A natriuretic vibrio strain capable of degrading biphenyl, and its preparation method and use
By inserting codon-optimized biphenyl degradation gene clusters into Vmax of sodium Vibrio, the VCOD-3 strain was constructed, which solved the problem of biphenyl degradation in high-salinated water bodies and achieved a fast and safe biphenyl degradation effect.
Patent Information
- Application Number
- CN202411060155.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-02
- Publication Date
- 2025-09-02
- Estimated Expiration
- 2044-08-02
AI Technical Summary
There is a lack of strains that can rapidly degrade biphenyl in a high salinity water environment in the prior art, and traditional methods have risks of secondary pollution and biosafety risks.
By inserting codon-optimized biphenyl degradation gene clusters into Vmax of sodium-degraded Vibrio, a VCOD-3 strain was constructed, and the gene clusters were integrated on chromosome 2 using a natural transformation system of Vibrio sodium, and the biphenyl degradation gene was expressed in a high-salt environment to prepare rest cells for degradation of biphenyl.
It achieves rapid degradation of biphenyls in seawater and high-salt industrial wastewater, avoids secondary pollution, and the strain grows quickly, is safe and does not carry antibiotic resistance genes.
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Figure CN118879603B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of bioengineering bacteria, and in particular to a natriuretic vibrio strain capable of degrading biphenyl, a preparation method thereof and applications thereof. Background Art
[0002] Biphenyl, an aromatic compound consisting of two benzene rings, is a member of the polycyclic aromatic hydrocarbons (PAHs). It occurs naturally in coal tar, crude oil, and natural gas, and enters the atmosphere, water bodies, and soil through the combustion of fossil fuels. Biphenyl is harmful to human and animal health. Long-term exposure can cause respiratory and cardiovascular diseases, and can also affect the immune, metabolic, and reproductive systems. It can also cause cancer, teratogenicity, and mutagenesis.
[0003] Existing methods for treating toxic organic pollutants are primarily chemical, physical, and biological. Chemical methods primarily treat toxic organic pollutants through oxidation, but often damage the physical and chemical properties of water and soil during the remediation process, causing secondary pollution. Physical methods cannot completely degrade toxic organic pollutants and also carry the risk of secondary pollution during the collection and transportation of pollutants.
[0004] Using microbial degradation to treat organic pollutants is an inexpensive, thorough treatment method that does not produce secondary pollution. An important limiting factor in the biological treatment of toxic organic pollutants is the resource of microbial strains. Naturally occurring biphenyl-degrading strains in the environment grow slowly. In addition, biphenyl-degrading strains screened from the environment may carry antibiotic resistance genes or pathogenic genes, thus creating biological risks. By using synthetic biology methods, biosafe strains can be used as chassis cells to construct strains with biphenyl-degrading gene clusters. This can break the time limit of natural evolution and quickly obtain biphenyl-degrading strains for the removal of biphenyls from seawater and saline industrial wastewater.
[0005] Therefore, those skilled in the art are committed to developing a bacterial strain that grows rapidly, has no biotoxicity, and can survive and degrade biphenyl in a water environment with high salinity, such as seawater. Summary of the Invention
[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to provide a bacterial strain that grows rapidly, has no biological toxicity, and can survive and degrade biphenyl in a water environment with high salinity such as seawater, and a method for using the same.
[0007] To achieve the above object, the present invention provides a method for preparing a natriuretic vibrio strain capable of degrading biphenyl, characterized in that the method comprises the following steps:
[0008] Step 1, preparing culture medium;
[0009] Step 2, inoculating and culturing Vmax bacteria;
[0010] Step 3, transcribing the biphenyl degradation metabolism artificial gene cluster into the bacteria cultured in step 2;
[0011] Step 4: The artificial gene cluster is installed on chromosome 2 of the Vibrio natriureticus Vmax strain through natural transformation, and the resulting engineered bacterium is named VCOD-3;
[0012] Step 5: inoculating the VCOD-3 strain obtained in step 4 into a liquid culture medium, inducing expression of the artificial gene cluster, and then starving the culture medium to obtain VCOD-3 resting cells, which are the Vibrio natriuresis strain capable of degrading biphenyl.
