Soil carbon sequestration microorganism separation culture method under medium and high petroleum pollution stress and application

Abstract

The invention discloses a soil carbon sequestration microorganism isolated culture method under medium and high petroleum pollution stress and application, and the isolated culture method comprises the following steps: dispersing medium and high petroleum pollution soil surface soil in sterile water to obtain an original bacterial suspension containing carbon sequestration microorganisms, and carrying out gradient dilution on the original bacterial suspension; respectively coating on a carbon-source-free solid culture medium for culturing, purifying a bacterial colony obtained by culturing, and carrying out step-by-step amplification culture on a single bacterial colony obtained by purifying in a carbon-source-free liquid culture medium. The method has the beneficial effects that the invention provides the method for enriching and screening the indigenous indigenous efficient carbon sequestration strains with the carbon sequestration function from the medium-high petroleum pollution soil sample, and the four strains which can survive in the medium-high petroleum pollution stress environment and have good carbon dioxide sequestration capability are obtained through the method; the strains have potential pollution reduction and carbon sequestration synergistic capability, and a new technical path can be provided for green remediation and carbon sequestration of petroleum-contaminated soil.

Description

Technical Field

[0001] The invention belongs to the technical field of bioremediation of petroleum-contaminated soil, and specifically relates to a method for isolating and culturing soil carbon-fixing microorganisms under medium or high petroleum pollution stress and an application thereof. Background Art

[0002] Global climate change is a common problem facing human society today. Soil is the largest carbon reservoir in terrestrial ecosystems, with carbon storage exceeding 80% of the global terrestrial carbon storage (about 2300Pg). Small changes in soil carbon balance will significantly affect atmospheric carbon concentration. Increasing soil carbon storage is one of the economically feasible and environmentally friendly ways to fix carbon, and is also an important measure to mitigate global climate change.

[0003] In 2017, Liang et al. proposed the concept of soil microbial carbon pump (MCP). Compared with the traditional theory of soil organic matter formation, this concept emphasizes that soil microbial assimilation and synthesis have a positive effect on soil carbon pool accumulation. Microbial residual carbon, as an important component of soil stable organic carbon pool, is of great significance to soil carbon capture and organic carbon sequestration. Biological carbon fixation has gradually become a new method for fixing CO2 due to its green economy and no secondary pollution. 2 The research focus can improve soil carbon sequestration potential and reduce atmospheric CO 2 Concentration provides new ideas.

[0004] Carbon-fixing microorganisms can be divided into autotrophic microorganisms and heterotrophic microorganisms according to their different nutritional modes. Most soil carbon-fixing microorganisms are autotrophic microorganisms, which have become a hot spot for biological carbon fixation due to their wide distribution, considerable number and strong environmental adaptability. In recent years, research on carbon-fixing microorganisms at home and abroad has mainly focused on the distribution and evaluation of carbon reserves in habitats such as oceans, rice fields, farmlands, and wetlands, the process and mechanism of microbial carbon fixation, and microalgae carbon fixation and bioenergy technology.

[0005] Carbon-fixing microorganisms refer to microorganisms that have the function of fixing carbon. They mainly achieve carbon fixation through photosynthetic CO assimilation. 2 Fixation. Soil carbon-fixing microorganisms are mostly photoautotrophic microorganisms such as cyanobacteria, photosynthetic bacteria, and microalgae, and chemoautotrophic microorganisms such as iron bacteria, sulfur bacteria, and hydrogen bacteria. Some carbon-fixing microorganisms have also been isolated from the above studies, but there are few reports on whether there are pollution-reducing and carbon-fixing microbial groups in soils under the stress of difficult-to-degrade organic matter such as petroleum hydrocarbons. Therefore, conducting research on pollution-reducing and carbon-fixing microorganisms in petroleum-contaminated soils is of great significance to soil organic carbon sequestration and mitigation of global climate change. However, there are certain difficulties in the purification, preservation, and application of strains, which are mainly reflected in how to utilize the characteristics of fast microbial reproduction, strong environmental adaptability, and short growth cycle, and to separate and screen carbon-fixing strains that are easy to culture and preserve.

