Bacillus velezensis, microbial inoculant and application thereof

Abstract

The present application relates to the fields of soil and groundwater pollution remediation technology and bioengineering technology, and discloses a bacillus velezensis, a bacterial agent and application thereof.The preservation number of the bacillus velezensis is CGMCC No.23199.The bacillus velezensis or the bacterial agent has a high degradation rate on crude oil, diesel oil, kerosene and the like with complex components, and can be used for remediation of petroleum hydrocarbon contaminated soil or groundwater, and has a good application prospect in green and sustainable remediation of petroleum hydrocarbon contaminated sites.

Description

Technical Field

[0001] This invention belongs to the fields of soil and groundwater pollution remediation technology and bioengineering technology, specifically relating to a method and application of Bacillus belyssus, bacterial agent, and degradation of petroleum hydrocarbons. Background Technology

[0002] With economic development, the demand for petroleum has increased significantly. Petroleum and its products continuously enter the environment through various pathways, polluting soil and water bodies. Petroleum hydrocarbons have become one of the most widespread organic pollutants in the environment. Therefore, remediation of petroleum hydrocarbon pollution is necessary, especially for soil and water bodies contaminated with petroleum hydrocarbons. Currently, commonly used methods for petroleum hydrocarbon pollution remediation mainly include physical, chemical, and biological remediation methods. Physical remediation methods include supercritical fluid extraction, evaporation extraction, and thermal desorption technologies. Chemical remediation methods include chemical oxidation and biochemical leaching. Biological remediation methods include protozoan remediation, microbial remediation, phytoremediation, and combined microbial-phytoremediation. Among these, compared to physical and chemical remediation, biological remediation has advantages such as low cost, simple operation, and minimal secondary pollution, making it a green and sustainable environmental governance technology. In recent years, numerous institutions both domestically and internationally have conducted extensive research on the screening, cultivation, and application of highly efficient petroleum hydrocarbon-degrading bacteria.

[0003] CN104004687B discloses a petroleum hydrocarbon degrading bacterium DLFJ1-1, its degradation genes, and its applications; the strain is Thioclava sp., and genome sequencing revealed many genes related to the degradation of alkanes or aromatic hydrocarbons. It can degrade 59.2% of a 5 g / L mixture of petroleum and diesel (V:V = 1:1) in an inorganic salt culture medium in 7 days.

[0004] Currently, the low degradation rate of petroleum hydrocarbons by existing strains in petroleum hydrocarbon-contaminated soil or groundwater is a bottleneck in the application of bioremediation technologies for petroleum hydrocarbon pollution. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low degradation rate of petroleum hydrocarbons and low remediation efficiency of existing petroleum hydrocarbon-contaminated soil by existing petroleum hydrocarbon-degrading bacteria, and to provide a method and application of Bacillus vesiculus, a bacterial agent, for degrading petroleum hydrocarbons. Using the Bacillus vesiculus or bacterial agent of this invention to degrade petroleum hydrocarbons achieves high degradation efficiency.

[0006] To achieve the above objectives, the first aspect of the present invention provides a Bacillus velezensis, which has the accession number CGMCC No. 23199.

[0007] A second aspect of the present invention provides a microbial agent containing Bacillus belye as described above.

[0008] A third aspect of the present invention provides the use of Bacillus vesiculosus as described above or the bacterial agent as described above in the degradation of petroleum hydrocarbons.

[0009] A fourth aspect of the present invention provides a method for degrading petroleum hydrocarbons, characterized in that the method comprises: contacting Bacillus berleissus or a bacterial agent as described above with pollutants containing petroleum hydrocarbons.

[0010] The fifth aspect of the present invention provides the application of Bacillus vesiculosus as described above or the bacterial agent as described above in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater.

[0011] The *Bacillus berberis* or its agent described in this invention exhibits a high petroleum hydrocarbon degradation rate. In an inorganic salt liquid culture medium with a petroleum hydrocarbon concentration of 10 g / L, at a temperature of 30.0 ± 2.0℃ and a pH of 7.0 ± 0.2, the *Bacillus berberis* provided by this invention achieves a petroleum hydrocarbon degradation rate of over 85% after 7 days. In contaminated soil with a petroleum hydrocarbon content of 10 g / kg, at a temperature of 30.0 ± 2.0℃ and a pH of 7.0 ± 0.2, the petroleum hydrocarbon degradation rate reaches over 80% after 30 days. The *Bacillus berberis* or its agent described in this invention also exhibits a high degradation rate for crude oil, diesel oil, kerosene, and other substances with complex compositions. The *Bacillus berberis* or its agent described in this invention can be used for the remediation of petroleum hydrocarbon-contaminated soil or groundwater, and has promising application prospects for the green and sustainable remediation of petroleum hydrocarbon-contaminated sites.

