A strain of Sphingobium sp.CJW01 and its application and degradation agent

Abstract

The present invention provides a strain of Sphingobium sp.CJW01 and its application and degradation agent, belonging to the field of microbial technology. A strain of Sphingobium sp.CJW01 was deposited in the China Center for Type Culture Collection on October 18, 2023, with a deposit number of CCTCC NO:M20231919. The strain provided by the present invention degrades chlorine-containing pollutants for two days at a pH value of 6.0 to 8.0 and a temperature of 30°C, and the degradation rate reaches more than 80%.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Sphingobium sp.CJW01 and an application and a degradation agent thereof. Background Art

[0002] 1. Selecting specific microorganisms: The key to the success of single-bacteria degradation technology is to select a microbial strain that can effectively degrade a specific pollutant. This usually involves screening microorganisms in the natural environment to find the strain that can best degrade a specific pollutant.

[0003] 2. Optimization of metabolic pathways: Studying the metabolic pathways of microorganisms can help understand how to most effectively degrade pollutants. Through genetic engineering techniques, scientists are able to enhance the metabolic pathways of specific microorganisms to make them more efficient in processing pollutants.

[0004] 3. Application areas: Single bacterial degradation technology is widely used in soil and water remediation. For example, specific strains can be used to degrade environmental pollution caused by oil spills or to treat pesticide residues generated in agricultural activities.

[0005] In addition to environmental remediation, the technology can also be used for industrial wastewater treatment in bioreactors to remove harmful chemicals through biodegradation.

[0006] 4. Advantages and Challenges: Advantages: Environmentally friendly, can reduce the concentration and harmfulness of pollutants at the source, and reduce the need for chemical and physical treatment.

[0007] Challenges: Each pollutant may require a specific strain of microorganism to degrade it, and this specificity may limit the flexibility of treating multiple pollutants. In addition, environmental factors such as pH, temperature and nutrient supply also greatly affect the degradation efficiency of microorganisms.

[0008] 5. Future development: Scientists are exploring the creation of more effective microbial strains through synthetic biology and gene editing technologies, which have the potential to improve degradation efficiency and broaden the types of pollutants that can be treated. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a Sphingobium sp. CJW01 and its application and degradation agent. The Sphingobium sp. CJW01 provided by the present invention can degrade chlorine-containing pollutants.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a Sphingobium sp. CJW01, which was deposited in the China Center for Type Culture Collection on October 18, 2023, with a deposit number of CCTCC NO: M20231919.

[0012] Preferably, the chlorine-containing pollutant comprises chlorinated phenol.

[0013] The present invention also provides the use of Sphingobium sp. CJW01 described in the above technical solution in degrading chlorine-containing pollutants.

[0014] Preferably, the chlorine-containing pollutant comprises chlorinated phenol.

[0015] Preferably, the degradation conditions include: pH value of 6.0-8.0 and temperature of 30°C.

[0016] The present invention also provides a degradation agent for degrading chlorine-containing pollutants, which contains the Sphingobium sp. CJW01 described in the above technical solution.

[0017] Preferably, the effective viable count of Sphingobium sp. CJW01 in the degradation agent is preferably 1×10 9 CFU / mL.

[0018] The beneficial effects of the present invention are:

[0019] The strain provided by the invention degrades chlorine-containing pollutants for two days at a pH value of 6.0-8.0 and a temperature of 30° C., and the degradation rate reaches more than 80%.

[0020] Biological Deposit Description

[0021] Sphingobium sp.CJW01 was deposited on October 18, 2023 in the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO:M20231919. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a strain with a high similarity to this strain;

[0023] Figure 2 is the phylogenetic tree of the strain;

[0024] Figure 3 It is a bar graph and a line graph showing the PCP degradation of the strain in MSN medium containing 10 mg / LPCP (pentachlorophenol) and the bacterial growth;

[0025] Figure 4This is a data table showing the degradation of PCP by the strain in MSN medium containing 10 mg / LPCP (pentachlorophenol) and the growth of bacteria. DETAILED DESCRIPTION

[0026] The present invention provides a Sphingobium sp. CJW01, which was deposited in China Center for Type Culture Collection on October 18, 2023, with a deposit number of CCTCC NO: M20231919. In the present invention, the chlorine-containing pollutant preferably includes chlorinated phenol.