[0013] In a preferred embodiment of the present invention, step 3 further includes:
[0014] The biphenyl degradation metabolism artificial gene cluster is bphA 1234 BCKHJID, consisting of 11 genes, was cloned from the genome of Pseudomonas putida B6-2 and obtained by chemical synthesis after codon optimization.
[0015] In another preferred embodiment of the present invention, step 3 further includes:
[0016] The gene cluster was cloned from the genome of Pseudomonas putida B6-2. T7 promoter and T7 terminator were added before bphA1 and after bphD respectively to regulate their transcription and expression. Ribosome binding site B0030 was added before each of the other genes. The base sequence of the gene cluster is shown in SEQ ID NO.3.
[0017] In another preferred embodiment of the present invention, step 4 specifically includes:
[0018] The tfoX gene regulated by the T7 promoter was inserted into the dns gene site on chromosome 1 of Vibrio natriuresis Vmax. At this time, the strain can absorb linear DNA fragments through natural transformation after IPTG induction and integrate the DNA fragments into the chromosome through homologous recombination. The artificial gene cluster is then delivered into the cell in the form of linear DNA fragments through natural transformation of Vibrio natriuresis and then integrated into the Chr2_297 site on its chromosome 2 by Vmax's own homologous recombination system to obtain the VCOD-3 strain.
[0019] In another preferred embodiment of the present invention, the step 5 further comprises:
[0020] Step 5.1, preparing NSS simulated seawater solution;
[0021] Step 5.2: Inoculate the VCOD-3 strain into LB3 liquid medium and culture at 30°C, 200 rpm for 45 minutes. Add IPTG to a final concentration of 1 mM to induce expression of the artificial gene cluster for 12 hours.
[0022] Step 5.3: Use the NSS simulated seawater solution to wash and resuspend the induced expression strain, resuspend the remaining cells in NSS simulated seawater or industrial wastewater, and starve them to obtain VCOD-3 resting cells.
[0023] In another preferred embodiment of the present invention, the formula of the NSS simulated seawater solution in step 5.1 is as follows:
[0024] Sodium chloride (NaCl) 7.6g / L, sodium sulfate (Na2SO4) 1.47g / L, sodium bicarbonate (NaHCO3) 0.08g / L, potassium chloride (KCl) 0.25g / L, potassium bromide (KBr) 0.04g / L, magnesium chloride hexahydrate (MgCl2·6H2O) 1.87g / L, calcium chloride dihydrate (CaCl2·2H2O) 0.45g / L, strontium chloride hexahydrate (SrCl2·6H2O) 0.01g / L, boric acid (H3BO3) 0.01g / L.
[0025] In another preferred embodiment of the present invention, the step 5.3 specifically includes:
[0026] The induced bacterial liquid was taken out and centrifuged at 4625g for 20 minutes. The supernatant was discarded and the cells were washed and resuspended with NSS solution. The washing operation was repeated twice. The remaining cells were resuspended in simulated seawater or industrial wastewater, and the OD600 was adjusted to about 10. The cells were starved at 30℃ and 200rpm for 2 hours to obtain VCOD-3 resting cells.
[0027] In another preferred embodiment of the present invention, the starvation treatment in step 5.3 specifically includes:
[0028] The supernatant was removed by centrifugation at 4000 g for 10 min, and the cells were resuspended in NSS-simulated seawater. This process was repeated twice, and the cells were diluted with NSS to an OD600 of 10.0. The cells were then cultured in a shaker at 30°C and 220 rpm for 2 h for starvation treatment.
[0029] The present invention also provides a strain prepared by the aforementioned method for preparing a Vibrio natriureticus strain capable of degrading biphenyl.
[0030] The present invention also provides the use of the aforementioned strain.
[0031] Technical Effects
[0032] 1. The present invention inserts a biphenyl degradation metabolic gene cluster into the chassis cell Vibrio natriuresis. Through codon optimization, the biphenyl degradation gene cluster from Pseudomonas putida can be expressed in Vibrio natriuresis. Vibrio natriuresis has a high salt tolerance. The obtained artificial degradation strain can grow and degrade pollutants in seawater and environments with salinity higher than seawater;
[0033] 2. Develop pollutant-degrading strains using Vibrio natriuresis as a base strain. The genome of Vibrio natriuresis has been sequenced, and it has been found that it does not contain virulence factor encoding genes or antibiotic resistance genes. Therefore, it will not spread to other strains in the environment through horizontal gene transfer, eliminating the steps of genome sequencing, antibiotic resistance testing, and virulence factor identification testing.
