Efficient carbon sequestration Brucella sourced from oil-containing solid waste residues and application of efficient carbon sequestration Brucella

Abstract

The invention discloses efficient carbon sequestration Brucella from an oil-containing solid waste residue source and application of the efficient carbon sequestration Brucella. The Brucella is Brucella sp A54-5 with the preservation number of CCTCC (China Center for Type Culture Collection) NO: M20241650. The Brucella is obtained through ARTP mutagenesis and screening by taking Brucella Brucella sp A54 separated from oil-containing solid waste residues of an oil field as a starting strain, has excellent characteristics of reducing pollution and sequestration carbon and relatively strong stress resistance, and has the advantages of deep recycling (soil transformation) of the oil-containing solid waste residues, improvement of the carbon sequestration capacity of petroleum-contaminated soil and improvement of the oil-containing solid waste residues. The method has a wide application prospect in the fields of petroleum-polluted soil remediation and the like, and has great significance in pollution reduction and convergence increase in petroleum and petrochemical industries under the background of supporting double carbon.

Description

Technical Field

[0001] The invention belongs to the technical field of solid waste resource utilization, and particularly relates to a high-efficiency carbon-fixing Brucella derived from oil-containing solid waste residue and application thereof. Background Art

[0002] The oil and gas industry is a traditional "big carbon emitter". According to the "2022 Carbon Dioxide Emissions Report" released by the International Energy Agency, global carbon dioxide emissions are about 36.8 billion tons / year, of which oil and gas emit about 11.3 billion tons / year, accounting for nearly 1 / 3 of the world's total carbon emissions. Carbon emissions are significantly linked to global warming. At present, major oil companies around the world have successively proposed "carbon neutrality goals" and corresponding carbon emission reduction measures, mainly including optimizing industrial structure, reducing direct carbon emissions, clean energy substitution, carbon capture and storage, and ecosystem carbon sinks.

[0003] Microbial carbon fixation is the process in which microorganisms absorb inorganic carbon and convert it into biomass. It is an important way for inorganic carbon in nature to be converted into organic carbon and enter the biosphere. It can reduce the content of carbon dioxide in the atmosphere and is of great significance for mitigating the atmospheric "greenhouse effect" and alleviating global warming. Carbon-fixing microorganisms can be divided into autotrophic microorganisms and heterotrophic microorganisms according to their different nutritional methods. Autotrophic microorganisms use CO2 as the only carbon source and use photosynthesis or chemosynthesis to convert CO2 into their own cell substances to achieve photosynthetic CO2 fixation; heterotrophic microorganisms use organic carbon compounds as carbon sources and achieve carbon fixation through carboxylation reactions in their own metabolic processes. Most soil carbon-fixing microorganisms are autotrophic microorganisms, which have become a hot spot for biological carbon fixation due to their strong environmental adaptability, rapid growth, and rapid reproduction. CN109536402A discloses Arthrobacter sp. with carbon fixation function screened from the soil on the southwestern edge of the Maowusu Sandy Land in Inner Mongolia, CN109182205A discloses Rhodococcus sp. with carbon fixation function screened from the above soil, and CN115851564A discloses that two recombinant bacteria that fix carbon dioxide were constructed by overexpressing phosphoenolpyruvate carboxykinase and acetyl-CoA carboxylase in Bacillus licheniformis, respectively. CN106967662A discloses that a recombinant vector expressing acetyl-CoA carboxylase gene accADBC, malonyl-CoA reductase gene mcr, propionyl-CoA synthase gene pcs, propionyl-CoA carboxylase gene pcc, methylmalonyl-CoA isomerase gene mcE, methylmalonyl-CoA mutase gene mcM, and succinyl-CoA synthetase gene sucCD is transformed into a host cell to obtain a recombinant bacterium that fixes carbon dioxide and synthesizes succinic acid. At present, carbon-fixing microorganisms are mainly isolated from unperturbed or low-pollution environments such as deep sea, craters, and desert soils, or obtained by introducing artificial methods such as genetic engineering technology, and their stress resistance is poor.