[0027] The present invention also provides the use of Sphingobium sp.CJW01 described in the above technical solution in the degradation of chlorine-containing pollutants. In the present invention, the chlorine-containing pollutants preferably include chlorine-containing phenols. In the present invention, the degradation conditions preferably include: pH value of 6.0-8.0, temperature of 30°C.

[0028] The present invention also provides a degradation agent for degrading chlorine-containing pollutants, comprising Sphingobium sp. CJW01 as described in the above technical solution. In the present invention, the effective viable count of Sphingobium sp. CJW01 in the degradation agent is preferably 1×10 9 CFU / mL.

[0029] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] Example 1

[0031] Isolation and identification of strains:

[0032] Bacterial isolation was performed from deep-sea mud in Shenzhen, my country. About 1.0 g of mud was mixed with 100 mL of MSN medium spiked with 100 mg / L PCP and M9 medium consisting of 0.85 g / L NaNO3, 17.1 g / L Na2HPO4·12H2O, 3 g / L KH2PO4, 0.5 g / L NaCl and 0.1 g / LMgSO4·7H2O at 30 °C at pH 7.2±0.1. The enriched culture was transferred to new R2A medium containing 100 mg / L PCP and incubated under the same conditions. For efficient isolation of candidate bacteria in PCP degradation, another round of transfer was performed from the medium spiked with 100 mg / L PCP to the medium spiked with 200 mg / L PCP. After such continuous cultivation, 50 μL of the established suspension was spread on R2A agar medium containing 500 mg / L PCP as the sole carbon source and incubated at 30°C. All colonies with different morphologies were picked out and incubated three more times to obtain pure strains.

[0033] All isolates were transferred to R2A liquid medium containing tryptone 0.25g / L acid hydrolyzed casein 0.5g / L yeast extract 0.5g / L soluble starch 0.5g / L dipotassium hydrogen phosphate 0.3g / L magnesium sulfate 0.1g / L sodium pyruvate 0.3g / L peptone 0.25g / L glucose 0.5g / L, and continued at 30°C, 150rpm for 24 hours to obtain sufficient bacterial biomass. Finally, the bacterial suspension was mixed with 50% glycerol (1:1, v:v) and stored at 20°C before identification. In order to observe the microscopic morphology of bacteria, the isolates were inoculated in R2A medium at 30°C, 150rpm for 24 hours. The bacterial suspension was centrifuged at 10000rpm for 5 minutes and fixed in 2.5% (v:v) glutaraldehyde for 4 hours. After centrifugation at 8000 rpm for 5 hours, the supernatant was decanted and washed three times with phosphate buffer solution (PBS, 19 mL 0.2 M NaH2PO4 + 81 mL 0.2 M Na2HPO4, pH 7.4).

[0034] The established biomass was dehydrated by ethanol with increasing ethanol concentrations of 30%, 50%, 70%, 85%, 90% (15 min each rinse) and 100% (twice, 15 min each rinse). To remove residual ethanol, the samples were thoroughly mixed with isoamyl acetate and centrifuged at 8000 rpm for 5 min. After continuous refrigeration at 20°C, 40°C and 80°C every 12 h, the bacterial biomass was freeze-dried on a vacuum freeze dryer for 12 h. After gold spraying treatment, the microscopic morphology of the strains was observed by scanning electron microscopy (. The isolates were incubated in R2A medium at 30°C, 150 rpm for 24 h. After centrifugation, the samples were centrifuged using Bacterial biomass was collected for DNA extraction using a bacterial DNA kit. The 16S rRNA gene was amplified by PCR using universal primers 27F and 1492R.

[0035] PCR amplification was performed as follows: initial denaturation at 95°C for 5 min, followed by 25 cycles at 95°C for 30 s, 56°C for 30 s and 72°C for 90 s, and finally extension for 10 min at 72°C. Once purified, the PCR products were sequenced by Shanghai Biotech Co., Ltd. The 16S rRNA gene sequence was analyzed by BLAST search against known sequences in the NCBI database and submitted to the GenBank database with accession number OQ621408. According to the alignment results of the EzBioCloud 16S database (such as Figure 1 The target sequence was further confirmed by comparing the target sequence with the following bacterial strains of the genus Sphingobium:

[0036] Sphingobium amiense NBRC 102518(T), with a similarity of 96.74%. Classification information: Proteobacteria>Alphaproteobacteria>Sphingomonadales>Sphingomonadaceae>Sphingobium.

