Composite microbial agent and application thereof

Abstract

This invention relates to the fields of soil and groundwater pollution remediation technology and bioengineering technology, and discloses a compound microbial agent and its application. The compound microbial agent provided by this invention exhibits high degradation efficiency for petroleum hydrocarbons. In an inorganic salt liquid culture medium with a petroleum hydrocarbon concentration of 10 g / L, under conditions of 20-40℃ and pH 6-9, the petroleum hydrocarbon degradation rate reaches over 85% in 7 days; in contaminated soil with a petroleum hydrocarbon content of 10 g / kg, under conditions of 20-40℃ and pH 6-9, the petroleum hydrocarbon degradation rate reaches over 80% in 30 days. The compound microbial agent provided by this invention can be used for the remediation of petroleum hydrocarbon-contaminated soil or groundwater, and has promising application prospects in the 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 compound microbial agent, a method for degrading petroleum hydrocarbons, and its application. Background Technology

[0002] With economic development, the demand for petroleum has increased significantly, and petroleum hydrocarbons have become one of the most widespread organic pollutants in the environment. Petroleum hydrocarbons (typically including one or more types of gasoline, kerosene, diesel, lubricating oil, paraffin wax, asphalt, etc.) can enter the human body through the food chain, seriously threatening human health. Therefore, it is necessary to degrade petroleum hydrocarbons in soil and water bodies and remediate petroleum hydrocarbon-contaminated soil and water bodies. Currently, commonly used methods for petroleum hydrocarbon pollution remediation mainly include physical, chemical, and biological remediation methods. Among these, compared with 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 at home and abroad have conducted extensive research on the screening, cultivation, and application of bacteria that efficiently degrade petroleum hydrocarbons.

[0003] CN110551662B discloses a strain of Arthrobacter ZJ-H and its application in degrading petroleum hydrocarbons under high salinity and alkalinity conditions; strain ZJ-H can survive normally under conditions of salinity of 1%-5% and pH of 7-11, and its degradation rate of crude oil in saline-alkaline environments reaches 74.45%; its degradation rate of n-alkanes in crude oil exceeds 83%.

[0004] In existing technologies, the low degradation rate of petroleum hydrocarbons by bacterial strains used for petroleum hydrocarbon degradation has become a bottleneck in the application of microbial remediation technology. Therefore, the preparation of compound bacterial agents with highly efficient petroleum hydrocarbon degradation function is of great significance for promoting the application of microbial remediation technology. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems existing in the prior art and provide a composite microbial agent with petroleum hydrocarbon degradation function, a method for degrading petroleum hydrocarbons, and its application. Using the microbial 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 composite microbial agent with petroleum hydrocarbon degradation function, the composite microbial agent comprising Bacillus velezensis and Microdochium sp.

[0007] The second aspect of this invention provides the application of the composite microbial agent with petroleum hydrocarbon degradation function as described above in the remediation of petroleum hydrocarbon contaminated soil or petroleum hydrocarbon contaminated groundwater.

[0008] A third aspect of the present invention provides a method for degrading petroleum hydrocarbons, characterized in that the method includes: contacting the composite microbial agent as described above with pollutants containing petroleum hydrocarbons.

[0009] The fourth aspect of this invention provides the application of the compound microbial agent as described above in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater.

[0010] The compound microbial agent with petroleum hydrocarbon degradation function provided by this invention achieves a petroleum hydrocarbon degradation rate of over 85% in 7 days under conditions of 10 g / L petroleum hydrocarbon concentration in an inorganic salt liquid culture medium at 20-40℃ and pH 6-9; and a petroleum hydrocarbon degradation rate of over 80% in 30 days in contaminated soil with a petroleum hydrocarbon content of 10 g / kg under the same conditions. This compound microbial agent with petroleum hydrocarbon degradation function 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.

[0011] Biological Preservation

[0012] 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.

[0013] Microdochium sp. 11-1B 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 CGMCC No. 23200. Detailed Implementation

[0014] 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.

