Petroleum hydrocarbon mixed bacteria liquid as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of environmental microorganisms, and discloses a petroleum hydrocarbon mixed bacteria solution as well as a preparation method and application thereof. The petroleum hydrocarbon mixed bacterial liquid disclosed by the invention is prepared from Desulfotomaculum, bacillus subtilis and actinomycetes, and is prepared from the following components: Desulfotomaculum, bacillus subtilis and actinomycetes. According to the petroleum hydrocarbon mixed bacterial liquid, petroleum hydrocarbon can be used as an electron donor, high-concentration iron and high-concentration sulfate are used as electron acceptors, and the petroleum hydrocarbon in a polluted site is degraded while a ferrous sulfide material with high reaction performance is synthesized in a composite organic polluted site in an in-situ environment-friendly manner; and the synthesized ferrous sulfide material can further degrade chlorides in a polluted site, comprehensive treatment of the composite pollutants is realized, the remediation efficiency is improved, and the method has important significance on bioremediation of the composite organic pollutants.

Description

Technical Field

[0001] The invention relates to the technical field of environmental microorganisms, and in particular to a petroleum hydrocarbon mixed bacterial liquid and a preparation method and application thereof. Background Art

[0002] As an important energy source and chemical raw material, petroleum is of great significance to the global economic and social development. However, with the continuous innovation and development of the petrochemical and organic chemical industries, the environmental problems caused by petroleum pollutants have become increasingly prominent. During the extraction, processing and utilization of petroleum, the components of petroleum hydrocarbon pollutants that are easily adsorbed by the soil will directly enter the soil; the components that are not easily adsorbed by the soil will penetrate into the ground with precipitation, pollute shallow groundwater, and then cause organic pollution of the environment. These pollutants pose a serious threat to the ecological environment and human health due to their toxicity, mutagenicity and carcinogenicity.

[0003] Trichloroethylene (TCE) is a colorless, transparent liquid with the chemical formula C2H3Cl3. It is widely used in industrial production. For example, it can be used as one of the raw materials for manufacturing polyvinyl chloride (PVC) plastic products, and is used in construction, wires and cables, automotive parts and other fields. In addition, trichloroethylene can dissolve many organic substances, such as resins, paints, rubber, etc. Therefore, trichloroethylene is often used as a solvent in industries such as printing and coating. However, at the same time, trichloroethylene is also one of the most widely used chlorinated organic pollutants. It has a strong migration ability in the soil and can penetrate the fine pores of the soil. This migration ability makes trichloroethylene very easy to migrate from the pollution source to the groundwater environment, causing groundwater pollution. As pointed out in the "Groundwater Environmental Quality" report in the "China Environmental Status Bulletin 2023", the main excessive indicators of groundwater are iron, sulfate and chloride.

[0004] It can be seen that when petroleum and trichloroethylene and other chemicals are used simultaneously in the production and processing of organic chemical enterprises, pollutants will flow into the soil and groundwater environment through various channels (such as leakage, discharge, improper treatment, etc.), not in the form of a single pollutant, but in a state of complex organic cross-pollution, which will pollute the soil and groundwater environment and cause a series of serious environmental problems. For example, complex organic pollutants can accumulate in organisms through the food chain, posing a potential threat to human health and the ecosystem. On the other hand, they may also change the physical and chemical properties of soil and groundwater, affect soil fertility and groundwater quality, and thus have an adverse impact on agricultural production and drinking water safety.

[0005] Therefore, it is urgent to develop a green and efficient method for the synergistic degradation of complex organic pollutants, which can synergistically degrade the soil or groundwater environment where complex organic pollution interacts, which is of great significance for protecting the ecological environment and human health. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a petroleum hydrocarbon mixed bacteria liquid and a preparation method and application thereof.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a petroleum hydrocarbon mixed bacterial liquid, comprising Desulfotomaculum with a relative abundance of 24.71%-25.28%, Bacillus with a relative abundance of 13.95%-14.77%, and Caproiciproducens with a relative abundance of 5.85%-6.50%.

[0009] The petroleum hydrocarbon mixed bacterial solution of the present invention includes a variety of bacteria, and these bacterial species can synergistically utilize petroleum hydrocarbons as electron donors, high concentrations of iron and high concentrations of sulfate as electron acceptors, and in situ green synthesize ferrous sulfide materials with high reactivity in the complex organic contaminated site while degrading the petroleum hydrocarbons in the complex organic contaminated site. Secondly, the ferrous sulfide materials with high reactivity synthesized in situ green by the petroleum hydrocarbon mixed bacterial solution of the present invention can further degrade chlorides in the complex organic contaminated site, thereby achieving comprehensive treatment of complex pollutants. It can be seen that the petroleum hydrocarbon mixed bacterial solution of the present invention has broad application prospects in treating complex organic pollution.