[0012] Biological Preservation

[0013] Bacillus velezensis 12-5B of the present invention was deposited on August 26, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode 100101) (abbreviation of depositary institution: CGMCC), with accession number CGMCCNo. 23199. Attached Figure Description

[0014] Figure 1 The colony morphology of Bacillus belyssus 12-5B on agar solid medium plates. Detailed Implementation

[0015] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0016] The first aspect of the present invention provides a Bacillus belyssus, the Bacillus belyssus having the accession number CGMCC No. 23199.

[0017] The Bacillus berleis of this invention was screened from soil contaminated with diesel fuel in the Beijing area.

[0018] The screening method can be any conventional method used in the field for screening new strains. For example, the screening method can be as follows: collect contaminated soil, add it to sterilized deionized water to prepare an inoculum; inoculate the inoculum into an inorganic salt culture medium with diesel as the sole carbon source, and subculture 3-5 times; perform serial dilution of the above culture medium and streak culture continuously until a single colony is isolated; scale up the above single colony culture using LB medium, centrifuge to obtain bacterial cells, which are the strains to be identified. Resuspend the obtained bacterial cells in sterilized physiological saline to obtain a bacterial suspension, mix the bacterial suspension with 70% glycerol at a 1:1 volume ratio, and store at -80°C.

[0019] The bacterial strain to be identified exhibited regular colony morphology on agar solid medium plates, appearing white, covered with long flagella, and with a dry, rough, and opaque colony surface. Figure 1 As shown.

[0020] The species of this strain was identified by 16S rDNA sequencing, and it was determined to be Bacillus belyssus, named 12-5B.

[0021] Bacillus belyes 12-5B was deposited at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China; accession number: CGMCC No. 23199; deposit date: August 26, 2021).

[0022] In this invention, the LB culture medium can be selected using conventional techniques in the art, and will not be described in detail here.

[0023] A second aspect of the present invention provides a microbial agent containing Bacillus belye as described above.

[0024] In some embodiments of the present invention, the microbial agent is a liquid microbial agent and / or a solid microbial agent.

[0025] In this invention, the concentration of *Bacillus belyceae* in the bacterial agent is not particularly limited and can be selected according to specific circumstances. In some embodiments of this invention, the viable count in the liquid bacterial agent is 10-1. 9 CFU / mL or higher. In some embodiments of the present invention, the viable count in the solid bacterial agent is 10-1. 9 CFU / g or higher.

[0026] In some embodiments of the present invention, the bacterial agent may also contain excipients. The excipients may be commonly used excipients in the art that will not affect the activity of *Bacillus belyssum*. Specific selections are well known to those skilled in the art, and will not be described in detail here.

[0027] Furthermore, the microbial agent provided by this invention can be prepared into different dosage forms depending on the intended use. Specific selections are well known to those skilled in the art, and will not be described in detail here.

[0028] The preparation method of the bacterial agent can be a conventional preparation method in the art. Preferably, the preparation method includes: activating the *Bacillus belye* as described above and culturing it in LB medium to obtain a bacterial stock solution; centrifuging the bacterial stock solution to obtain bacterial cells, and preparing a bacterial suspension with sterile water to achieve a viable count of 102. 9 A bacterial agent is obtained when the concentration of cfu / mL is above a certain level.

[0029] In this invention, the culture conditions can be the conventional conditions known in the art for the culture of Bacillus belesii, which will not be described in detail here.

[0030] A third aspect of the present invention provides the use of Bacillus vesiculosus as described above or the bacterial agent as described above in the degradation of petroleum hydrocarbons.

[0031] In this invention, the petroleum hydrocarbon refers to at least one of the hydrocarbon compounds in petroleum, or a mixture of multiple hydrocarbons (n-alkanes, branched alkanes, cycloalkanes, aromatics) and small amounts of other organic compounds, such as sulfides, nitrides, and cycloalkanoates. In this invention, the petroleum hydrocarbon can be a complex mixture of hydrocarbon compounds in petroleum, such as crude oil, diesel oil, and kerosene, or it can be a hydrocarbon compound such as n-dodecane, n-hexadecane, n-eicosane, and n-octacosane. In some embodiments of this invention, the petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel oil, crude oil, and kerosene.

