A Flavobacterium and a method for synthesizing biological protein using the same

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to a strain of Xanthobacter flavus and a method for synthesizing biological protein using the same. The specific technical solution is as follows: a strain of Xanthobacter flavus CIB1, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 19, 2022, with the deposit number: CGMCC NO. 24923. The present invention provides a method for a hydrogen-oxidizing bacterium to synthesize biological protein using CO2 and air. Under the condition of no addition of nitrogen source compounds, it can not only achieve the capture and reduction of CO2, but also obtain high-value-added biological protein, having the advantages of low cost and environmental friendliness.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and particularly relates to a strain of Xanthobacter flavus and a method for synthesizing biological protein using the same. Background Art

[0002] Hydrogen-oxidizing bacteria (HOB) are a type of bacteria that use hydrogen as an electron donor to reduce CO2 to organic matter. There are research reports that some pure HOB strains can synthesize high-value-added products. For example, Cupriavidus eutrophus can synthesize polyhydroxyalkanoates (PHA), Cupriavidus necator can synthesize terpenes, and Ralstonia eutropha can synthesize isopropanol, etc. However, there is little in-depth research on N2 fixation based on HOB. There is no report on using HOB to simultaneously fix CO2 and N2 (using CO2 as the sole carbon source and N2 as the sole nitrogen source) to prepare biological protein.

[0003] Air contains abundant and inexpensive N2 and the O2 required by HOB. If CO2 and air are used as raw materials and HOB is used to produce high-value-added biological protein, it can achieve the capture and emission reduction of high carbon emissions in industries such as petrochemical and coal-fired. The synthesized biological protein can replace high-protein feeds such as fish meal and soybean meal, alleviate the problem that the feed sources in China's aquaculture and livestock farming industries highly rely on imports, and can also indirectly save arable land resources, which undoubtedly has very important significance. Summary of the Invention

[0004] The purpose of the present invention is to provide a strain of Xanthobacter flavus and a method for synthesizing biological protein using the same.

[0005] To achieve the above-mentioned invention purpose, the technical solution adopted by the present invention is: a strain of Xanthobacter flavus CIB1, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 19, 2022, and the deposit number is: CGMCC NO.24923.

[0006] Preferably, the 16S rDNA sequence of the Xanthobacter flavus is as shown in SEQ ID NO.1.

[0007] Correspondingly, the application of the Xanthobacter flavus in the preparation of biological protein.

[0008] Preferably, in the above application, the carbon source utilized by the Xanthobacter flavus comes from CO2.

[0009] Preferably, in the above application, the nitrogen source utilized by the Xanthobacter flavus comes from N2.

[0010] Preferably, in the above application, the energy source for the fermentation of Xanthobacter flavus comes from H2 and / or electricity.

[0011] The present invention has the following beneficial effects: The present invention provides a method for a hydrogen-oxidizing bacterium to synthesize biological protein using CO2 and air. Under the condition of no addition of nitrogen-containing compounds, it can not only achieve the capture and reduction of CO2, but also obtain high-value-added biological protein, with the advantages of low cost and environmental friendliness. The present invention can use H2 or electricity as the only input energy source, CO2 as the only carbon source, and N2 as the only nitrogen source. Compared with the production of soybean protein, milk protein, etc., it not only saves production land space, but also can achieve the capture and reduction of high carbon emissions in industries such as petrochemical and coal-fired. Description of the Drawings

[0012] Figure 1 Schematic structural diagram of the fermentation device used in Example 2;

[0013] Figure 2 Schematic diagram of the change in the OD value of Xanthobacter flavus in Group 2 of Example 2;

[0014] Figure 3 Schematic diagram of the change in the total organic carbon of Xanthobacter flavus in Group 2 of Example 2;

[0015] Figure 4 Schematic structural diagram of the fermentation device used in Example 3;

[0016] Figure 5 Schematic diagram of the change in the OD value of Xanthobacter flavus in Group 2 of Example 3. Detailed Embodiments

[0017] A strain of Xanthobacter flavus CIB1 of the present invention has its 16S rDNA sequence shown in SEQ ID NO.1. This microorganism was deposited on May 19, 2022 at the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is: CGMCC NO.24923.

[0018] The Xanthobacter flavus can use CO2 and N2 to prepare biological protein. Therefore, based on this microorganism, the present invention further provides a method for synthesizing biological protein, which specifically includes the following steps:

[0019] 1. Inoculate 2% - 10% (v / v) of the said Flavobacterium in the culture medium, and the OD value of Flavobacterium after inoculation is 0.02 - 0.10. The components of the culture medium are: K2HPO4 0.8 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.50 g / L, CaSO4·2H2O 0.05 g / L, sodium molybdate 2.5 mg / L, FeSO4 15 mg / L, vitamin 10 mL / L, trace elements 10 mL / L. Unless otherwise specified, the vitamin and trace element formulations used in the present invention are the same as those in DSMZ 141 culture medium.