[0004] In recent years, there are reports on microorganisms under the stress of oil pollution. CN108996707A discloses that various strains that can form dominant bacterial communities are formulated into efficient microbial preparations for polycyclic aromatic hydrocarbons in groundwater of oil-contaminated sites to accelerate the degradation of polycyclic aromatic hydrocarbons by microorganisms. CN109576186A discloses that the highly efficient microbial preparations formed with oil-contaminated soil / groundwater as the research object have good degradation effects on polycyclic aromatic hydrocarbons; CN117106608A discloses a mutagenized amyloliquefaciens and its application in the degradation of petroleum hydrocarbons; CN114752517A discloses a paralicheniformis ARTP rapid mutagenesis oil degradation and its preparation method and application, which adopts the ARTP rapid mutagenesis method to carry out directed evolution of petroleum hydrocarbon degradation strains, and the mutagenized bacteria have strong environmental adaptability and high degradation efficiency, and can be used as a bioenhancer. In the above disclosed technologies, these microorganisms can be applied to oil-contaminated environments to improve stress resistance, but none of them have carbon fixation effects.

[0005] Therefore, the prior art lacks relevant reports on microorganisms that have strong stress resistance and excellent properties of pollution reduction and carbon fixation. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a highly efficient carbon-fixing Brucella derived from oil-containing solid waste residues, which is a new carbon-fixing microorganism with strong environmental resistance and is easy to separate, purify and preserve, and is suitable for deep resource conversion of oil-containing solid waste residues.

[0007] One aspect of the present invention provides a high-efficiency carbon-fixing Brucella derived from oil-containing solid waste residue, which is Brucella sp. A54-5 with a preservation number of CCTCC NO: M20241650.

[0008] Another aspect of the present invention provides a fermentation culture of the efficient carbon-fixing Brucella derived from the aforementioned oil-containing solid waste residue.

[0009] Another aspect of the present invention provides a bacterial agent containing the highly efficient carbon-fixing Brucella or fermentation culture derived from the aforementioned oil-containing solid waste residue.

[0010] Another aspect of the present invention provides the use of the highly efficient carbon-fixing Brucella or fermentation culture or bacterial agent derived from the aforementioned oil-containing solid waste residue in pollution reduction and carbon fixation.

[0011] Another aspect of the present invention provides the use of the aforementioned highly efficient carbon-fixing Brucella or fermentation culture or bacterial agent derived from the oil-containing solid waste residue in the deep resource conversion of the oil-containing solid waste residue.

[0012] Another aspect of the present invention provides a method for pollution reduction and carbon fixation based on microorganisms, comprising: mixing and culturing oily solid waste residues with efficient carbon-fixing Brucella or fermentation culture or bacterial agent derived from the aforementioned oily solid waste residues.

[0013] According to a specific embodiment of the present invention, the oil-containing solid waste residue is oil-containing solid waste residue with an oil content of less than 2% after being treated by physical and chemical methods.

[0014] According to a specific embodiment of the present invention, the physical and chemical treatment includes but is not limited to hot water washing and pyrolysis treatment.

[0015] According to a specific embodiment of the present invention, the culture temperature is 31° C. and the culture time is 5-7 days.

[0016] The beneficial effects of the present invention are:

[0017] The Brucella provided by the present invention is obtained by ARTP mutagenesis and screening using Brucella sp. A54 isolated from oil-containing solid waste residues in oil fields as a starting strain. The Brucella sp. A54 has excellent characteristics of pollution reduction and carbon fixation and has strong stress resistance. It has broad application prospects in the fields of deep resource utilization (soilization) of oil-containing solid waste residues, improving the carbon sink capacity of petroleum-contaminated soil, and repairing petroleum-contaminated soil. It is of great significance to support pollution reduction and carbon sink increase in the petroleum and petrochemical industry under the background of "dual carbon". BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a Gram staining photo of strain A54;