[0032] In some embodiments of the present invention, the liquid alkane may include at least one of the following common alkanes in the art that are liquid at room temperature (25°C) with 10-16 carbon atoms: straight-chain or branched alkanes, such as n-dodecane or n-hexadecane. The solid alkane may include at least one of the following common alkanes in the art that are solid at room temperature (25°C) with 17-40 carbon atoms: straight-chain or branched alkanes, such as n-eicosane or n-octacosane.

[0033] A fourth aspect of the present invention provides a method for degrading petroleum hydrocarbons, characterized in that the method comprises: contacting Bacillus berleissus or a bacterial agent as described above with pollutants containing petroleum hydrocarbons.

[0034] In some embodiments of the present invention, the petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel oil, crude oil, and kerosene.

[0035] In some embodiments of the present invention, the liquid alkane may include at least one of the following common alkanes in the art that are liquid at room temperature (25°C) with 10-16 carbon atoms: straight-chain or branched alkanes, such as n-dodecane or n-hexadecane. The solid alkane may include at least one of the following common alkanes in the art that are solid at room temperature (25°C) with 17-40 carbon atoms: straight-chain or branched alkanes, such as n-eicosane or n-octacosane.

[0036] In some embodiments of the present invention, the pollutant containing petroleum hydrocarbons is petroleum hydrocarbon-containing soil or petroleum hydrocarbon-containing groundwater.

[0037] In some embodiments of the present invention, when the pollutant containing petroleum hydrocarbons is soil containing petroleum hydrocarbons, petroleum hydrocarbons (C) are used. 10 -C 40 The petroleum hydrocarbon content is calculated to be 2-50 g / kg soil, for example, it can be any one of the following values, or a value within the range of any two of the above: 2 g / kg soil, 5 g / kg soil, 7 g / kg soil, 10 g / kg soil, 12 g / kg soil, 15 g / kg soil, 17 g / kg soil, 20 g / kg soil, 22 g / kg soil, 25 g / kg soil, 27 g / kg soil, 30 g / kg soil, 32 g / kg soil, 35 g / kg soil, 37 g / kg soil, 40 g / kg soil, 42 g / kg soil, 45 g / kg soil, 47 g / kg soil, and 50 g / kg soil. The soil weight is on a dry basis.

[0038] In this invention, the amount of the bacterial strain or the bacterial agent used can be selected within a wide range. In some embodiments of this invention, when the petroleum hydrocarbon-containing pollutant is petroleum hydrocarbon-containing soil, the amount of the bacterial strain or the bacterial agent used is such that the viable bacteria count in the petroleum hydrocarbon-containing soil is 10. 6 -10 8 cfu / g. The soil weight is on a dry basis.

[0039] In some embodiments of the present invention, the contact conditions include a temperature of 20-40°C and a pH value of 5-10. The temperature can be any one of 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, or 40°C, or a value within the range of any two of these values. The pH can be any one of 5, 5.2, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.5, 9.8, or 10, or a value within the range of any two of these values. Preferably, to further improve the petroleum hydrocarbon degradation rate, the contact conditions are: a temperature of 28-32°C and a pH of 6.8-7.2. The inventors have found that when the contact conditions are within the above-preferred range, the petroleum hydrocarbon degradation rate can be further improved.

[0040] In some embodiments of the present invention, when the pollutant containing petroleum hydrocarbons is petroleum hydrocarbon-containing groundwater, petroleum hydrocarbons (C...) are used... 10 -C 40 The concentration of the petroleum hydrocarbon is calculated to be 2-50 g / L of groundwater, for example, it can be any one of the following values ​​or a value within the range of any two of the above values: 2 g / L, 5 g / L, 7 g / L, 10 g / L, 12 g / L, 15 g / L, 17 g / L, 20 g / L, 22 g / L, 25 g / L, 27 g / L, 30 g / L, 32 g / L, 35 g / L, 37 g / L, 40 g / L, 42 g / L, 45 g / L, 47 g / L, and 50 g / L of groundwater.

[0041] In some embodiments of the present invention, when the petroleum hydrocarbon-containing pollutant is petroleum hydrocarbon-containing groundwater, the amount of the bacterial strain or the bacterial agent used is such that the viable bacteria count in the petroleum hydrocarbon-containing groundwater is 10. 6 -10 8 cfu / mL.