[0037] Sphingobiumphenoxybenzoativorans SC_3(T), similarity 96.55%. Classification information: Same as above.

[0038] Sphingobium olei IMMIB HF-1(T), similarity 96.65%. Classification information: Same as above.

[0039] Sphingobium fontiphilum Chen 16-4(T), similarity 96.55%. Classification information: Same as above.

[0040] Sphingobium nicotianae H33 (TMZ266333), similarity 96.52%. Classification information: Same as above.

[0041] Sphingobium cloacaeNBRC 102517(T), similarity 96.24%. Classification information: Same as above.

[0042] Sphingobium limneticum JN591313(T), similarity 96.24%. Classification information: Same as above.

[0043] These results show that the target sequence has a high similarity with multiple strains of the genus Sphingobium, all of which are above 96%. Based on these similarity data, the target sequence is likely to belong to a known strain of the genus Sphingobium.

[0044] Finally, the phylogenetic tree of 16S rRNA gene was constructed based on the neighbor joining algorithm in MEGA6.0, as shown in Figure 2 As shown, it was determined that the strain belonged to the genus Sphingobium, and CJW-1 was the strain CJW01 of the present invention.

[0045] Example 2

[0046] Verify the strain's ability and effect in degrading PCP.

[0047] The strain CJW01 obtained in Example 1 was streaked onto an R2A solid plate for activation, and the grown single colonies were inoculated into R2A liquid culture medium after about 24 hours, and the seed solution was obtained by overnight culture at 30°C and 150rpm. Then, the collected biomass was washed three times with PBS and resuspended in sterile water. An aliquot of the suspension was transferred to an MSN culture medium containing 10 mg / L PCP (pentachlorophenol) to produce a final OD600 of 0.01, and cultured for 24 hours at 30°C and 150rpm. During the culture process, OD600 was recorded regularly by a spectrophotometer to assess bacterial growth. After the culture was completed, the biomass was collected and extracted. The culture solution was centrifuged at 10000rpm for 5 minutes, and after collecting the supernatant, it was extracted three times with an equal volume of ethyl acetate to remove impurities in the culture medium. Subsequently, the solution was evaporated at 40°C to remove ethyl acetate, and the residue was resuspended in 2mL 50% methanol (v:v). Centrifuge again at 10000rpm for 5 minutes, and filter the collected supernatant through a 0.22μm filter membrane. Finally, the concentration of PCP was determined by high performance liquid chromatography (HPLC). Reverse phase separation was performed using a ZORBAX SB-C18 column (particle size 5μm, pore size 4.6×150mm), and the conditions for HPLC analysis included a flow rate of 0.3mL / min, an ultraviolet wavelength of 247nm, and a mobile phase consisting of 100% methanol (solvent A) and 0.1% (v:v) formic acid (solvent B) in a ratio of 30:70.

[0048] The results are as follows Figure 3 , Figure 4 As shown in the figure, strain CJW01 showed good growth during the culture process, and the OD600 data measured by the spectrophotometer showed that it grew rapidly. During the extraction and analysis stage, the PCP concentration results measured by HPLC showed that strain CJW01 could effectively degrade PCP in the culture medium. The specific degradation rate and time relationship will be listed in detail in the subsequent data. This shows that strain CJW01 has good PCP degradation ability under specific conditions and is suitable for environmental pollution control.

[0049] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A Sphingobium sp. CJW01, characterized in that: It was deposited in the China Center for Type Culture Collection on October 18, 2023, with the deposit number CCTCCNO:M20231919.

2. Use of the sphingobacterium CJW01 described in claim 1 in degrading pentachlorophenol.

3. The use according to claim 2, characterized in that: The degradation conditions include: pH value of 6.0-8.0 and temperature of 30°C.

4. A degradation agent for degrading pentachlorophenol, characterized in that: Contains the sphingobacterium CJW01 according to claim 1.

5. The degradation agent according to claim 4, characterized in that The effective viable bacteria count of Sphingobacterium CJW01 in the degradation agent is 1×10 9 CFU / mL.

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