Carbon fiber fixing vibrio and application thereof

Abstract

The invention discloses a carbon fiber fixing vibrio and application thereof, and belongs to the technical field of microorganisms. According to the invention, a strain of Cellvibrio sp. Is separated, the strain is preserved in the China General Microbiological Culture Collection Center on May 9, 2025, and the preservation number is CGMCC No.34469. The invention also discloses a method for preparing the Cellvibrio sp. The Cellvibrio sp GT-5 shows a remarkable carbon sequestration capability in a culture medium without a carbon source. The activity of the RubisCO enzyme reaches 78.8 U / mL, and the RubisCO enzyme can catalyze the combination of C5 and CO2 and can be applied to the field of microbial carbon sequestration, so that the biological function cognition of Cellvibrio is widened, and a new method is provided for immobilizing CO2.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a type of Vibrio sclerotiorum and its applications. Background Technology

[0002] Cellulose-degrading bacteria have long been a research focus in the utilization of biomass resources, among which Vibrio cellulose ( Cellvibrio It has attracted much attention due to its highly efficient cellulase system. For example, Cellvibrio japonicus By secreting multi-module cellulases (such as Cel5A and Cel6B), lignocellulose can be efficiently converted into fermentable sugars, which are widely used in biofuel production. However, research on strains of this genus has long been limited to their carbon source decomposition function, and their carbon assimilation potential, especially their carbon fixation ability, has never been revealed.

[0003] Existing literature on Cellvibrio The analysis of the genera almost entirely focuses on the degradation mechanisms of complex polysaccharides such as cellulose and xylan. For example, the carbon-fixing strains disclosed in the patent application CN 119432607 A regarding the isolation and culture method and application of soil carbon-fixing microorganisms under high petroleum pollution stress are all Pseudomonas ( ). Pseudomonas ) and Bacillus ( Bacillus ),and Cellvibrio Genera not included in the screening scope; genotype databases (such as KEGG, NCBI) Cellvibrio The strain annotations only cover cellulase genes (such as the GH5 and GH6 families), and do not involve key carbon fixation genes such as the Calvin cycle or cbbL / cbbS.

[0004] Although some studies speculate that fiber-degrading bacteria may indirectly affect the carbon cycle through metabolic byproducts (such as the production of short-chain fatty acids), their ability to directly fix CO2 has remained unverified. For example, Cellvibrio mixtus The rate of CO2 release in cellulose media has been quantified, but there is no data to support whether it possesses the ability to reverse CO2 absorption. Cellvibrio The application of engineered strains is mainly concentrated in the field of biorefining (such as pulp degumming and agricultural waste treatment), while their potential in carbon sequestration and soil remediation remains untapped. In particular, in the remediation of saline-alkali land, strains with both cellulose degradation and CO2 fixation capabilities can simultaneously increase soil organic matter and carbon storage, but such multifunctional strains have not yet been reported. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a *Vibrio fibrous* strain and its application. This invention isolates a *Vibrio fibrous* strain (… Cellvibrio sp. RubisCO can grow well in carbon-free medium. The RubisCO enzyme activity reached 78.8 U / mL, a finding that broadens the scope of research in this field. CellvibrioThe biological function cognition of the genus provides a new method for fixing CO2.

[0006] To achieve the above object, the application adopts the following technical solutions. In a first aspect, the application provides a fiber Arcobacter (A. Cellvibrio sp ) GT-5, which was preserved in the China General Microbiological Culture Collection Center on May 9, 2025, with a preservation number of CGMCC No. 34469.

[0007] In a second aspect, the application provides an application of the fiber Arcobacter (A. Cellvibrio sp ) GT-5 in producing a carbon fixation enzyme.

[0008] Preferably, the carbon fixation enzyme is a ribulose-1,5-bisphosphate carboxylase / oxygenase.

[0009] In a third aspect, the application provides a microbial agent containing the fiber Arcobacter (A. Cellvibrio sp ) GT-5.