[0015] The first aspect of the present invention provides a compound microbial agent with petroleum hydrocarbon degradation function, the compound microbial agent comprising Bacillus velezensis and Microdochium sp.

[0016] In some embodiments of the present invention, the compound microbial agent is a liquid compound microbial agent and / or a solid compound microbial agent.

[0017] In this invention, the concentrations of *Bacillus bellis* and *Micronodularia* in the compound microbial agent are not particularly limited and can be selected according to specific circumstances. In some embodiments of this invention, the viable count in the liquid compound microbial 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.

[0018] 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 belyssae* and *Micronodularia*. Specific selections are well known to those skilled in the art, and will not be described in detail here.

[0019] In some embodiments of the present invention, the Bacillus belyssus has the accession number CGMCC No. 23199.

[0020] In some embodiments of the present invention, the micronodular mold is preserved with the accession number CGMCC No. 23200.

[0021] The *Bacillus belyssae* and *Micronodularia* strains described in this invention were screened from diesel-contaminated soil in the Beijing area. The screening method can be a conventional method for screening new strains in the art. For example, the screening method may include: collecting contaminated soil, adding it to sterilized deionized water to prepare an inoculum; inoculating the inoculum into an inorganic salt culture medium with diesel as the sole carbon source, and subculturing 3-5 times; serially diluting the above culture medium and streaking it continuously until a single colony is isolated; scaling up the above single colony using LB medium and centrifuging to obtain the bacterial cells. The two strains to be identified obtained according to the above screening method were respectively identified as *Bacillus belyssae* (named 12-5B) and *Micronodularia* (named 11-1B).

[0022] Bacillus belyes 12-5B is deposited at the China General Microbiological Culture Collection Center (CGMCC), No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China, accession number CGMCC No. 23199, deposited on August 26, 2021.

[0023] Micronodularia 11-1B is deposited at the China General Microbiological Culture Collection Center (CGMCC) (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China) with accession number CGMCC No. 23200 and deposit date of August 26, 2021.

[0024] In this invention, the LB culture medium can be selected using conventional techniques in the art, and will not be described in detail here.

[0025] In this invention, the preparation method of the compound microbial agent can be a conventional preparation method in the art. The inventors have discovered that the *Micronodularia 11-1B* described in this invention can be cultured in LB medium. Therefore, preferably, considering production cost and ease of operation, the preparation method includes: culturing *Bacillus belye* and *Micronodularia* as described above separately in LB medium to obtain a stock solution of the microbial solution; centrifuging the stock solution of the microbial solution separately to prepare bacterial suspensions with sterile water, ensuring that the viable cell count in the bacterial suspension reaches 10-1. 9 The concentration of cfu / mL or higher is then used to mix the two bacterial suspensions. The amounts of Bacillus bellis suspension and Micronodular mold suspension are such that the ratio of viable Bacillus bellis to Micronodular mold in the prepared compound bacterial agent is 10:1-1:10, preferably 3:1-1:3.

[0026] In this invention, the culture conditions can be the commonly used culture conditions for Bacillus belyssus and Micronodularia in the art, which will not be elaborated here.

[0027] In some embodiments of the present invention, the ratio of viable Bacillus bellis to Micronodella fuciformis in the composite microbial agent is 10:1 to 1:10. The inventors have found that when the ratio of viable Bacillus bellis to Micronodella fuciformis in the composite microbial agent is 3:1 to 1:3, the degradation rate of petroleum hydrocarbons by the composite microbial agent can be further improved. More preferably, the ratio of viable Bacillus bellis to Micronodella fuciformis in the composite microbial agent is 2:1 to 1:3.

[0028] The second aspect of this invention provides the application of the composite microbial agent with petroleum hydrocarbon degradation function as described above in the remediation of petroleum hydrocarbon contaminated soil or petroleum hydrocarbon contaminated groundwater.