[0042] The fifth aspect of the present invention provides the application of Bacillus vesiculosus as described above or the bacterial agent as described above in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater.

[0043] In some embodiments of the present invention, the petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel oil, crude oil, and kerosene.

[0044] In some embodiments of the present invention, the liquid alkane may include at least one of the following common alkanes in the art that are liquid at room temperature (25°C) with 10-16 carbon atoms: straight-chain or branched alkanes, such as n-dodecane or n-hexadecane. The solid alkane may include at least one of the following common alkanes in the art that are solid at room temperature (25°C) with 17-40 carbon atoms: straight-chain or branched alkanes, such as n-eicosane or n-octacosane.

[0045] The present invention will be described in detail below through examples. Unless otherwise specified, all reagents and raw materials used in the following examples are commercially available, and all methods used are conventional methods in the art.

[0046] In the following embodiments:

[0047] LB medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, deionized water 1 L.

[0048] Inorganic salt culture medium: NH4Cl 0.67g / L, NaNO3 1.06g / L, MgSO4·7H2O 0.1g / L, CaCl2 0.1g / L, K2HPO4·3H2O 1.5g / L, KH2PO4 0.5g / L, FeCl3·6H2O 0.1g / L, deionized water 1L.

[0049] The crude oil was taken from the Beijing Yanshan Branch of China Petroleum & Chemical Corporation.

[0050] The diesel fuel was purchased from Beijing Petroleum Branch of China Petroleum & Chemical Corporation (Sinopec Sales Co., Ltd.), and its grade was 0#.

[0051] The kerosene was purchased from Beijing Innocare Technology Co., Ltd.

[0052] n-Dodecane was purchased from Beijing Innocare Technology Co., Ltd.

[0053] hexadecane was purchased from Beijing Innocare Technology Co., Ltd.

[0054] The n-eicosane was purchased from Beijing Innocare Technology Co., Ltd.

[0055] The n-octadecane was purchased from Beijing Innocare Technology Co., Ltd.

[0056] Bacillus belye 12-5B was deposited on August 26, 2021, at the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) (CGMCC accession number CGMCC No. 23199).

[0057] The detection method for petroleum hydrocarbon content in contaminated soil shall refer to the "Petroleum Hydrocarbons (C6H2O) Detection Method for Soils and Sediments". 10 -C 40 The determination of petroleum hydrocarbons by gas chromatography (HJ1021-2019) was performed. Specifically, hexane was used for pressurized fluid extraction of petroleum hydrocarbons. When the final volume was 1.0 mL and the injection volume was 1.0 μL, the method detection limit for petroleum hydrocarbons was 6 mg / kg, and the lower limit of quantification was 24 mg / kg. The petroleum hydrocarbons were determined using (C... 10 -C 40 )count.

[0058] The method for detecting the concentration of petroleum hydrocarbons in inorganic salt culture media should refer to the "Water Quality - Extractable Petroleum Hydrocarbons (C6H2O)" standard. 10 -C 40 The determination of ) was carried out by gas chromatography (HJ894-2017).

[0059] The degradation rate (%) of petroleum hydrocarbons in contaminated soil = (initial petroleum hydrocarbon content - remaining petroleum hydrocarbon content) / initial petroleum hydrocarbon content. Wherein, the initial petroleum hydrocarbon content refers to the petroleum hydrocarbon content in the contaminated soil before inoculation with the bacterial suspension.

[0060] The degradation rate (%) of petroleum hydrocarbons in inorganic salt culture medium = (initial petroleum hydrocarbon concentration - remaining petroleum hydrocarbon concentration) / initial petroleum hydrocarbon concentration. Wherein, the initial petroleum hydrocarbon concentration refers to the petroleum hydrocarbon concentration in the inorganic salt culture medium without inoculated bacterial suspension.

[0061] Example 1

[0062] This example illustrates the degradation effect of Bacillus vesiculosus 12-5B on crude oil.

