Pseudomonas chlorophenolate with carmine degrading function and application of pseudomonas chlorophenolate

Abstract

The invention discloses pseudomonas chlorophenolate with a carmine degrading function and application of the pseudomonas chlorophenolate, and belongs to the technical field of microorganisms. The preservation number of the pseudomonas chlorophenol is GDMCC (China General Microbiological Culture Collection Center) NO: 65476. The viable bacteria and cell-free extracts of the strain can degrade carmine acid, and have wide application prospects. The strain has the characteristics of broad-spectrum pH adaptability and no harm to the environment, has the advantages of being low in cost, safe in component, natural, energy-saving, environment-friendly and the like when being used for industrially preparing a microbial preparation, can be used as a potential microbial resource in treatment of waste water containing carmine acid dye, and provides a new way for treating the waste water through a microbiological method.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a strain of Pseudomonas chlorophenolicus capable of degrading carminic acid and application thereof. Background Art

[0002] Carminic acid is a glycosylated anthraquinone compound. As a red colorant, it has the characteristics of heat resistance, light resistance and antioxidant, and is widely used in food, daily chemicals, pharmaceuticals, textiles and other fields. At the same time, carminic acid also has the characteristics of high color rendering and poor coloring. It is easily discharged with production water and accumulates within a certain range, causing abnormal color of land and water. In addition, the accumulation of carminic acid in water bodies will hinder the diffusion of sunlight required for the photosynthesis process, which is detrimental to aquatic plants and animals; accumulation in land may also block the pores of the soil, resulting in the loss of soil productivity. Therefore, it is necessary to explore innovative and environmentally friendly water treatment methods for the textile printing and dyeing industry.

[0003] In the water treatment methods of the textile printing and dyeing industry, physical or chemical methods are used to pre-treat dye wastewater, which is costly, energy-intensive, and produces toxic sludge. Microbial degradation is an eco-friendly and cost-effective method that can solve the bottlenecks associated with the physical and chemical treatment of dye wastewater. Using microorganisms to treat industrial wastewater, by participating in the degradation of natural compounds and some artificial compounds, the resulting material resources are returned to nature, which is another open-source way to achieve resource utilization. At the same time, the coupling of different microorganisms is beneficial to carbon-nitrogen conversion. The reasonable structure and abundance of microbial communities determine the efficiency of sewage treatment, making sewage treatment a controllable switch.

[0004] Pseudomonas chlorophenols is widely found in soil and is often studied for the microbial degradation of phenolic compounds. It is a reliable biological resource. However, there are no reports on the use of Pseudomonas chlorophenols for the degradation of glycosylated anthraquinone compounds. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Pseudomonas chlorophenols having the function of degrading carminic acid and its application, so as to solve the problems existing in the above-mentioned prior art. The Pseudomonas chlorophenols TR03 and its cell-free extract provided by the present invention can degrade carminic acid, have broad application prospects, can be used as a potential microbial resource in the treatment of carminic acid-containing dye wastewater, and provide a new way for microbial method to treat dye wastewater.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a strain of Pseudarthrobacter chlorophenolicus TR03 having the function of degrading carminic acid. The Pseudarthrobacter chlorophenolicus TR03 was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 12, 2024, with a deposit number of GDMCC NO: 65476.

[0008] The present invention also provides the use of the Pseudomonas chlorophenols TR03 and / or its cell-free extract in preparing a wastewater treatment agent.

[0009] The present invention also provides a wastewater treatment agent, wherein the active ingredients include the Pseudomonas chlorophenols TR03 and / or a cell-free extract thereof.

[0010] The invention also provides application of the wastewater treatment agent in degrading carminic acid.

[0011] The invention also provides application of the wastewater treating agent in treating wastewater, wherein the wastewater contains carminic acid.

[0012] The invention also provides a method for treating wastewater containing carminic acid, comprising the step of treating the wastewater with the wastewater treating agent.

[0013] Furthermore, the pH value of the wastewater is 8-10.

[0014] Furthermore, the temperature of the wastewater treatment agent when treating the wastewater is 30°C to 40°C.