[0010] Preferably, the fiber Arcobacter (A. Cellvibrio sp ) GT-5 exists in the form of a cultured live bacterium, a bacterial suspension, or a fermentation broth in the microbial agent.

[0011] Preferably, the bacterial suspension is prepared by the following method: The fiber Arcobacter (A. Cellvibrio sp ) GT-5 is inoculated into a carbon-free liquid medium, and after being cultured in a constant-temperature shaking incubator at 37℃ and 200 r / min for 2-4 days, the bacteria are collected by centrifugation, the bacterial body is resuspended with sterile distilled water, and the bacterial suspension is prepared.

[0012] Preferably, the preparation method of the carbon-free liquid medium is as follows: 0.02 g of MnSO4, 1 g of Na2HPO4, 1.8 g of KH2PO4, 0.4 g of MgSO4, 0.05 g of CaCl2, 1 g of NaCl, 0.5 g of NH4Cl, 0.02 g of FeCl3, and 10 g of Na2S2O3 are mixed, distilled water is added to 1000 mL, and sterilization is performed at 121℃ for 20 min.

[0013] Preferably, the enzyme activity of the ribulose-1,5-bisphosphate carboxylase / oxygenase in the bacterial suspension is at most 78.8 U / mL.

[0014] In a fourth aspect, the application provides an application of the above microbial agent in fixing CO2 or preparing a carbon fixation agent.

[0015] Preferably, the application includes fixing CO2, preparing a carbon fixation agent, or increasing the organic matter content in the soil.

[0016] In a fifth aspect, the present application provides a use of the cell of the genus Marinobacterium Cellvibrio sp. GT-5 in fixing CO2 or preparing a carbon sequestration agent.

[0017] In a sixth aspect, the present application provides a carbon sequestration agent containing the above-mentioned microbial agent.

[0018] Advantages of the present application: The present application isolates a cell of the genus Marinobacterium Cellvibrio which exhibits a significant carbon sequestration ability in a carbon-free medium. The RubisCO enzyme activity reaches 78.8 U / mL, and the RubisCO enzyme can catalyze the combination of C5 and CO2, which can be applied in the field of microbial carbon sequestration. This discovery broadens the biological function cognition of the genus Marinobacterium and provides a new method for fixing CO2. Figure 1 Advantages of the present application: BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 2 : Growth of strain GT-5 on a carbon-free medium plate; Figure 3 : Phylogenetic tree of strain GT-5 based on 16S rDNA sequence; Figure 4 : Comparison of RubisCO enzyme activity of soil samples after applying the carbon sequestration bacteria liquid; Figure 1 : Comparison of organic matter content of soil samples after applying the carbon sequestration bacteria liquid. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs.

[0021] Explanation: The carbon-free inorganic liquid medium used in the present application: MnSO40.02 g, Na2HPO41 g, KH2PO41.8 g, MgSO40.4 g, CaCl20.05 g, NaCl 1 g, NH4Cl 0.5 g, FeCl30.02 g, Na2S2O310 g, distilled water to 1000 mL, sterilized at 121 ℃ for 20 min.

[0022] The carbon-free inorganic solid medium is obtained by adding 2% agar powder to the carbon-free inorganic liquid medium.

[0023] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0024] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0025] Example 1: Isolation and Identification of Strains 1. Isolation of strains Weigh 10.0 g of soil collected from the saline-alkali land of Dongying (37°32'21”N, 118°36'19''E), add it to an Erlenmeyer flask containing 90 mL of sterile water, mix thoroughly, and dilute to a concentration of 10. -1 The soil suspension was placed on a shaker at 30℃ and 200 r / min for 30 min, and then diluted sequentially to a dilution of 10 under aseptic conditions. -2 10 -3 10 -4 A dilution of 10 was selected. -2 10 -3 10 -4 0.1 mL of each dilution solution was injected into a sterile culture dish containing carbon-free inorganic solid culture medium, spread evenly, and incubated upside down at 30°C for 5-7 days. Single colonies were picked, labeled, streaked to isolate and purify, and then transferred to slant agar and stored at 4°C for later use.