[0034] 3. Currently reported natural biphenyl-degrading strains are mostly from the genera Pseudomonas putida, Burkholderia, and Sphingomonas. However, the biphenyl-degrading strain VCOD-3, from the genus Sodium Vibrio, grows significantly faster than other strains, with the fastest growth rate being controlled within 10 minutes. VCOD-3 also exhibits excellent salt tolerance and can maintain normal growth in liquid environments supplemented with 50g / L of sodium chloride. This makes it advantageous for the remediation of biphenyl-contaminated seawater or high-salinity industrial wastewater.
[0035] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0036] Figure 1 This is a growth curve of VCOD-15 bacteria in a preferred embodiment of the present invention in LB3 culture medium supplemented with different concentrations of biphenyl;
[0037] Figure 2 This is a schematic diagram of a biphenyl degradation metabolic gene cluster according to a preferred embodiment of the present invention;
[0038] Figure 3 This is a schematic diagram of the experimental results of the degradation of biphenyl in simulated seawater by the strain VCOD-3 according to a preferred embodiment of the present invention. DETAILED DESCRIPTION
[0039] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.
[0040] The present invention provides an artificial degradation strain VCOD-3 capable of efficiently degrading biphenyl in seawater or industrial saline wastewater. In a resting cell state, the strain can degrade all pollutants within 48 hours in simulated seawater containing 1 mM biphenyl.
[0041] The present invention requires the use of Vibrio natriuresis, and the tfoX gene regulated by the T7 promoter is inserted into the dns gene site on chromosome 1 of Vibrio natriuresis Vmax. At this time, the strain can absorb linear DNA fragments through natural transformation after IPTG induction and integrate the DNA fragments into the chromosome through homologous recombination. At this time, it is the VCOD-2 strain. The artificial gene cluster is then delivered into the cell in the form of a linear DNA fragment through natural transformation of Vibrio natriuresis, and then integrated into the Chr2_297 site on chromosome 2 by Vmax's own homologous recombination system (the sequence information of this site can be obtained from the NCBI website, with the sequence number WP_014233456.1), thereby obtaining the VCOD-3 strain.
[0042] Example 1: Utilizing the Natural Transformation Characteristics of Vibrio natriuresis to Obtain VCOD-2 Strain Capable of Expressing Exogenous Fragments
[0043] 1. Cloning of the global regulatory factor VchtfoX of natural transformation in Vibrio cholerae
[0044] As shown in Table 1, the tfoX gene on the Vibrio cholerae genome was codon-optimized based on the codon frequency of Vibrio natriuresis, resulting in the gene sequence shown in SEQ ID NO. 1. The T7 promoter and T7 terminator were added before and after the tfoX gene, respectively, to obtain the complete VchtfoX expression cassette sequence. The sequence of the expression cassette gene is shown in SEQ ID NO. 2. The VchtfoX expression cassette DNA fragment was obtained by chemical synthesis. The VchtfoX fragment was ligated into the pET28a plasmid using Gibson assembly, amplified, and extracted in Escherichia coli.
[0045] Table 1: Statistics of codon usage frequency of Vmax genome of Vibrio natriuresis
[0046]
[0047] Prepare LB3 medium according to the following formula: 10 g / L tryptone, 5 g / L yeast extract, and 30 g / L sodium chloride (NaCl). Inoculate 100 μL of the Vmax strain seed solution into 5 mL of the medium and culture in a constant temperature shaker at 30°C and 220 rpm.
[0048] 2. Preparation of Vmax Electroporation Competent Cells
[0049] Cultivate in 50 mL of LB3 medium until Vmax reaches OD600 = 0.4-0.6, centrifuge at 4200 rpm for 8 min, remove the supernatant, and resuspend in 25 mL of sucrose buffer. Repeat three times and finally resuspend in 5 mL of sucrose buffer (the formula of sucrose buffer is 232.8 g / L sucrose and 1.6 g / L potassium dihydrogen phosphate (K2HPO4)). Aliquot 50 μL into a 1.5 mL centrifuge tube, quickly freeze in liquid nitrogen, and store at -80°C.