[0015] Furthermore, the wastewater contains Mg 2+ , Ba 2+ or K + .

[0016] Furthermore, the wastewater contains dimethyl sulfoxide or methanol.

[0017] The present invention discloses the following technical effects:

[0018] The invention provides a soil-derived Pseudarthrobacterchlorophenolicus TR03, the live bacteria and cell-free extract of which can degrade carminic acid, and has broad application prospects. The strain has a broad spectrum of pH adaptability and is harmless to the environment. The industrial preparation of microbial preparations using the strain has the advantages of low cost, safe ingredients, natural energy saving and environmental protection, etc. The strain can be used as a potential microbial resource in the treatment of carminic acid-containing dye wastewater, and provides a new approach for treating dye wastewater by microbial method. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is the standard curve for measuring carminic acid standard by liquid chromatography;

[0021] Figure 2 The liquid chromatography method was used to measure the degradation of carminic acid by different strains;

[0022] Figure 3 Comparison of the degradation rates of carminic acid by cell-free extracts of different strains;

[0023] Figure 4 This is a study on the enzyme activity of the cell-free extract of Pseudomonas chlorophenols TR03, where A is the effect of different pH on the enzyme activity, B is the effect of different metal ions on the enzyme activity, C is the effect of different temperatures on the enzyme activity, and D is the effect of different organic solvents on the enzyme activity;

[0024] Figure 5 This is the electrophoresis result of 16SrRNA amplification product of Pseudomonas chlorophenols TR03;

[0025] Figure 6 Phylogenetic tree constructed for the 16SrRNA of Pseudomonas chlorophenols TR03;

[0026] Figure 7 This is the growth curve of Pseudomonas chlorophenols TR03. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example

[0033] 1. Preparation of culture medium

[0034] Enriched medium (W / V): Luria-Bertani medium.

[0035] Screening medium (W / V): 0.1% carminic acid, 0.05% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, and 0.05% magnesium sulfate.

[0036] Purification medium (W / V): 0.1% carminic acid, 0.05% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate and 2% agar powder.

[0037] Fermentation medium (W / V): 0.02% carminic acid, 2% anhydrous glucose, 1% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate and 0.05% magnesium sulfate.

[0038] The above culture media were sterilized at 121°C for 15 min.

[0039] 2. Isolation and purification of strains

[0040] From the organic matter-rich area of ​​Guangxi, a total of 4 soil samples (sugar factory waste pool sludge, flower bed soil, rice field sludge and orchard soil) were collected as samples. The collection method was to select 200g of soil 5-20cm deep from the surface, collect it in a sampling bag, label it and place it in a cool and ventilated place. After grinding and filtering the soil, weigh 10g of soil and add it to 90mL of LB culture medium. The culture medium was placed in a shake flask for 12h (200rpm, 30℃), and the supernatant was diluted to 10 with physiological saline. -1 —10 -6 Concentration, take 0.1 μL of diluted bacterial solution of each concentration and evenly spread it on the solid medium with carminic acid as the sole carbon source. Use an inoculation loop to pick the non-mold morphological strain with good growth status on the solid medium and inoculate it into the enrichment medium.

[0041] Dip the enriched bacterial solution with an inoculation loop, streak it on the screening medium, and invert the plate in a 37°C incubator until a distinct single colony appears. Select colonies with different morphologies and further streak them on the purification medium. The non-mold morphological strains were initially screened and enriched in LB medium, and then 2% was inoculated into the carminic acid fermentation medium for fermentation. Twelve strains that can grow on the carminic acid sole carbon source medium were isolated and screened from the soil samples, of which 6 strains showed carminic acid degradation ability in the fermentation medium, recorded as TR01, TR02, TR03, TR04, TR05 and TR06.