[0019] Figure 2 This is a Gram staining photo of strain A54-5;

[0020] Figure 3 The results of the RubisCO enzyme activity test;

[0021] Figure 4 These are the test results of the effect of strains on the RubisCO enzyme activity of oily solid waste residues. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Example 1 Strain Isolation, Purification and Screening

[0024] 1. Collection of oily solid waste residue samples

[0025] We selected Liaohe Oilfield, Daqing Oilfield and other major oilfields in my country for oil and gas development to collect oil-containing solid waste residues after hot water washing and pyrolysis treatment, and obtained a total of 12 samples. We used sterile iron shovels to scoop about 500g of the residue samples and put them into sterile sealed bags. After removing animal and plant residues and gravel, we put them into 4℃ ice boxes and transported them back to the laboratory for further processing.

[0026] 2. Culture medium preparation

[0027] 2.1 Carbon source-free inorganic salt liquid culture medium, as shown in Table 1:

[0028] Table 1 Carbon-free inorganic salt liquid culture medium formula

[0029]

[0030] 2.2 Carbon-free inorganic salt solid culture medium

[0031] Add 20-25 g / L agar powder to the formula of the carbon-free inorganic salt liquid culture medium. The pH of the culture medium is adjusted to 7-8. The preparation method is to add each component into ultrapure water, stir evenly, and sterilize by high temperature wet method at 121°C for 15 minutes.

[0032] 3. Strain isolation, purification and screening

[0033] Take 1g of soil from each of the 12 samples collected, mix them evenly, put them into a triangular flask containing 100mL of sterile water, mix them thoroughly, place them in a shaker at 30℃ and 150r / min for about 20min, and make a soil suspension, which is then diluted in a gradient. -4 , 10 -5 , 10 -6 Concentration, respectively aspirate 10 μL of soil suspension of different dilution concentrations and inject it into a sterile culture dish containing a carbon source-free inorganic salt solid culture medium, spread it evenly, and culture it upside down at 35°C for 3-5 days. Pick a single colony and streak it for isolation and purification for multiple times. After the purified single colony is expanded and preserved, the original strain is obtained, and ARTP mutagenesis and screening are performed.

[0034] ①ARTP mutagenesis

[0035] a) Inoculate the purified single colony into an LB medium shake flask for overnight culture. After centrifugation, wash both sides with sterile water and then dilute with sterile water (add 5% glycerol as a humectant) to OD600 = 0.6.

[0036] b) Place the slide in the clean bench under the flame of an alcohol lamp and burn for 30 seconds. After cooling, place it in a sterilized culture dish and evenly spread 10 μL of the diluted bacterial solution on the surface of the slide.

[0037] c) Move the culture dish containing the sample slide to the operation room of the ARTP mutagenesis breeding instrument and place the slide into the corresponding groove using sterile tweezers.

[0038] d) Adjust the mutagenesis parameters (distance 2 mm; treatment power 120 W; gas flow 10 SL) and treatment time (0 s, 30 s, 40 s, 60 s, 80 s, 100 s) and start processing the samples after the settings are completed.

[0039] e) After all samples have been processed, use sterile tweezers to place the slides into EP tubes containing 1 mL of sterile water.

[0040] f) Place the EP tube on an oscillator and oscillate for 1 minute to elute the microorganisms attached to the slide into a sterile solution to form a new bacterial suspension.

[0041] g) Dilute the new bacterial suspension appropriately [time: dilution factor (0s:10 1 ,10 2 ,10 3 ; 30s:10 1 ,10 2 ,10 3 ; 40s:10 1 ,10 2 ,10 3 ; 60s:10 1 ,10 2 ,10 3 ; 80s:10 1 ,10 2 ,10 3 ; 100s:10 1 ,10 2 ,10 3 )], take 100 μL of the dilution and spread it on a carbon source-free inorganic salt solid culture medium for screening.

[0042] ② Screening

[0043] a) Place the coated plate in a 30°C incubator for 4-6 days, and select colonies with larger diameters for enrichment in a carbon-free inorganic salt solid culture medium.