[0006] In the prior art, the Chinese utility model patent "A microbial carbon fixation reaction system (CN202220654808.1)" discloses a microbial carbon fixation reaction system, including a water quality and quantity adjustment pool and a microbial carbon fixation separation system, which is mainly used in the field of sewage treatment and does not involve the screening of carbon-fixing microbial strains; there are many invention patents related to the screening of petroleum-degrading microbial strains, such as "Preparation and application of a bacteria-enzyme composite preparation for petroleum hydrocarbon degradation (CN202011083706.0)", "A halophilic petroleum hydrocarbon-degrading bacterium and its application (CN202110482736.7)", "A high-salt degradation wastewater COD strain and screening method and application (CN202110976522.5)", etc. However, the above patents all focus on the degradation of pollutants such as petroleum hydrocarbons, but there are few reports on the screening of carbon-fixing microorganisms under the stress of petroleum pollution. Summary of the invention

[0007] In view of the above problems, the present invention provides a method for isolating and culturing soil carbon-fixing microorganisms under medium-to-high petroleum pollution stress and its application. By adopting a technical means combining environmental self-screening with alternating illumination and shading with bioinformatics, four strains that are easy to culture and preserve and have high carbon fixation efficiency are separated and screened. This isolation and cultivation method is of great significance for conducting research on pollution reduction and carbon-fixing microorganisms in petroleum-contaminated soil and green remediation of petroleum-contaminated soil.

[0008] The first aspect of the present invention provides a method for isolating and culturing soil carbon-fixing microorganisms under the stress of medium-to-high petroleum pollution, comprising: (1) dispersing the surface soil of medium-to-high petroleum pollution in sterile water to obtain an original bacterial suspension containing carbon-fixing microorganisms; (2) gradient diluting the original bacterial suspension containing carbon-fixing microorganisms, respectively applying them on a solid culture medium without a carbon source, and culturing them by first culturing under light and then culturing in the dark; purifying the colonies obtained by culture to obtain a plurality of single colonies; (3) using each single colony as a starting strain, respectively, and performing step-by-step expansion culture in a liquid culture medium without a carbon source to obtain a bacterial solution of each single colony, thereby completing the isolation and cultivation of soil carbon-fixing microorganisms under the stress of medium-to-high petroleum pollution.

[0009] As a specific embodiment of the present invention, in step (1), the topsoil of medium-high petroleum contaminated soil is added with sterile water, and the mixture is thoroughly mixed and then shaken; preferably, the mass volume ratio of the topsoil of medium-high petroleum contaminated soil to sterile water is 10 g:90 mL; preferably, the mixture is shaken on a shaker at 30°C and 150 r / min for 30 to 40 min.

[0010] As a specific embodiment of the present invention, in step (2), the gradient dilution is to dilute to 10% of the original bacterial suspension concentration. -5 , 10-6 , 10 -7 and 10 -8 times.

[0011] As a specific embodiment of the present invention, in step (2), the cells are first cultured in an inverted state at 30-35°C under light for 12 hours and then cultured in the dark for 12 hours; preferably, the intensity of the light is 4000-7000 lux.

[0012] As a specific embodiment of the present invention, in step (2), the purification is to pick different single colonies from the cultured colonies, repeatedly streak them on a carbon source-free solid culture medium, and culture them under light conditions; preferably, streaking is performed 4 to 6 times; preferably, the intensity of the light is 4000 to 7000 lux.

[0013] As a specific embodiment of the present invention, in step (2), the carbon source-free solid culture medium comprises the following components in the following concentrations: 20 g / L agar, 0.02 g / L manganese sulfate, 1 g / L disodium hydrogen phosphate, 1.8 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate, 0.05 g / L calcium chloride, 1 g / L sodium chloride, 0.5 g / L ammonium chloride, 0.02 g / L ferric chloride, 10 g / L sodium thiosulfate, and the balance is water, pH 7-8.

[0014] As a specific embodiment of the present invention, in step (3), the carbon source-free liquid culture medium includes the following components in the following concentrations: manganese sulfate 0.02 g / L, disodium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1.8 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.05 g / L, sodium chloride 1 g / L, ammonium chloride 0.5 g / L, ferric chloride 0.02 g / L, sodium thiosulfate 10 g / L, and the balance is water, pH 7-8.