[0010] As a preferred embodiment of the petroleum hydrocarbon mixed bacterial liquid of the present invention, the petroleum hydrocarbon mixed bacterial liquid also includes Clostridium with a relative abundance of 21.46%-22.64%, Lachnoclostridium with a relative abundance of 7.8%-9.71%, unclassified Lutispora with a relative abundance of 6.37%-6.6%, Fonticella with a relative abundance of 4.87%-5.58%, Sporomusa with a relative abundance of 4.23%-4.85%, uncultured Firmicutes bacterium with a relative abundance of 2.35%-2.57%, Desulfitobacterium with a relative abundance of 1.14%-1.22%, and Herbinix with a relative abundance of 1.35%-1.43%.

[0011] Preferably, the Clostridium includes Clostridium_sensu_stricto_1, Clostridium_sensu_stricto_8, Clostridium_sensu_stricto_10, Clostridium_sensu_stricto_12, and Clostridium_sensu_stricto_13.

[0012] In a second aspect, the present invention provides a method for preparing a petroleum hydrocarbon mixed bacterial liquid, comprising the following steps:

[0013] (1) activating dormant bacteria in petroleum hydrocarbon-contaminated soil to obtain an activated bacterial solution;

[0014] (2) enriching the activated bacterial solution in an enrichment medium to obtain an enriched bacterial solution;

[0015] (3) placing the enriched bacterial liquid in an acclimation medium for acclimation to obtain the petroleum hydrocarbon mixed bacterial liquid; the carbon source of the acclimation medium comprises at least one of gasoline, kerosene, diesel, lubricating oil, paraffin and asphalt.

[0016] The preparation method of the petroleum hydrocarbon mixed bacterial liquid of the present invention comprises the following steps: collecting mixed bacterial samples from soil contaminated by petroleum hydrocarbons, and subjecting the samples to enrichment and specific domestication so that the bacterial strains gradually adapt to an environment in which petroleum hydrocarbons are used as electron donors, and forming a mixed bacterial liquid with high-efficiency degradation capability; at the same time, the preparation method is completely based on biological processes, does not produce toxic intermediates, is environmentally friendly, is easy to operate, has low cost, and is suitable for large-scale applications.

[0017] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial liquid of the present invention, the enrichment culture medium comprises the following components in parts by weight: 8-15 parts of carbon source, 8-12 parts of nitrogen source, 0.5-2 parts of sodium thioglycolate, 30-40 parts of inorganic salt, 3-8 parts of buffer, and 0.8-1.5 parts of antioxidant.

[0018] Preferably, the enrichment medium comprises the following components in parts by weight: 11.2 parts of carbon source, 10 parts of nitrogen source, 1 part of sodium thioglycolate, 35.5 parts of inorganic salt, 5 parts of buffer, and 1 part of antioxidant.

[0019] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial liquid of the present invention, the culture temperature of the enrichment culture medium is 35° C.-40° C., and the culture time is 5 days-10 days.

[0020] Preferably, the culture temperature of the enriched medium is 37° C. and the culture time is 7 days.

[0021] Preferably, the inorganic salt comprises the following components in parts by weight: 8-15 parts of sulfate, 3-8 parts of ferrous salt, 15-25 parts of magnesium salt, and 0.2-1 part of calcium salt.

[0022] Further preferably, the inorganic salt comprises the following components in parts by weight: 10 parts of sulfate, 5 parts of ferrous salt, 20 parts of magnesium salt, and 0.5 parts of calcium salt.

[0023] Preferably, the carbon source is sodium DL-lactate; the nitrogen source is yeast extract; the buffer is potassium dihydrogen phosphate; and the antioxidant is ascorbic acid.

[0024] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial liquid of the present invention, the carbon source of the acclimation culture medium includes diesel.

[0025] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial liquid of the present invention, the acclimation culture medium also includes the following components in parts by weight: 8-12 parts of nitrogen source, 0.5-2 parts of sodium thioglycolate, 30-40 parts of inorganic salt, 3-8 parts of buffer, and 0.8-1.5 parts of antioxidant.

[0026] Preferably, the acclimation medium comprises the following components in parts by weight: 10 parts of nitrogen source, 1 part of sodium thioglycolate, 35.5 parts of inorganic salt, 5 parts of buffer, and 1 part of antioxidant.

[0027] Further preferably, the inorganic salt comprises the following components in parts by weight: 8-15 parts of sulfate, 3-8 parts of ferrous salt, 15-25 parts of magnesium salt, and 0.2-1 part of calcium salt.

[0028] More preferably, the inorganic salt comprises the following components in parts by weight: 10 parts of sulfate, 5 parts of ferrous salt, 20 parts of magnesium salt, and 0.5 parts of calcium salt.

