Highly efficient carbon-fixing Brucella bacteria derived from oily solid waste residues and their applications

Abstract

This invention discloses a highly efficient carbon-fixing Brucella bacillus derived from oily solid waste residue and its applications. The Brucella bacillus is Brucella sp A54-5, with accession number CCTCC NO: M20241650. This Brucella bacillus strain was obtained through ARTP mutagenesis and screening, starting with Brucella sp A54 isolated from oily solid waste residue in oil fields. It possesses excellent pollution reduction and carbon fixation characteristics and strong resilience, showing broad application prospects in the deep resource utilization (soil conversion) of oily solid waste residue, improving the carbon sequestration capacity of petroleum-contaminated soils, and remediating petroleum-contaminated soils. It is of great significance for supporting pollution reduction and carbon sequestration in the petroleum and petrochemical industry under the "dual carbon" background.

Description

Technical Field

[0001] This invention belongs to the field of solid waste resource utilization technology, and specifically relates to a highly efficient carbon-fixing Brucella bacteria derived from oily solid waste residue and its application. Background Technology

[0002] The oil and gas industry is a traditional major contributor to carbon emissions. According to the International Energy Agency's 2022 CO2 Emissions Report, global CO2 emissions are approximately 36.8 billion tons per year, of which oil and gas emissions account for about 11.3 billion tons per year, nearly one-third of global carbon emissions. Carbon emissions are significantly linked to global warming. Currently, major global oil companies have successively proposed "carbon neutrality targets" and corresponding carbon 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 by which microorganisms absorb inorganic carbon and convert it into biomass. It is a crucial pathway for the conversion of inorganic carbon into organic carbon and its entry into the biosphere, reducing atmospheric carbon dioxide levels and playing a significant role in mitigating the greenhouse effect and global warming. Carbon-fixing microorganisms can be classified into autotrophic and heterotrophic microorganisms based on their nutritional modes. Autotrophic microorganisms use CO2 as their sole carbon source, converting it into their own cellular material through photosynthesis or chemosynthesis to achieve photosynthetic CO2 fixation. Heterotrophic microorganisms use organic carbon compounds as their carbon source, achieving carbon fixation through carboxylation reactions during their metabolism. Most soil carbon-fixing microorganisms are autotrophic, and their strong environmental adaptability, rapid growth, and fast reproduction have made them a current focus in biological carbon fixation. CN109536402A discloses Arthrobacter sp. with carbon fixation activity screened from soil on the southwestern edge of the Mu Us Desert in Inner Mongolia. CN109182205A discloses Rhodococcus sp. with carbon fixation activity screened from the same soil. CN115851564A discloses two recombinant bacteria that fix carbon dioxide by overexpressing phosphoenolpyruvate carboxylkinase and acetyl-CoA carboxylase in Bacillus licheniformis, respectively. CN106967662A discloses a recombinant bacterium that fixes carbon dioxide and synthesizes succinic acid, obtained by transforming a recombinant vector expressing the following genes into host cells: 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 synthase gene sucCD. Currently, carbon-fixing microorganisms are mainly isolated from undisturbed or low-pollution environments such as the deep ocean, volcanic craters, and desert soils, or obtained through artificial means such as genetic engineering. However, their resistance to adverse conditions is relatively poor.

[0004] In recent years, the following reports have been published on microorganisms under the stress of petroleum pollution: CN108996707A discloses a highly efficient microbial preparation formulated from various strains capable of forming dominant bacterial communities to accelerate the degradation of polycyclic aromatic hydrocarbons (PAHs) in groundwater from petroleum-contaminated sites; CN109576186A discloses a highly efficient microbial preparation developed using petroleum-contaminated soil / groundwater as the research object, showing good degradation effects on PAHs; CN117106608A discloses a mutagenic Bacillus amyloliquefaciens strain and its application in the degradation of petroleum hydrocarbons; CN114752517A discloses an ARTP-induced rapid mutagenesis strain for petroleum degradation, its preparation method, and its application. This strain uses the ARTP rapid mutagenesis method to conduct directed evolution of petroleum hydrocarbon-degrading strains. The mutagenic bacteria exhibit strong environmental adaptability and high degradation efficiency, and can be used as a biofortifier. Among the technologies disclosed above, these microorganisms can be applied to petroleum-contaminated environments, improving stress resistance, but none of them have carbon fixation capabilities.