[0029] In this invention, the term "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, kerosene, etc., or it can be hydrocarbon compounds such as n-dodecane, n-hexadecane, n-eicosane, n-octacosane, etc., and mixtures of any of them. In some embodiments of this invention, the petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel oil, crude oil, and kerosene.

[0030] 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.

[0031] A third aspect of the present invention provides a method for degrading petroleum hydrocarbons, the method comprising: contacting the aforementioned compound microbial agent with pollutants containing petroleum hydrocarbons.

[0032] In some embodiments of the present invention, the contact conditions include: a temperature of 20-40°C and a pH of 6-9. The temperature can be any one of 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, 37°C, and 40°C, or a value within the range of any two of the above values. The pH can be any one of 6, 6.2, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, and 9, or a value within the range of any two of the above values.

[0033] 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.

[0034] 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.

[0035] In some embodiments of the present invention, the pollutant containing petroleum hydrocarbons is petroleum hydrocarbon-containing soil or petroleum hydrocarbon-containing groundwater.

[0036] 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.

[0037] In this invention, the dosage of the compound microbial agent 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 dosage of the compound microbial agent 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. The inventors have found that when the ratio of viable *Bacillus levesiculosus* to *Micronodularia nigra* in the compound microbial agent is 1:8-7:1, the degradation rate of petroleum hydrocarbons in the petroleum hydrocarbon-containing soil can be further improved. More preferably, the ratio of viable *Bacillus levesiculosus* to *Micronodularia nigra* in the compound microbial agent is 3:1-1:3, and more preferably 2:1-1:3.

[0038] 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 petroleum hydrocarbon content 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.

[0039] In some embodiments of the present invention, when the pollutant containing petroleum hydrocarbons is petroleum hydrocarbon-containing groundwater, the amount of the compound microbial agent used is such that the viable bacteria count in the petroleum hydrocarbon-containing groundwater is 10. 6 -10 8 cfu / mL. The inventors discovered that when the ratio of viable *Bacillus belyssioides* to *Micronodularia pyrenoidosa* in the compound microbial agent is 1:8-7:1, the degradation rate of petroleum hydrocarbons in the petroleum hydrocarbon-containing groundwater can be further improved. More preferably, the ratio of viable *Bacillus belyssioides* to *Micronodularia pyrenoidosa* in the compound microbial agent is 1:8-1:1, and even more preferably 1:2-1:1.

[0040] In some preferred embodiments of the present invention, the ratio of viable Bacillus vesiculosus and Micronodularia micrantha in the compound bacterial agent is 1:2 to 1:1, and the contact conditions include: temperature 25-35℃ and pH 6-8.5.

[0041] The fourth aspect of this invention provides the application of the aforementioned compound microbial agent in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In the following embodiments:

[0046] LB medium: yeast extract 5 g / L, peptone 10 g / L, NaCl 10 g / L, deionized water 1 L.

[0047] 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.

[0048] The crude oil was taken from the Beijing Yanshan Branch of China Petroleum & Chemical Corporation.

[0049] The diesel fuel was purchased from Beijing Petroleum Branch of China Petroleum & Chemical Corporation (Sinopec), and its grade is 0#.

[0050] The kerosene was purchased from Beijing Innocare Technology Co., Ltd.

[0051] n-Dodecane was purchased from Beijing Innocare Technology Co., Ltd.

[0052] hexadecane was purchased from Beijing Innocare Technology Co., Ltd.

[0053] The n-eicosane was purchased from Beijing Innocare Technology Co., Ltd.

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

[0055] 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), with accession number CGMCC No. 23199.

[0056] Microdochium sp. 11-1B 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. 23200).

[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 was carried out according to the "Gas Chromatography Method" (HJ1021-2019), which uses n-hexane for pressurized fluid extraction. The detection standards are as follows: when the contaminated soil sample size is 10.0 g, the final volume is 1.0 mL, and the injection volume is 1.0 μL, the method detection limit for petroleum hydrocarbons is 6 mg / kg, and the lower limit of quantification is 24 mg / kg; when the inorganic salt culture medium sample size is 1000 mL, the method detection limit is 0.01 mg / L, and the lower limit of quantification is 0.04 mg / L.

