Sphingosine cyanidin and application thereof
By isolating and accumulating Yanoguchi Sphingosine bacteria, using its biodegradation function to efficiently degrade RDX, the problem of high cost of RDX pollution treatment and possible secondary pollution in the prior art has been solved, and efficient, economical and environmentally friendly pollution repair effect has been achieved .
Patent Information
- Application Number
- CN202411321673.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-23
- Publication Date
- 2024-12-20
AI Technical Summary
The prior art has problems such as high treatment cost, complex operation and possible secondary pollution when dealing with RDX pollution, and lacks efficient, economical and environmentally friendly pollution treatment methods.
By isolating and accumulating a Sphingobium yanoikuyae LXC-RD-4, it uses its biodegradation function to convert RDX into a harmless or low-toxic substance to achieve efficient degradation.
Achieving a 77.7% degradation rate of RDX provides a practical bioremediation solution that reduces the cost and environmental impact of contamination treatment.
Smart Images
Figure CN119162031A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a Sphingobacterium yanostomum strain and application thereof. Background Art
[0002] Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common cyclic nitramine explosive, which is widely used in military and civilian blasting due to its high energy, high stability and good explosive performance. However, RDX may cause environmental pollution during its production, use and disposal, especially in the case of accidental leakage or improper handling, and its potential harm to soil and groundwater has attracted widespread attention.
[0003] Traditional RDX pollution treatment methods, such as physical adsorption, chemical oxidation and soil elution, can reduce pollution to a certain extent, but they often have problems such as high treatment costs, complex operations, and possible secondary pollution. Therefore, it is of great practical significance to develop an efficient, economical and environmentally friendly RDX pollution treatment technology.
[0004] In recent years, biodegradation technology has become a hot topic of research due to its environmental friendliness and sustainability. Using microorganisms to biotransform pollutants into harmless or low-toxic substances is a strategy with great potential for environmental pollution control. In particular, biodegradation shows unique advantages when dealing with difficult-to-degrade pollutants, such as explosive compounds.
[0005] To use biological methods to treat RDX components, it is necessary to isolate, culture or domesticate specific microbial strains with efficient degradation functions. Microorganisms grow and metabolize RDX as a nitrogen source to achieve the purpose of degradation. It is urgent to find a microorganism that can degrade RDX. Summary of the invention
[0006] The purpose of the present invention is to provide a Sphingobacterium Yanoguchii that can degrade RDX.
[0007] The present invention provides a strain of Sphingobium yanoikuyae, the name of the Sphingobium yanoikuyae is LXC-RD-4, which is deposited in the China General Microbiological Culture Collection Center on November 19, 2020, and the deposit number is: CGMCC NO.21207.
[0008] The present invention provides a microbial preparation containing the above-mentioned Sphingobacterium Yanoguchii.
[0009] The present invention provides an application of the above-mentioned Sphingobacterium Yanoguchii or the above-mentioned microbial preparation in resisting RDX.
[0010] The present invention provides an application of the above-mentioned Sphingobacterium Yanoguchii or the above-mentioned microbial preparation in degrading RDX.
[0011] The present invention provides an application of the above-mentioned Sphingobacterium Yanoguchii or the above-mentioned microbial preparation in purifying and repairing soil or industrial wastewater.
[0012] It is further defined that the application is the degradation of RDX.
[0013] The invention provides a method for degrading RDX. The method comprises adding the above-mentioned Sphingobacterium Yanoorhiza into a sample containing RDX and reacting the sample at 30°C for at least 3 days.
[0014] Furthermore, the amount of Sphingobacterium Yanoguchii added is limited to 2%.
[0015] It is further defined that the sample is an inorganic salt culture medium or soil.
[0016] It is further defined that the concentration of RDX is 16 mg / L.
[0017] Beneficial effects: The present invention targets wastewater containing RDX, utilizes the Sphingobacterium yanogamii flora to achieve biodegradation under a common matrix, utilizes biological methods to treat RDX components, and performs special cultivation, domestication and separation of specific microbial strains with efficient degradation functions. This patent obtains a strain of RDX-degrading bacteria through targeted domestication and separation, achieves biodegradation of RDX, and provides a practical solution for the bioremediation of RDX pollution, with an RDX degradation rate of 77.7%.