[0005] Therefore, there is a lack of reports on microorganisms with strong stress resistance and excellent pollution reduction and carbon sequestration properties in the current technology. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention provides a highly efficient carbon-fixing Brucella from oily solid waste residue. It is a new carbon-fixing microorganism with strong environmental resistance, easy to isolate, purify and preserve, and is suitable for the deep resource transformation of oily solid waste residue.

[0007] In one aspect, the present invention provides a highly efficient carbon-fixing Brucella source from oily solid waste residue, which is Brucella sp. A54-5 with accession number CCTCC NO: M20241650.

[0008] In another aspect of the present invention, a fermentation culture of Brucella a highly efficient carbon-fixing bacillus derived from the aforementioned oily solid waste residue is provided.

[0009] In another aspect, the present invention provides a microbial agent containing highly efficient carbon-fixing Brucella or fermentation culture derived from the aforementioned oily solid waste residue.

[0010] In another aspect of the present invention, the application of highly efficient carbon-fixing Brucella bacteria or fermentation cultures or agents derived from the aforementioned oily solid waste residues in pollution reduction and carbon fixation is provided.

[0011] In another aspect, the present invention provides the application of highly efficient carbon-fixing Brucella bacteria or fermentation cultures or agents derived from the aforementioned oily solid waste residues in the deep resource utilization and transformation of oily solid waste residues.

[0012] In another aspect of the present invention, a method for pollution reduction and carbon sequestration based on microorganisms is provided, comprising: mixing and culturing oily solid waste residue with highly efficient carbon-fixing Brucella bacteria or fermentation culture or bacterial agent derived from the aforementioned oily solid waste residue.

[0013] According to a specific embodiment of the present invention, the oily solid waste residue is an oily solid waste residue with an oil content of <2% after physicochemical treatment.

[0014] According to specific embodiments of the present invention, physicochemical 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 this invention are as follows:

[0017] The Brucella bacteria provided by this invention are obtained by ARTP mutagenesis and screening using Brucella sp. A54, which was isolated from oil-containing solid waste residues in oil fields, as the starting strain. It has excellent pollution reduction and carbon sequestration characteristics and strong stress resistance. It has broad application prospects in the fields of deep resource utilization (soil conversion) of oil-containing solid waste residues, improving the carbon sequestration capacity of oil-contaminated soils, and remediation of oil-contaminated soils. It is of great significance to support pollution reduction and carbon sequestration increase in the petroleum and petrochemical industry under the background of "dual carbon". Attached Figure Description

[0018] Figure 1 Photographs of Gram staining of strain A54;

[0019] Figure 2 Photographs of Gram staining of strain A54-5;

[0020] Figure 3 The results are for RubisCO enzyme activity assay.

[0021] Figure 4 The results show the effects of the strain on RubisCO enzyme activity in oily solid waste residue. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Isolation, Purification, and Screening of Strains

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

[0025] Twelve oil-containing solid waste residues were collected from major oilfields in my country, such as Liaohe Oilfield and Daqing Oilfield, after hot water washing and pyrolysis treatment. Approximately 500g of each residue sample was scooped out with a sterile shovel, placed in a sterile sealed bag, and after removing animal and plant remains and gravel, it was placed in a 4°C ice box and transported back to the laboratory for further processing.

[0026] 2. Culture medium preparation

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

[0028] Table 1. Formulation of Carbon-Free Inorganic Salt Liquid Culture Medium

[0029]

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

[0031] Agar powder of 20–25 g / L was added to a carbon-free inorganic salt liquid culture medium formula. The pH of the culture medium was adjusted to 7–8. The preparation method was to add all components to ultrapure water, stir evenly, and then sterilize by high-temperature wet sterilization at 121°C for 15 min.

[0032] 3. Strains are isolated, purified, and screened.