[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. Here, the initial petroleum hydrocarbon content refers to the petroleum hydrocarbon content in the contaminated soil before inoculation with the compound microbial agent.

[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 before inoculation with the compound bacterial agent.

[0061] Example 1

[0062] This example illustrates the degradation effect of the compound microbial agent 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] 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.

[0065] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0066] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B suspension and Nodularia micronodosa 11-1B suspension at a colony ratio of 1:1 (the viable bacteria content of the compound microbial agent was 10%). 9CFU / mL). Inoculate the compound bacterial agent at a rate of 1 vol.% (10... 9 CFU was inoculated with compound microbial agent to a crude oil concentration (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the concentration of residual crude oil (as petroleum hydrocarbons) was periodically measured. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of crude oil after 7 days was 94.8% using the compound microbial agent. The compound microbial agent was inoculated at a dosage of 1% (v / w) until the crude oil content reached a level where the petroleum hydrocarbon content was [calculated as C]. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 30.0±2.0℃ and pH 7.0±0.2, the residual crude oil content (as petroleum hydrocarbons (C4H2O)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of crude oil by the compound microbial agent after 30 days was 90.1%.

[0067] The results show that when the ratio of the compound microbial agent is Bacillus belyssus 12-5B: Micronodella 11-1B at 1:1, under the conditions of 30.0±2.0℃ and pH 7.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 94% within 7 days, approaching complete degradation. This indicates that the ratio of the compound microbial agent and the temperature and pH conditions are superior for the remediation of petroleum hydrocarbon-contaminated groundwater. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 90% within 30 days, indicating that the ratio of the compound microbial agent and the temperature and pH conditions are superior for the remediation of petroleum hydrocarbon-contaminated groundwater. However, due to the adsorption properties of soil particles, the degradation of petroleum hydrocarbon-contaminated soil is more difficult than that of petroleum hydrocarbon-contaminated groundwater.

[0068] Example 2

[0069] This example illustrates the degradation effect of the compound microbial agent 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] 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.

[0072] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0073] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B suspension and Nodularia micronodosa 11-1B suspension at a colony ratio of 1:2 (the viable bacteria content of the compound microbial agent was 10%). 9 CFU / mL). Inoculate at a rate of 1 vol.% (10... 9 CFU was inoculated with a compound microbial agent to a diesel concentration (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 20.0 ± 2.0 °C and a pH of 7.5 ± 0.2, the residual diesel concentration (as petroleum hydrocarbons (C50)) was periodically measured. 10 -C 40 )Calculate the petroleum hydrocarbon degradation rate. The diesel degradation rate of the compound microbial agent after 7 days is 89.1%; based on an inoculum amount of 1 (v / w)% (10 9 CFU was inoculated with compound microbial agent to a diesel content (based on petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 20.0 ± 2.0℃ and pH 7.5 ± 0.2, the residual crude oil content (as petroleum hydrocarbons (C4H2O)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of crude oil by the compound microbial agent after 30 days was 85.4%.

[0074] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B suspension and Nodularia micronodosa 11-1B suspension at a colony ratio of 3:1 (the viable bacteria content of the compound microbial agent was 10%). 9 CFU / mL). Inoculate at a rate of 1 vol% (10... 9 CFU was inoculated with a compound microbial agent to a diesel concentration (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 40.0 ± 2.0 °C and a pH of 8.0 ± 0.2, the residual diesel concentration (as petroleum hydrocarbons (C4)) was periodically measured. 10 -C 40 )Calculated the petroleum hydrocarbon degradation rate, the diesel degradation rate of the compound microbial agent after 7 days was 85.7%; based on an inoculum amount of 1 (v / w)% (10 9 CFU was inoculated with compound microbial agent to a diesel content (based on petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 40.0±2.0℃ and pH 8.0±0.2, the residual diesel content (as petroleum hydrocarbons (C4)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the diesel degradation rate of the compound microbial agent after 30 days was 81.9%.