[0015] The second aspect of the present invention provides a carbon-fixing bacterial strain combination, comprising: Pseudomonas sp. CQ-1, Rhizobium petrolearium DG-1, Pseudomonas kunmingensis DG-2 and Bacillus cereus CQ-2, all strains were deposited in the China Center for Type Culture Collection on July 10, 2023, and the preservation address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation numbers are: CCTCC NO: M 20231235, CCTCC NO: M 20231236, CCTCCNO: M 20231237, CCTCC NO: M 20231238; the carbon-fixing strain combination was isolated and cultured by the above-mentioned method.

[0016] The third aspect of the present invention provides a method for improving the carbon fixation capacity of petroleum contaminated soil, comprising: mixing the petroleum contaminated soil with the aforementioned carbon fixation strain combination in a certain proportion, adding water to maintain a certain moisture content, and culturing statically; preferably, the mass ratio of the petroleum contaminated soil to the carbon fixation strain combination is 100:1; and / or, adjusting the moisture content to 20-25%; and / or, culturing at 25±5°C for more than 60 days.

[0017] A fourth aspect of the present invention provides an application of the aforementioned separation and culture method. Specifically, the separation and culture method is used to study pollution-reducing and carbon-fixing microorganisms in petroleum-contaminated soil.

[0018] The beneficial effects of the present invention are: The present invention provides a method for enriching and screening original indigenous high-efficiency carbon-fixing bacterial strains with carbon fixation function from medium- and high-petroleum-contaminated soil samples, and through this method, four bacterial strains are obtained that can survive in medium- and high-petroleum pollution stress environments and have good carbon dioxide fixation capabilities. These strains have potential synergistic capabilities for pollution reduction and carbon fixation, and can provide a new technical path for green remediation and synergistic carbon fixation in petroleum-contaminated soils. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1A This is the bacterial culture condition under light culture conditions; Figure 1B This is the culture condition of bacteria under light-shielding culture conditions; Figure 1C It is the culture status of bacteria under the alternating light-shading culture conditions; Figure 2 For the present invention Pseudomonas sp. CQ-1 plate colony morphology; Figure 3 For the present invention Rhizobium petrolearium DG-1 plate colony morphology; Figure 4 For the present invention Pseudomonas kunmingensis Colony morphology on DG-2 plates; Figure 5 For the present invention Bacillus cereus CQ-2 plate colony morphology; Figure 6 Based on 16S rRNA gene sequence homology Pseudomonas sp. CQ-1 phylogenetic tree; Figure 7 Based on 16S rRNA gene sequence homology Rhizobium petrolearium DG-1 phylogenetic tree; Figure 8 Based on 16S rRNA gene sequence homology Pseudomonas kunmingensis DG-2 phylogenetic tree; Figure 9 Based on 16S rRNA gene sequence homology Bacillus cereus CQ-2 phylogenetic tree; Figure 10 Comparison of RubisCO enzyme activity in soil samples after applying carbon-fixing bacteria solution in the present invention; wherein: bacteria 1: Pseudomonas sp. CQ-1; bacteria 2: Rhizobium petrolearium DG-1; bacteria 3: Pseudomonas kunmingensis DG-2; bacteria 4: Bacillus cereus CQ-2. DETAILED DESCRIPTION

[0021] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0022] 1. Sample source Soil samples were taken from the surface soil (0-20 cm) of a well site in an oil field in North China. About 500 g of soil sample was scooped with a sterile iron shovel and placed in a sterile sealed bag. After removing animal and plant remains and gravel, the sample was placed in a 4 ℃ ice box and transported back to the laboratory for further processing.

[0023] 2. Determination method of total petroleum hydrocarbons in soil The determination of total petroleum hydrocarbons in soil was carried out according to the "Gas Chromatographic Method for the Determination of Petroleum Hydrocarbons (C10-C40) in Soil and Sediment" (HJ1021-2019). Weigh 10 g of soil sample in a mortar, add an appropriate amount of diatomaceous earth and grind it into loose particles. After grinding, the sample was extracted using a rapid solvent extractor. The extraction conditions were: pressure 1200 psi, extraction temperature 100 °C, static extraction 5 min, elution volume 60%, nitrogen purge 60 s, cycle 2 times, and the extractant was acetone-n-hexane mixed solvent (chromatographic grade, volume ratio 1:1). The extract was washed twice with 100 mL of ultrapure water, and the upper organic phase was retained. After dehydration with anhydrous sodium sulfate (superior grade), it was concentrated to 1.00 mL by rotary evaporation to obtain the sample to be tested.