[0033] From each of the 12 collected samples, 1g of soil was taken, mixed thoroughly, and placed in an Erlenmeyer flask containing 100mL of sterile water. The mixture was then thoroughly mixed and placed in a shaker at 30℃ and 150rpm for approximately 20min to prepare a soil suspension. This suspension was subsequently serially diluted. Under aseptic conditions, the soil suspension was diluted to 10... -4 10 -5 10 -6 To determine the concentration, 10 μL of soil suspension at different dilutions was injected into sterile culture dishes containing carbon-free inorganic salt solid culture medium, spread evenly, and incubated upside down at 35°C for 3-5 days. Single colonies were picked, streaked, and purified multiple times. The purified single colonies were then expanded and preserved to obtain the original strain, and ARTP mutagenesis and screening were performed.

[0034] ①ARTP mutagenesis

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

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

[0037] c) Transfer the culture dish containing the sample slides to the ARTP mutation breeding instrument operating chamber, and use sterile forceps to place the slides into the corresponding grooves.

[0038] d) After adjusting the mutagenesis parameters (distance 2mm; processing power 120W; gas flow rate 10SL) and processing time (0s, 30s, 40s, 60s, 80s, 100s) and setting them, start processing the sample.

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

[0040] f) Place the EP tube on a shaker and shake for 1 minute to wash the microorganisms attached to the slide into a sterile solution to form a new bacterial suspension.

[0041] g) Dilute the fresh 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 diluted solution and spread it on a carbon-free inorganic salt solid culture medium for screening.

[0042] ② Filtering

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

[0044] b) Inoculate different colonies into LB tubes and incubate overnight. Then dilute the bacterial solution to the same concentration and inoculate it into carbon-free inorganic salt liquid culture medium tubes, and incubate for 4-6 days.

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

[0046] d) Continue to culture the vigorous bacteria in LB medium, wash and dilute them to different concentrations, drop them onto carbon-free inorganic salt solid medium, select vigorous bacteria for culture and preservation.

[0047] The best-growing (largest colony diameter) mutant strain of Brucella was finally obtained through screening, with sample number A54-5. It was deposited on July 22, 2024, at the China Center for Type Culture Collection (CCTCC; address: China Center for Type Culture Collection, Wuhan University, Wuhan, China, 430072, China), with accession number CCTCC NO: M 20241650.

[0048] Example 2: Strain Identification

[0049] The original strain and the mutant strain were placed in carbon-free inorganic salt liquid medium and cultured at 35℃ and 150r / min for 7-10 days. When the bacterial solution became turbid, 4mL of the bacterial solution was taken for bacterial identification. The bacterial identification platform was provided by Shanghai Meiji Biomedical Technology Co., Ltd.

[0050] 1. Morphological characteristics of the strain

[0051] Gram staining microscopy: Take a clean glass slide, and briefly heat the smeared bacterial area over a flame to remove grease. Spread a small amount of bacterial suspension evenly on the slide, allow it to air dry naturally, and then, with the bacterial membrane facing upwards, quickly pass the slide back and forth over the flame 2-3 times to fix the bacteria. Use a Gram staining kit (Changde Bickman Biotechnology Co., Ltd.). Add ammonium oxalate crystal violet solution to the fixed bacteria, stain for 1 minute, wash away the remaining stain with distilled water, allow to dry, add 1 drop of iodine solution, mordant for 1 minute, wash away the remaining stain with distilled water, allow to dry, add 95% ethanol for 30 seconds to decolorize, and wash with water. Finally, add safranin counterstain for 1 minute, wash away the remaining stain with distilled water. Allow to air dry completely before microscopic examination.

[0052] 2. Physiological and biochemical characteristics of the strain

[0053] Starch hydrolysis test: Divide a plate containing starch solid medium (3g peptone, 1.5g NaCl, 1.5g beef extract, 0.76g soluble starch, and 6g agar in 300mL distilled water, pH adjusted to 7.6, sterilized at 121℃ for 20min) into three parts. Use an inoculation loop to streak the experimental strains in different parts, labeling each strain with a marker. Invert the plate and incubate at 37℃ for 24h. Observe the growth of various bacteria. Add a small amount of Lugol's iodine solution to the plate, gently rotate the plate to evenly distribute the iodine solution across the entire plate. 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, pH adjusted to 7.0–7.2, sterilized at 121℃ for 20min), and incubate at 37℃ with shaking for 48h. Then, add 2 drops of methyl red reagent to each culture medium. A red color indicates a positive result, while a yellow color indicates a negative result.