[0042] 3. Screening of bacterial strains with carminic acid degradation ability

[0043] 3.1 Screening of carminic acid-reducing strains

[0044] Drawing of the standard curve of carminic acid: Weigh 4.063 mg of carminic acid standard, dilute to 10 mL with pure water, and dilute it to 0.875, 0.75, 0.5, 0.375, 0.25, 0.125 and 0.0625 times the original value (configured into 400 mg / L, 350 mg / L, 300 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 50 mg / L and 25 mg / L). Use a 1 mL syringe to draw 1 mL of the standard solution, filter with a 0.22 μm syringe filter, and detect in a high performance liquid phase. The liquid phase conditions are a. 0.1% formic acid: b. acetonitrile containing 0.1% formic acid = 89:11, flow rate 0.8 mL / min, column temperature 40°C, detection wavelength 494 nm (LC), injection volume 20 μL. The drawn standard curve is as follows Figure 1 shown.

[0045] 3.2 Measurement of fermentation broth

[0046] Take 1 mL of TR01, TR02, TR03, TR04, TR05 and TR06 bacterial suspension and inoculate into 100 mL LB medium for activation, then adjust the strain OD 600 Around 1.0. Take 1 mL of the adjusted mother solution and inoculate it into carminic acid medium (fermentation medium (W / V): 0.02% carminic acid, 2% anhydrous glucose, 1% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate) and ferment for 4 days.

[0047] Take 1 mL of fermented carminic acid bacteria liquid and blank control with a 1 mL syringe, filter with a 0.22 μm syringe filter, and detect in high performance liquid chromatography. The liquid phase conditions are a. 0.1% formic acid: b. acetonitrile containing 0.1% formic acid = 89:11, flow rate 0.8 mL / min, column temperature 40°C, detection wavelength 494 nm (LC), injection volume 20 μL.

[0048] The results of the experimental study on the degradation rate of carminic acid by 6 strains of bacteria are shown in Figure 2 Thus, three strains of bacteria with higher carminic acid-reducing ability, TR01, TR05 and TR03, were obtained and used in the following experiments.

[0049] 3.3 Collection of cell-free extracts

[0050] The strain cultured for 16 hours was inoculated into LB medium at 1% inoculum, and cultured continuously for 16 hours in a shaker at 30°C and 200 rpm, then the culture medium was centrifuged at 4°C and 8000 rpm for 20 minutes, washed twice with 20 mM Tris·HCl, pH 8, and resuspended in the same buffer. The resuspended bacterial solution was ultrasonically destroyed at 150 W for 10 minutes, and the cell debris was removed by centrifugation (4°C, 8000 rpm, 10 minutes), and filtered through a 0.22 μm syringe filter to prepare a cell-free extract, i.e., a crude enzyme.

[0051] The crude enzyme of the strain with carminic acid degradation ability screened above was reacted with carminic acid. The mixed reaction system was 200 μL crude enzyme solution plus 20 μL 4.925 g / L carminic acid for 48 h. The degradation ability of the mixed reaction system was compared as follows: Figure 3 As shown in the figure, it can be seen that strain TR03 has the best degradation efficiency, and strain TR03 is used as the experimental object for the next experiment.

[0052] 3.4 Study on the Enzyme Activity of Pseudomonas chlorophenols TR03 Cell-free Extract

[0053] Effect of different pH values: The mixed reactants were placed in different pH substrates (pH 6, 7, 8, 9 and 10) at room temperature for 4 days, and the absorbance at 494 nm was measured. The reaction system was: 1 mL pH gradient buffer, 400 μL cell-free extract and 20 μL 4.925 g / L carminic acid solution.

[0054] Effect of different metal ions: The mixed reactants were placed in different metal ion solutions (KCl, BaCl2 and MgCl2) at room temperature for 4 days, and the absorbance at 494nm was measured. The reaction system was: 20μL of each metal solution was added with 400μL of cell-free extract and 20μL of 4.925 g / L carminic acid solution.

[0055] Effect of different temperatures: The mixed reactants were placed at different temperatures (20℃, 30℃, 40℃, 50℃ and 60℃) for 4 days, and the absorbance at 494nm was measured. The reaction system was: 400μL cell-free extract and 20μL 4.925 g / L carminic acid solution.

[0056] Effect of different organic solvents: The mixed reactants were placed in different organic solvents (methanol, ethanol and dimethyl sulfoxide) for 4 days at room temperature, and the absorbance at 494 nm was measured. The reaction system was: 20 μL of each organic solvent was added with 400 μL of cell-free extract and 20 μL of 4.925 g / L carminic acid solution.