[0044] b) Different bacterial colonies were inoculated into LB test tubes for overnight culture, and then the bacterial solution was diluted to the same concentration and inoculated into test tubes of carbon source-free inorganic salt liquid culture medium for 4-6 days.

[0045] c) Dilute the culture solution to different concentrations and drop it onto a carbon-free inorganic salt solid culture medium to observe the growth.

[0046] d) Continue to culture the bacteria with good growth with LB, wash, dilute to different concentrations and drop them on a carbon source-free inorganic salt solid culture medium, select the bacteria with good growth, culture and preserve.

[0047] The final screened mutant strain of Brucella with the best growth potential (large colony diameter) was sample numbered A54-5, which was deposited in the China Center for Type Culture Collection (CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan, China, Postal Code 430072) on July 22, 2024, with the deposit number CCTCC NO: M 20241650.

[0048] Example 2 Strain Identification

[0049] The original strain and the induced strain were placed in a carbon-free inorganic salt liquid culture medium respectively, and cultured at 35°C and 150 r / min on a shaker for 7-10 days. When the bacterial solution became turbid, 4 mL of the bacterial solution was taken for strain identification. The strain identification platform was provided by Shanghai Meiji Biotechnology Co., Ltd.

[0050] 1. Morphological characteristics of strains

[0051] Gram staining microscopic test: Take a clean glass slide, bake the part where the bacteria are smeared over a flame to remove grease. Take a small amount of bacterial solution and evenly apply it on the glass slide. After it is naturally dried, the bacterial film faces upward, and the glass slide is quickly passed back and forth through the flame 2-3 times to fix the bacteria. The staining reagent is a Gram staining kit (Changde Beekman Biotechnology Co., Ltd.), and ammonium oxalate crystal violet solution is added to the fixed bacteria. The initial staining is 1 minute, and the remaining staining solution is washed off with distilled water. After drying, 1 drop of iodine solution is added, and the mordanting is 1 minute. The remaining staining solution is washed off with distilled water. After drying, 95% ethanol is added to decolorize for 30 seconds, and then washed with water. Finally, add a drop of safflower red staining solution for 1 minute, and the remaining staining solution is washed off with distilled water. Microscopic examination can be performed after natural drying.

[0052] 2. Physiological and biochemical characteristics of strains

[0053] Starch hydrolysis test: Divide the plate containing starch solid culture medium (3g peptone, 1.5g NaCl, 1.5g beef extract, 0.76g soluble starch and 6g agar into 300mL distilled water, adjust pH to 7.6, and sterilize at 121℃ for 20min) into three parts, use an inoculation loop to streak the experimental strains in different parts, mark the strain names with a marker, and invert the plate in a 37℃ biochemical incubator for 24h; observe the growth of various bacteria, drop a small amount of Lugol's iodine solution into the plate, and gently rotate the plate to evenly cover the entire plate with iodine solution. Methyl red test reference: Inoculate the experimental strains into glucose peptone medium (1.5g peptone, 1.5g glucose and 0.6g K2HPO4 dissolved in 1L water, adjust pH to 7.0-7.2, and sterilize at 121℃ for 20min), and culture at 37℃ with shaking for 48h. Then add 2 drops of methyl red reagent to each culture medium. If the culture medium turns red, it is positive, and if it turns yellow, it is negative.

[0054] VP experiment: After 48 hours of incubation after inoculation, add liquid A and liquid B and shake. The experimental results are as follows: compared with the blank, a red color change is positive, and no color change is negative.

[0055] Sugar alcohol fermentation: The strains were fermented with sugar alcohols, and the phenomena were observed and recorded after 20h and 46h. Sugar alcohol fermentation medium: 5g peptone, 5g beef extract, 5g yeast extract, 0.5mL Tween-80, 10g sugar or alcohol, 5-6g agar, 1000mL tap water, 1.4mL 1.6% bromocresol purple was added to adjust the pH to 6.8-7.0, divided into test tubes, and steam sterilized at 112℃ for 30min.