[0054] VP test: After inoculation and culture for 48 hours, solution A and solution B were added and shaken. The results showed that, compared with the blank, a red color indicated a positive result, and no color change indicated a negative result.

[0055] Sugar alcohol fermentation: The culture was subjected to sugar alcohol fermentation, 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, and the pH was adjusted to 6.8-7.0. The mixture was dispensed into test tubes and sterilized by steam at 112℃ for 30min.

[0056] The above experimental results are shown in Figure 1 , Figure 2 And Table 2:

[0057] Table 2. Strain identification

[0058]

[0059]

[0060] 3. 16S rDNA sequencing

[0061] The 16S rDNA gene was amplified from the isolated and screened mutagenic strains. PCR amplification of the V3–V4 variable region of the 16S rRNA gene was performed using upstream primer 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and downstream primer 806R (5'-GGACTACHVGGGTWTCTAAT-3') carrying barcode sequences. The PCR reaction mixture consisted of: 4 μL 5×TransStart FastPfu buffer, 2 μL 2.5 mM dNTPs, 0.8 μL upstream primer (5 μM), 0.8 μL downstream primer (5 μM), 0.4 μL TransStartFastPfu DNA polymerase, 10 ng template DNA, and a final volume of 20 μL. The amplification program was as follows: pre-denaturation at 95℃ for 3 min, 27 cycles (denaturation at 95℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 30 s), followed by a stable extension at 72℃ for 10 min, and finally storage at 4℃. The obtained product was sequenced by Shanghai Meiji Biotechnology Co., Ltd. The 16S rDNA sequence of the strain is shown in SEQ ID NO: 1.

[0062]

[0063] Then, using the NCBI database, BLAST analysis was performed based on the 16S rDNA gene sequence of the strain. Taking into account the morphological, structural, and physiological and biochemical characteristics of the strain, the strain was finally identified as Brucella sp.

[0064] 4. Whole genome sequencing and alignment

[0065] Whole-genome sequencing was performed on both the original strain and the mutant strain. The sequencing alignment results of mutant strain A54-5 and original strain A54 are shown in Table 4. It can be seen that mutant strain A54-5 has one SNP compared to original strain A54, proving that the genome composition of the mutant strain is different from that of the original strain, and that the mutation type affects gene function.

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

[0067]

[0068] Example 3 RubisCO enzyme activity test

[0069] RubisCO enzyme is the rate-limiting enzyme that catalyzes CO2 fixation in autotrophic microorganisms using the Calvin cycle. High RubisCO enzyme activity indicates that autotrophic microorganisms have a high carbon assimilation capacity.

[0070] The activity of RubisCO enzyme was determined using a soil ribulose 1,5-bisphosphate carboxylase / oxygenase (RubisCO) ELISA kit (Shanghai Ruifan Biotechnology Co., Ltd.). The determination method strictly followed the instructions of the kit, as follows: (1) Set up standard wells: Set up 6 standard wells in a 96-well microplate, add 50 μL of standard to each well, and set up 5 replicates for each gradient as follows: 0 U / L, 5 U / L, 10 U / L, 20 U / L, 40 U / L and 80 U / L. (2) Sample addition: Set up 16 sample wells and 1 blank well. Add the bacterial culture sample to be tested to the sample wells (add the original strain or the mutant strain to 10 mL of phosphate buffer (0.01 mol / L, pH 7.2-7.4), place it in a multi-tube vortex shaker (UMV-1 type, Beijing Yousheng United Technology Co., Ltd.) and shake for 30 s, then centrifuge at 4000 r / min (5427-R type, Eppendorf, Germany) for 15 min, and take the supernatant). Take 10 μL of supernatant and then add 40 μL of sample diluent. Set up 5 replicates for each sample. (3) Incubation: Add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to both the standard wells and the sample wells. After sealing with a sealing film, incubate at 37℃ (HH-4 type, Youlian Instrument Research Institute, Jintan City, Jiangsu Province) for 60 min. The blank wells are the same except that no sample or enzyme-labeled antibody is 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 type, Beijing Weixin Yi'ao Technology Development Co., Ltd.) and wash 5 times. (5) Color development: Add 50 μL each of substrate A and B to each well, and incubate at 37℃ in the dark for 15 min. (6) Measurement: Add 50 μL of stop solution, and measure the absorbance (OD value) at 450 nm wavelength in each well within 15 min (Synergy H1 type, BioTek, USA). (7) Calculation of RubisCO enzyme activity value of samples: Plot a linear regression curve of the standard with the concentration of the standard as the abscissa and the corresponding OD value as the ordinate. Calculate the concentration value of each sample according to the curve equation, which is the RubisCO enzyme activity value of the sample. See the results below. Figure 3 .