[0026] 2. Identification of strains (1) Morphological identification The strain was cultured at 28℃ on carbon-free inorganic solid medium for 4 days, and its colony characteristics and cell growth were observed. Single colonies of strain GT-5 were streaked onto carbon-free inorganic solid medium plates, which were then inverted and incubated at 37℃ for 48 hours. Figure 2 As shown, the colonies it produces on solid culture medium are small, round, pale yellow, opaque, with a smooth and viscous surface, neat edges, and low convexity.

[0027] (2) Molecular biological identification: The genus and species of the isolated and screened strain GT-5 were identified using 16S rDNA gene sequence analysis. The bacterial 16S rDNA sequence was obtained by amplification using universal primers 27F (SEQ ID NO: 1, 5'-TACGGYTACCTTGTTACGACTT-3') and 1492R (SEQ ID NO: 2, 5'-AGAGTTTGATCMTGGCTCAG-3'), and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Alignment was performed using BLAST in NCBI, and a phylogenetic tree was constructed, as shown below. Cellvibrio sp As shown.

[0028] The nucleotide sequence of the bacterial GT-5 is shown as SEQ ID NO: 3:

[0029] (3) Physiological and biochemical characteristics According to the standard operating procedures provided in the Common Bacteria Identification Manual and Bergey's Manual of Bacterial Identification, the physiological and biochemical characteristics of the strain were identified. The isolated and screened strain GT-5 was subjected to physiological and biochemical reactions such as starch hydrolysis, gelatin hydrolysis, methyl red reaction, and indole reaction. The reaction results were observed and recorded. The results are shown in Table 1.

[0030] Table 1. Physiological and biochemical reactions of carbon-fixing strain GT-5 Note: "+" represents positive, and "-" represents negative.

[0031] Based on the comprehensive morphological, molecular biological, and physiological and biochemical characteristics, strain GT-5 was identified as Vibrio fibronectin (…). Cellvibrio sp The isolated Vibrio cytogenes GT-5 was deposited at the China General Microbiological Culture Collection Center (CGMCC, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China). The deposit information is as follows: Referenced biological material (strain): GT-5; Suggested classification and nomenclature: Vibrio fibrosus Cellvibrio sp ; Registration number: CGMCC No. 34469; Preservation date: May 9, 2025.

[0032] Example 2: RubisCO enzyme activity assay Vibrio fibrinolyticus ( Figure 3 GT-5 was inoculated into carbon-free liquid culture medium and cultured at 37℃ and 200 r / min in a constant temperature shaking incubator for 2-4 days. After centrifugation, the bacterial cells were collected and resuspended in sterile distilled water to prepare a bacterial suspension. The activity of RubisCO enzyme produced by strain GT-5 was determined using a microbial ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) ELISA kit (Yancheng Junxing Biotechnology Co., Ltd.). The RubisCO enzyme activity of the GT-5 bacterial suspension was as high as 78.8 U / mL.

[0033] Example 3: Carbon fixation investigation The RubisCO enzyme activity of soil samples was determined using a soil ribulose-1,5-bisphosphate carboxylase / oxygenase (RubisCO) enzyme-linked immunosorbent assay kit provided by Yancheng Junxing Biotechnology Co., Ltd.; meanwhile, the soil organic matter content was determined using the potassium dichromate titration method-external heating method.

[0034] The preparation method of the carbon fixation bacteria agent is as follows: GT-5 single colony is streaked into a carbon-free inorganic liquid medium, and cultured at 28°C for 7 days to obtain a bacterial solution. Then, 1 mL of the bacterial solution is added to 50 g of soil taken from the Dongying saline-alkali soil (37°32'21"N, 118°36'19''E, EC value of 3.03 mS / cm), and indoor culture is carried out at room temperature for 14 days. Two treatment groups are set: normal soil (CK) and soil added with the carbon fixation bacteria GT-5 solution (GT-5).