[0056] The above experimental results can be found in Figure 1 , Figure 2 and Table 2:

[0057] Table 2 Identification of strains

[0058]

[0059]

[0060] 3. 16S rDNA sequencing

[0061] The 16S rDNA gene was amplified for the isolated and screened mutagenic strains. 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 regions of the 16SrRNA 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 (5 μM), 0.8 μL of downstream primer (5 μM), 0.4 μL of TransStartFastPfu DNA polymerase, and 10 ng of template DNA, which was supplemented to 20 μL. The amplification procedure was as follows: 95°C pre-denaturation for 3 min, 27 cycles (95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 30 s), then 72°C stable extension for 10 min, and finally stored at 4°C. The obtained product was sequenced by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The 16SrDNA sequence of the strain was shown in SEQ ID NO: 1.

[0062]

[0063] Then, the NCBI database was used to perform BLAST analysis based on the strain's 16S rDNA gene sequence, and the strain was finally identified as Brucella sp. based on its morphological, structural, physiological and biochemical characteristics.

[0064] 4. Whole genome sequencing comparison

[0065] The original strain and the induced strain were sequenced for the whole genome, and the sequencing comparison results of the induced strain A54-5 and the original strain A54 are shown in Table 4. It can be seen that compared with the original strain A54, the induced strain A54-5 has 1 SNP, which proves that the genome composition of the mutant strain is different from that of the original strain, and the mutation type affects the gene function.

[0066] Table 4 Comparison of whole genome sequencing results of A54 and A54-5

[0067]

[0068] Example 3 RubisCO enzyme activity test

[0069] RubisCO is the rate-limiting enzyme that catalyzes CO2 fixation by autotrophic microorganisms using the Calvin cycle. Higher RubisCO enzyme activity indicates that autotrophic microorganisms have higher carbon assimilation capacity.

[0070] The soil 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) ELISA kit (Shanghai Ruifan Biotechnology Co., Ltd.) was used to determine the activity of RubisCO. The determination method strictly followed the instructions of the kit, as follows: (1) Setting up standard wells: Set up 6 standard wells in a 96-well ELISA plate, add 50 μL of standard to each well, and the concentration gradient is 0 U / L, 5 U / L, 10 U / L, 20 U / L, 40 U / L, and 80 U / L, respectively, with 5 parallel wells for each gradient. (2) Sample addition: Set up 16 sample wells and 1 blank well, add the test bacterial solution sample to the sample well (add the original strain or the induced strain to 10mL phosphate buffer (0.01mol / L, pH 7.2-7.4), place it in a multi-tube vortex oscillator (UMV-1, Beijing Yousheng United Technology Co., Ltd.) and shake it for 30s, then centrifuge it at 4000r / min (5427-R, Eppendorf, Germany) for 15min, and take the supernatant.) Take 10μL of the supernatant and add 40μL of the sample diluent. Set up 5 parallel wells for each sample. (3) Incubation: Add 100μL of the 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 it at 37℃ (HH-4, Jintan Youlian Instrument Research Institute, Jiangsu Province) for 60min. The blank wells follow the same steps except that no sample and enzyme-labeled antibody are added. (4) Washing: Inject 350 μL of washing solution into each well, soak for 1 min, and then place in an automatic plate washer (RT-3000, Beijing Weixin Yiao Technology Development Co., Ltd.) to wash 5 times. (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: Add 50 μL of stop solution and measure the absorbance (OD value) of each well at 450 nm within 15 min (Synergy H1, BioTek, USA). (7) Calculation of sample RubisCO enzyme activity: 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, which is the activity value of the sample RubisCO enzyme. Results are shown in Figure 3 .