[0050] 3. Electrotransformation of pET28a-VchtfoX plasmid
[0051] Electroporation was performed using an electroporator at 1.4 kV / 1.6 kV, 200 Ω, and 25 μF. The clone was plated on LB3 solid medium (i.e., LB3 medium supplemented with 18 g / L agar powder) supplemented with 200 ng / μL kanamycin and cultured overnight at 30°C. The resulting positive clone was Vmax-pET28a-VchtfoX.
[0052] 4. Insertion and expression of the VchtfoX gene in the Vmax genome of Vibrio natriuresis
[0053] Take a Vmax-pET28a-VchtfoX bacterial culture and streak it onto LB3 solid medium supplemented with 200 ng / μL kanamycin. Transfer the culture to a 37°C incubator and incubate for approximately 8 hours until a single colony is visible. In a 14 mL Falcon tube, use a pipette to precisely add 2.997 mL of LB3 medium and 3 μL of 1 M IPTG solution. Pick a single colony of Vmax-pET28a-VchtfoX and culture it. Incubate the culture in a shaker at 30°C at 220 rpm for 4-4.5 hours.
[0054] After the bacterial solution becomes turbid, measure its OD600. When OD600 reaches 3.9-4.6, the competent cells can be used. Take a 1.5 mL centrifuge tube (sterilized) and add 1 μL of the donor linear fragment.
[0055] Prepare IOM medium: IOM (Instant Ocean Medium) is a commercial solid seawater medium used for aquarium fish farming (Aquarium Systems Inc., Catalog No. SS15-10). Dissolve it in a liquid at a concentration of 28 g / L and sterilize it at 121°C for 20 minutes before use.
[0056] The T7 RNA polymerase locus on the Vmax 1 genome of Vibrio natriuresis was selected as the insertion site for VchtfoX. A premix system was prepared according to the ratio of each fragment to be transformed: 350 μL 1OM medium + 0.35 μL 1M IPTG + 3.5 μL competent cells. 350 μL of the premix system was then drawn and mixed with the VchtfoX linear fragment (i.e., a fragment containing the expression cassette, sequence SEDID NO.2) solution. The cells were cultured at 30°C for 6 hours. 1 mL of LB3 culture medium was added for recovery, cultured at 30°C and 220 rpm for 1.5 hours, and then cultured in a 30°C incubator. A single colony was picked for PCR detection, and the single bacterium with the VchtfoX gene inserted was named VCOD-2.
[0057] Example 2 Composition and Induced Expression Method of Complex Pollution Degradation Gene Cluster
[0058] 1. Composition of the artificial gene cluster bph
[0059] like Figure 2 As shown, the biphenyl degradation gene cluster bphA1234BCKHJID consists of 11 genes. This gene cluster was cloned from the genome of Pseudomonas putida B6-2 and obtained by chemical synthesis after codon optimization. A T7 promoter and T7 terminator were added before bphA1 and after bphD, respectively, to regulate their transcription and expression. The ribosome binding site B0030 was added before each of the remaining genes. The sequence of the gene cluster is SEQ ID NO. 3. This gene cluster was installed on chromosome 2 of the Vmax strain of Vibrio natriuresis through multiple iterative gene editing. The resulting Vmax engineered strain was named VCOD-3. Vmax is a commercial strain of Vibrio natriuresis that is commercially available.
[0060] 2. Preparation of VCOD-3 strain
[0061] (1) Using VCOD-2 to insert foreign genes into the genome of Vibrio natriuresis
[0062] Remove the VCOD-2 from the glycerol tube stored at -20°C and streak onto LB3 solid medium. Transfer to a 37°C incubator and incubate for approximately 8 hours until a single colony is visible. In a 14mL Falcon tube, accurately add 2.997mL of LB3 medium and 3μL of 1M IPTG solution using a pipette. Pick a single colony and culture. Incubate at 30°C in a shaker at 220 rpm for 4-4.5 hours.
