microbial composite nano-bactericide have small particle size, good dispersibility and stability, and can effectively inhibit harmful bacteria such as sulfate-reducing bacteria in oilfield environments.

[0113] The above technical features constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. A method for preparing Bacillus subtilis fermentation broth, characterized in that... Includes the following steps: Bacillus subtilis strains were cultured in a culture medium to obtain Bacillus subtilis fermentation broth; The culture medium is either a fermentation medium or a hydrocarbon degradation medium.

2. The method for preparing Bacillus subtilis fermentation broth according to claim 1, characterized in that... The fermentation medium consists of: liquid paraffin 1 g / L to 10 g / L, yeast extract 0.1 g / L to 1 g / L, NaNO3 5 g / L to 20 g / L, KH2PO4 0.1 g / L to 0.5 g / L, Na2HPO4 0.5 g / L to 2 g / L, and MgSO4·7H2O. 0.1 g / L to 0.5 g / L, trace element solution 5 mL / L to 20 mL / L; wherein, the trace element solution is prepared by nitrogen triacetic acid, diethylenetriaminepentaacetic acid, MgSO4·7H2O, MnSO4·2H2O, NaCl, FeSO4·7H2O, CoSO4·7H2O, CaCl2·2H2O, ZnSO4·7H2O, CuSO4·5H2O, KAl(SO4)2·12H2O, H3BO3, Na2MoO4·2H2O, NiCl2·6H2O, Na2SeO3·5H2O and water.

3. The method for preparing Bacillus subtilis fermentation broth according to claim 2, characterized in that... The trace element solution consists of: nitrogen triacetic acid 1.000 g / L, diethylenetriaminepentaacetic acid 0.600 g / L, MgSO4·7H2O 3.500 g / L, MnSO4·2H2O 0.580 g / L, NaCl 1.000 g / L, FeSO4·7H2O 0.180 g / L, CoSO4·7H2O 0.180 g / L, CaCl2·2H2O 0.130 g / L, ZnSO4·7H2O 0.180 g / L, CuSO4·5H2O 0.011 g / L, KAl(SO4)2·12H2O 0.023 g / L, H3BO3 0.012 g / L, Na2MoO4·2H2O 0.010 g / L, NiCl2·6H2O 0.026 g / L, Na2SeO3·5H2O 0.330 g / L, the remainder is water.

4. The method for preparing Bacillus subtilis fermentation broth according to any one of claims 1 to 3, characterized in that... The culture temperature was 35℃ to 37℃, and the pH tolerance range of the Bacillus subtilis strain was 7.0 to 7.

5.

5. A Bacillus subtilis fermentation broth prepared by the method according to any one of claims 1 to 4.

6. The application of the Bacillus subtilis fermentation broth according to claim 5 in the preparation of a bactericide.

7. The application of the Bacillus subtilis fermentation broth according to claim 5 in the preparation of nanoparticle materials.

8. A method for preparing a microbial composite nano-bactericide using the Bacillus subtilis fermentation broth as described in claim 5 as a raw material, characterized in that... Includes the following steps: Step 1: Mix the required amounts of silver nitrate solution and sodium borohydride solution to obtain the metal precursor solution; Step 2: Mix the metal precursor solution with the Bacillus subtilis fermentation broth and then culture to obtain a microbial composite nano-bactericide.

9. The preparation method of the microbial composite nano-bactericide according to claim 8, characterized in that... In step one, the molar concentration of silver nitrate solution is 0.01 mol / L to 0.05 mol / L, the molar concentration of sodium borohydride solution is 0.0011 mol / L to 0.021 mol / L, and the volume ratio of silver nitrate solution to sodium borohydride solution is 100:

1. Or / and, in step two, the culture temperature is 40℃ to 80℃, and the culture time is 1h to 3h; Or / and, in step two, the volume ratio of the metal precursor solution to the Bacillus subtilis fermentation broth is 1:

1.

10. A microbial composite nano-bactericide prepared by the preparation method of the microbial composite nano-bactericide according to claim 8 or 9.

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