[0063] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: Crude oil was added to an inorganic salt culture medium to achieve a crude oil concentration (in terms of petroleum hydrocarbons (C)). 10 -C 40 The concentration of crude oil (calculated as petroleum hydrocarbons (C)) was 10 g / L, yielding the crude oil concentration (calculated as petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0064] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0065] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into the above-mentioned crude oil concentration (in petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and with temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual crude oil (as petroleum hydrocarbons (C50)) was measured periodically. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for crude oil in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were a temperature of 30.0±2.0℃ and a pH of 7.0±0.2. The degradation effect of crude oil by Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0066] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then naturally air-dried. Crude oil was added to the uncontaminated soil to achieve a crude oil content (based on petroleum hydrocarbon concentration). 10 -C 40 The soil weight was calculated to be 10 g / kg (wherein, the weight of the soil is on a dry basis), and the crude oil content (in terms of petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0067] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned crude oil content (in terms of petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with Bacillus beleales 12-5B). The residual crude oil content (as petroleum hydrocarbons) was periodically measured. 10 -C 40The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for crude oil in contaminated soil by Bacillus bellis 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus bellis 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The crude oil degradation effect of Bacillus bellis 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 30 days is shown in Table 1.

[0068] Example 2

[0069] This example illustrates the degradation effect of Bacillus belye 12-5B on diesel fuel.

[0070] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: Diesel fuel was added to an inorganic salt culture medium to achieve a diesel fuel concentration (in terms of petroleum hydrocarbon concentrations (C)). 10 -C 40 The concentration of diesel fuel (calculated as petroleum hydrocarbons (C)) was 10 g / L, yielding a diesel fuel concentration (calculated as petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0071] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0072] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated into the above-mentioned diesel fuel concentration (in the form of petroleum hydrocarbons (C)). 10 -C 40 An inorganic salt culture medium with a concentration of 10 g / L was used, with temperature and pH as influencing factors. Experimental and control groups were set up (the control group was not inoculated with *Bacillus belyssiensis* 12-5B). The concentration of residual diesel fuel (as petroleum hydrocarbons) was periodically monitored. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for diesel fuel in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The diesel fuel degradation effect of Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0073] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. Diesel fuel was added to the uncontaminated soil to achieve a diesel fuel content (based on petroleum hydrocarbon concentrations (C)). 10 -C 40 The soil weight was calculated to be 10 g / kg (on a dry basis), and the diesel content (in terms of petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0074] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned diesel fuel content (in petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with Bacillus belye 12-5B). The residual diesel content (as petroleum hydrocarbons) was periodically measured. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for diesel fuel in contaminated soil by Bacillus belyssus 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyssus 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The diesel fuel degradation effect of Bacillus belyssus 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 30 days is shown in Table 1.

[0075] Example 3

[0076] This example illustrates the degradation effect of Bacillus belyssus 12-5B on kerosene.

[0077] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: Kerosene was added to an inorganic salt culture medium to achieve a kerosene concentration (as expressed by petroleum hydrocarbons (C2)). 10 -C 40 The concentration of kerosene was calculated to be 10 g / L, yielding a kerosene concentration (based on petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0078] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0079] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9CFU was inoculated with the above-mentioned kerosene concentration (in the form of petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and with temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual kerosene was periodically measured (as petroleum hydrocarbons (C)). 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for kerosene in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The kerosene degradation effect of Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0080] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then naturally air-dried. Kerosene was added to the uncontaminated soil to achieve a kerosene content (based on petroleum hydrocarbon concentration). 10 -C 40 The kerosene content (calculated as petroleum hydrocarbons (C)) was 10 g / kg (where the weight of soil is on a dry basis), and the kerosene content (calculated as petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0081] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned kerosene content (in terms of petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with Bacillus beleales 12-5B). The residual kerosene content (as petroleum hydrocarbons) was periodically measured. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for kerosene in contaminated soil by Bacillus bellis 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus bellis 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The kerosene degradation effect of Bacillus bellis 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 30 days is shown in Table 1.

[0082] Example 4

[0083] This example illustrates the degradation effect of Bacillus belye 12-5B on n-dodecane.

[0084] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: n-Dodecane was added to an inorganic salt culture medium to achieve a n-dodecane concentration (as expressed by petroleum hydrocarbons (C1)). 10 -C 40 The concentration of n-dodecane was calculated to be 10 g / L, yielding a concentration of n-dodecane (based on petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0085] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0086] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated with the above-mentioned n-dodecane concentration (as petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and considering temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual n-dodecane was periodically measured (as petroleum hydrocarbons (C)). 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-dodecane in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The n-dodecane degradation effect of Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0087] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. n-Dodecane was added to the uncontaminated soil to achieve a n-dodecane content (based on petroleum hydrocarbon concentrations (C1)). 10 -C 40 The soil weight was calculated to be 10 g / kg (on a dry basis), and the n-dodecane content (based on petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0088] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned n-dodecane content (in petroleum hydrocarbons (C)). 10 -C 40In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with *Bacillus belyssae* 12-5B). The residual n-dodecane content was periodically measured (as petroleum hydrocarbons (C...). 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-dodecane in contaminated soil by Bacillus bellis 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus bellis 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of n-dodecane by Bacillus bellis 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) for 30 days is shown in Table 1.