[0020] 2. Before the reaction, evacuate the air in the reactor; the main purpose of evacuation is to reduce the O2 content, and too high O2 content is not conducive to protein synthesis. Subsequently, introduce gaseous substrates CO2, N2 and O2 into the reactor; because N2 accounts for a very high proportion in the air, for cost savings, air can also be used instead of N2 in production. Among them, CO2 and N2 are the carbon source and nitrogen source respectively, accounting for 20 - 60% and 10 - 50% of the total gas volume respectively, and O2 accounts for 1 - 10% of the total gas substrate volume.

[0021] 3. Introduce H2 into the reactor (such that H2:O2 is 16:1 - 4:1) or provide electricity as the energy source. When choosing electricity as the energy source, the preferred solution is: provide electrical energy to the reaction system in the reactor, that is, apply a voltage of 2 - 5 V or adjust the cathode potential to -0.7 - -1.1 V (vs. Ag / AgCl, saturated KCl). It should be noted that: H2 can also be provided simultaneously and electricity can be provided as the energy source.

[0022] 4. Set the operating conditions as 25 - 35 °C, pH 6 - 8, the stirrer speed is 200 - 800 rpm, and culture for 7 - 20 days. If the pH is too high or too low, adjust it with a small amount of HCl or NaOH.

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0024] Example 1: Screening and Identification of Microorganisms

[0025] 1. Microbial source and screening. Take 10 mL of mixed microbial strains (originally from activated sludge) from the laboratory hydrogen autotrophic reactor and add them to a 500 mL anaerobic bottle containing 100 mL of inorganic salt medium without organic carbon and nitrogen. The composition of this medium is: K2HPO4 0.8 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.50 g / L, CaSO4·2H2O 0.05 g / L, sodium molybdate 2.5 mg / L, FeSO4 15 mg / L, vitamins 10 mL / L, trace elements 10 mL / L. Pass a mixed gas of H2, N2, CO2, and O2 (volume ratio 16:4:2:1) into the headspace of the anaerobic bottle, and then culture for 1 month at room temperature. Then transfer it to a 1 L fermenter and continuously pass a mixed gas of H2, N2, CO2, and O2 (volume ratio 8:4:2:1). After observing the phenomenon of rapid proliferation of the microbial community, use the dilution plating method to screen out the autotrophic bacteria in the bacterial solution. Pick single colonies to obtain purified microbial cells.

[0026] 2. Molecular identification of strains. Pick single colonies into LB medium (2 mL) and culture overnight on a shaker. Extract DNA from the microbial samples, use universal bacterial 16S primers for amplification and sequencing (completed by Beijing Qingke Biotechnology Co., Ltd., Chengdu Branch), and use the sequencing results for comparison in the NCBI database to preliminarily identify the samples. After comparison by the Blast tool, the strain is Xanthobacter flavus. Its 16S rDNA sequence is shown in SEQ ID NO.1. This microorganism was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on May 19, 2022, with the deposit number: CGMCC NO.24923.

[0027] Example 2: Demonstration of the effect of Xanthobacter flavus in synthesizing biological proteins under a certain implementation mode

[0028] 1. Construct the reaction device according to Figure 1 the method. Figure 1It is a biological protein fermentation device, including a fermentation tank 10. The fermentation tank 10 is in a sealed state, and air is introduced into the interior of the fermentation tank 10 through a first gas mixer 12. The preferred solution is that the exhaust pipe of the first gas mixer 12 extends into the bottom of the fermentation tank 12, in a circular shape, and the circular size is adapted to the size of the bottom of the fermentation tank 12, so as to evenly input gas into the fermentation tank 10 from bottom to top. The first gas mixer 12 is also connected to gas cylinders of H2, CO2 and air, and the volume ratio of each gas in the first gas mixer 12 is controlled by the volume of the gas cylinder, the ventilation speed, etc. The fermentation tank 10 is filled with a culture medium and inoculated with microorganisms. A stirrer 11 is also arranged in the fermentation tank 10 to mix the culture medium, microorganisms and gas evenly. In addition, a pH electrode 13, a thermometer 14 extending into the interior of the fermentation tank 10, and a tail gas collection device 15 are also arranged at the top of the fermentation tank 10. Among them, the pH electrode 13 is used to measure the pH of the fermentation system.

[0029] 6 groups of experiments were set up, and the reaction devices used in each group were the same and were all constructed according to Figure 1 1 L of the culture medium was added to the fermentation tank 10 and inoculated with the yellow xanthobacteria. The stirring device was turned on, and then the gas cylinders were opened to introduce a mixed gas of H2, CO2 and air (volume ratio 3:1:1), and the ventilation state was maintained. The operating conditions were set at 28 °C, pH 6.5, and the stirrer rotation speed was 200 rpm for a certain period of time. During the operation, the culture solution in the reaction device was taken every day to measure the OD value of the yellow xanthobacteria, calculate the growth of total organic carbon (TOC, mg / L, the total amount of carbon contained in the organic matter in the culture solution during the cycle, measured by a TOC analyzer), and after the culture was completed, the cell biomass (CDW, g / L, CDW = 1000m / 50, m represents the mass of the precipitate after drying 50 mL of the culture solution, unit g; 50 represents the volume of the taken culture solution, unit mL), crude protein concentration (MP, mg / L, the amount of crude protein in the microbial cells during the fermentation cycle, measured by the Kjeldahl method), and the protein content in the cells (calculated by the ratio of the crude protein content to the cell dry weight, (MP / CDW)×10) were measured. The experimental conditions set for each group are shown in Table 1.