[0018] [Biological Deposit]: A strain of Sphingobium yanoikuyae, the name of which is LXC-RD-4, deposited in the China General Microbial Culture Collection Center, the deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is November 19, 2020, and the deposit number is: CGMCC NO.21207. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 Phylogenetic tree constructed for Sphingobium yanoikuyae LXC-RD-4 and strains with high homology included in GenBank;
[0020] Figure 2 This is a colony image of Sphingobium yanoikuyae LXC-RD-4;
[0021] Figure 3 This is the growth curve of Sphingobium yanoikuyae LXC-RD-4 in an inorganic salt liquid medium containing RDX;
[0022] Figure 4 This is the degradation curve of Sphingobium yanoikuyae LXC-RD-4 in RDX solution;
[0023] Figure 5 This is the tolerance curve of Sphingobium yanoikuyae LXC-RD-4 in RDX solution. DETAILED DESCRIPTION
[0024] Example 1: Isolation of Sphingobacterium Yanoguchii
[0025] The present invention adopts a method of gradually increasing the substrate concentration to screen out RDX-degrading bacteria from polluted wastewater through enrichment, domestication, separation and purification. The specific implementation method is as follows:
[0026] The inorganic salt medium used includes Na2HPO 4· 12H2O 15.2g / L, KH2PO4 3g / L, NaCl 0.5g / L, NH4Cl1g / L, glucose 2g / L; add 2% agar to the solid culture medium; the formula of trace element solution is MnSO4 0.039g / L, ZnSO4·H2O 0.043g / L, (NH4)2MoO4·4H2O 0.035g / L, take 1mL of trace elements and add them to the pollutant inorganic salt culture medium, and adjust the pH to about 7. Take 100mL of the above culture medium with the solvent as 300mL conical flask, sterilize it at 121℃ for 20min for use. Use ultrapure water to make a solution with an RDX content of 1g / L, sterilize it through a 0.22μm filter membrane, and add it to the inorganic salt culture medium in proportion to prepare enrichment and acclimation culture medium of different concentrations. Take 10mL of wastewater and inoculate it into the enrichment screening medium with an initial RDX concentration of 5mg / L, and culture it in a constant temperature shaker at 30℃ and 180r / min for 7d. Then, inoculate it into the enrichment screening medium containing a higher concentration of pollutants at a transfer volume of 5% for the second enrichment and acclimation culture. Repeat the culture 5 times in total, and the concentration of the transferred pollutants each time is 5mg / L, 10mg / L, 15mg / L, 20mg / L, and 25mg / L. Then, the culture solution of the last enrichment and acclimation was transferred to the solid inorganic salt medium containing RDX by the dilution plate method to separate the strains by streaking, and cultured at a constant temperature of 30℃ for 5-7d. Pick out single colonies with different colors, sizes and shapes and good growth and streak them into LB medium for purification culture.
[0027] This strain is a Gram-negative bacterium, and the colony morphology is shown in Figure 2 , characterized by: the colony is round, light yellow and opaque, with a moist, smooth and raised surface.
[0028] Example 2: Identification of Sphingobacterium yanogamicin
[0029] The present invention uses 16S rDNA (27F: AGTTTGATCMTGGCTCAG, SEQ ID NO.1) and 1492R: GGTTACCTTGTTACGACTT, SEQ ID NO.2) to perform homology analysis on the gene sequence of the screened strains, performs sequence comparison through the NCBI (National Center for Biotechnology Information) database, and constructs a phylogenetic tree of each strain using MEGA-X software and the neighbor joining method. The confidence of each branch of the phylogenetic tree is obtained by repeated experiments of 1000 times using the resampling method. The comparison results are shown in FIG. Figure 1 As shown, the strain was identified as Sphingobiu myanoikuyae, named LXC-RD-4, and deposited in the China General Microbiological Culture Collection Center on November 19, 2020, with the deposit number: CGMCC No.21207.