[0071] Example 4: Experimental study on the effect of strain on RubisCO enzyme activity in oily solid waste residue

[0072] Oil-containing solid waste residue collected from Liaohe Oilfield was air-dried, and after removing impurities such as stones, it was sieved through a 2mm sieve. The basic nutrient content (pH 8.21, total N 0.31g / kg, available phosphorus 15.32mg / kg, organic matter 30.55g / kg) and petroleum hydrocarbon content (1.41×10⁻⁶) of the solid waste residue were determined. 3(mg / kg), for later use. Use plastic basins with an inner diameter of 20cm and a depth of 30cm, each containing 5kg. Add the mutant bacterial solution (diluted with distilled water to a viable bacterial concentration of 1×10⁻⁶) with the water. 8 CFU / mL), applied at a concentration of 0.5 mL / kg soil, with replenishment of bacterial solution every 15 days. Water was periodically added to the oily solid waste residue, and the mixture was cultured for 60 days. After culture, the RubisCO enzyme activity in the oily solid waste residue was measured. A blank treatment was used without bacterial solution, and a control treatment was used with sterilized bacterial solution. Each treatment was replicated in 5 batches. The RubisCO enzyme activity of the solid waste residue samples was determined using a ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) ELISA kit provided by Shanghai Ruifan Biotechnology Co., Ltd. (Shanghai Ruifan Biotechnology Co., Ltd.). The results are shown below. Figure 4 .

[0073] Depend on Figure 4 It is evident that the RubisCO enzyme activity of oily solid waste residues after the application of bacterial solution was significantly higher than that without bacterial solution or with sterilized bacterial solution, indicating that the application of bacterial solution can improve the RubisCO enzyme activity of oily solid waste residues, giving them better carbon fixation function. Oily solid waste residues from oilfields are special oily solid waste residues formed by the mixture of crude oil and oily wastewater from oil wells brought to the surface during downhole operations, along with surface soil and steelmaking slag used to cover the well site. These residues are large in volume, and direct discharge without treatment not only occupies a large amount of arable land but also pollutes the surrounding soil, water, and air. By co-culturing these residues with the mutagenic bacteria provided in this invention, the deep resource-based transformation and utilization of oily solid waste residues is greatly promoted.

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

Claims

1. A highly efficient carbon-fixing Brucella sp. A54-5 derived from oily solid waste residue, characterized in that, The Brucella A54-5 was deposited at the China Center for Type Culture Collection on July 22, 2024, with accession number CCTCC NO: M 20241650.

2. A microbial agent, characterized in that, The bacterial agent contains highly efficient carbon-fixing Brucella bacteria derived from the oily solid waste residue described in claim 1.

3. The application of the highly efficient carbon-fixing Brucella bacteria derived from oily solid waste residue as described in claim 1 or the bacterial agent as described in claim 2 in pollution reduction and carbon fixation.

4. The application of the highly efficient carbon-fixing Brucella bacteria from the oily solid waste residue as described in claim 1 or the bacterial agent as described in claim 2 in the deep resource utilization and transformation of oily solid waste residue.

5. A method for pollution reduction and carbon sequestration based on microorganisms, characterized in that, The method includes: mixing and culturing oily solid waste residue with Brucella aerosols derived from the oily solid waste residue of claim 1 or the bacterial agent of claim 2.

6. The method according to claim 5, characterized in that, The oily solid waste residue is oily solid waste residue with an oil content of <2% after physicochemical treatment.

7. The method according to claim 6, characterized in that, The physicochemical treatment includes either hot water washing or pyrolysis treatment.

8. The method according to claim 5, characterized in that, The culture temperature was 31°C.

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

Images