Carbon sequestration fiber vibrio and application thereof

Abstract

The application discloses a carbon-fixing Vibrio fibrius and application thereof, and belongs to the technical field of microorganisms. Cellvibrio sp. The Vibrio fibrius is preserved in the China General Microbiological Culture Collection Center on May 9, 2025, and the preservation number is CGMCC No. 34469. The Vibrio fibrius GT-5 shows a significant carbon-fixing capacity in a carbon-free culture medium. Cellvibrio sp The RubisCO enzyme activity reaches 78.8 U / mL, the RubisCO enzyme can catalyze the combination of C5 and CO2, can be applied to the field of microbial carbon fixation, and the discovery widens the biological function cognition of the genus and provides a new method for fixing CO2. Cellvibrio ​

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 from 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. CellvibrioUnderstanding the biological functions of the genus provides a new approach to CO2 fixation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Vibrio fibrosus ( Cellvibrio sp GT-5, this strain was deposited on May 9, 2025 at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 34469.

[0008] A second aspect of the invention provides Vibrio fibrosus ( Cellvibrio sp Application of GT-5 in the production of carbon fixation enzymes.

[0009] Preferably, the carbon-fixing enzyme is ribulose-1,5-bisphosphate carboxylase / oxygenase.

[0010] A third aspect of the present invention provides a microbial agent containing Vibrio fibrosus (… Cellvibrio sp GT-5.

[0011] Preferably, the bacterial agent contains Vibrio fibrosum ( Cellvibrio sp GT-5 exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.

[0012] Preferably, the bacterial suspension is prepared by the following method:

[0013] 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.

[0014] Preferably, the preparation method of the carbon-free liquid culture 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 sterilized at 121 °C for 20 min.

[0015] Preferably, the highest enzyme activity of ribulose-1,5-bisphosphate carboxylase / oxygenase in the bacterial suspension is 78.8 U / mL.

[0016] In a fourth aspect, the present invention provides the use of the above-mentioned microbial agent in CO2 fixation or in the preparation of carbon fixation agents.

[0017] Preferably, the application includes fixing CO2, preparing carbon fixatives, or increasing the organic matter content in the soil.

[0018] In a fifth aspect, the invention provides Vibrio fibrosus ( Cellvibrio sp Application of GT-5 in CO2 fixation or preparation of carbon fixation agents.

[0019] In a sixth aspect, the present invention provides a carbon-fixing agent containing the aforementioned microbial agent.

[0020] The beneficial effects of this invention are:

[0021] This invention isolated a strain of Vibrio fibrosus ( Cellvibrio sp. RubisCO enzyme exhibited significant carbon fixation ability in carbon-free media. With an activity of 78.8 U / mL, RubisCO enzyme catalyzes the binding of C5 to CO2, and can be applied to the field of microbial carbon fixation. This discovery broadens the scope of research in this area. Cellvibrio Understanding the biological functions of the genus provides a new approach to CO2 fixation. Attached Figure Description

[0022] Figure 1 Growth of strain GT-5 on carbon-free medium plates;

[0023] Figure 2 Phylogenetic tree of strain GT-5 constructed based on 16S rDNA sequence;

[0024] Figure 3 Comparison of RubisCO enzyme activity in soil samples after application of carbon-fixing bacteria inoculum solution;

[0025] Figure 4 Comparison of organic matter content in soil samples after application of carbon-fixing bacteria solution. Detailed Implementation

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this 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 this application pertains.

[0027] illustrate:

[0028] The carbon-free inorganic liquid culture medium used in this invention consists of: 0.02 g MnSO4, 1 g Na2HPO4, 1.8 g KH2PO4, 0.4 g MgSO4, 0.05 g CaCl2, 1 g NaCl, 0.5 g NH4Cl, 0.02 g FeCl3, and 10 g Na2S2O3, with distilled water added to a final volume of 1000 mL. The medium is then sterilized at 121 °C for 20 min.