[0024] A gas chromatograph was used to determine the TPH of the sample to be tested. The detector was a flame ionization detector, and the chromatographic column was a capillary column (DB-5MS, Agilent Technologies; column length 30 mm × inner diameter 0.32 mm × film thickness 0.25 μm). Instrument conditions: injection port temperature 300 ℃, detector temperature 325 ℃, column temperature 320 ℃. Temperature program: initially 50 ℃ for 2 min, heated to 230 ℃ at 40 ℃ / min, heated to 320 ℃ at 20 ℃ / min and held for 20 min; gas flow rates were 99.999% high-purity nitrogen 1.5 mL / min, 99.99% high-purity hydrogen 30 mL / min, and air 300 mL / min; splitless injection, injection volume 1 μL.

[0025] The soil samples were numbered S1 to S8 according to the total petroleum hydrocarbon content from low to high. The total petroleum hydrocarbon content is shown in Table 1.

[0026] Table 1 Total petroleum hydrocarbon content in soil samples

[0027] 3. Culture medium 3.1 Carbon-free liquid medium The carbon-free liquid culture medium is used for carbon-fixing microorganism cultivation, and the medium formula is shown in Table 2. The preparation method is to add each component drug into ultrapure water, stir evenly, adjust the pH to 7-8, and sterilize by high temperature wet method at 121 °C for 15 min.

[0028] Table 2 Carbon-free liquid culture medium formula

[0029] 3.2 Carbon-fixing medium without carbon source The solid culture medium is the original carbon-free liquid culture medium with 20-25 g / L agar powder added. The pH of the culture medium is adjusted to 7-8. The preparation method is to add each component to ultrapure water, stir evenly, and sterilize by high temperature wet sterilization at 121°C for 15 min.

[0030] 4. Identification of carbon-fixing strains The 16S rDNA gene of the isolated and screened carbon-fixing strains was amplified. The upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and the downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying the Barcode sequence were used to PCR amplify the variable region of the 16S rRNA gene V3~V4. The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5uM), 0.8 μL of downstream primer (5uM), 0.4 μL of TransStart FastPfu DNA polymerase, and 10 ng of template DNA, which was supplemented to 20 μL. The amplification program was as follows: pre-denaturation at 95 °C for 3 min, 27 cycles (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s), followed by stable extension at 72 °C for 10 min, and finally storage at 4 °C.

[0031] 5. RubisCO enzyme activity assay 5.1 Cultivation of carbon-fixing bacteria in soil Surface soil with medium to high petroleum pollution (0-20 cm, TPH concentration 200-1000 mg / kg) was collected, and after removing stones, plant roots, etc., it was air-dried and passed through a 2 mm sieve. The culture soil was placed in plastic pots, with each pot containing about 5-6 kg of soil. Four carbon-fixing bacteria solutions were added with water (the addition ratio was 0.5-1.5 mL / kg of the original solution of the bacteria solution), and the solution was supplemented every 15-20 days. Water was added to the soil regularly (the moisture content of the culture soil was kept at about 25%), and the RubisCO enzyme activity in the soil was measured after the culture was completed.