[0089] Example 5

[0090] This example illustrates the degradation effect of Bacillus belyssus 12-5B on n-hexadecane.

[0091] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: n-Hexadecane was added to an inorganic salt culture medium to achieve a n-hexadecane concentration (as expressed by petroleum hydrocarbons (C1)). 10 -C 40 The concentration of n-hexadecane was calculated to be 10 g / L, yielding a concentration of n-hexadecane (based on petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0092] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0093] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated with the above-mentioned concentration of n-hexadecane (as petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and considering temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual n-hexadecane (as petroleum hydrocarbons (C...)) was periodically measured. 10 -C 40The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-hexadecane in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The n-hexadecane degradation effect of Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0094] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. Hexadecane was added to the uncontaminated soil to achieve a hexadecane content (based on petroleum hydrocarbon concentrations). 10 -C 40 The soil weight was calculated to be 10 g / kg (on a dry basis), and the hexadecane content (based on petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0095] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned hexadecane content (in petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with *Bacillus belyssae* 12-5B). The residual n-hexadecane content was periodically measured (as petroleum hydrocarbons (C...). 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-hexadecane in contaminated soil by Bacillus belyss 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of n-hexadecane by Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 30 days is shown in Table 1.

[0096] Example 6

[0097] This example illustrates the degradation effect of Bacillus belye 12-5B on n-eicosane.

[0098] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: n-eicosane was added to an inorganic salt culture medium to achieve a n-eicosane concentration (as expressed by petroleum hydrocarbons (C1)). 10 -C 40 The concentration of n-eicosane was calculated to be 10 g / L, yielding a concentration of n-eicosane (based on petroleum hydrocarbons (C)). 10 -C40 The inorganic salt culture medium is 10 g / L.

[0099] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0100] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated with the above-mentioned concentration of n-eicosane (in petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and considering temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual n-eicosane (as petroleum hydrocarbons (C)) was periodically measured. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-eicosane in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The n-eicosane degradation effect of Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0101] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. Eicosane was added to the uncontaminated soil to achieve an eicosane content (based on petroleum hydrocarbon concentrations). 10 -C 40 The soil weight was calculated to be 10 g / kg (on a dry basis), and the n-eicosane content (based on petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0102] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated into the above-mentioned n-eicosane content (in petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with *Bacillus beleales* 12-5B). The residual n-eicosane content was periodically measured (as petroleum hydrocarbons (C...)). 10 -C 40The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-dodecane in contaminated soil by Bacillus bereaves 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus bereaves 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of n-eicosane by Bacillus bereaves 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) for 30 days is shown in Table 1.

[0103] Example 7

[0104] This example illustrates the degradation effect of Bacillus belye 12-5B on n-octacosan.

[0105] Preparation of simulated petroleum hydrocarbon-contaminated groundwater: Octacosandecane was added to an inorganic salt culture medium to achieve an octacosandecane concentration (as expressed by petroleum hydrocarbons (C1)). 10 -C 40 The concentration of octadecane added was 10 g / L, yielding a concentration of n-octadecane (based on petroleum hydrocarbons (C)). 10 -C 40 The inorganic salt culture medium is 10 g / L.

[0106] After activating the preserved Bacillus belye 12-5B cells, a stock solution of bacteria was obtained by culturing in LB medium. The bacterial cells obtained after centrifugation were then used to prepare a bacterial suspension with sterile water, with a viable cell content of approximately 10%. 9 cfu / mL.

[0107] Inoculate the bacterial suspension at a rate of 1 vol.% (10 9 CFU was inoculated with the above-mentioned concentration of n-octadecane (as petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, and considering temperature and pH as influencing factors, experimental and control groups were set up (the control group was not inoculated with Bacillus belyssae 12-5B). The concentration of residual n-octacosan (as petroleum hydrocarbons (C...)) was periodically measured. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-octacosanol in inorganic salt culture medium by Bacillus belyss 12-5B were determined, along with the degradation effect after 7 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus belyss 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of n-octacosanol by Bacillus belyss 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) after 7 days is shown in Table 1.