[0035] After 14 days of culture, the RubisCO carbon fixation enzyme activity of the two groups of soil is determined using a microbial 1,5-diphosphoribulokinase / oxygenase (RuBisCO) ELISA detection kit. According to the manufacturer's instructions, the soil sample is diluted to 1:10 with the dilution buffer, and 100 μL of the diluted sample is added to the microplate well. After incubation at 37°C for 1 hour, the reaction is terminated by adding 100 μL of the stop solution, and the microplate is incubated at room temperature for 10 minutes. Then, 100 μL of the detection solution is added to each well, and the microplate is incubated at 37°C for 30 minutes. After incubation, the microplate is washed 5 times with the washing buffer, and 100 μL of the substrate solution is added to each well. After incubation at room temperature for 30 minutes, the reaction is terminated by adding 100 μL of the stop solution, and the microplate is incubated at room temperature for 10 minutes. Finally, the absorbance at 450 nm is measured using a microplate reader. Figure 4 As can be seen, the carbon fixation enzyme activity of the normal soil is 8.095 U / L, and the RubisCO carbon fixation enzyme activity of the soil added with the carbon fixation bacteria solution is significantly increased to 12.905 U / L. This result shows that after applying the carbon fixation bacteria GT-5 solution, the carbon fixation enzyme activity of the soil sample is significantly enhanced.

[0036] The organic matter content after the end of the culture is determined by the potassium dichromate volumetric method-external heating method, and the determination result is as follows: ​ The organic matter content of the normal soil is 9.7779 g·kg -1， and the soil added with the carbon fixation bacteria GT-5 solution reaches 10.8087 g·kg -1 This result shows that after applying the carbon fixation bacteria GT-5 solution, the organic matter content of the soil is also significantly increased. The above determination results provide a theoretical basis for the application of the carbon fixation bacteria GT-5, and further verify its potential in improving the carbon fixation capacity of saline-alkali soil.

[0037] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A type of Vibrio fibrosus ( Cellvibrio sp GT-5, with accession number CGMCC No.34469.

2. The cell of the Fibrobacter succinogenes according to claim 1, Cellvibrio sp ) Use of GT-5 in the production of carbon sequestering enzymes.

3. Use according to claim 2, characterized in that, The carbon-fixing enzyme is ribulose-1,5-bisphosphate carboxylase / oxygenase.

4. An inoculant characterized in that, The bacterial agent contains the Vibrio fibrius of claim 1 Cellvibrio sp ) GT-5.

5. The bacterial agent of claim 4, wherein The bacterial agent is in the form of a live bacterium, a bacterial suspension or a fermentation broth of Fibrobacter succinogenes (F. succinogenes) Cellvibrio sp GT-5.

6. The bacterial agent of claim 5, characterized in that, The bacterial suspension is prepared by the following method: Vibrio fibrinolyticus ( Cellvibrio sp GT-5 was inoculated into carbon-free liquid culture medium and cultured in a constant temperature shaking incubator at 37℃ and 200 r / min for 2-4 days. After centrifugation, the bacterial cells were collected and resuspended in sterile distilled water to prepare a bacterial suspension.

7. The bacterial agent of claim 6, wherein The preparation method of the carbon-free liquid medium is as follows: 0.02 g of MnSO4, 1 g of Na2HPO4, 1.8 g of KH2PO4, 0.4 g of MgSO4, 0.05 g of CaCl2, 1 g of NaCl, 0.5 g of NH4Cl, 0.02 g of FeCl3, and 10 g of Na2S2O3 are mixed, distilled water is added to 1000 mL, and sterilization is performed at 121 ℃ for 20 min.

8. The bacterial agent of claim 6, wherein The enzyme activity of ribulose-1,5-bisphosphate carboxylase / oxygenase in the bacterial suspension is at most 78.8 U / mL.

9. Use of the bacterial agent of any one of claims 4-8 in fixing CO2 or preparing a carbon-fixing agent.

10. A carbon sequestering agent characterized by, The carbon-fixing agent contains the bacterial agent of any one of claims 4-8.

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