[0063] After the bacterial solution becomes turbid, measure its OD600. Competent cells can be used when OD600 reaches 3.9-4.6. Prepare a premixed system in a sterile 1.5 mL centrifuge tube (350 μL of 1OM medium + 0.35 μL of 1M IPTG + 3.5 μL of competent cells per experimental group of fragments to be transformed). Then, pipette 350 μL of this premixed system and mix it with 50-200 ng of a linear donor DNA fragment solution. This linear fragment represents the biphenyl degradation gene cluster bphA1234BCKHJID, whose sequence is shown in SEQ ID NO. 3. Incubate at 30°C for at least 6 hours (this can be extended, not shortened). Add 1 mL of LB3 medium to resuscitate the culture, incubate at 30°C, 220 rpm, for 1.5 hours, and pipette the bacterial solution onto a plate (20-100 μL. If the transformation efficiency is extremely low, increase the concentration to 1 mL by centrifugation and remove the supernatant to 100 μL before plating). Incubate in a 30 or 37°C incubator.
[0064] Add 6 μL of LB3 medium to the eight-tube strip. Pick a single bacterium from the overnight culture plate and dissolve it in 6 μL of LB3 medium by rinsing with a pipette tip. Add 3 μL of the bacterial solution to another corresponding resistance plate and incubate at 37°C until colonies grow noticeably. Pick bacteria from the edge of the colony and dissolve them in 20 μL of ddH2O. Heat at 95°C for 3 minutes, cool at 4°C for 2 minutes, and repeat 5 cycles. Take 3 μL of the high-temperature treated bacterial solution as a template and perform PCR using Taq thermostable DNA polymerase to verify whether the exogenous gene has been inserted into the designated site.
[0065] 3. Culture method of VCOD-3
[0066] (1) Preparation of culture medium
[0067] LB3 liquid culture medium was prepared according to the following formula: 10 g / L tryptone, 5 g / L yeast extract, and 30 g / L sodium chloride (NaCl).
[0068] Add 1.8 g / L agar powder to LB3 liquid medium and sterilize at 121°C for 20 minutes to obtain a solid medium. After heating, antibiotics and inducers can be added as needed for cultivation.
[0069] (2) Inoculation of VCOD-3
[0070] A small amount of VCOD-3 bacterial suspension was picked from the glycerol tube stored at -80℃, streaked onto an LB3+5ng / μL chloramphenicol plate, and cultured overnight at 30℃. A single colony was picked and cultured in LB3+5ng / μL chloramphenicol+1mM IPTG liquid medium at 30℃ and 200rpm. The growth curve is shown in the figure below. Figure 1shown.
[0071] 4. Inducible Expression of the Artificial Gene Cluster bphA1234BCKHJID on the VCOD-3 Genome and Preparation of Resting Cells
[0072] A simulated seawater solution (NSS) was prepared according to the following formula: sodium chloride (NaCl) 7.6 g / L, sodium sulfate (Na2SO4) 1.47 g / L, sodium bicarbonate (NaHCO3) 0.08 g / L, potassium chloride (KCl) 0.25 g / L, potassium bromide (KBr) 0.04 g / L, magnesium chloride hexahydrate (MgCl2·6H2O) 1.87 g / L, calcium chloride dihydrate (CaCl2·2H2O) 0.45 g / L, strontium chloride hexahydrate (SrCl2·6H2O) 0.01 g / L, and boric acid (H3BO3) 0.01 g / L.
[0073] VCOD-3 cells were inoculated into LB3 liquid medium and cultured at 30°C, 200 rpm, for 45 minutes. IPTG was added to the container to a final concentration of 1 mM to induce expression of the artificial gene cluster. After 12 hours, the culture was removed and centrifuged at 4625g for 20 minutes. The supernatant was discarded, and the cells were washed and resuspended in NSS solution. This washing process was repeated twice. The remaining cells were resuspended in simulated seawater or industrial wastewater, adjusted to an OD600 of approximately 10, and starved for 2 hours at 30°C, 200 rpm, to obtain VCOD-3 resting cells.
[0074] Example 3 HPLC quantitative detection method of biphenyl
[0075] Biphenyl was analyzed using an Agilent 1200 Series high-performance liquid chromatograph (HPLC) equipped with a UV detector and an Agilent Eclipse XDB-C18 column. The mobile phase consisted of 80% methanol and 20% HCl (0.1% formic acid for pH adjustment) at a flow rate of 0.8 mL / min. The column was maintained at 30°C, and the analysis time for a single sample was 15 minutes. The 280 nm wavelength channel was used for biphenyl detection.