[0108] Preparation of simulated petroleum hydrocarbon contaminated soil: Soil uncontaminated with petroleum hydrocarbons was taken, pretreated by sieving to remove small stones, grass roots, and other impurities, and then air-dried. Octacosandecane was added to the uncontaminated soil to achieve an octacosandecane content (based on petroleum hydrocarbon concentrations (C1)). 10 -C 40 The content of n-octacosanane (calculated as petroleum hydrocarbons (C)) was 10 g / kg (wherein, the weight of soil is on a dry basis), and the content of n-octacosanane (calculated as petroleum hydrocarbons (C)) was obtained. 10 -C 40 The amount of contaminated soil is calculated to be 10 g / kg.

[0109] Inoculate the bacterial suspension at a rate of 1 (v / w) (10 9 CFU was inoculated with the above-mentioned n-octadecane content (in petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, temperature and pH were used as influencing factors. An experimental group and a control group were established (the control group was not inoculated with Bacillus belyssae 12-5B). The residual n-octacosan content was periodically measured (as petroleum hydrocarbons (C...). 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the optimal degradation conditions for n-octacosanol in contaminated soil by Bacillus bereaves 12-5B were determined, along with the degradation effect after 30 days under these optimal conditions. The results showed that the optimal degradation conditions for Bacillus bereaves 12-5B were 30.0±2.0℃ and pH 7.0±0.2. The degradation effect of n-octacosanol by Bacillus bereaves 12-5B under the optimal conditions (temperature 30.0±2.0℃, pH 7.0±0.2) for 30 days is shown in Table 1.

[0110] Table 1. Petroleum hydrocarbon degradation effect of Bacillus belyssus 12-5B

[0111]

[0112] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A type of Bacillus belesii ( Bacillus velezensis ), characterized in that, The preservation number of this Bacillus belyss is CGMCC No. 23199.

2. A microbial agent, characterized in that, The bacterial agent contains Bacillus belye as described in claim 1.

3. The microbial agent according to claim 2, wherein, The microbial agent is a liquid microbial agent and / or a solid microbial agent.

4. The microbial agent according to claim 3, wherein, The liquid bacterial agent contains 10 live bacteria. 9 cfu / mL or higher; The number of viable bacteria in the solid bacterial agent is 10. 9 CFU / g or higher.

5. The application of Bacillus belyssus of claim 1 or the bacterial agent of any one of claims 2-4 in the degradation of petroleum hydrocarbons; wherein the petroleum hydrocarbons are at least one of liquid alkanes, solid alkanes, diesel, crude oil and kerosene; wherein the liquid alkanes are at least one of straight-chain or branched alkanes with 10-16 carbon atoms that are liquid at 25°C; wherein the solid alkanes are at least one of straight-chain or branched alkanes with 17-40 carbon atoms that are solid at 25°C.

6. A method for degrading petroleum hydrocarbons, characterized in that, The method comprises: contacting the Bacillus belyei of claim 1 or the bacterial agent of any one of claims 2-4 with a pollutant containing petroleum hydrocarbons; wherein the petroleum hydrocarbons are at least one of liquid alkanes, solid alkanes, diesel, crude oil, and kerosene; wherein the liquid alkanes are at least one of straight-chain or branched alkanes having a carbon number of 10-16 that are liquid at 25°C; and wherein the solid alkanes are at least one of straight-chain or branched alkanes having a carbon number of 17-40 that are solid at 25°C.

7. The method according to claim 6, wherein, The pollutants containing petroleum hydrocarbons are petroleum hydrocarbon-containing soil or petroleum hydrocarbon-containing groundwater.

8. The method according to claim 6, wherein, The contact conditions include a temperature of 20-40°C and a pH value of 5-10.

9. The application of Bacillus belyssus of claim 1 or the bacterial agent of any one of claims 2-4 in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater; wherein the petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel, crude oil, and kerosene; wherein the liquid alkanes are at least one of straight-chain or branched alkanes with 10-16 carbon atoms that are liquid at 25°C; and wherein the solid alkanes are at least one of straight-chain or branched alkanes with 17-40 carbon atoms that are solid at 25°C.

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