[0057] The experimental results are as follows Figure 4 shown.

[0058] 4. 16S rRNA gene sequence analysis

[0059] 4.1 Template DNA extraction

[0060] The TR03 strain liquid stored in 100 μL cryopreservation tube was inoculated into LB medium and cultured in a shaker at 37°C and 200 rpm for 6 hours. The strain was separated and purified by the plate streak method. After two subcultures, a single bacterium was picked up with an inoculation loop and transferred to a 10% resin that had been sterilized. After being broken by vortex oscillator, it was placed in a 100°C metal bath for 10 minutes and centrifuged in a 10,000 rpm centrifuge for 10 minutes.

[0061] 4.2 PCR amplification of 16S rDNA

[0062] The total DNA of the strain extracted by the resin method was used as a template, and the 16S rDNA sequence of the strain was amplified by PCR using 16S rDNA universal primers. Primer sequence:

[0063] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1;

[0064] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3', SEQ ID NO. 2.

[0065] PCR reaction system: 40 μL PCR reaction system contains 0.6 μL rTaq enzyme, 2 μL template DNA, 1 μL primers 27F and 1492R, 4 μL 10× Buffer, 2 μL dNTPs, and 29.4 μL ddH2O. The PCR reaction program is: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 1 min, 56℃ annealing for 1 min, 72℃ extension for 3 min, 30 cycles; 72℃ extension for 5 min. The PCR products were subjected to agarose gel electrophoresis and sent to Shanghai Biotech for sequencing. Figure 5 shown.

[0066] 4.316S rDNA phylogenetic tree construction

[0067] The phylogenetic tree was constructed using the strain 16S rDNA sequence. The sequencing results were aligned on NCBI and the phylogenetic tree was constructed using MEGA software, as shown in Figure 6 shown.

[0068] Depend on Figure 6 It can be seen that strain TR03 and Pseudarthrobacter chlorophenolicus strain MRHB1-129 are clustered together, and the homology with Pseudarthrobacter chlorophenolicus strain A6 reaches 98.57%. Therefore, strain TR03 is identified as Pseudarthrobacter chlorophenolicus, which was deposited in Guangdong Microbial Culture Collection Center on November 12, 2024. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC NO: 65476.

[0069] 5. Growth curve of Pseudomonas chlorophenols TR03

[0070] The strain TR03 was inoculated into LB liquid medium at a 2% inoculation rate, and cultured in a shaking incubator at 30°C and 200 rpm. Samples were taken every 4 hours, and the absorbance of the fermentation liquid at 600 nm was measured, and then a growth curve was drawn. The experimental results are shown in Figure 7 , Pseudomonas chlorophenols TR03 has excellent growth performance.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Pseudarthrobacterchlorophenolicus TR03 having the function of degrading carminic acid, characterized in that: The Pseudomonas chlorophenols TR03 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on November 12, 2024, with the collection number GDMCC NO: 65476.

2. Use of Pseudomonas chlorophenols TR03 and / or its cell-free extract as claimed in claim 1 in preparing a wastewater treatment agent.

3. A wastewater treatment agent, characterized in that The active ingredient comprises the Pseudomonas chlorophenols TR03 and / or a cell-free extract thereof as described in claim 1.

4. Use of the wastewater treatment agent as claimed in claim 3 in degrading carminic acid.

5. The use of the wastewater treatment agent according to claim 3 in treating wastewater, characterized in that: The wastewater contains carminic acid.

6. A method for treating wastewater containing carminic acid, characterized in that: The method comprises the step of treating the wastewater using the wastewater treating agent according to claim 3.

7. The method according to claim 6, characterized in that The pH value of the wastewater is 8-10.

8. The method according to claim 6, characterized in that The temperature of the wastewater treatment agent when treating the wastewater is 30°C to 40°C.

9. The method according to claim 6, characterized in that The wastewater contains Mg 2+ , Ba 2+ or K + .

10. The method according to claim 6, characterized in that The wastewater contains dimethyl sulfoxide or methanol.

Images