[0030]
[0031] Example 3: Degradation of RDX by Sphingobacterium Yanoguchii
[0032] The invention determines the degradation characteristics of Sphingobacterium yanostomum LXC-RD-4 on RDX, and the specific steps are: inoculating the strain into an inorganic salt culture medium containing RDX with an inoculation amount of 2%, placing the culture medium in a constant temperature shaker at 30 DEG C and 180 r / min in the dark for 8 days, observing the change of the bacterial solution, and regularly determining the growth status of the strain in the solution and the residual amount of RDX every day.
[0033] from Figure 3 It can be seen that the delay period of Sphingobium yanoikuyae LXC-RD-4 is relatively long. After 3 days of culture, it enters the logarithmic growth phase. Subsequently, the substances in the culture medium for the growth of the strain are continuously consumed, the growth rate of the strain slows down, and its activity gradually decreases. Figure 4 As shown, the RDX content in the solution decreased slowly from 0 to 3 days, and the RDX content in the solution decreased significantly after 3 days of cultivation. When the culture reached the 8th day, the concentration of RDX decreased from 16 mg / L to 1.2 mg / L. According to the formula of bacterial degradation rate (%) = (RDX content of uninoculated solution - RDX content of inoculated solution) / RDX content of uninoculated solution × 100, the degradation rate of RDX by the bacterial strain reached 92.5%.
[0034] Example 4: Tolerance of Sphingobacterium yanogamicin
[0035] The present invention determines the acid and alkali resistance, temperature resistance and salt resistance of Sphingobacterium yanostomum LXC-RD-4. The specific steps are: inoculate the seed liquid of the degradation bacteria into a liquid inorganic salt culture medium with an RDX concentration of 7 mg / L at an inoculum amount of 2%, control the variable to be unique, culture at 180r / min for 4 days under different pH (5, 6, 7, 8, 9), different temperatures (20°C, 25°C, 30°C, 35°C, 40°C) and different salinities (in terms of NaCl, the concentrations are 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, respectively), and measure the growth amount OD of the strain every 24 hours. 600 .
[0036] from Figure 5It can be seen that Sphingobacterium yanostomum cannot grow in acidic solutions, and its bacterial activity is low in alkaline solutions. Its optimal pH growth range is 7-8. When the temperature is 20-30℃, the biomass of the bacteria gradually increases with the increase of temperature; when the temperature is 30-40℃, the biomass of the bacteria decreases significantly with the increase of temperature. It can be seen that the optimal growth temperature of Sphingobacterium yanostomum is 30℃, and the biomass of the strain gradually decreases with the increase of salt content in the aqueous solution, and its salinity growth range is 0-10g / L.
Claims
1. A strain of Sphingobium yanoikuyae, characterized in that The name of the Sphingobacterium Yanoguchii is LXC-RD-4, which is deposited in the China General Microbiological Culture Collection Center. The deposit date is November 19, 2020, and the deposit number is: CGMCC NO.21207.
2. A microbial preparation containing the Sphingobacterium Yanoguchii according to claim 1.
3. Use of the Sphingobacterium Yanoorhiza described in claim 1 or the microbial preparation described in claim 2 in anti-RDX.
4. Use of the Sphingobacterium Yanoorhiza described in claim 1 or the microbial preparation described in claim 2 in degrading RDX.
5. Use of the Sphingobacterium Yanoguchii described in claim 1 or the microbial preparation described in claim 2 in purifying or repairing soil or industrial wastewater.
6. The use according to claim 5, characterized in that: The application is to degrade RDX.
7. A method for degrading RDX, characterized in that: The Sphingobacterium Yanoorhizium of claim 1 is added to a sample containing RDX and reacted at 30° C. for at least 3 days.
8. The method according to claim 7, characterized in that The amount of Sphingobacterium Yanoguchii added was 2%.
9. The method according to claim 7, characterized in that: The sample is an inorganic salt culture medium or soil.
10. The method according to claim 7, characterized in that The concentration of RDX is 16 mg / L.