[0029] Preferably, the nitrogen source is yeast extract; the inorganic salts include sulfates, ferrous salts, magnesium salts, and calcium salts; the buffer is potassium dihydrogen phosphate; and the antioxidant is ascorbic acid.

[0030] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial liquid of the present invention, the culture temperature of the acclimation culture medium is 20° C.-40° C., and the culture time is 15 days-30 days.

[0031] As a preferred embodiment of the method for preparing the petroleum hydrocarbon mixed bacterial solution of the present invention, the domestication comprises the following steps: placing the enriched bacterial solution in a first domestication medium, a second domestication medium, and a third domestication medium in sequence for domestication.

[0032] Preferably, the volume of the carbon source of the first acclimation medium is 1%-4% of the total volume of the first acclimation medium; the volume of the carbon source of the second acclimation medium is 4%-6% of the total volume of the second acclimation medium; the volume of the carbon source of the second acclimation medium is 6%-10% of the total volume of the second acclimation medium.

[0033] Further preferably, the volume of the carbon source of the first acclimation medium is 3% of the total volume of the first acclimation medium; the volume of the carbon source of the second acclimation medium is 5% of the total volume of the second acclimation medium; the volume of the carbon source of the second acclimation medium is 8% of the total volume of the second acclimation medium.

[0034] Preferably, the culture temperature of the first acclimation medium is 35°C-40°C, and the culture time is 5d-10d; the culture temperature of the second acclimation medium is 15°C-25°C, and the culture time is 5d-10d; the culture temperature of the third acclimation medium is 35°C-40°C, and the culture time is 5d-10d.

[0035] Further preferably, the culture temperature of the first acclimation medium is 37°C, and the culture time is 6 days; the culture temperature of the second acclimation medium is 20°C, and the culture time is 6 days; the culture temperature of the third acclimation medium is 37°C, and the culture time is 6 days.

[0036] In a third aspect, the present invention provides a petroleum hydrocarbon mixed bacteria liquid, wherein the petroleum hydrocarbon mixed bacteria liquid is prepared by the preparation method of the petroleum hydrocarbon mixed bacteria liquid.

[0037] In a fourth aspect, the present invention provides a bacterial agent, which includes the petroleum hydrocarbon mixed bacterial solution and / or metabolites of the petroleum hydrocarbon mixed bacterial solution.

[0038] The bacterial agent of the present invention comprises a petroleum hydrocarbon mixed bacterial liquid and / or its metabolites. The mixed bacterial liquid is obtained by a specific preparation method, collected from soil contaminated by petroleum hydrocarbons, and gradually adapted to an environment with petroleum hydrocarbons as electron donors after enrichment and domestication, and the multiple bacteria in the mixed bacterial liquid can synergistically and efficiently degrade petroleum hydrocarbons; at the same time, the metabolites produced in the metabolic process of the mixed bacterial liquid using petroleum hydrocarbons as electron donors and high concentrations of iron and high concentrations of sulfate as electron acceptors have excellent surface properties and high dispersibility, and can degrade chlorides efficiently and greenly.

[0039] As a preferred embodiment of the bacterial agent of the present invention, the metabolite includes ferrous sulfide.

[0040] In a fifth aspect, the present invention provides the use of the petroleum hydrocarbon mixed bacterial solution and the bacterial agent in the bioremediation of complex organic pollutants.

[0041] As a preferred embodiment of the application of the present invention, the composite organic pollutants include petroleum hydrocarbons and / or chlorides.