[0032] 5.2 RubisCO enzyme activity assay RubisCO enzyme activity was determined using the soil (Soil) 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) ELISA kit (Shanghai Ruifan Biotechnology Co., Ltd.). The determination method strictly followed the instructions of the kit, as follows: (1) Setting standard wells: Take a 96-well ELISA plate and set 6 standard wells. Add 50 μL of standard to each well. The concentration gradient is 0 U / L, 5 U / L, 10 U / L, 20 U / L, 40 U / L, and 80 U / L. Each gradient has 3 parallels. (2) Sample addition: Set 12 sample wells and 1 blank well. Add 10 μL of the sample to be tested to the sample well and then add 40 μL of sample diluent. Set 3 parallels. (3) Incubation: Add 100 μL of detection antibody labeled with Horseradish Peroxidase (HRP) to both the standard well and the sample well. Seal the plate with a sealing film and incubate at 37 °C for 60 min. The blank wells follow the same steps except that no sample or enzyme-labeled reagent is added. (4) Washing: Inject 350 μL of washing solution into each well, soak for 1 min, and then wash the plate 5 times in an automatic plate washer. (5) Color development: Add 50 μL of substrate A and B to each well and incubate at 37 °C in the dark for 15 min. (6) Determination: After adding 50 μL of stop solution, measure the absorbance (OD value) of each well at 450 nm within 15 min. (7) Calculate the RubisCO enzyme activity value of the sample: Draw a linear regression curve of the standard with the concentration of the standard as the horizontal axis and the corresponding OD value as the vertical axis. Calculate the concentration value of each sample according to the curve equation to obtain the RubisCO enzyme activity value of the sample.

[0033] Example 1 Isolation and cultivation of indigenous carbon-fixing bacteria from highly petroleum-contaminated soil This embodiment provides a method for isolating and culturing soil carbon fixation microorganisms under medium to high petroleum pollution stress, comprising the following steps: (1) Take 10 g of topsoil from medium-to-high petroleum-contaminated soil (TPH content 200 mg / kg-1000 mg / kg), remove impurities such as plant roots and stems, add to a conical flask containing 90 mL of sterile water, mix thoroughly, and place on a shaker at 30 °C and 150 rpm for 30-40 min to obtain a bacterial suspension. (2) The bacterial suspension was aseptically diluted in a clean bench to obtain a diluted bacterial suspension; the dilution gradients were 10 -5 , 10 -6 , 10 -7 , 10 -8 ; (3) Take 1-2 mL of the diluted bacterial suspension and inject it into sterile culture dishes containing carbon-free solid culture medium for coating. Cultivate the strains at 30-35 ℃ using three methods: pure illumination, pure shading, and alternating illumination and shading (inverted culture for 12 hours, then inverted culture for 12 hours under shading). Among them, the number of bacterial strains in the culture dish with the illumination-shading alternating culture method is the largest (as shown in Figure 1). According to the small shape, color, transparency and other characteristics of the colonies after the illumination-shading alternating culture, select several different single colonies, streak them on the carbon-free solid culture medium, and activate and culture for 24-48 hours to obtain the activated strains; (4) After 3 to 5 streaking and smearing cultures, the activated strains are cultured until complete single colonies with a diameter of 1 to 5 mm are seen, which are the activated single strains. The purified single colonies are picked and preserved; (5) The activated single strain was cultured on a shaker at 150 r / min to obtain a seed solution with an OD600 of 1 to 1.5; (6) The seed solution is transferred to the expansion culture medium according to a step percentage of 8-10%, and after expansion culture, a carbon-fixing bacteria solution is obtained.

[0034] Example 2 Identification and preservation of strains The strains were preliminarily identified based on their Gram color reaction results, morphological characteristics, and physiological properties.

[0035] strain Pseudomonas sp. The colony morphology of CQ-1 plate is as follows Figure 2 Its main biological characteristics are as follows: on the solid plate without carbon source, the colonies are round, white, with raised, rough, moist and opaque surfaces, Gram-positive, negative for methyl red reaction, negative for amylase hydrolysis, no lactic acid production, and no lactic acid gas production.

[0036] Rhizobium petrolearium The colony morphology of DG-1 plate is as follows Figure 3 Its main biological characteristics are as follows: on the solid plate without carbon source, the colonies are round, white, smooth, moist and opaque without protrusions, Gram-positive, negative for methyl red reaction, negative for amylase hydrolysis, no lactic acid production, no lactic acid gas production.

[0037] Pseudomonas kunmingensis The colony morphology of DG-2 plate is as follows Figure 4 Its main biological characteristics are as follows: on the solid plate without carbon source, the colonies are round, white, with raised, smooth, moist and opaque surface, Gram-positive, methyl red-negative, amylase-positive, lactic acid-free, lactic acid-free and lactic acid-free.