[0075] The results show that when the ratio of the compound bacterial agent is *Bacillus belyssus* 12-5B to *Micronodularia 11-1B* at 1:2, under conditions of 20.0±2.0℃ and pH 7.5±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater approaches 90% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 85% within 30 days. When the ratio of the compound bacterial agent is *Bacillus belyssus* 12-5B to *Micronodularia 11-1B* at 3:1, under conditions of 40.0±2.0℃ and pH 8.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 85% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 81% within 30 days.

[0076] Based on the above degradation effects, it can be seen that controlling the ratio of viable Bacillus bellis and Micronodella fuciformis in the compound microbial agent within an optimal range results in better remediation of petroleum hydrocarbon-contaminated groundwater. Furthermore, the microbial agent of this invention exhibits even better degradation effects on petroleum hydrocarbon-contaminated groundwater.

[0077] Example 3

[0078] This example illustrates the degradation effect of the compound microbial agent on kerosene.

[0079] 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.

[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] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0082] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:3 (the viable bacteria content of the compound microbial agent was 10%). 9 CFU / mL). Inoculate at a rate of 1 vol.% (10... 9 CFU was inoculated with a compound bacterial agent to a kerosene concentration (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 35.0 ± 2.0℃ and a pH of 6.0 ± 0.2, the concentration of residual kerosene (as petroleum hydrocarbons (C6H2O)) was periodically measured. 10 -C 40 )Calculate the petroleum hydrocarbon degradation rate. The kerosene degradation rate of the compound bacterial agent after 7 days is 85.2%; based on an inoculum amount of 1 (v / w)% (10 9 CFU was inoculated with a compound microbial agent to a kerosene content (based on petroleum hydrocarbons (C)). 10 -C 40In contaminated soil with a concentration of 10 g / kg, under conditions of 35.0±2.0℃ and pH 6.0±0.2, the residual kerosene content (as petroleum hydrocarbons (C4)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the kerosene degradation rate of the compound microbial agent after 30 days was 80.1%.

[0083] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 2:1 (the viable bacteria content of the compound microbial agent was 10). 9 CFU / mL). Inoculate at a rate of 1 vol.% (10... 9 CFU was inoculated with a compound bacterial agent to a kerosene concentration (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 25.0 ± 2.0 °C and a pH of 8.5 ± 0.2, the concentration of residual kerosene (as petroleum hydrocarbons) was periodically measured. 10 -C 40 )Calculate the petroleum hydrocarbon degradation rate. The kerosene degradation rate of the compound bacterial agent after 7 days is 89.3%; based on an inoculum amount of 1 (v / w)% (10 9 CFU was inoculated with a compound microbial agent to a kerosene content (based on petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 25.0 ± 2.0℃ and pH 8.5 ± 0.2, the residual kerosene content (as petroleum hydrocarbons (C4)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the kerosene degradation rate of the compound microbial agent after 30 days was 85.1%.

[0084] The results show that when the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 1:3, under conditions of 35.0±2.0℃ and pH 6.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater approaches 85% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 80% within 30 days. When the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 2:1, under conditions of 25.0±2.0℃ and pH 8.5±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 89% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 85% within 30 days.

[0085] Based on the above degradation effects, it can be seen that the ratio of viable Bacillus bellis and Micronodularia in the compound microbial agent, the degradation temperature, and the degradation pH value have a synergistic effect. Within the preferred range of viable bacterial ratio, degradation temperature, and degradation pH value, the compound microbial agent has a superior remediation effect on petroleum hydrocarbon-contaminated groundwater. Furthermore, the microbial agent of this invention has an even better degradation effect on petroleum hydrocarbon-contaminated groundwater.