[0042] The petroleum hydrocarbon mixed bacterial liquid of the present invention can synergistically utilize petroleum hydrocarbon substances as electron donors, high concentrations of iron and high concentrations of sulfate as electron acceptors, and degrade petroleum hydrocarbon substances while synthesizing ferrous sulfide materials with high reactivity. Secondly, the synthesized ferrous sulfide materials with high reactivity can further degrade chlorides. Therefore, the petroleum hydrocarbon mixed bacterial liquid and bacterial agent of the present invention can be applied to the bioremediation of composite organic pollutants, such as being used in soil or groundwater environments with composite pollution of petroleum hydrocarbon pollutants and chlorinated pollutants, while degrading petroleum hydrocarbons in the contaminated site in the in-situ green synthesis of ferrous sulfide materials with high reactivity, and the in-situ green synthesis of ferrous sulfide materials with high reactivity in the contaminated site can further degrade chlorides in the contaminated site, achieving comprehensive treatment of composite pollutants and improving repair efficiency. The method does not need to transfer contaminated soil or groundwater to other places for treatment, but directly performs in-situ repair at the contaminated site. This greatly reduces the processing cost and reduces secondary pollution to the environment.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows: first, the petroleum hydrocarbon mixed bacterial solution of the present invention can rely on sufficient electron donors and sufficient electron acceptors in the environment to metabolize while degrading petroleum hydrocarbons in a contaminated site with complex organic pollution interaction through the three mechanisms of microbial action, precipitation process and chemical conversion in the biogeochemical conversion process, and reduce sulfate and iron in the environment to produce ferrous sulfide with high reactivity to degrade excessive chloride, thereby achieving comprehensive treatment of complex pollutants. Secondly, the ferrous sulfide metabolite of the petroleum hydrocarbon mixed bacterial solution of the present invention has excellent surface properties and high dispersibility, and can degrade chloride efficiently and greenly. At the same time, the preparation method of the petroleum hydrocarbon mixed bacterial solution of the present invention is completely based on biological processes, does not produce toxic intermediates, is environmentally friendly, simple to operate, has low cost, and is suitable for large-scale application. In addition, the petroleum hydrocarbon mixed bacterial solution and bacterial agent of the present invention can be applied to the bioremediation of complex organic pollutants, which can realize the process of biogeochemical conversion of multiple pollutants in the contaminated site without high energy consumption and high cost ex situ remediation, and the pollutants can form ferrous sulfide, a chemical remediation material with high reactivity, after conversion. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is a schematic diagram of the soil extract and sulfate-reducing bacteria culture medium of Example 1 of the present invention;

[0045] Figure 2 This is a schematic diagram of enriching bacterial liquid in Example 1 of the present invention;

[0046] Figure 3This is a diagram of microbial diversity analysis of the enriched bacterial solution in Example 1 of the present invention;

[0047] Figure 4 This is a schematic diagram of the petroleum hydrocarbon mixed bacterial solution and the acclimation culture medium in Example 1 of the present invention;

[0048] Figure 5 This is a distribution diagram of microbial species in the petroleum hydrocarbon mixed bacterial solution of Example 1 of the present invention;

[0049] Figure 6 This is a graph showing the concentration variation of n-hexadecane degraded by a petroleum hydrocarbon mixed bacterial solution in Verification Example 1 of the present invention;

[0050] Figure 7 This is a scanning electron microscope image of ferrous sulfide under a 200nm lens in Verification Example 2 of the present invention;

[0051] Figure 8 This is a scanning electron microscope image of ferrous sulfide under a 20 μm lens in Verification Example 2 of the present invention;

[0052] Fig. 9 This is the X-ray diffraction pattern of ferrous sulfide of Verification Example 2 of the present invention;

[0053] Fig.10 This is the Fourier infrared spectrum of ferrous sulfide of Verification Example 2 of the present invention;

[0054] Fig.11 This is the Fourier infrared spectrum of ferrous sulfide of Verification Example 2 of the present invention;

[0055] Fig.12 This is the Fourier infrared spectrum of ferrous sulfide of Verification Example 2 of the present invention;

[0056] Fig.13 This is a graph showing the concentration variation of trichloroethylene degradation by ferrous sulfide in Verification Example 3 of the present invention;

[0057] Fig.14 This is a schematic diagram of the degradation rate of trichloroethylene by ferrous sulfide in Verification Example 3 of the present invention;

[0058] Fig.15 This is a diagram showing the hydrogenolysis reaction mechanism of ferrous sulfide degrading trichloroethylene in Verification Example 3 of the present invention;

[0059] Fig.16 This is the β-elimination reaction mechanism diagram of ferrous sulfide degradation of trichloroethylene in Verification Example 3 of the present invention. DETAILED DESCRIPTION

[0060] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0061] The following is an elaboration in conjunction with specific embodiments to illustrate the practical effects of the solutions of the present invention.

[0062] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, equipment, etc. used, unless otherwise specified, can be obtained from commercial sources.

[0063] The purity of the pharmaceutical reagents used in the present invention is not less than AR grade, and the glassware needs to be ultrasonically cleaned and sterilized under high pressure before use and then sealed and stored.

[0064] The experimental instruments used in the present invention include: a high-pressure sterilizing pot, an anaerobic workstation, a microbial shaking table, an X-ray diffraction, an X-ray photoelectron spectrum, a Fourier infrared spectrum, a microscopic electron scanning electron microscope, a nitrogen generator and a gas chromatograph.

[0065] Example 1: Preparation of petroleum hydrocarbon mixed bacterial liquid

[0066] The petroleum hydrocarbon contaminated soil of the present invention is taken from soil near an oil refinery in Dingbian County, Yulin City, Shaanxi Province. The petroleum hydrocarbon contamination depth exceeds 1m. Drilling sampling is performed using a mechanical soil sampling device. GC detection shows that the total petroleum hydrocarbon concentration of the soil is 14035 mg / L.