[0038] Bacillus cereus The colony morphology of CQ-2 plate is as follows Figure 5Its main biological characteristics are as follows: on the solid plate without carbon source, the colonies are round, white, with raised, smooth, moist and opaque surface, Gram-positive, negative for methyl red reaction, negative for amylase hydrolysis, no lactic acid production, no lactic acid gas production.

[0039] Identification of strain 16S rDNA: Single colonies of four strains, CQ-1, DG-1, DG-2, and CQ-2, were taken for PCR sequencing, using common references. The PCR reaction system was: 4 μL of 5×TransStart FastPfu buffer, 2μL of 2.5 mM dNTPs, 0.8 μL of upstream primer (5uM), 0.8 μL of downstream primer (5uM), 0.4 μL of TransStart FastPfu DNA polymerase, 10 ng of template DNA, and made up to 20 μL. The amplification procedure was as follows: pre-denaturation at 95 °C for 3 min, 27 cycles (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s), and then stable extension at 72 °C for 10 min. Then the 16S rDNA gene sequence was sequenced and compared with the NCBI database, showing that CQ-1 and pseudomonas sp. The homology of DG-1 and Rhizobium petrolearium The homology of DG-2 and Pseudomonas kunmingensis The homology of CQ-2 and Bacillus cereus The homology of the four strains was 99.927%, and the four strains were identified as Pseudomonas species, Rhizobium petroleum species, Pseudomonas kunmingensis species, and Sword species. The phylogenetic tree of the four strains was constructed based on the homology of the 16S rRNA gene sequence. Figures 6 - 9 shown.

[0040] The four carbon-fixing bacteria screened from medium and high petroleum contaminated soils were all deposited in the China Center for Type Culture Collection on July 10, 2023, with the address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with strain deposit numbers: CCTCC NO: M20231235, CCTCC NO: M 20231236, CCTCC NO: M 20231237, and CCTCC NO: M 20231238.

[0041] Example 3: Verification of carbon fixation effect of bacterial strains The carbon fixation effect of the bacterial agents prepared from the above four strains of indigenous carbon fixation bacteria in medium- and high-petroleum contaminated soils was evaluated in the following steps: 1. The petroleum-contaminated soil with an oil content of 5.33±0.59% and the four carbon-fixing bacteria prepared in Example 1 (purified single colonies were selected for preservation) were mixed evenly at a mass ratio of 100:1, and the moisture content of the bacteria-soil mixture was adjusted to 20-25% with sterile distilled water. At this time, the concentration of live bacteria was 5.2×10 8 CFU / mL, 4.9×10 8 CFU / mL, 5.5×10 8 CFU / mL, 5.1×10 8 CFU / mL.

[0042] 2. Keep the moisture content of the bacterial soil mixture at 20-25%, the room temperature at 25±5℃, and apply bacterial solution every 15-20 days. Regularly add water to the soil and cultivate for 60 days.

[0043] After the test, RubisCO enzyme enzyme-linked immunosorbent assay was used to measure the RubisCO enzyme activity in the soil. The results showed that the concentrations of the four carbon-fixing bacteria were 3.5×10 8 CFU / mL, 3.1×10 8 CFU / mL, 3.7×10 8 CFU / mL, 3.2×10 8 CFU / mL, indicating that the petroleum-contaminated soil had little effect on the growth of the four carbon-fixing bacteria. After 60 days of cultivation, the four bacteria could maintain strong growth activity. Figure 10 As shown, the RubisCO enzyme activity of soil samples after adding the four carbon-fixing bacteria solution was significantly higher than that of soil without adding the bacteria solution.

[0044] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for isolating and culturing soil carbon fixation microorganisms under medium and high petroleum pollution stress, characterized in that: The separation and culture method comprises: (1) Dispersing the topsoil of medium- and high-petroleum contaminated soil in sterile water to obtain an original bacterial suspension containing carbon-fixing microorganisms; (2) diluting the original bacterial suspension containing carbon-fixing microorganisms in a gradient manner, applying the diluted suspension on a solid culture medium without a carbon source, culturing the suspension in a light-induced culture followed by a dark-induced culture, purifying the colonies obtained by the culture, and obtaining a plurality of single colonies; (3) Using each single colony as the starting strain, the strain is gradually expanded and cultured in a carbon-free liquid culture medium to obtain a bacterial liquid of each single colony, thereby completing the isolation and culture of soil carbon fixation microorganisms under medium-to-high petroleum pollution stress.