[0076] Example 4 Experiment on degradation of composite pollutants in simulated seawater by composite pollution degradation strain VCOD-3
[0077] The VCOD-3 bacteria were resuspended into resting cells with OD600=10 using simulated seawater NSS and starved. Each 10 mL was divided into 50 mL ground-mouth conical flasks. Three parallels were made for each sampling point. A mixed pollutant mother liquor dissolved in DMF was added to make the initial biphenyl concentration 1 mM. Using Vibrio natriuresis Vmax as the negative control group, resting cells of the same concentration were prepared and subsequently subpackaged and treated with pollutants. Samples were taken at 0, 6, 12, 24, and 48 hours. 10 mL of ethyl acetate was added, vortexed at 45 W for 2 minutes, centrifuged at 8000 rpm for 10 minutes, and the supernatant was taken for subsequent testing. The degradation results are shown as follows. Figure 3 shown.
[0078] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.
Claims
1. A method for preparing a natriuretic vibrio strain capable of degrading biphenyl, characterized in that: The method comprises the following steps: Step 1, preparing culture medium; Step 2, inoculating and culturing the Vmax strain of Vibrio natriuresis; Step 3: inserting the tfoX gene regulated by the T7 promoter into the dns gene site on chromosome 1 of the Vibrio natriureticus Vmax strain obtained in step 2 to obtain the VCOD-2 strain; Step 4: The artificial gene cluster was installed on chromosome 2 of the VCOD-2 strain obtained in step 3 through natural transformation. The resulting engineered strain was named VCOD-3. bphA 1234 BCKHJID , consisting of 11 genes, this gene cluster was cloned from the genome of Pseudomonas putida B6-2 and obtained by chemical synthesis after codon optimization; the method for obtaining the gene cluster is as follows: a T7 promoter and a T7 terminator are added before bphA1 and after bphD, respectively, to regulate their transcription and expression, and a ribosome binding site B0030 is added before each of the remaining genes. The sequence of the gene cluster is SEQ ID NO. 3; Step 5: inoculating the VCOD-3 strain obtained in step 4 into a liquid culture medium, inducing expression of the artificial gene cluster, and then starving the culture medium to obtain VCOD-3 resting cells, which are the Vibrio natriuresis strain capable of degrading biphenyl; The step 4 specifically includes: The artificial gene cluster is delivered into the cell in the form of a linear DNA fragment through natural transformation of Vibrio natriuresis and then integrated into the Chr2_297 site of chromosome 2 by the Vmax self-homologous recombination system to obtain the VCOD-3 strain.
2. The preparation method according to claim 1, wherein The step 5 further comprises: Step 5.1, prepare NSS simulated seawater solution; the formula of the NSS simulated seawater solution is as follows: Sodium chloride 7.6 g / L, sodium sulfate 1.47 g / L, sodium bicarbonate 0.08 g / L, potassium chloride 0.25 g / L, potassium bromide 0.04 g / L, magnesium chloride hexahydrate 1.87 g / L; calcium chloride dihydrate 0.45 g / L, strontium chloride hexahydrate 0.01 g / L, boric acid 0.01 g / L; Step 5.2: Inoculate the VCOD-3 strain into LB3 liquid medium and culture at 30°C, 200 rpm for 45 minutes. Add IPTG to a final concentration of 1 mM to induce expression of the artificial gene cluster for 12 hours. The LB3 liquid medium contains 10 g / L tryptone, 5 g / L yeast extract, and 30 g / L sodium chloride. Step 5.3: Use the NSS simulated seawater solution to wash and resuspend the induced expression strain, resuspend the remaining cells in the NSS simulated seawater or industrial wastewater, and starve them to obtain VCOD-3 resting cells.
3. The preparation method according to claim 2, wherein The step 5.3 specifically includes: The induced bacterial liquid was removed and centrifuged at 4625 g for 20 minutes. The supernatant was discarded and the cells were washed and resuspended with NSS solution. The washing operation was repeated twice. The remaining cells were resuspended in simulated seawater or industrial wastewater, and the OD600 was adjusted to about 10. The cells were starved at 30°C and 200 rpm for 2 hours to obtain VCOD-3 resting cells.
4. A strain prepared by the method for preparing a biphenyl-degrading Vibrio natriureticus strain according to any one of claims 1 to 3.