[0071] Example 4 Experimental study on the effect of strains on RubisCO enzyme activity in oil-containing solid waste residues

[0072] The oil-containing solid waste residues collected from Liaohe Oilfield were air-dried, and after removing impurities such as stones, they were sieved through a 2 mm sieve to determine the basic nutrient content (pH 8.21, total nitrogen 0.31 g / kg, available phosphorus 15.32 mg / kg, organic matter 30.55 g / kg) and petroleum hydrocarbon content (1.41 × 10 3mg / kg) and set aside. Use a plastic basin with an inner diameter of 20 cm and a depth of 30 cm, and put 5 kg in each basin. Add the mutagenic bacterial solution (diluted with distilled water to a live bacterial concentration of 1×10 8 CFU / mL), and the bacterial solution was applied at 0.5mL / kg soil, and the bacterial solution was supplemented every 15 days. Water was added to the oil-containing solid waste residue regularly, and the culture was carried out for 60 days. After the culture was completed, the RubisCO enzyme activity in the oil-containing solid waste residue was measured. The blank treatment was without the application of bacterial solution, and the control was the application of sterilized bacterial solution. Each treatment was repeated for 5 pots. The RubisCO enzyme activity of the solid waste residue samples was determined using the 1,5-bisphosphate ribulose carboxylase / oxygenase (RubisCO) ELISA kit (Shanghai Ruifan Biotechnology Co., Ltd.) provided by Shanghai Ruifan Biotechnology Co., Ltd., and the measurement results are shown in the table. Figure 4 .

[0073] Depend on Figure 4 It can be seen that the RubisCO enzyme activity of the oil-containing solid waste residue after the application of the bacterial solution is significantly higher than that of the residue without the bacterial solution and the residue with the sterilized bacterial solution, indicating that the application of the bacterial solution can improve the RubisCO enzyme activity of the oil-containing solid waste residue, making it have a better carbon fixation function. The oil-containing solid waste residue of the oil field is a special operating well site oil-containing solid waste residue formed by the crude oil and oily wastewater brought to the surface of the ground by the oil wells during the underground operation of the oil field, and the surface soil and steelmaking waste slag paved on the ground of the well site. The volume is huge. If it is not treated and discharged directly, it will not only occupy a large amount of arable land, but also cause pollution to the surrounding soil, water and air. By mixing it with the mutagenic bacteria provided by the present invention for cultivation, the deep resource conversion and utilization of the oil-containing solid waste residue is greatly promoted.

[0074] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. An efficient carbon-fixing Brucella derived from oily solid waste residue, characterized in that: The strain is Brucella sp. A54-5 with a deposit number of CCTCCNO: M 20241650.

2. The fermentation culture of the efficient carbon-fixing Brucella derived from the oil-containing solid waste residue according to claim 1.

3. A bacterial agent, characterized in that: The bacterial agent contains the efficient carbon-fixing Brucella derived from the oil-containing solid waste residue according to claim 1 or the fermentation culture according to claim 2.

4. Use of the efficient carbon-fixing Brucella derived from oil-containing solid waste residues as described in claim 1, the fermentation culture as described in claim 2, or the bacterial agent as described in claim 3 in pollution reduction and carbon fixation.

5. Use of the efficient carbon-fixing Brucella derived from the oil-containing solid waste residue according to claim 1, the fermentation culture according to claim 2, or the bacterial agent according to claim 3 in the deep resource conversion of oil-containing solid waste residue.

6. A method for reducing pollution and fixing carbon based on microorganisms, characterized in that: The method comprises: mixing and culturing the oil-containing solid waste residue with the efficient carbon-fixing Brucella derived from the oil-containing solid waste residue according to claim 1 or the fermentation culture according to claim 2 or the bacterial agent according to claim 3.

7. The method according to claim 6, characterized in that The oil-containing solid waste residue is oil-containing solid waste residue with an oil content of less than 2% after being treated by physical and chemical methods.

8. The method according to claim 7, characterized in that The physical and chemical treatment includes any one of hot water washing and pyrolysis treatment.

9. The method according to claim 6, characterized in that The culture temperature was 31°C.

10. The method according to claim 6, characterized in that The culture time is 5-7 days.

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