Pseudomonas chlororaphis with function of degrading carmine acid and application thereof

Abstract

The application discloses a chlorophenol pseudomonas with a function of degrading carmine acid and an application thereof, and belongs to the technical field of microorganisms. The chlorophenol pseudomonas has a preservation number of GDMCC NO: 65476. The live bacteria and the cell-free extract of the strain can degrade carmine acid, and have a wide application prospect. The strain has the characteristics of wide pH adaptability and harmlessness to the environment, and industrial preparation of a microbial preparation by using the strain has the advantages of low cost, safe composition, natural energy saving and environmental protection, and can be used as a potential microbial resource in treatment of wastewater containing carmine acid dyes, and provides a new way for treatment of dye wastewater by using microorganisms.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a chlorophenol pseudomonas with the function of degrading carmine acid and application thereof. BACKGROUND

[0002] Carmine acid is a glycosylated anthraquinone compound. As a red colorant, it has the characteristics of heat resistance, light resistance and oxidation resistance, and is widely used in food, daily chemical, pharmaceutical and textile fields. At the same time, carmine acid also has the characteristics of high color development and poor color intensity, and is easy to be discharged with production water, gathered in a certain range, causing abnormal color of land and water area. In addition, the gathering of carmine acid in water body will hinder the diffusion of sunlight required in the process of photosynthesis, which is not conducive to aquatic plants and animals; the gathering in the 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 method of the textile printing and dyeing industry, physical or chemical methods are used for pretreatment of dye wastewater, which has high cost, large energy consumption and produces toxic sludge. Microbial degradation is an ecological friendly and cost-effective method, which can solve the bottleneck related to 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 for resource utilization, which is another open source approach to realize resource utilization. At the same time, the coupling of different microorganisms is conducive to carbon-nitrogen conversion, and reasonable microbial community structure and abundance determine the efficiency of wastewater treatment, so that wastewater treatment becomes a controllable switch.

[0004] Chlorophenol pseudomonas widely exists in soil and is often used for microbial degradation of phenolic compounds, which is a reliable biological resource. At present, there is no related report that chlorophenol pseudomonas can be used for degrading glycosylated anthraquinone compounds. SUMMARY

[0005] The purpose of the present application is to provide a chlorophenol pseudomonas with the function of degrading carmine acid and application thereof, in order to solve the problems existing in the prior art. The chlorophenol pseudomonas TR03 and its cell-free extract provided by the present application can both degrade carmine acid, have wide application prospects, and can be used as potential microbial resources in the treatment of dye wastewater containing carmine acid, thereby providing a new way for microbial treatment of dye wastewater.

[0006] To achieve the above purpose, the present application provides the following solutions.

[0007] The application provides a Pseudarthrobacter chlorophenolicus TR03 having the function of degrading carmine acid, which is preserved in the Guangdong Microbial Culture Collection Center on November 12, 2024, and has a preservation number of GDMCC NO: 65476.

[0008] The application also provides application of the Pseudarthrobacter chlorophenolicus TR03 and / or cell-free extract thereof in preparation of a wastewater treatment agent.

[0009] The application also provides a wastewater treatment agent, and active ingredients of the wastewater treatment agent comprise the Pseudarthrobacter chlorophenolicus TR03 and / or cell-free extract thereof.

[0010] The application also provides application of the wastewater treatment agent in degradation of carmine acid.

[0011] The application also provides application of the wastewater treatment agent in treatment of wastewater containing carmine acid.

[0012] The application also provides a method for treating wastewater containing carmine acid, which comprises the step of treating the wastewater by using the wastewater treatment agent.

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

[0014] Further, the temperature for treating the wastewater by using the wastewater treatment agent is 30-40 DEG C.

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

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

[0017] The application discloses the following technical effects:

[0018] The application provides a Pseudarthrobacter chlorophenolicus TR03 from soil, and the living bacteria and cell-free extract of the strain can degrade carmine acid, and have wide application prospects. The strain has the characteristics of wide pH adaptability and harmlessness to the environment, and industrial preparation of the microbial preparation by using the strain has the advantages of low cost, safe components, natural energy saving and environmental protection, and can be used as a potential microbial resource in treatment of wastewater containing carmine acid dyes, and provides a new way for treatment of dye wastewater by using the microbial method. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only illustrate some of the embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative effort.

[0020] Figure 1 Standard curve for determination of carminic acid by liquid chromatography;

[0021] Figure 2 Determination of degradation of carminic acid by different strains by liquid chromatography;

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

[0023] Figure 4 Study on the enzyme activity of cell-free extract of Pseudomonas chlororaphis TR03, wherein 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 Electrophoresis result of 16S rRNA amplification product of Pseudomonas chlororaphis TR03;

[0025] Figure 6 Phylogenetic tree of 16S rRNA of Pseudomonas chlororaphis TR03;

[0026] Figure 7 Growth curve of Pseudomonas chlororaphis TR03. DETAILED DESCRIPTION

[0027] The various illustrative embodiments of the present application will now be described in detail below. This detailed description is not intended to limit the present application, but rather to explain certain aspects, features, and embodiments of the present application.