[0086] Example 4

[0087] This example illustrates the degradation effect of the compound microbial agent on n-dodecane.

[0088] 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.

[0089] 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.

[0090] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0091] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:5 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL); at an inoculum of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-dodecane (based on petroleum hydrocarbons (C)). 10 -C40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 20.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the residual n-dodecane concentration (as petroleum hydrocarbons (C)) was periodically measured. 10 -C 40 )Calculate the degradation rate of petroleum hydrocarbons. The degradation rate of n-dodecane by the compound microbial agent after 7 days is 88.4%; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the n-dodecane content was reached (as shown by petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 20.0 ± 2.0℃ and pH 7.0 ± 0.2, the residual n-dodecane content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-dodecane by the compound microbial agent after 30 days was 85.2%.

[0092] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 7:1 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL); at an inoculum of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-dodecane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 20.0 ± 2.0 °C and a pH of 7.5 ± 0.2, the residual n-dodecane concentration (as petroleum hydrocarbons (C)) was periodically measured. 10 -C 40 )Calculate the degradation rate of petroleum hydrocarbons. The degradation rate of n-dodecane by the compound microbial agent after 7 days is 88.1%; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the n-dodecane content was reached (as shown by petroleum hydrocarbons (C)). 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 30.0±2.0℃ and pH 7.5±0.2, the residual n-dodecane content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-dodecane by the compound microbial agent after 30 days was 85.9%.

[0093] The results show that when the ratio of the compound bacterial agent is *Bacillus belyssioides* 12-5B to *Micronodularia 11-1B* at 1:5, under conditions of 20.0±2.0℃ and pH 7.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches nearly 88% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 85% within 30 days. When the ratio of the compound bacterial agent is 7:1, under conditions of 20.0±2.0℃ and pH 7.5±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 88% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 85% within 30 days.

[0094] Example 5

[0095] This example illustrates the degradation effect of the compound microbial agent on n-hexadecane.

[0096] 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.

[0097] 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.

[0098] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0099] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:8 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL); at an inoculum of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-hexadecane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 30.0 ± 2.0 °C and a pH of 8.0 ± 0.2, the concentration of residual n-hexadecane (as petroleum hydrocarbons (C6H2O)) was periodically measured. 10 -C 40 )Calculate the petroleum hydrocarbon degradation rate, the compound bacterial agent achieved a hexadecane degradation rate of 91.6% after 7 days; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the hexadecane content reached a petroleum hydrocarbon (C) concentration. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 30.0±2.0℃ and pH 8.0±0.2, the residual n-hexadecane content (as petroleum hydrocarbons (C6H2O)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-hexadecane by the compound microbial agent after 30 days was 86.6%.

[0100] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 10:1 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL); inoculate at a rate of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-hexadecane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 35.0 ± 2.0 °C and a pH of 9.0 ± 0.2, the concentration of residual n-hexadecane (as petroleum hydrocarbons (C6H2O)) was periodically measured. 10 -C 40 )Calculate the petroleum hydrocarbon degradation rate. The compound bacterial agent showed a hexadecane degradation rate of 85.0% after 7 days; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the hexadecane content reached a petroleum hydrocarbon (C) concentration. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 35.0±2.0℃ and pH 9.0±0.2, the residual n-hexadecane content (as petroleum hydrocarbons (C6H2O)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-hexadecane by the compound microbial agent after 30 days was 82.3%.

[0101] The results show that when the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 1:8, under conditions of 30.0±2.0℃ and pH 8.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches nearly 91% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 86% within 30 days. When the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 10:1, under conditions of 35.0±2.0℃ and pH 9.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 85% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 82% within 30 days.

[0102] Example 6

[0103] This example illustrates the degradation effect of the compound microbial agent on n-eicosane.

[0104] 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 -C 40 The inorganic salt culture medium is 10 g / L.

[0105] 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.