[0067] (1) Soil extract: The petroleum hydrocarbon contaminated soil was air-dried and sieved to obtain dust-like dry soil. 15 g of the dust-like dry soil was added to 300 mL of physiological saline and stirred thoroughly. The dormant microorganisms were activated in a microbial shaker at a temperature of 37°C and a rotation speed of 200 rpm for 7 days.

[0068] (2) Preparation of sulfate-reducing bacteria culture medium: 0.5 g KH2PO4, 1 g Na2SO4, 1 g NH4Cl, 2 g MgCl2·6H2O, 0.05 g CaCl2, 0.5 g FeSO4·7H2O, 1 g yeast extract, 0.1 g ascorbic acid and 0.1 g sodium thioglycolate were added to a 1000 mL volumetric flask with deoxygenated deionized water (pH = 7) and shaken thoroughly. The solution in the volumetric flask was then drained into a 1000 mL anaerobic bottle using a glass rod in an anaerobic workstation. The solution was sterilized in a high pressure steam cooker at 121°C for 30 min. After the solution was cooled, it was sealed and stored in an anaerobic workstation filled with nitrogen.

[0069] (3) Enrichment: After the dormant organisms in the petroleum hydrocarbon-contaminated soil were activated after 7 days, the soil extract and 1000 mL of sulfate-reducing bacteria culture medium were transferred to an anaerobic workstation to allow them to fully contact the nitrogen environment. Then, 10 mL of the supernatant of the soil extract and 1.12 g of DL-sodium lactate were extracted using a disposable sterile syringe, respectively, and added to 1000 mL of sulfate-reducing bacteria culture medium through a 0.45 μm microporous filter. After addition, the chitin stopper, aluminum cap and sealing film were quickly sealed. The sealed culture medium was fixed in a microbial shaker for 7 days of bacterial enrichment at 37°C, 200 rpm, and an enriched bacterial solution was obtained.

[0070] (4) Domestication:

[0071] According to the above medium configuration scheme, a new sulfate-reducing bacteria medium solution is prepared, and the prepared sulfate-reducing bacteria medium solution is sterilized by high pressure and stored anaerobically.

[0072] (I) First domestication: After 7 days of strain enrichment and the culture medium solution changes from yellow-brown to black, the sulfate-reducing bacteria culture medium solution in the shaker is taken out and transferred to the anaerobic workstation, and the supernatant is added to the newly prepared 1000 mL sulfate-reducing bacteria culture medium solution in the workstation. At the same time, 30 mL of diesel is added to the culture medium as a microbial carbon source, and the chitin plug, aluminum cap and sealing film are quickly sealed. The sealed culture medium is fixed in a microbial shaker for the first domestication of petroleum hydrocarbon microorganisms at 37°C, 200 rpm, and 6 days.

[0073] According to the above medium configuration scheme, a new sulfate-reducing bacteria medium solution is prepared, and the prepared sulfate-reducing bacteria medium solution is sterilized by high pressure and stored anaerobically.

[0074] (II) Second acclimatization: After the first acclimatization of petroleum hydrocarbon microorganisms for 6 days is completed, the sulfate-reducing bacteria culture medium solution in the shaker is taken out and transferred to the anaerobic workstation, and the supernatant is added to the newly prepared 1000 mL culture medium solution in the workstation, and 50 mL diesel is added to the culture medium as a microbial carbon source, and the chitin plug, aluminum cap and sealing film are quickly sealed. The sealed sulfate-reducing bacteria culture medium is fixed in the microbial shaker for the second acclimatization of petroleum hydrocarbon microorganisms at 20°C, 200 rpm, and 6 days.

[0075] According to the above medium configuration scheme, a new sulfate-reducing bacteria medium solution is prepared, and the prepared sulfate-reducing bacteria medium solution is sterilized by high pressure and stored anaerobically.

[0076] (III) Third domestication: After the second domestication of petroleum hydrocarbon microorganisms is completed after 6 days, the sulfate-reducing bacteria culture medium solution in the shaker is taken out and transferred to the anaerobic workstation. The supernatant is added to the newly configured 1000mL culture medium solution in the workstation, and 80mL diesel is added to the culture medium as a microbial carbon source. The chitin plug, aluminum cover and sealing film are quickly sealed. The sealed culture medium is fixed in a microbial shaker for 6 days of petroleum hydrocarbon microbial domestication at 37°C and 200rpm to obtain a domesticated bacterial solution. The domesticated bacterial solution is centrifuged at 6000rpm in a high-speed centrifuge, and the supernatant is collected to obtain a petroleum hydrocarbon (diesel) mixed bacterial solution, which is transferred to the anaerobic workstation for storage.