[0028] It should be understood that the terms used in the present application merely describe particular embodiments, and are not intended to limit the present application. In addition, for the numerical ranges in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range within any stated range or within any stated intermediate value, as well as any other stated value or intermediate value within the stated range, is also included in the present application. The upper limit and the lower limit of these smaller ranges can be included or excluded independently from the range.

[0029] All technical and scientific terms used herein have, unless otherwise defined, the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned in this specification are herein incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. In case of conflict, the content of the present specification will control.

[0030] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative only.

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0032] Examples

[0033] 1. Preparation of culture media

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

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

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

[0037] Fermentation medium (W / V): 0.02% carmine acid, 2% anhydrous glucose, 1% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% monopotassium 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 region of Guangxi, 4 kinds of 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 ground, collect it into a sampling bag, label it and place it in a cool and ventilated place. After grinding and filtering the soil, 10g of soil was added to 90mL of LB medium. The medium was placed in a shaking flask and cultured for 12h (200rpm, 30°C), and the supernatant was diluted with physiological saline to 10 -1 -10 -6 concentrations, and 0.1μL of each concentration of diluted bacterial solution was evenly coated on the solid medium with carminic acid as the sole carbon source. The non-mold morphological strains with better growth state on the solid medium were picked with an inoculation loop and inoculated into the enrichment medium.

[0041] The enrichment bacterial solution was dipped with an inoculation loop and streaked on the screening medium, and the plate was inverted and cultured in a 37°C incubator until obvious single colonies appeared. The colonies with different morphologies were selected and further streaked on the purification medium. The non-mold morphological strains obtained by preliminary screening were enriched in LB medium and inoculated into 2% carminic acid fermentation medium for fermentation. Twelve strains capable of growing on carminic acid as the sole carbon source were screened from the soil samples, of which 6 strains showed carminic acid degradation ability in the fermentation medium, designated as TR01, TR02, TR03, TR04, TR05 and TR06.

[0042] 3. Screening of carminic acid-degrading strains

[0043] 3.1 Screening of carminic acid-degrading strains

[0044] Preparation of carminic acid standard curve: 4.063mg of carminic acid standard was weighed and diluted with purified water to 10mL. The solution was diluted to 0.875, 0.75, 0.5, 0.375, 0.25, 0.125 and 0.0625 times (prepared to 400mg / L, 350mg / L, 300mg / L, 200mg / L, 150mg / L, 100mg / L, 50mg / L and 25mg / L) of the original concentration. 1mL of the sample solution was taken with a 1mL syringe and filtered with a 0.22μm needle filter. The liquid phase was detected in high performance liquid chromatography. The liquid phase conditions were a. 0.1% formic acid: b. 0.1% formic acid acetonitrile = 89:11, flow rate 0.8mL / min, column temperature 40°C, detection wavelength 494nm (LC), injection volume 20μL. The standard curve is shown in Figure 1 .

[0045] 3.2 Measurement of fermentation broth

[0046] The 1 mL TR01, TR02, TR03, TR04, TR05 and TR06 bacterial liquid was inoculated into 100 mL LB medium for activation, and then the OD of the bacterial strain was adjusted to about 1.0. The adjusted mother liquor was inoculated into 1 mL carmine acid medium (fermentation medium (W / V): 0.02% carmine acid, 2% anhydrous glucose, 1% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate) for fermentation for 4 days. 600 The adjusted mother liquor was inoculated into 1 mL carmine acid medium (fermentation medium (W / V): 0.02% carmine acid, 2% anhydrous glucose, 1% ammonium sulfate, 0.05% dipotassium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate) for fermentation for 4 days.

[0047] The fermented carmine acid bacterial liquid and the blank control were each taken 1 mL with a 1 mL syringe, filtered with a 0.22 μm needle filter, and detected in high performance liquid. The liquid phase conditions were a. 0.1% formic acid: b. containing 0.1% formic acid acetonitrile = 89:11, flow rate 0.8 mL / min, column temperature 40°C, detection wavelength 494 nm (LC), sample size 20 μL.

[0048] The degradation rate of 6 strains of bacteria on carmine acid is shown in Table 1. Figure 2 Thus, three strains TR01, TR05 and TR03 with high carmine acid degradation ability were obtained, which participated in the following experiments.