[0029] Carbon-free inorganic solid culture medium is made by adding 2% agar powder to carbon-free inorganic liquid culture medium.

[0030] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0031] 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.

[0032] Example 1: Isolation and Identification of Strains

[0033] 1. Isolation of strains

[0034] 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.

[0035] 2. Identification of strains

[0036] (1) Morphological identification

[0037] 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 1 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.

[0038] (2) Molecular biological identification:

[0039] 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. Figure 2 As shown.

[0040] The nucleotide sequence of bacteria GT-5 is shown in SEQ ID NO: 3:

[0041]

[0042] (3) Physiological and biochemical characteristics

[0043] 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.

[0044] Table 1. Physiological and biochemical reactions of carbon-fixing strain GT-5

[0045]

[0046] Note: "+" represents positive, and "-" represents negative.

[0047] 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:

[0048] Referenced biological material (strain): GT-5;

[0049] Suggested classification and nomenclature: Vibrio fibrosus Cellvibrio sp ;

[0050] Registration number: CGMCC No. 34469;

[0051] Preservation date: May 9, 2025.

[0052] Example 2: RubisCO enzyme activity assay

[0053] Vibrio fibrinolyticus ( Cellvibrio sp 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.

[0054] Example 3: Carbon fixation investigation

[0055] The RubisCO enzyme activity of soil samples was determined using the 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.

[0056] The preparation method of the carbon-fixing bacterial agent was as follows: a single colony of GT-5 was streaked onto a carbon-free inorganic liquid culture medium and incubated at 28°C for 7 days to obtain the bacterial suspension. Subsequently, 1 mL of the bacterial suspension was added to 50 g of soil taken from the Dongying saline-alkali land (37°32'21”N, 118°36'19''E, EC value 3.03mS / cm) and incubated indoors at room temperature for 14 days. Two treatment groups were set up in the experiment: normal soil (CK) and soil with added carbon-fixing bacterial suspension (GT-5).

[0057] After 14 days of cultivation, the RubisCO carbon fixation enzyme activity in the two soil groups was measured using a microbial ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCO) ELISA kit. Figure 3 It can be seen that the carbon fixation enzyme activity in normal soil is 8.095 U / L, while the RubisCO carbon fixation enzyme activity in soil with added carbon fixation bacteria solution is significantly increased to 12.905 U / L. This result shows that the application of carbon fixation bacteria GT-5 solution significantly enhances the carbon fixation enzyme activity of soil samples.

[0058] The organic matter content after cultivation was determined using the potassium dichromate titration method-external heating method. The results... Figure 4 The data shows that the organic matter content of normal soil is 9.7779 g·kg⁻¹. -1， The soil with added GT-5 carbon-fixing bacteria solution reached 10.8087 g·kg⁻¹. -1 This result indicates that the application of GT-5 carbon-fixing bacteria solution significantly increased the organic matter content of the soil. These findings provide a theoretical basis for the application of GT-5 and further validate its potential in improving the carbon sequestration capacity of saline-alkali soils.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

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

2. The fiber arcus (Cell Vibrio) according to claim 1, wherein the fiber arcus (Cell Vibrio) is Cell Vibrio GT-5. Cellvibrio sp. ) GT-5 for use in the production of a carbon-fixing enzyme; the carbon-fixing enzyme is a ribulose-1,5-bisphosphate carboxylase / oxygenase.

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

4. The bacterial agent of claim 3, characterized in that, The bacterial agent is in the form of live bacteria of Cell Vibrio sp. Cellvibrio sp. GT-5 is in the form of live bacteria.

5. The bacterial agent of claim 4, 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.

6. The bacterial agent of claim 5, characterized in that, The preparation method of the carbon-free liquid culture 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.

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

8. Use of the microbial agent according to any one of claims 3-7 in fixing CO2 or preparing a carbon fixation agent.

9. A carbon sequestering agent characterized in that, The carbon fixation agent contains the microbial agent according to any one of claims 3-7.

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