[0106] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9 The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0107] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:10 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL). Inoculate at a rate of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-eicosane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 40.0 ± 2.0 °C and a pH of 6.0 ± 0.2, the residual n-eicosane concentration (as petroleum hydrocarbons (C)) was periodically measured. 10 -C 40 )Calculate the degradation rate of petroleum hydrocarbons. The degradation rate of n-eicosane by the compound microbial agent after 7 days is 88.6%; based on an inoculum amount of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the content of n-eicosane (as per petroleum hydrocarbons (C)) was reached. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 40.0±2.0℃ and pH 6.0±0.2, the residual n-eicosane content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-eicosane by the compound microbial agent after 30 days was 83.6%.

[0108] A compound microbial agent was prepared by mixing Bacillus belye 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 9:1 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL). Inoculate at a rate of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-eicosane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 20.0 ± 2.0 °C and a pH of 6.5 ± 0.2, the concentration of residual n-eicosane (as petroleum hydrocarbons (C...)) was periodically measured. 10 -C 40 )Calculate the degradation rate of petroleum hydrocarbons. The degradation rate of n-eicosane by the compound microbial agent after 7 days is 85.3%; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound bacterial agent until the content of n-eicosane (as per petroleum hydrocarbons (C)) was reached. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 20.0 ± 2.0℃ and pH 6.5 ± 0.2, the residual n-eicosane content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-eicosane by the compound microbial agent after 30 days was 80.2%.

[0109] The results show that when the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 1:10, under conditions of 40.0±2.0℃ and pH 6.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches nearly 88% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 83% within 30 days. When the ratio of the compound bacterial agent is *Bacillus belyssiensis* 12-5B to *Micronodularia 11-1B* at 9:1, under conditions of 20.0±2.0℃ and pH 6.5±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 85% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 80% within 30 days.

[0110] Example 7

[0111] This example illustrates the degradation effect of the compound microbial agent on n-octacosane.

[0112] 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.

[0113] 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.

[0114] Bacillus bellis 12-5B and Nodularia micronodosa 11-1B were cultured in LB medium to obtain stock solutions of Bacillus bellis 12-5B and Nodularia micronodosa 11-1B, respectively. The bacterial cells obtained after centrifugation of the two stock solutions were used to prepare Bacillus bellis 12-5B and Nodularia micronodosa 11-1B suspensions, respectively, with sterile water. The viable cell count of the Bacillus bellis 12-5B suspension was 10⁻⁶. 9The cfu / mL count of the micronodular mold 11-1B suspension was 10. 9 cfu / mL.

[0115] A compound microbial agent was prepared by mixing Bacillus belye 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:1 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL). Inoculate at a rate of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-octadecane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 30.0 ± 2.0 °C and a pH of 7.0 ± 0.2, the concentration of residual n-octacosan (as petroleum hydrocarbons (C...)) was periodically measured. 10 -C 40 )Calculate the degradation rate of petroleum hydrocarbons. The degradation rate of n-octacosanol by the compound microbial agent after 7 days is 95.6%; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound microbial agent until the content of n-octadecane (as expressed by petroleum hydrocarbons (C50)) reached a concentration of n-octadecane. 10 -C 40 In contaminated soil with a concentration of 10 g / kg, under conditions of 30.0±2.0℃ and pH 7.0±0.2, the residual n-octacosan content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-octacosanol by the compound microbial agent after 30 days was 90.9%.