2. The method for isolating and culturing soil carbon fixation microorganisms under medium-high petroleum pollution stress according to claim 1, characterized in that: In step (1), the topsoil of medium-high petroleum contaminated soil is added with sterile water, mixed thoroughly and then shaken; Preferably, the mass volume ratio of the medium-to-high petroleum contaminated topsoil to sterile water is 10 g:90 mL; Preferably, the mixture is shaken at 30°C and 150 rpm for 30-40 min.

3. The method for isolating and culturing soil carbon fixation microorganisms under medium and high petroleum pollution stress according to claim 1, characterized in that: In step (2), the gradient dilution is to dilute to 10% of the original bacterial suspension concentration. -5 , 10 -6 , 10 -7 and 10 -8 times.

4. The method for isolating and culturing soil carbon fixation microorganisms under medium and high petroleum pollution stress according to claim 1, characterized in that: In step (2), the culture is performed at 30-35°C, in an inverted state, first under light for 12 h, and then in dark for 12 h; preferably, the intensity of the light is 4000-7000 lux.

5. The method for isolating and culturing soil carbon fixation microorganisms under medium-high petroleum pollution stress according to claim 1, characterized in that: In step (2), the purification is to pick different single colonies from the cultured colonies, repeatedly streak them on a carbon source-free solid culture medium, and culture them under light conditions; preferably, streaking is performed 4 to 6 times; preferably, the intensity of the light is 4000 to 7000 lux.

6. The method for isolating and culturing soil carbon fixation microorganisms under medium-high petroleum pollution stress according to claim 1, characterized in that: In step (2), the carbon-free solid culture medium comprises the following components in the following concentrations: 20 g / L agar, 0.02 g / L manganese sulfate, 1 g / L disodium hydrogen phosphate, 1.8 g / L potassium dihydrogen phosphate, 0.4 g / L magnesium sulfate, 0.05 g / L calcium chloride, 1 g / L sodium chloride, 0.5 g / L ammonium chloride, 0.02 g / L ferric chloride, 10 g / L sodium thiosulfate, and the balance is water, pH 7-8.

7. The method for isolating and culturing soil carbon fixation microorganisms under medium-high petroleum pollution stress according to claim 1, characterized in that: In step (3), the carbon source-free liquid culture medium comprises the following components in the following concentrations: manganese sulfate 0.02 g / L, disodium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1.8 g / L, magnesium sulfate 0.4 g / L, calcium chloride 0.05 g / L, sodium chloride 1 g / L, ammonium chloride 0.5 g / L, ferric chloride 0.02 g / L, sodium thiosulfate 10 g / L, and the balance is water, pH 7-8.

8. A carbon-fixing bacterial strain combination, characterized in that: The carbon-fixing strain combination comprises: Pseudomonas sp. CQ-1, Rhizobium petrolearium DG-1, Pseudomonas kunmingensis DG-2 and Bacillus cereus CQ-2, the preservation numbers of each strain are: CCTCC NO: M 20231235, CCTCC NO: M 20231236, CCTCC NO: M20231237, CCTCC NO: M 20231238; the carbon-fixing strain combination is isolated and cultured by the method according to any one of claims 1 to 7.

9. A method for improving the carbon sequestration capacity of petroleum-contaminated soil, characterized in that: The method comprises: mixing the petroleum-contaminated soil with the carbon-fixing bacterial strain combination according to claim 8 in a certain proportion, adding water to maintain a certain moisture content, and culturing statically; Preferably, the mass ratio of the petroleum-contaminated soil to the carbon-fixing bacterial strain combination is 100:1; Preferably, the moisture content is adjusted to 20-25%; Preferably, the culture is carried out at 25±5°C for more than 60 days.

10. Use of the method according to any one of claims 1 to 7, characterized in that: The application is to study the pollution reduction and carbon fixation microorganisms in petroleum-contaminated soil.

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