[0030] Table 1 Comparison table of reaction conditions for each group

[0031]

[0032] 2. The test results after the reaction of each group are shown in Table 2. Among them, the change in the OD value of the yellow xanthobacteria in Group 2 every day is as Figure 2 shown, and the growth of total organic carbon in Group 2 is as Figure 3 shown.

[0033] Table 2 Comparison table of results display for each group

[0034]

[0035] The inventors found that under the same conditions, when the culture time was extended, the protein content of the bacteria decreased while the crude protein concentration increased. This might be because after the amount of bacteria increased, Flavobacterium flavum was more inclined to synthesize extracellular polymers.

[0036] Example 3: Demonstration of the effect of Flavobacterium flavum on synthesizing biological protein under another implementation mode

[0037] 1. Construct the reaction device as Figure 4 shown. Compared with Example 2, in this example, the energy was changed from introducing H2 to inputting electric energy, that is, an electric current was applied externally to the reaction system. Figure 4 It is an electro-synthesis device, including an anode chamber 20 provided with an anode 22 and a cathode chamber 21 provided with a cathode 23. The anode chamber 20 and the cathode chamber 21 are connected through a proton exchange membrane 28, and the anode 22 and the cathode 23 are electrically connected through an electric power device 25. Both the anode chamber 20 and the cathode chamber 21 are sealed tanks, and channels for inserting reference electrodes are provided on the side walls. A reference electrode 24 is inserted into the cathode chamber 21 through a channel on the side wall of the cathode chamber 21, and the reference electrode 24 extends into the liquid in the cathode chamber 21. A second gas mixer 26 is provided at the top of the cathode chamber 21, and the setting method is the same as that of the first gas mixer 12, both of which are externally connected to gas cylinders and can introduce the required mixed gas. It should be understood that: Figure 4 the construction method is for illustration rather than limitation, and those skilled in the art can introduce an electric current into the reaction system using other structures or methods according to the actual situation.

[0038] Set 8 groups of experiments. The reaction devices used in each group are the same and are all constructed as Figure 4 shown. Add 100 mL of culture medium to each reaction device and inoculate 2% (v / v) of the Flavobacterium flavum. After inoculation, the initial OD value of the culture medium is 0.02. Then turn on the stirring device, and then turn on the gas cylinder to introduce a mixed gas of CO2 and air (volume ratio 3:1) of 1.5 L. After starting to run, introduce 1.5 L of fresh mixed gas every 2 days. Set the operating conditions as 28 °C, pH 6.5, and the stirring speed of the stirrer is 400 rpm.

[0039] During the operation, 0.5 L of hydrogen gas was introduced every 2 days in Group 1 and Group 4 as the energy source. Other groups controlled different cathode potentials. Regularly take the culture solution in the reaction device to measure the OD value of the microorganisms, and after the culture ends, measure the biomass of the bacteria, the total organic carbon content, the crude protein concentration, and the protein content in the bacteria. The experimental conditions set for each group are shown in Table 3.

[0040] Table 3 Comparison table of reaction conditions for each group

[0041]

[0042]

[0043] 2. The test results after each group reaction are shown in Table 4. Among them, the change in the OD value of Flavobacterium per day in Group 2 is as Figure 5 shown.

[0044] Table 4 Comparison Table of Results Displayed for Each Group

[0045]

[0046] For Group 6 in Table 4, since the biomass of the bacteria was too low, the total organic carbon value was not measured and recorded as "--". In addition, the small reactor used in this example had its gas replaced every 2 days, while the large reactor was used in Example 2 with fresh gas continuously supplied, resulting in the protein yields of Groups 1 and 4 in this example being lower than the output value of Example 2.

[0047] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, variations, modifications, and substitutions made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Flavobacterium sp. ( Xanthobacter flavus ), CIB1, characterized in that: The said Xanthobacter flavus was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on May 19, 2022, with the deposit number: CGMCC NO. 24923.

2. Use of the Xanthobacter flavus CIB1 described in claim 1 in the preparation of biological proteins.

3. The application according to claim 2, characterized in that: In the said use, the carbon source utilized by the Xanthobacter flavus is CO2.

4. The application according to claim 2, wherein: In the said use, the nitrogen source utilized by the Xanthobacter flavus is N2.

5. The application according to claim 2, wherein: In the said use, the energy sources used for the fermentation of the Xanthobacter flavus are H2 and / or electricity.

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