[0077] On the 3rd, 12th and 18th days of acclimation, 10 mL of bacterial solution was extracted and centrifuged, and then frozen at -80°C for 16sr RNA microbial community structure analysis. The microbial samples tested on the 3rd day were numbered A3, B3, and C3, the microbial samples tested on the 12th day were numbered A12, B12, and C12, and the microbial samples tested on the 18th day were numbered A18, B18, and C18.

[0078] The relative abundance percentage of microbial species by genus is shown in Table 1, and the distribution map of microbial species by genus is shown in Figure 5 The results show that on the third day, Clostridium is the dominant bacterial genus in the microbial community. This is because the presence of low molecular organic matter in the culture medium promotes the enrichment of Clostridium with hydrogen production ability. As time goes by, Desulfotomaculum and Bacillus become the dominant bacterial genera. Through the domestication operation, the microbial community structure of the mixed bacterial liquid cultured in the present invention includes Desulfotomaculum, Bacillus, Clostridium_sensu_stricto_12, Lachnoclostridium, Clostridiu m_sensu_stricto_13, Caproiciproducens, unclassified_Lutispora, Fonticella, Sporomusa, Clostridium_sensu_stricto_10, uncultured_Firmicutes_bacterium, Herbinix, Desulfitobacterium, Clostridium_sensu_stricto_1, Clostridium_sensu_stricto_8.

[0079] Table 1 Relative abundance percentage of microbial species in petroleum hydrocarbon (diesel) mixed bacterial solution

[0080]

[0081]

[0082] Example 2: Extraction of ferrous sulfide material

[0083] Add 1L of the domesticated bacterial solution prepared in Example 1 to the centrifuge tubes respectively, centrifuge at 6000rpm in a high-speed centrifuge, discard the supernatant in the centrifuge tube with a rubber-tipped dropper, add deionized water to the centrifuge tube for centrifugation, discard the supernatant, repeat 3 times, and wash the free metal ions; add anhydrous ethanol to the centrifuge tube for centrifugation, discard the supernatant, repeat 3 times, and wash the organic matter in the solution; after washing, add deionized water to the centrifuge tube and freeze it in a -80℃ refrigerator for 24 hours. After the deionized water and the diesel solution adsorbed on the surface of ferrous sulfide in the centrifuge tube solidify, transfer them to a freeze dryer. In a vacuum environment and at -50℃, the solidified deionized water and diesel will be sublimated to obtain 1.53g of ferrous sulfide material. The collected ferrous sulfide material is sealed and stored anaerobically. Verification Example 1:

[0084] This verification example verifies the degradation effect of the petroleum hydrocarbon (diesel) mixed bacterial liquid prepared in Example 1 on petroleum hydrocarbon.

[0085] Petroleum hydrocarbon degrading bacteria culture medium: A solution of 0.5 g KH2PO4, 1 g Na2SO4, 1 g NH4Cl, 2 g MgCl2·6H2O, 0.05 g CaCl2, 0.5 g FeSO4·7H2O, 1 g yeast extract, 0.1 g ascorbic acid, 0.1 g sodium thioglycolate and 1000 mL deoxygenated deionized water (pH = 7) was placed in a 1 L anaerobic bottle, sterilized at 121 ° C in a high pressure steam cooker for 30 min, and then transferred to an anaerobic workstation.

[0086] The hexadecane solution and 10 mL of the mixed bacterial solution of petroleum hydrocarbon (diesel)-sulfate-reducing bacteria of Example 1 were added to the petroleum hydrocarbon degradation bacteria culture medium, and the concentrations of the hexadecane solution, sulfate concentration and ferrous iron concentration in the solution were respectively 1000 mg / L, 850 mg / L and 100 mg / L, and the anaerobic bottle was sealed with a chitin stopper, an aluminum cap and a sealing film. The sealed petroleum hydrocarbon degradation bacteria solution was fixed in a microbial shaking incubator for 21 days of petroleum hydrocarbon degradation and ferrous sulfide precipitation at 25°C, 160 rpm.

[0087] The petroleum hydrocarbon degradation experiment set up two control groups, a blank experiment and a microbial experiment, and each control group conducted three repeated experiments. The sampling time was day 0, day 3, day 6, day 9, day 12, day 15, day 18 and day 21. The sampling method was to use n-hexane solution to organically extract the n-hexadecane solution in the experimental solution, and then use a vortex instrument to evenly mix the n-hexadecane in the petroleum hydrocarbon degradation bacteria culture medium and centrifuge it. Centrifugal extraction was performed at 6000rpm and 5min, and the non-aqueous phase liquid in the lower layer was taken out by a rubber-tipped dropper, and NaSO4 was added to dry for 24 hours. After the samples were collected, the concentration of n-hexadecane was detected by gas chromatography.