[0116] A compound microbial agent was prepared by mixing Bacillus bellis 12-5B and Nodularia micronodosa 11-1B bacterial suspensions at a colony ratio of 1:2 (the viable bacteria content of the compound microbial agent was 10%). 9 cfu / mL). Inoculate at a rate of 1 vol.% (10 9 CFU was inoculated with a compound bacterial agent to a concentration of n-octadecane (based on petroleum hydrocarbons (C)). 10 -C 40 In an inorganic salt culture medium with a concentration of 10 g / L, at a temperature of 35.0 ± 2.0 °C and a pH of 7.5 ± 0.2, the concentration of residual n-octacosan (as petroleum hydrocarbons (C...)) was periodically measured. 10 -C 40 )Calculation of petroleum hydrocarbon degradation rate, the compound bacterial agent achieved a 92.9% degradation rate of n-octadecane after 7 days; based on an inoculum size of 1 (v / w)% (10 9 CFU was inoculated with a compound microbial agent until the content of n-octadecane (as expressed by petroleum hydrocarbons (C50)) reached a concentration of n-octadecane. 10 -C 40In contaminated soil with a concentration of 10 g / kg, under conditions of 35.0 ± 2.0℃ and pH 7.5 ± 0.2, the residual n-octacosan content (as petroleum hydrocarbons (C)) was periodically tested. 10 -C 40 The degradation rate of petroleum hydrocarbons was calculated, and the degradation rate of n-octacosanol by the compound microbial agent after 30 days was 89.6%.

[0117] The results show that when the ratio of the compound bacterial agent is *Bacillus bellis* 12-5B to *Micronodularia 11-1B* at 1:1, under conditions of 30.0±2.0℃ and pH 7.0±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater approaches 95% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 90% within 30 days. When the ratio of the compound bacterial agent is *Bacillus bellis* 12-5B to *Micronodularia 11-1B* at 1:2, under conditions of 35.0±2.0℃ and pH 7.5±0.2, the crude oil degradation rate in the inorganic salt culture medium simulating petroleum hydrocarbon-contaminated groundwater reaches over 92% within 7 days. Under the same conditions, the crude oil degradation rate in the simulated petroleum hydrocarbon-contaminated soil reaches over 89% within 30 days.

[0118] As can be seen from the above embodiments, the composite microbial agent of the present invention has a good degradation effect on petroleum hydrocarbons and a good remediation effect on petroleum hydrocarbon-contaminated groundwater and soil. Furthermore, compared to petroleum hydrocarbon-contaminated soil, the composite microbial agent of the present invention has a better remediation effect on petroleum hydrocarbon-contaminated groundwater. In addition, in the present invention, the strain ratio, degradation temperature, and degradation pH of the composite microbial agent have a synergistic effect, resulting in even better remediation effects on petroleum hydrocarbon-contaminated groundwater and soil when within the preferred range.

[0119] 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 complex microbial agent having a petroleum hydrocarbon degradation function, characterized by, The complex microbial agent comprises Bacillus velezensis (Bacillus velezensis) Bacillus velezensis ) and Microcnidium sp. (Microcnidium sp. Microdochium sp. ); the preservation number of the Bacillus velezensis is CGMCC No. 23199, and the preservation number of the Microcnidium sp. is CGMCC No. 23200.

2. The complex bacterial agent according to claim 1, wherein The ratio of viable Bacillus vesiculosus and Micronodularia in the compound microbial agent is 10:1 to 1:

10.

3. The complex bacterial agent of claim 2, wherein The ratio of viable Bacillus bellis and Micronodularia in the compound microbial agent is 3:1 to 1:

3.

4. The application of the composite microbial agent with petroleum hydrocarbon degradation function as described in any one of claims 1-3 in the remediation of petroleum hydrocarbon contaminated soil or petroleum hydrocarbon contaminated groundwater.

5. A method of degrading petroleum hydrocarbons, characterized by, The method includes contacting the composite microbial agent according to any one of claims 1-3 with a pollutant containing petroleum hydrocarbons.

6. The method of claim 5, wherein, The petroleum hydrocarbon is at least one of liquid alkanes, solid alkanes, diesel, crude oil, and kerosene.

7. The method of claim 5 or 6, wherein, The pollutants containing petroleum hydrocarbons are petroleum hydrocarbon-containing soil or petroleum hydrocarbon-containing groundwater.

8. The application of the compound microbial agent according to any one of claims 1-3 in the remediation of petroleum hydrocarbon-contaminated soil or petroleum hydrocarbon-contaminated groundwater.