[0049] 3.3 Collection of cell-free extract

[0050] The 16 h cultured bacterial strain was inoculated into LB medium at an inoculation amount of 1%, and continuously cultured in a 30°C, 200 rpm shaker for 16 h. Then the medium was centrifuged at 4°C, 8000 rpm for 20 min, washed twice with 20 mM pH 8 Tris-HCl, and resuspended in the same buffer. The resuspended bacterial liquid was destroyed by ultrasonic wave at 150 W for 10 min, the cell debris was removed by centrifugation (4°C, 8000 rpm, 10 min), and the cell-free extract, i.e. crude enzyme, was prepared by filtration through a 0.22 μm needle filter.

[0051] The crude enzyme of the above screened strain with carmine acid degradation ability was reacted with carmine acid, and the mixed reaction system was 200 μL crude enzyme liquid added with 20 μL 4.925 g / L carmine acid for 48 h. The degradation ability comparison of the mixed reaction system is shown in Table 2. Figure 3 Thus, it can be known that the strain TR03 has the best degradation efficiency, and the strain TR03 is taken as the experimental object for the next step experiment.

[0052] 3.4 Study on the enzyme activity of Pseudomonas chlororaphis TR03 cell-free extract

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

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

[0057] The experimental results are shown in Table 1. Figure 4

[0058] 4. 16S rRNA gene sequence analysis

[0059] 4.1 Template DNA extraction

[0060] The TR03 strain liquid stored in a 100 μL cryovial was inoculated in LB medium and cultured at 37℃, 200 rpm on a shaker for 6h. The strain was isolated and purified by plate streaking method, and after two passages, a single bacterium was picked up with a inoculation loop and transferred into 10% resin that had been sterilized. After vortexing to break up, it was placed in a 100℃ metal bath for 10min and centrifuged at 10000 rpm for 10min.

[0061] 4.2 PCR amplification of 16S rDNA

[0062] The total DNA of the strain was extracted by resin method, and the 16S rDNA sequence of the strain was amplified by PCR with 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 rTaq enzyme 0.6 μL, template DNA 2 μL, primer 27F and 1492R 1 μL each, 10 x Buffer 4 μL, dNTPs 2 μL, ddH2O 29.4 μL. PCR reaction program: 94°C pre-denaturation 5 min; 94°C denaturation 1 min, 56°C annealing 1 min, 72°C extension 3 min, 30 cycles; 72°C extension 5 min. The PCR product was subjected to agarose gel electrophoresis, and was sent to Shanghai Shenguo Biotechnology for sequencing, as shown in Figure 5 .

[0066] 4. Construction of 316S rDNA phylogenetic tree

[0067] The 16S rDNA sequence of the strain was used to construct a phylogenetic tree. The sequencing results were subjected to multiple sequence alignment on NCBI, and a phylogenetic tree was constructed on MEGA software, as shown in Figure 6 .

[0068] As shown in Figure 6 , strain TR03 was clustered with Pseudarthrobacter chlorophenolicus strain MRHB1-129, and had 98.57% homology with Pseudarthrobacter chlorophenolicus strain A6. Therefore, strain TR03 was identified as Pseudarthrobacter chlorophenolicus. The strain was deposited at Guangdong Microbial Culture Collection Center on November 12, 2024. The deposit address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the deposit number is GDMCC NO: 65476.

[0069] 5. Growth curve of Pseudarthrobacter chlorophenolicus TR03

[0070] Strain TR03 was inoculated in LB liquid medium at an inoculation amount of 2%, and was cultured in a 30°C, 200 rpm shaker. Samples were taken every 4 h, and the absorbance value of the fermentation broth at 600 nm was measured, and then the growth curve was plotted. The experimental results are shown in Figure 7 , and Pseudarthrobacter chlorophenolicus TR03 has excellent growth performance.

[0071] The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the protection scope of the present application as defined by the claims.

Claims

1. A Pseudarthrobacter chlorophenolicus TR03 strain having the function of degrading carmine acid, characterized in that, The Pseudomonas chloraphis TR03 is preserved in the Guangdong Microbial Culture Collection Center on November 12, 2024, and the preservation number is GDMCC NO: 65476.

2. The use of the Pseudomonas chloraphis TR03 in claim 1 in the preparation of a wastewater treatment agent, wherein the wastewater contains carmine acid.

3. A wastewater treatment agent, characterized by, The active ingredient comprises the Pseudomonas chloraphis TR03 in claim 1.

4. The use of the wastewater treatment agent in claim 3 in the degradation of carmine acid.

5. Use of the wastewater treatment agent according to claim 3 in treating wastewater, characterized by, The wastewater contains carmine acid.

6. A method of treating wastewater containing carmine acid, characterized by, The method comprises the step of treating the wastewater with the wastewater treatment agent in claim 3.

7. The method of claim 6, wherein, The pH value of the wastewater is 8-10.

8. The method of claim 6, wherein, The temperature when the wastewater treatment agent treats the wastewater is 30-40℃.

9. The method of claim 6, wherein, The wastewater contains Mg 2+ , Ba 2+ , or K + .

10. The method of claim 6, wherein, The wastewater contains dimethyl sulfoxide or methanol.

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