[0088] like Figure 6 As shown, the concentration of n-hexadecane was detected by gas chromatography, and the experimental results showed that the concentration of n-hexadecane pollutant in the microbial experimental group decreased with the increase of days, indicating that the petroleum hydrocarbon (diesel) mixed bacterial liquid of the present invention can utilize and degrade petroleum hydrocarbon pollutants and produce ferrous sulfide materials.

[0089] Verification Example 2:

[0090] This verification example verifies the microstructure, crystal composition and chemical bond information of the ferrous sulfide material of the present invention. The ferrous sulfide material collected in Example 2 was subjected to microscopic electron scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy characterization and analysis.

[0091] like Figure 7 and Figure 8 As shown, the ferrous sulfide material of the present invention has excellent surface properties and high dispersibility at both nanoscale and microscale perspectives.

[0092] like Fig. 9 As shown, the XRD spectrum of the ferrous sulfide material of the present invention shows characteristic peaks of Mackinawite (JCPDS file NO.15-0037) at 2θ=18.4°, 29.6°, 39.2°, 50.5° and 59.7°. Due to the low crystallinity of the microbial metabolic material, it is reflected in the XRD spectrum as blunt diffraction lines, which also shows that the ferrous sulfide prepared by the technical solution of the present invention is an iron-sulfur compound material mainly composed of Mackinawite produced by the metabolism of sulfate-reducing bacteria.

[0093] like Figure 10-12 As shown, the FTIR spectrum of the ferrous sulfide material of the present invention shows that at 3440 cm -1 There is a significant stretching vibration peak of -CH at 2947 cm -1 and 2985cm -1 It is the antisymmetric stretching vibration of -CH3 and -CH2-, 2927cm -1 The methyl vibration peak of the fatty acid of sulfate-reducing bacteria is 1442 cm-1 -CH2- bending vibration of lipids or proteins in sulfate-reducing bacteria, 1741 cm -1 and 1639cm -1 The results show that sulfate-reducing bacteria are attached to the surface of ferrous sulfide prepared by the technical scheme of the present invention, and the metabolism of sulfate-reducing bacteria is closely related to the production of ferrous sulfide materials.

[0094] It can be seen that the material prepared by the technical solution of the present invention is determined to be a ferrous sulfide material produced by the metabolism of sulfate-reducing bacteria through microscopic electron scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and other test analyses, and has the surface properties of nano-scale materials. In addition, through the above material characterization result analysis and the petroleum hydrocarbon pollutant degradation concentration analysis of Verification Example 1, it can be determined that the petroleum hydrocarbon (diesel) mixed bacterial liquid of the present invention can successfully prepare ferrous sulfide materials while having the ability to degrade petroleum hydrocarbon pollutants.

[0095] Verification Example 3:

[0096] This verification example verifies the degradation effect of ferrous sulfide materials obtained by different preparation methods on trichloroethylene (TCE).

[0097] Experimental Materials:

[0098] Bio-FeS: The ferrous sulfide material in the sulfate-reducing bacteria culture medium enriched with lactic acid in step (3) of Example 1 is collected by centrifugation according to the collection method of Example 2;

[0099] Oil-Bio-FeS: ferrous sulfide material collected in Example 2;

[0100] Abio-FeS: a purchased chemically synthesized ferrous sulfide material (purchased from: Sigma-Aldrich).

[0101] All experimental operations in this verification example were carried out in an anaerobic workstation, all experimental solutions were prepared with ultrapure water, and the three ferrous sulfide materials were deoxygenated by nitrogen for 40 minutes before being transferred to the anaerobic workstation.

[0102] Trichloroethylene degradation experiment:

[0103] (1) FeS suspension: 0.5 g Bio-FeS, 0.5 g Bio-oil-FeS and 0.5 g Abio-FeS were weighed respectively and added into 5 mL of 0.02 mol / L Bis-Tris propane buffer (0.5 mL Tris buffer + 4.5 mL deoxygenated deionized water), and the pH of the solution was maintained at pH = 7 (adjusted by HCl and NaOH) to obtain three FeS suspensions prepared by different methods.

[0104] (2) TCE stock solution: Prepare 30 mL of 200 mg / L TCE stock solution (4 μL TCE + 29.996 ml deionized water) and oscillate in a water bath to evenly disperse TCE in the solution.

[0105] (3) Degradation of trichloroethylene: 16 mL of deoxygenated deionized water (pH = 7) was added to each reaction bottle in advance, and then TCE stock solution and FeS suspension were added to the reaction bottle, so that the final concentration of Tris buffer in all experimental groups was 0.002 mol / L, the FeS concentration was 10 g / L, and the TCE concentration was 20 mg / L; then the bottle was sealed and sent to a shaker for oscillation reaction.

[0106] (4) TCE concentration determination: prepare standard substance stock solutions of trichloroethylene (TCE), 1,1-dichloroethane (1,1DCE), cis-1,2-dichloroethane (cis-1,2DCE), trans-1,2-dichloroethane (trans-1,2DCE), and vinyl chloride at concentrations of 100 mg / L and 10 mg / L, then prepare standard solutions of TCE and its intermediates at concentrations of 20 mg / L, 15 mg / L, 10 mg / L, 5 mg / L, 2 mg / L, and 1 mg / L by adding methanol to dilute the stock solutions. Use manual headspace injection to draw 100 uL of headspace gas with a gas-tight needle for gas chromatography determination.

[0107] The headspace gas is extracted from the reaction bottle by a universal gas-tight injection every 24 hours, and the TCE concentration is measured on a gas chromatograph. After the gas is extracted, the reaction bottle is quickly sealed and then returned to the shaker to continue the experimental reaction.

[0108] like Fig.13 and Fig.14As shown, the ferrous sulfide materials in the three groups of experiments all achieved more than 80% degradation of TCE. Among them, the microbial product ferrous sulfide material showed better reaction performance in degrading TCE than the chemically synthesized material. In addition, the experimental results of the two microbial materials showed that the TCE concentration decreased significantly on the first day and then rebounded slightly. This phenomenon is due to the fact that the microbial material has a high surface property and can quickly adsorb a large amount of TCE in the initial stage. As the reaction proceeds, part of the adsorbed TCE is gradually degraded, but there is also a desorption phenomenon, which causes the TCE concentration to rebound slightly. At the same time, ethylene and acetylene, the intermediate products of TCE degradation, were also detected in the TCE degradation experiment. The appearance of these products indicates that TCE has undergone specific chemical reactions during the degradation process.

[0109] like Fig.15 and Fig.16 The reaction pathways shown, the generation of ethylene and acetylene can be attributed to the hydrogenolysis reaction and β-elimination reaction of the C-Cl bond in the TCE molecule. The existence of these reaction channels further confirms that the ferrous sulfide material prepared by the present invention has a variety of electronic reaction capabilities and can effectively degrade TCE.

[0110] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A petroleum hydrocarbon mixed bacterial liquid, characterized in that: Including Desulfotomaculum with a relative abundance of 24.71%-25.28%, Bacillus with a relative abundance of 13.95%-14.77%, and Caproiciproducens with a relative abundance of 5.85%-6.50%.

2. A method for preparing a petroleum hydrocarbon mixed bacterial liquid, characterized in that: The following steps are involved: (1) activating dormant bacteria in petroleum hydrocarbon-contaminated soil to obtain an activated bacterial solution; (2) enriching the activated bacterial solution in an enrichment medium to obtain an enriched bacterial solution; (3) placing the enriched bacterial liquid in an acclimation medium for acclimation to obtain the petroleum hydrocarbon mixed bacterial liquid; the carbon source of the acclimation medium comprises at least one of gasoline, kerosene, diesel, lubricating oil, paraffin and asphalt.

3. The method for preparing a petroleum hydrocarbon mixed bacteria liquid as claimed in claim 2, characterized in that: The carbon source of the acclimation medium includes diesel.

4. The method for preparing a petroleum hydrocarbon mixed bacteria liquid as claimed in claim 2, characterized in that: The domestication comprises the following steps: placing the enriched bacterial liquid in a first domestication medium, a second domestication medium, and a third domestication medium in sequence for domestication.

5. The method for preparing a petroleum hydrocarbon mixed bacteria liquid as claimed in claim 4, characterized in that: The culture temperature of the first acclimation medium is 35°C-40°C, and the culture time is 5d-10d; the culture temperature of the second acclimation medium is 15°C-25°C, and the culture time is 5d-10d; the culture temperature of the third acclimation medium is 35°C-40°C, and the culture time is 5d-10d.

6. A petroleum hydrocarbon mixed bacterial liquid, characterized in that: Prepared by the preparation method according to any one of claims 2 to 5.

7. A bacterial agent, characterized in that: The bacterial agent comprises the petroleum hydrocarbon mixed bacterial solution according to claim 6 and / or metabolites of the petroleum hydrocarbon mixed bacterial solution.

8. The bacterial agent according to claim 7, characterized in that The metabolites include ferrous sulfide.

9. Use of the petroleum hydrocarbon mixed bacterial solution according to claim 6 and the bacterial agent according to any one of claims 7 to 8 in the bioremediation of complex organic pollutants.

10. The use according to claim 9, characterized in that The composite organic pollutants include petroleum hydrocarbons and / or chlorides.

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